key: cord- -dp im authors: otting, nel; doxiadis, gaby g. m.; bontrop, ronald e. title: definition of mafa-a and -b haplotypes in pedigreed cynomolgus macaques (macaca fascicularis) date: - - journal: immunogenetics doi: . /s - - - sha: doc_id: cord_uid: dp im the major histocompatibility complex (mhc) class i b gene/allelic repertoire was investigated in a pedigreed population of cynomolgus macaques of mixed indonesian/malaysian origin. the mafa-b alleles detected in this cohort are mostly specific for a given geographic area, and only a small number of alleles appears to be shared with other populations. this suggests the fast evolution of mafa-b alleles due to adaptation to new environments. in contrast to humans, the b locus in old world monkeys displays extensive copy number variation. the mafa-b and previously defined -a gene combinations segregate in families and thus allowed the definition of extended haplotypes. in many cases it was possible to assign a particular mafa-i allele to one of these mafa-a/b haplotypes as well. the presence of a large number of stable haplotypes in this cohort of animals, which was pedigreed for up to eight generations, looks promising for developing discriminative mhc typing tools that are less cumbersome. furthermore, the discovery of unreported mafa-b sequences expands the lexicon of alleles significantly, and may help in understanding the complex organisation of the macaque b region. the cynomolgus macaque (macaca fascicularis), also known as the crab-eating or long-tailed macaque, is widely used as an animal model in biomedical studies. currently this species is applied as often as the commonly used rhesus macaque (macaca mulatta). cynomolgus monkeys are used as models for infectious diseases, such as aids, sars and tuberculosis, as well as for transplantation research (mcauliffe et al. ; wiseman et al. ; aoyama et al. ; mee et al. ; reed et al. ). owing to use of macaques in immune-related research, thorough investigations of their major histocompatibility complexes (mhc) are required. the mhc represents a multigene family in which the proteins play a key role in the generation of adaptive immune responses in vertebrate species. the class i and ii genes of the mhc display abundant polymorphism that has a profound impact on features such as disease susceptibility, organ transplantation, and reproduction success. the mhc systems in humans (hla) and in other primate species have been studied extensively (bontrop ) . the orthologues of the classical hla-a and -b genes, which are involved in the presentation of intracellularly processed peptides to cytotoxic t cells, are present in the rhesus and cynomolgus macaque (boyson et al. ; krebs et al. ) . however, in these animals the genes have undergone several rounds of duplication and display copy number variation (anzai et al. ; daza-vamenta et al. ; otting et al. ) . whereas in humans only one copy of the hla-a and -b genes is present, in macaques seven a-like genes are distinguished. on each haplotype, one polymorphic gene is observed, named mamu-a or mafa-a , in combination with one or two oligomorphic genes designated mamuor mafa-a up to -a , respectively (otting et al. ; pendley et al. ; campbell et al. ; kita et al. ). the same organisation is also applicable to the pigtailed macaque (macaca nemestrina) (lafont et al. ; wu et al. ) . the situation for the hla-b orthologues in macaque species is even more complicated. in one rhesus macaque, the mhc region was completely sequenced, yielding one complete haplotype of . megabase-pairs. on this haplotype, distinct mamu-b genes were present, of which genes have the potential to code for bonafide proteins (anzai et al. ; daza-vamenta et al. ; bonhomme et al. ; doxiadis et al. ). for the mhc of cynomolgus macaque a bac-based contig map was constructed (watanabe et al. ) . although the degree of gene multiplication is less than in the rhesus macaque, this contig map still contains distinct mafa-b like loci. sequencing studies at the cdna level, however, have shown that only two or three genes per haplotype are transcribed at considerable levels (majors) in rhesus-and in cynomolgus macaques (krebs et al. ; otting et al. otting et al. , pendley et al. ) . at least one other b-like gene, characterised by low levels of polymorphism and transcription (minors), is present on all haplotypes. it has been designated mamu-i, mafa-i, and mane-i in the respective species of macaques (urvater et al. ; robinson et al. ) . on the completely sequenced mhc-region of the rhesus macaque, this locus is designated as the mamu-b gene (daza-vamenta et al. ; doxiadis et al. ). the sequencing of macaques from different geographic areas has shown that each population has its own characteristic set of mamu/mafa-a and -b alleles, and only a few alleles are shared between cohorts/populations (krebs et al. ; otting et al. ; campbell et al. ). this is in contrast to the data that were observed for the mhc class ii sequences obtained from these species (otting et al. ; doxiadis et al. ; o'connor et al. ; de groot et al. ) . moreover, the interspecies sharing of mhc class i alleles in rhesus and cynomolgus macaques is in the same order of magnitude as the intraspecies sharing (otting et al. ) . we have access to cynomolgus macaques that have been pedigreed for eight generations, and the origin of the animals was determined based on mtdna analyses. in an earlier study, we showed that the mafa-a alleles are mostly unique for this population. hence, a unique set of mafa-b alleles is expected to be present in the same animals. the question is whether these b-alleles segregate in a stable linkage to the already described mafa-a sequences in these animals. should this be the case, mhc typing on this cohort may be then performed using less cumbersome techniques: for instance, those based on microsatellite or snp analyses. furthermore, expanding the lexicon of mafa-b alleles may provide more insight into the organisation of the macaque b-region, and may help in the definition of different lineages and loci, resulting in a more appropriate nomenclature. the cynomolgus macaques used in this study had originally been kept at the university of utrecht, where the animals were housed in social groups for up to eight generations. recently, however, the colony of animals was transferred to the new facilities at the biomedical primate research centre (bprc), for the purpose of behavioural studies. the bprc had access to blood samples drawn during health-checks, and lymphoblastoid cell-lines were established. the origin of the animals was determined by mitochondrial dna ( s rrna) analyses (doxiadis et al. ; , and the founder animals appear to have originated either in the indonesian islands or in continental malaysia. cdna, cloning, and sequencing for all animals used in this study rna was isolated from lymphoblastoid b-cells (rneasy kit, qiagen) and subjected to one-step rt-pcr, as recommended by the supplier (qiagen or promega). the primers ′mbs: aattcatggcgccccgaaccctcctcctgc and ′ mbs: ctagaccacacaagacagttgtctcag were used that anneal specifically to mhc-b transcripts in macaques (boyson et al. ) . furthermore, for a subset of the animals the generic class i primers ′ ggactca g a a t c t c c c c a g a c g c c g a g a n d ′ tctcagtccctcacaaggcagctgtc were used. the final elongation step was extended to min to generate a ′da overhang. the rt-pcr products were cloned using the inst/aclone kit (fermentas) or the pcr cloning kit (qiagen). after transformation, to colonies were picked for plasmid isolation. sequencing reactions were performed using the bigdye terminator cycle sequencing kit, and samples were run on an automated capillary sequencing system (applied biosystems genetic analyzer ). sequences were analysed using sequence navigator software version . . (applied biosystems) and macvector™ version . . (oxford molecular group), followed by manual adjustments. after the alignments of all mamuand mafa-b exon - sequences using the macvector software, version . . , phylogenetic analysis was performed with the phylogeny.fr pipeline (dereeper et al. ) using maximum likelihood (ml) of the software phyml . with the substitution model hky with categories, the gamma shape parameter of . , a transition/transversion ratio of . , and a sh-like approximate likelihood-ratio test (alrt) for statistical test of branch support. for tree rendering the pipeline uses the program treedyn , and the output tree is rooted using the mid-point rooting method. the mafa-b alleles have been named according to published nomenclature proposals (klein et al. ; ellis et al. ) ; however, the number of mafa-b lineages has exceeded , and for the lineage numbers three digits have been introduced. in this document, the designations that were published previously are extended with a zero, and contain five ciphers. two alleles that are highly similar receive the same lineage number, but any difference is indicated by the allele number (fourth and fifth cipher). if two alleles have synonymous base-pair differences, they receive an identical allele number, and the difference is indicated by a sixth and seventh digit. for example, mafa-b* has synonymous differences in comparison to mafa-b* and non-synonymous ones as compared to mafa-b* . the novel alleles were submitted to the embl-ebi database (accession numbers fm -fm , fm -fm , fn , fn , and fn ) and to the non-human primate section of the imgt/mhc immuno polymorphism database (robinson et al. ) . in the cohort of cynomolgus macaques, three to eight different mafa-b sequences per animal were detected, with varying levels of transcription. the sequences, of which at least three identical clones were present, were reported as alleles. in most cases, these alleles were also confirmed in different animals. in total, mafa-b alleles were present, of which were previously described by other research groups (uda et al. ; lafont et al. ; pendley et al. ; wu et al. ; campbell et al. ; kita et al. ). the other sequences have been submitted to embl-ebi and to the mhc-nhp database (robinson et al. ) , and have been catalogued. a list of the mafa-b alleles is provided, including the accession numbers, and the reference animals (table ) . in most animals, sequences that are alleles of the mafa-i gene, the equivalent of the oligomorphic mamu-i locus, were detected (urvater et al. ) . the mamu-i/mafa-i locus has the characteristics of a nonclassical, with low levels of polymorphism and transcription. only mafa-i sequences met the criterion of three identical clones, and eight of them have been submitted as novel alleles (table ) . nevertheless, the mafa-i gene appears to be present on all haplotypes. the mhc-a region-derived class i alleles of the animals in this cohort were sequenced in an earlier study (otting et al. ). in those analyses, primers were used that are specific for mhc-a alleles in macaques. however, in some animals the mafa-a locus was not amplified by this primer set. in the present study, rt-pcr was performed with macaque mhc-b-specific primers, and with the generic class i primers for those animals previously lacking a mafa-a sequence. the use of these generic primers resulted in the detection of six new alleles for the mafa-a locus, and one for both the mafa-a and the -a genes (table ) , and as such extends the earlier reported data. since pedigree data were available, it was possible to determine the combinations of mafa-b alleles on one chromosome (haplotype). most haplotypes contain two major alleles, in combination with one or two alleles with lower levels of transcription, or minors, as based on the number of picked clones within a pcr sample. unfortunately, it can not be excluded that some alleles are incorrectly considered as minors due to primer inconsistencies. haplotypes with only one or three majors were also observed. moreover, it was possible to extend these mafa-b combinations with specific -a region configurations/haplotypes that were described in an earlier study (otting et al. ). combinations of mafa-a and -b alleles that are segregating, and are observed in at least two related animals are listed (table ) . although more than one mafa-a gene is present on the cynomolgus chromosome, only alleles of the highly polymorphic mafa-a locus are provided for sake of convenience. six additional mafa-a/b haplotypes were seen in only one animal, whereas nine sequence combinations were ambiguous, and are not listed in the table. further analyses on these animals, and on their offspring are needed to find out if these are recombinations. the number of at least distinct haplotypes is high, though they appear to be stable entities in this population of macaques; recombination was seldom observed. only one case of crossing over between the mhc-a and -b region is seen; mafa-a * is present in conjunction with two different mafa-b combinations ( and in table ). for eleven of the mafa-a/-b haplotypes it was possible to add an associated mafa-i allele. preliminary studies with drb-microsatellites de groot et al. ) indicate that the mafa-a/b haplotypes are also linked to drb-str patterns. further investigation should reveal whether in the future these animals and their offspring can be typed for the class i alleles by means of this extremely fast and accurate typing technique. in our cohort of animals, mafa-b alleles were detected that were already described in studies on other cynomolgus populations. to determine whether these alleles were arranged in haplotypes that are shared between populations a comparison was made. three of these combinations were observed. pendley and coworkers have already described the sharing of b* /b* / and b* /b* allele-combinations in indonesian and mauritian cohorts (pendley et al. ) , which illustrates that the mauritian animals originate in the archipelago. probably the animals were introduced to the island by merchant ships in the dutch golden age (sussman and tattersall ) . both haplotypes are present in our animals, and are listed, respectively, as and in table . the first one was extended to a * / b* /b* /b* . in pendley's cohort this combination of mafa-b alleles is seen in an animal that also transcribes mafa-a * . this allele differs by two basepairs from mafa-a * . with genotyping based on reference-strand conformational analysis (rsca), krebs and co-workers found the combination b* / b* /b* in a cohort of mauritian animals (krebs et al. ) . in our animals, however, we observed this set without b* (table , haplotype ). it is possible that this allele is a minor, and therefore was probably missed in our cloning procedures. in the rsca study the b* peak is also low in comparison to the peaks of the other two alleles. the three shared haplotypes were present in cohorts that, like most of our animals at the bprc, originate in the indonesian islands. sharing of haplotypes with the recently described filipino cynomolgus macaques was not observed (campbell et al. ). the restricted sharing of alleles in different populations of cynomolgus macaques, and moreover the recombination of similar alleles into other haplotypes in these populations, suggests that the diversity within the mafa-b region has been the result of recombination and reshuffling of b-like loci during evolution. the cohort under study has been pedigreed for up to generations, however, the haplotypes listed are observed in maximal five generations of related animals. the finding that within this cohort only one crossing between mafa-a and mafa-b is observed may be due to this relatively small number of generations. the fact that each cohort of animals has its own set of alleles and haplotypes necessitates investigation of the mhc for each cohort under study. only within a breeding colony are the haplotypes more or less stable and predictable, and once the haplotypes are inventoried, robust mhc typing based on microsatellite analyses may be performed. recombinations can be traced by using microsatellites spanning the whole mhc region. fig. . the phylogenetic tree shows that cynomolgus and rhesus sequences are fully intertwined, and each clade contains alleles of both species. in total, sets of alleles were observed that are identical for all exons (table ) , and this number is in the same order of magnitude as the shared alleles among different populations of cynomolgus macaques. for instance, mafa-b alleles were described in the cohort filipino cynomolgus macaques, of which three alleles were shared with animals of indonesian origin (campbell et al. ). next to these shared alleles are several that differ by only one or two basepairs. identity at the predicted amino-acid level for these alleles was not investigated. the sharing of haplotypes between cynomolgus and rhesus macaques was also investigated. only one combination of three mafa-b sequences was present in the rhesus macaque, and interestingly this was the b* / b* /b* combination mentioned above. moreover, this mamu-b* / /b* / /b* haplotype is one of four combinations that is shared by indian and chinese rhesus macaques, apart from a few basepair differences. the cynomolgus version differs by three basepairs in mafa-b* from the indian rhesus macaque haplotype. the presence of the haplotype in different cohorts of rhesus monkeys and in the indonesian/mauritian cynomolgus macaques suggests that its ancestor was already present before the separation of both species. the stability of the haplotype during macaque evolution may have been caused by a significant advantage in the combat of intercellular pathogens. it is also possible that the sharing of the haplotype results from hybridisation. molecular studies have revealed that introgression from rhesus macaques to cynomolgus monkeys has occured into the indo-chinese peninsula ). fig. phylogenetic analyses of mafa-b alleles detected in this study and mamu-b alleles in known haplotypes of indian and chinese rhesus monkeys. the analyses are based on the exon , and sequences. alleles that seem identical in this three may have basepair differences in other exons. the two species of macaques are indicated by different colors. mafa/mamu-b alleles of the one shared haplotype are depicted in green. the extension of allele-names with mi means that these are minors; alleles with relatively low transcription similarities in the genetics of the macaque mhc class i regions are at this stage unfortunately not reflected in the nomenclature for mhc-b alleles. the recent renaming of mhc-a alleles has led to a nomenclature in which the distinct a loci and lineage numbers are compatible for all macaque species. for the mhc-b region in macaques, however, it is not yet possible to assign the transcribed alleles to distinct b loci on the chromosome (e.g. mamu-b , -b , -b etc.). however, it would be useful to adjust the lineage numbers (first three ciphers after the asterisk) so that the same numbers refer to similar sequences in different macaque species. should more information become available in the near future on the number and order of b genes/loci on the macaque haplotypes, the allele designations may then easily be extended by a cipher following the b, without affecting the lineage numbers. comparative sequencing of human and chimpanzee mhc class i regions unveils insertions/deletions as the major path to genomic divergence comparison of lung and kidney allografts in induction of tolerance by a mixed-chimerism approach in cynomolgus monkeys genomic plasticity of the immune-related mhc class i b region in macaque species assessing natural introgression in biomedical model species, the rhesus macaque (macaca mulatta) and the long-tailed macaque (macaca fascicularis) comparative genetics of mhc polymorphisms in different primate species: duplications and deletions the mhc class i genes of the rhesus monkey. different evolutionary histories of mhc class i and ii genes in primates table shared alleles in cynomolgus and rhesus macaques mafa-b mamu-b b* b* b* the alleles that were published earlier are depicted in bold. the shaded mafa-b alleles were published earlier and are not detected in our cohort of animals. characterization of mhc class i sequences in filipino cynomolgus macaques genetic divergence of the rhesus macaque major histocompatibility complex comparative genetics of a highly divergent drb microsatellite in different macaque species phylogeny.fr: robust phylogenetic analysis for the non-specialist evolutionary stability of mhc class ii haplotypes in diverse rhesus macaque populations extensive sharing of mhc class ii alleles between rhesus and cynomolgus macaques a highly divergent microsatellite facilitating fast and accurate drb haplotyping in humans and rhesus macaques compound evolutionary history of the rhesus macaque mhc class i b region revealed by microsatellite analysis and localization of retroviral sequences isag/iuis-vic comparative mhc nomenclature committee report mhc class i a loci polymorphism and diversity in three southeast asian populations of cynomolgus macaque nomenclature for the major histocompatibility complexes of different species: a proposal unusually high frequency mhc class i alleles in mauritian origin cynomolgus macaques the locus encoding an oligomorphic family of mhc-a alleles (mane-a* /mamu-a* ) is present at high frequency in several macaque species replication of sars coronavirus administered into the respiratory tract of african green, rhesus and cynomolgus monkeys mhc haplotype h is associated with sustained control of sivmac infection in mauritian cynomolgus macaques comprehensive characterization of mhc class ii haplotypes in mauritian cynomolgus macaques extensive mhc-dqb variation in humans and non-human primate species unparalleled complexity of the mhc class i region in rhesus macaques mhc class i a region diversity and polymorphism in macaque species a snapshot of the mamu-b genes and their allelic repertoire in rhesus macaques of chinese origin mhc class i characterization of indonesian cynomolgus macaques defined tuberculosis vaccine, mtb f/as a, evidence of protection in cynomolgus monkeys imgt/hla and imgt/mhc: sequence databases for the study of the major histocompatibility complex distribution, abundance, and putative ecological strategy of macaca fascicularis on the island of mauritius, southwestern indian ocean identification of the mhc class i b locus in cynomolgus monkeys mamu-i: a novel primate mhc class i b-related locus with unusually low variability a bac-based contig map of the cynomolgus macaque (macaca fascicularis) major histocompatibility complex genomic region simian immunodeficiency virus sivmac infection of major histocompatibility complex-identical cynomolgus macaques from mauritius allelic diversity within the high frequency mamu-a * /mane-a * (mane-a* )/mafa-a * family of macaque mhc-a loci acknowledgements the authors wish to thank donna devine for editing the manuscript.open access this article is distributed under the terms of the creative commons attribution noncommercial license which permits any noncommercial use, distribution, and reproduction in any medium, provided the original author(s) and source are credited. key: cord- -zzongyfa authors: oany, arafat rahman; pervin, tahmina; mia, mamun; hossain, motaher; shahnaij, mohammad; mahmud, shahin; kibria, k. m. kaderi title: vaccinomics approach for designing potential peptide vaccine by targeting shigella spp. serine protease autotransporter subfamily protein siga date: - - journal: j immunol res doi: . / / sha: doc_id: cord_uid: zzongyfa shigellosis, a bacillary dysentery, is closely associated with diarrhoea in human and causes infection of million people worldwide per year. casein-degrading serine protease autotransporter of enterobacteriaceae (spate) subfamily protein siga, an outer membrane protein, exerts both cytopathic and enterotoxic effects especially cytopathic to human epithelial cell type- (hep- ) and is shown to be highly immunogenic. in the present study, we have tried to impose the vaccinomics approach for designing a common peptide vaccine candidate against the immunogenic siga of shigella spp. at first, siga proteins from different variants of s. flexneri, s. dysenteriae, s. boydii, and s. sonnei were assessed to find the most antigenic protein. we retrieved peptides based on the highest score for human leukocyte antigen (hla) supertypes analysed by netctl. initially, these peptides were assessed for the affinity with mhc class i and class ii alleles, and four potential core epitopes vtaraglgy, fhtvtvntl, httwtltgy, and ielagtltl were selected. from these, fhtvtvntl and ielagtltl peptides were shown to have % conservancy. finally, ielagtltl was shown to have the highest population coverage ( . %) among the whole world population. in vivo study of the proposed epitope might contribute to the development of functional and unique widespread vaccine, which might be an operative alleyway to thwart dysentery from the world. shigella is a gram-negative, facultative anaerobic, nonmotile, nonspore forming, and rod-shaped true bacteria closely related to salmonella and escherichia coli. the resulting infection by this organism called shigellosis, also known as bacillary dysentery or marlow syndrome, is most typically associated with diarrhoea and other gastrointestinal symptoms in humans. this pathogen is usually found in water that is contaminated with human feces within the setting of poor hygiene among kids of underneath years old and is transmitted via the fecal-oral route. the infection will occur even if there is just a bodily function of only ten to one hundred microorganisms [ ] . in each year, million cases of shigella infection are accounted worldwide, of that, million take place in developing countries and ultimately result in millions of death [ ] . bangladesh has got the top rates of shigellosis according to the recent global enteric multicenter study (gems) in asia. the output of this study has revealed that the shigella is the third leading reason behind diarrhoea in children [ , ] . shigella species are usually classified into four serogroups: s. dysenteriae ( serotypes), s. flexneri ( serotypes), s. boydii ( serotypes), and s. sonnei (one serotype) based on the biochemical properties and group-specific o antigens within the outer portion of the semipermeable membrane. s. dysenteriae, s. flexneri, and s. boydii are physiologically similar in distinction to s. sonnei. among them, s. flexneri is the most frequently isolated species globally and accounts for % of cases in the unindustrialized countries; s. sonnei causes % of cases in the industrialized countries [ ] . the underlying therapeutic challenge to manage shigella is its accrued resistance to most often used antibiotics like ampicillin, tetracycline, streptomycin, nalidixic acid, and sulfamethoxazole-trimethoprim [ ] . earlier, ciprofloxacin, a third-generation fluoroquinolone antibiotic, has been used effectively for the treatment of bacillary dysentery [ ] . however, this antibiotic is no longer helpful for the treatment of bacillary dysentery in south asian countries together with bangladesh, because of the dissemination of fluoroquinolone-resistant variety and its equivalent clones across the countries [ , ] . hence, it is essential to find a sustainable approach like vaccinomics, which can elicit long-term and consistent immunological responses to fight against shigella. siga is annotated in the she pathogenicity island of shigella, encoding siga protein which belongs to the serine protease autotransporter of enterobacteriaceae (spate) subgroup proteins. the autotransporter proteins of gramnegative bacteria exhibit an n-terminal signal sequence, required for secretion across the inner membrane, and a cterminal domain that forms an amphipathic β-barrel pore that allows passage of the functional domain across the outer membrane. this type of exporter proteins either remains attached to the cell surface or is released from the cell by proteolytic cleavage [ ] . siga is a multifunctional protein, able to degrade casein with cytotoxic and enterotoxic effects. moreover, siga is cytopathic for human epithelial type- (hep- ) cells, causing morphological changes and loss of integrity of the cell monolayers, important for the pathologic process of shigella [ ] . the position of siga in the chromosome made them less vulnerable to loss compare to the other virulence factors harbouring within the plasmid, and more exposure to the immune cells occurred by this secreted toxin [ ] . most importantly, this protein has been shown to be immunogenic following infection with shigella [ ] . the generalized modules of membrane antigen-(gmma-) based outer membrane proteins including siga were also shown to be highly immunogenic [ ] , which prompted us to target siga as one of the best vaccine candidates and to design potential peptide vaccine covering all the shigella spp. and most of the regions of the world. epitope-based immunizing agents are often an inexpensive choice to thwart enteric shigella infection. the identification of specific epitopes derived from infectious pathogens has considerably advanced the event of epitopebased vaccines (evs). higher understanding of the molecular basis of substance recognition and human leukocyte antigen-(hla-) binding motifs has resulted in the advancement of rationally designed vaccines that solely depends on algorithms predicting the peptide's binding to human hla. the traditional process for the development of a vaccine is very complex compared to that of the epitope-based vaccine, and additionally, it is chemically stable, more specific, and free of any infectious or oncogenic potential hazard [ ] . however, the invention of a wet laboratory-based candidate epitope is expensive and laborious that requires varied medicine experiments in the laboratory for the ultimate choice of epitopes. hence, the interest for predicting epitopes by computational strategies, alternate in silico approaches among researchers, is growing bit by bit with reduced efforts. vaccinomics is the application of integrated knowledge from different disciplines including immunogenetics and immunogenomics to develop candidate next-generation vaccine and understand its immune response [ ] . currently, various vaccinomics databases are accessible for identification of distinctive b lymphocyte epitopes and hla ligands with high sensitivity and specificity [ ] [ ] [ ] . the vaccinomics approach has already proven its potency in identifying the conserved epitope in the case of human immunodeficiency virus [ ] , multiple sclerosis [ ] , tuberculosis [ ] , and malaria [ ] with desired results. in our study, we have applied vaccinomics approaches for the screening of potentially conserved epitopes by targeting protein siga. the flow chart summarizing the protocols for the complete epitope prediction is illustrated in figure . the siga protein sequences of different strains of shigella species were retrieved from the ncbi genbank [ ] database and analysed in the vaxijen v . [ ] server for the determination of the most potent antigenic protein. additionally, the target protein was crosschecked against human pathogens and other similar pathogens to ensure the orthologous entry by using blast-p [ ] and orthomcl [ ] databases [ ] . the epitope prediction for the respective protein and their affinity score with mhc class i and class ii allele was measured following previously used approach [ , ] . concisely, the netctl v . server [ ] was used for predicting potential cytotoxic t-lymphocyte (ctl) epitopes from the most antigenic protein. a combined algorithms including mhc-i binding, transporter of antigenic peptide (tap) transport efficiency, and proteasomal c-terminal cleavage prediction were employed for the t-cell epitope prediction. the epitope with the highest score for mhc class i supertypes was selected. t cell epitope prediction tools from immune epitope database and analysis resource (iedb-ar) were used for the prediction of affinity with mhc class i [ ] and mhc class ii [ , ] . the stabilized matrix method (smm) was used to calculate the half-maximal inhibitory concentration (ic ) of peptide binding to mhc class i with a preselected . -mer epitope. the peptides were also assessed for hla i binding affinity by the software, episoft. for the analyses of mhc class ii binding, the iedb-recommended method was used for the specific hla-dp, hla-dq, and hla-dr loci. fifteen-mer epitopes were designed for mhc class ii binding analysis considering the preselected -mer epitope and its conserved region in the shigella strains. for the mhc class i and mhc class ii alleles, the epitopes consisting ic < nm and ic < nm, respectively, were selected for further analysis. the mhc class ii binding prediction tool predivac was also used to assess their affinity with hla_drb_ . alleles. furthermore, the mhccluster v . server [ ] was used for the identification of cluster of mhc restricted allele with appropriate peptides to further strengthen our prediction. this is the additional crosscheck of the predicted mhc restricted allele analysis from the iedb analysis resources. the output from this server is a static heat map and a graphical tree for describing the functional relationship between peptides and hlas. epitope conservancy of the candidate epitopes was examined using a web-based epitope conservancy tool available in iedb analysis resource [ ] . the conservancy level of each potential epitope was calculated by considering identities in all siga protein sequences of different strains retrieved from the database. multiple sequence alignment (msa) was employed to understand the positions of the table : epitopes for cd + t-cell along with their interacting mhc class i alleles with affinity < nm. interacting mhc-i allele (ic color key epitopes within the sequences. as spate family is very much specific for the enterobacteria, specifically, e. coli and shigella, we also include two e. coli sequences (gi| | and gi| |) along with those of four species of shigella for msa construction. the jalview (http://www.jalview.org/) tool was used for this analysis. the conservancy of the selected peptides was also substantiated by the protein variability software (pvs) [ ] . population coverage for the epitope was assessed by the iedb population coverage calculation tool [ ] . the combined score for mhc classes i and ii was assessed for the analysis of the population coverage. a homology model of the conserved region was obtained by modeller v [ ] , and the predicted model was assessed by the procheck [ , ] server. for the disorder prediction among the amino acid sequences, disopred v [ ] was used. the protein frustratometer server [ ] was employed for the detection of the stability and energy differences of the d structure of the protein. docking studies were also performed using the best possible epitope following the strategy used in previous studies [ , ] . autodock vina [ ] was used for the docking analysis. in our study, we have selected the hla-e * : molecule as a candidate for mhc class i and the hla-dqa as a candidate for mhc class ii for docking analysis because they are the available hits in the protein data bank (pdb) database. the pdb structure esv, human cytomegalovirus complexes with t-cell receptors, vmaprtlil peptide, and pl -structure of autoimmune tcr hy. b in complex with hla-dq -were retrieved from the research collaboratory for structural bioinformatics (rcsb) protein database [ ] . then, the structures were simplified by using pymol (the pymol molecular graphics system, version . . . , schrödinger, llc) for the final docking purpose. the pep-fold server [ ] was used for the conversion of the d structure of the epitope "ielagtltl" for mhc i and the epitope "kaielagtltltgtp" for the mhc ii molecule in order to analyse the interaction with hla alleles. the allerhunter server [ ] was used to predict the allergenicity of our proposed epitope for further securing the prediction, and the support vector machine (svm) algorithm was used for the prediction within the server [ ] . the predicted t-cell epitope ( -mer) was screened by iedb-ar using a number of web-based tools for the suitability as the b-cell epitope [ ] [ ] [ ] . a total of siga proteins from different variants of s. flexneri, s. dysenteriae, s. boydii, and s. sonnei were retrieved from the genbank database (table s in supplementary material available online at https://doi.org/ . / / ). thereafter, analyses with the vaxijen v . server showed the protein with the accession number of gi| | to have the highest antigenicity of . (table s ). this highly antigenic protein was further analysed to detect the highly immunogenic epitope. no significant entry was found in the orthologous entry search of our targeted protein. identification. the netctlv . server identified the t-cell epitopes, where the epitope prediction was confined to mhc class i supertypes. based on the combined score, the top twelve epitopes (table ) were listed for further analysis. analysis. iedb analysis resource predicted both mhc class i and mhc class ii restricted allele on the basis of the ic value. all the predicted epitopes in table were assessed for the mhc interaction analysis. epitopes for the mhc class i alleles are presented in table . the peptide ielagtltlt was predicted to have the highest number of mhc class i binding. this peptide was predicted to have the binding affinity with five mhc class i alleles including hla-e * : , hla-b * : , hla-b * : , hla-c * : , and hla-c * : . furthermore, the interacted alleles were reassessed by cluster analysis and are shown in figure (a) , as a heat map, and in figure s a , as a dynamic tree. the peptides were reassessed by the episopt software for the hla i binding, and ielagtltl was found to have affinity with six hla i alleles (table s ) . from this analysis, we selected top four peptides vtaraglgy, fhtvtvntl, httwtltgy, and ielagtltl depending on the affinity with most mhc class i. epitopes for the mhc class ii alleles are presented in table . depending on the ic values as well as on the number of mhc class ii alleles, three -mer peptide candidates were selected. the peptides nsgfhtvtvntldat, kaielagtltltgtp, and aaksymsgnykaflt were predicted to have high affinity with mhc-ii allele, which can interact with , , and mhc class ii alleles. the data has been validated by another software predivac. the predivac scores of the two core peptides fhtvtvntl and ielagtltl have been shown to be promising for their binding to hla_drb_ (table ) . accumulating both mhc class i allele-and mhc class ii allele-based analyses, we showed fhtvtvntl and ielagtltl peptides to have the best score to be a vaccine potential. conservancy of all the proposed epitopes was assessed by the iedb conservancy analysis tool and is summarized in table . fhtvtvntl, ielagtltl, nyawvngni, and smyntlwrv were shown to have % conserved regions across all the siga proteins. the position of all the predicted epitopes is shown in a multiple sequence alignment of siga proteins in figure . here, we used only our desired sequences for the proper annotation. so, from the most potential candidates, only two, that is, fhtvtvntl and ielagtltl, were found to be fully conserved. the top four epitopes were shown within the protein in figure . the conservancy of both of these peptides were crosschecked by pvs software, and it was found that they were located in the conserved region of the siga protein ( figure s ). the epitopes are precisely positioned on the surface of the protein indicating that they would be accessible to the immune system, especially by b-cells. y k s n nq y k s n nq y k s n nq y k s n nq y k s n nq y k s n nq modeller modelled the three-dimensional structure of the targeted protein through the best multiple templatebased modelling approach. the validation of the model was measured by the procheck server through the ramachandran plot and is depicted in figure s , where . % amino acid residues were found within the favoured region. furthermore, the predicted model was also assessed for the frustration analysis and is depicted in figure . the disopred server likewise assessed the disorder of the protein sequences in order to get an understanding about the disorder among the targeted sequences, which is shown in figure s . . . population coverage analysis. iedb analysis resource predicted both mhc class i-and mhc class ii-based coverage of the selected epitopes for the world population to assess the feasibility of being a potential vaccine candidate. the combined prediction was also assessed. the epitope "ielagtltlt" has the highest population coverage of . % for the whole world population (shown graphically in figure ); however, another potential epitope "fhtvtvntl" was shown to have . % population coverage (table s ) . . . molecular docking analysis. the core epitope (ielagtltl) with . mer and its -mer extension (kaie-lagtltltgtp) were bound in the groove of the hla-e * : and hla-dqa with an energy of − . and − . kcal/mol, respectively. autodock vina generated different poses of the docked peptide, and the best one was picked for the final calculation at an rmsd (root-mean-square deviation) value of . . the docking interface was visualized with the pymol molecular graphics system. the . -mer epitope interacted with arg- , asn- , and glu- through steric interaction and formed hydrogen bonding with the glu- amino acid residues. on the other hand, the -mer epitope interacted with asp- through electrostatic interaction and glu- through steric interaction and formed hydrogen bonding with the gly- , arg- , asn- , and asn- amino acid residues. the docking output and the interacted residues are shown in figures and with different orientations. furthermore, the control docking energy was found to be − . kcal/mol and is illustrated in figure s . . . allergenicity analysis. the allerhunter web server predicted the sequence-based allergenicity calculation very precisely. the allergenicity of the queried core epitope (ielagtltlt) was . (sensitivity = . %, specificity = . %), and the allergenicity of the -mer epitope (kaielagtltltgtp) was . (sensitivity = . %, specificity = . %). . . b-cell epitope prediction. b-cell epitope prediction was obtained for the peptide kaielagtltltgtp ( mer) through the sequence-based approaches, and values are anticipated with different parameters, ranging from − . to . . these values are the different propensity scores and predicted with a threshold ranging from − . to . ( figure ). the kolaskar and tongaonkar antigenicity scale was employed for evaluating the antigenic property of the peptide with a maximum of . . the antigenic plot is showed in figure (a). peptide surface accessibility is another important benchmark to meet up the criteria of a potential b-cell epitope. henceforth, emini surface accessibility prediction was employed, with a maximum propensity score of . (figure (b)). to reinforce our provision for the prediction of the epitope to elicit b-cell response, the parker hydrophilicity prediction was also employed with a maximum score of . and is depicted in figure (c). enteric infections are the foremost cause of sickness and impermanence throughout the world, and only the shigella infections resulted in over a million deaths annually [ ] . the ever rising multidrug-resistant (mdr) strains of the shigella bacteria area unit are another international concern for the researchers to search out a brand new resolution for preventing the deaths [ , ] . recently, there are several studies that focus on the development of the vaccine against shigella and continue in the clinical trial. most of them use attenuated and inactivated preparation of the bacteria for eliciting immune responses which has some potential escape risk [ ] [ ] [ ] . in this study, we have tried to find out alternatives to treat this global burden through vaccinomics approaches and targeting the immunogenic and toxic protein siga. the sequences of different strains of shigella showed that there is a little island of conserved sequence throughout the species [ ] , and we have focused on that target for designing the vaccine candidate. the orthologous entry search of our targeted protein revealed no significant similarity with human pathogens and other closely related pathogens. these results further strengthen our prediction through confirming no cross immunity. in recent time, most of the vaccines are grounded on bcell immunity; vaccines based on a t-cell epitope have been invigorated lately. this is often as a result of body substance response from memory b-cells which may be overawed basically by matter drift as time goes on, whereas cell-mediated immunity repeatedly delivers long-run immunity [ , ] . as a consequence, a t-lymphocyte epitope elicits a robust and distinctive immune response through the cytotoxic lymphocyte-(ctl-) mediated pathway and impedes the spreading of the infectious agents by the ctl through recognizing and killing the infected cells or by secreting specific cytokines [ ] . the epitopes vtaraglgy, fhtvtvntl, httw tltgy, and ielagtltl are primarily selected for the designing of vaccine from the initial analysis depending on the affinity with mhc class i and additionally confirmed their presence along with those of the ancestral homologue in e. coli (figure ). finally, through substantiation with different parameters, the core epitopes ielagtltl and fhtvtvntl (in . -mer form, kaielagtltltgtp and nsgfhtvtvntldat, resp.) were found to be the most potential and highly interacting hla candidates for mhc class ii molecule. furthermore, we have used psortb to predict the subcellular localization of siga and found that there is a score of . for localization in the outer membrane and another score of . for extracellular localization. the result was quite similar with that for the localization of other spate proteins in the bacterial cell surface as well as in secreted forms. the three-dimensional model built through model-ler and validated by the ramachandran plot with an acceptable range resulted in the display of the perfect position of the epitope on the surface of the structure. as the epitope was found on the surface (figure ) of the model, it would increase the possibility to interact with the immune system earlier. furthermore, the analysis from the dis-opred and frustration analysis servers strengthen our prediction, though there are no disorder and energy frustration in the epitope region of the sequences and model, respectively ( figure and figure s ). to get the acceptability, vaccine candidates must have wider population coverage. this is very much important before designing. in our analysis, we have found that our proposed epitope ielagtltl had combined population coverage of . %, whereas the other most potential candidate fhtvtvntl had combined population coverage of . %. this output revealed that the proposed epitopes would have wider coverage in vitro. molecular docking upkeeps the prediction with a higher docking score and the perfectly oriented interactions between the both mhc and the predicted . -mer and -mer epitopes. additionally, comparative analysis with the experimentally known peptide-mhc complex-has also revealed the precision of our prediction through the similar binding energy and interacted residues. another significant finding is the conservancy result. through analysis of the whole retrieved sequences, it was found that our predicted epitopes have a % conservancy and hopefully they would be potential candidates for treating all of the shigella spp. our proposed epitopes are nonallergenic in nature according to the fao/who allergenicity evaluation scheme. finally, the core epitope "ielagtltl" was also found to be more potential b-cell epitope candidates that were proposed through the sequence-based approaches including the kolaskar and tongaonkar antigenicity scale, emini surface accessibility prediction, and parker hydrophilicity prediction. from the overhead analysis, we envisage that our suggested epitope would also elicit an immune response in vitro. the improved knowledge about antigen recognition at molecular level led us to the development of rationally designed peptide vaccines. the idea of peptide vaccines is based on detecting and chemical synthesis of immunodominant b-cell and t-cell epitopes capable of evoking specific immune responses. in this study, we used different computational tools to identify potential epitope targets against shigella which will help to decrease the cost and time of wet lab experiments more successfully. our bioinformatic analyses speculate that the selected part of the outer membrane and highly immunogenic protein, siga, is a potential candidate for a peptide vaccine. it might also contribute to the reduction in the siga-mediated pathogenicity to the host. however, further wet lab validation is necessary to confirm the efficiency of our identified peptide sequence as an epitope vaccine against shigella. cytotoxic t-lymphocyte tap: transporter of antigenic peptide smm: stabilized matrix method hla: human leukocyte antigen. availability of data and materials. information about the data and their availability is intricately described in methods. as samples from human or animals had not been used in this study, ethical clearance is not applicable. patient consent is not applicable. no potential competing interest was reported by the authors. arafat rahman oany conceived, designed, and guided the study; drafted the manuscript; and analysed the data. tahmina pervin, mamun mia, and motaher hossain carried out the molecular genetic studies, participated in the sequence alignment, and drafted the manuscript. mohammad shahnaij helped in the design of the study. shahin mahmud helped in drafting the manuscript. k. m. kaderi kibria participated in the design and coordination, performed critical revision, and helped in drafting the manuscript. all authors read and approved the final manuscript. university of texas medical branch at galveston global burden of shigella infections: implications for vaccine development and implementation of control strategies a multicentre study of shigella diarrhoea in six asian countries: disease burden, clinical manifestations, and microbiology burden and aetiology of diarrhoeal disease in infants and young children in developing countries (the global enteric multicenter study, gems): a prospective, case-control study shifting serotypes, plasmid profile analysis and antimicrobial resistance pattern of shigellae strains isolated from kolkata, india during randomised comparison of ciprofloxacin suspension and pivmecillinam for childhood shigellosis genetic relatedness of ciprofloxacin-resistant shigella dysenteriae type strains isolated in south asia fluoroquinolone resistance linked to both gyra and parc mutations in the quinolone resistance-determining region of shigella dysenteriae type the great escape: structure and function of the autotransporter proteins the immunogenic siga enterotoxin of shigella flexneri a binds to hep- cells and induces fodrin redistribution in intoxicated epithelial cells the siga gene which is borne on the shepathogenicity island of shigella flexneri a encodes an exported cytopathic protease involved in intestinal fluid accumulation high yield production process for shigella outer membrane particles optimizing vaccine design for cellular processing application of pharmacogenomics to vaccines major histocompatibility complex linked databases and prediction tools for designing vaccines mhcpep, a database of mhc-binding peptides: update syfpeithi: database for mhc ligands and peptide motifs development of a dna vaccine designed to induce cytotoxic t lymphocyte responses to multiple conserved epitopes in hiv- a highly immunogenic trivalent t cell receptor peptide vaccine for multiple sclerosis t cell vaccines for microbial infections a synthetic malaria vaccine elicits a potent cd + and cd + t lymphocyte immune response in humans. implications for vaccination strategies genbank vaxijen: a server for prediction of protective antigens, tumour antigens and subunit vaccines basic local alignment search tool orthomcl-db: querying a comprehensive multi-species collection of ortholog groups genome-wide prediction of vaccine candidates for leishmania major: an integrated approach design of an epitope-based peptide vaccine against spike protein of human coronavirus: an in silico approach highly conserved regions in ebola virus rna dependent rna polymerase may be act as a universal novel peptide vaccine target: a computational approach large-scale validation of methods for cytotoxic t-lymphocyte epitope prediction sensitive quantitative predictions of peptide-mhc binding by a 'query by committee' artificial neural network approach a systematic assessment of mhc class ii peptide binding predictions and evaluation of a consensus approach peptide binding predictions for hla dr, dp and dq molecules mhccluster, a method for functional clustering of mhc molecules development of an epitope conservancy analysis tool to facilitate the design of epitope-based diagnostics and vaccines pvs: a web server for protein sequence variability analysis tuned to facilitate conserved epitope discovery predicting population coverage of t-cell epitope-based diagnostics and vaccines evaluation of comparative protein modeling by model-ler aqua and procheck-nmr: programs for checking the quality of protein structures solved by nmr the swiss-model workspace: a web-based environment for protein structure homology modelling the disopred server for the prediction of protein disorder protein frustratometer: a tool to localize energetic frustration in protein molecules autodock vina: improving the speed and accuracy of docking with a new scoring function, efficient optimization, and multithreading the protein data bank pep-fold: an updated de novo structure prediction server for both linear and disulfide bonded cyclic peptides allerhunter: a svm-pairwise system for assessment of allergenicity and allergic cross-reactivity in proteins combining pairwise sequence similarity and support vector machines for detecting remote protein evolutionary and structural relationships a semi-empirical method for prediction of antigenic determinants on protein antigens induction of hepatitis a virus-neutralizing antibody by a virus-specific synthetic peptide new hydrophilicity scale derived from high-performance liquid chromatography peptide retention data: correlation of predicted surface residues with antigenicity and x-ray-derived accessible sites the role of integrons in antibiotic resistance gene capture arginine-dependent acid-resistance pathway in shigella boydii new possibilities for the development of a combined vaccine against etec and shigella safety and immunogenicity of a candidate bioconjugate vaccine against shigella flexneri a administered to healthy adults: a singleblind, randomized phase i study safety and immunogenicity of an intranasal shigella flexneri a invaplex vaccine genome sequence of shigella flexneri a: insights into pathogenicity through comparison with genomes of escherichia coli k and o cd + regulatory t cells: mechanisms of induction and effector function antibody regulation of t-cell immunity: implications for vaccine strategies against intracellular pathogens role of cd + t cells in control of west nile virus infection key: cord- - c d d authors: larcher, clara; recheis, heidrun; sgonc, roswitha; göttinger, wolfgang; huemer, hartwig p.; irschick, eveline u. title: influence of viral infection on expression of cell surface antigens in human retinal pigment epithelial cells date: journal: graefes arch clin exp ophthalmol doi: . /bf sha: doc_id: cord_uid: c d d • background: subacute viral infection is known to change the phenotype of infected cells, thereby causing immune-mediated tissue damage. the aim of this study was to investigate the expression of different cell surface molecules on human retinal pigment epithelial cells (rpec) following viral infection, with special emphasis on those having immuneregulatory functions. • methods: cultured rpec were infected with cytomegalovirus (cmv), coxsackievirus b (cvb) or herpes simplex virus type i (hsv). double-staining fluorescence technique was used for visualization of virus infection and cell surface markers in the same cells by laser microscopy. • results: cmv down-regulated mhc class i antigens on rpec, whereas cvb and hsv did not alter mhc class i antigen expression. no induction of class antigens was observed in rpec infected with cvb, hsv or cmv. the intercellular adhesion molecule icam- (cd ) was strongly expressed in uninfected rpec, and a slight increase was observed after virus infection. vascular cell adhesion molecule (vcam- ) was expressed in low amounts in both uninfected and infected rpec. no expression of intercellular adhesion molecule (icam- ), e-selectin elam- or lymphocyte-function-associated antigen (lfa- ) was observed on rpec before or after virus infection. • conclusion: down-modulation of immune-regulating cell surface antigens has been suggested to provide a means of long-term survival of viruses in the infected cell, favoring establishment of persistent infection. our observation in cultured human rpec indicates that this mechanism might indeed contribute to the development of disease affecting retinal tissue. abstract • background: subacute viral infection is known to change the phenotype of infected cells, thereby causing immune-mediated tissue damage. the aim of this study was to investigate the expression of different cell surface molecules on human retinal pigment epithelial cells (rpec) following viral infection, with special emphasis on those having immuneregulatory functions. • methods: cultured rpec were infected with cytomegalovirus (cmv), coxsackievirus b (cvb) or herpes simplex virus type i (hsv). double-staining fluorescence technique was used for visualization of virus infection and cell surface markers in the same cells by laser microscopy. • results: cmv downregulated mhc class i antigens on rpec, whereas cvb and hsv did not alter mhc class i antigen expression. no induction of class ii antigens was observed in rpec infected with cvb, hsv or cmv. the intercellular adhesion molecule icam- (cd ) was strongly expressed in uninfected rpec, and a slight increase was observed after virus infection. vascular cell adhesion molecule (vcam- ) was expressed in low amounts in both uninfected and infected rpec. no expression of intercellular adhesion molecule (icam- ), e-selectin elam- or lymphocyte-function-associated antigen (lfa- ) was observed on rpec before or after virus infection. • conclusion: downmodulation of immune-regulating cell surface antigens has been suggested to provide a means of long-term sur- the etiologic factors leading to retinitis are often unknown. whereas autoantigens [review ] and immune mechanisms have been clearly shown to play a destructive role, suggesting a self-perpetuating mechanism [ , , , ] , the initial damaging event leading to exposure to autoantigens in vivo is rather unclear. host t-cells require major histocompatibility complex (mhc) molecules in order to respond to antigens. mhc class i molecules are involved in the presentation of cytoplasmic antigens to cytotoxic t-cells (ctl) and are im-portant for the destruction of antigenically altered (e.g. virus-infected) cells. mhc class ii antigens are responsible for presenting circulating antigens to helper t-cells [review ]. therefore, alteration of expression of human mhc molecules could be important in the development of autoimmune disease [ ] . adherence molecules are expressed especially on inflamed retinal tissue [ , ] and seem to be involved in the autoimmune process [ ] . cytokine-mediated activation of retinal pigment epithelial cells (rpec) may be important for autoimmune disease facilitated by mhc class ii expression [ ] . particularly interferon-gamma (ifn-y) produced by t lymphocytes has been shown to induce expression of mhc class ii (ia) antigens on rpec and vascular endothelial cells [ , ] and seems to play a crucial role in the inflammatory process [ ], most likely by enhancing presentation of retinal antigens [ ] . possible antigenic cross-reactivity of microbial antigens and retinal antigens has also been suggested [ , , . the intercellular molecules and (icam- = cd and icam- = cd ) and vascular cell adhesion molecule (vcam- = cd ) are known as cellular adhesion molecules. icam- was initially identified as a widely distributed cytokine-counterreceptor for the lymphocyte-function-associated antigen (lfa- ). the highest levels of icam- antigen are found on activated endothelium, with lower levels that increase in inflammation on multiple other cell types. high levels of icam- antigens are found on endothelium and lower levels on hemopoietic cells, including platelets, and on thymic stroma cells. unlike icam- , icam- is constitutively expressed and not responsive to lipopolysaccharides or cytokines. vcam- was shown to be expressed by dendritic cells and crypt epithelium (basal part). the e-selectin elam- (=cd e) was first identified as a cytokine-inducible surface antigen on endothelial cells. expression of elam- has been clearly documented on acutely activated endothelium, where it participates in the recruitment of leukocytes at sites of acute inflammation. this adhesion molecule has also been identified in chronic inflammatory lesions of the skin and synovium. lfa- is the receptor for three members of the ig supergene family of proteins icam- , icam- and icam- (=cd ). lfa- has been shown to play a central role in homotypic and heterotypic cellular adhesion in immune and inflammatory responses and is expressed on lymphocytes, monocytes, and, more weakly, on neutrophils. lfa- is a heterodimer consisting of two subunits, alpha (cdlla) and beta (cd ). for further details see reference [ ] . we did not expect the latter antigen to be expressed on human cultured rpec but used the antibody as control to exclude contamination of the cultures. various viruses have been shown to affect retinal tissue in animals [review ]. the most interesting virus in this respect is the murine coronavirus mouse hepatitis virus, which has been shown to induce retinitis in infected animals [ , ] . in humans, virally induced chorioretinitis is known in congenital rubella and cytomegalovirus (cmv) infection. the latter virus is also the most common cause of retinitis in adults, primarily because of the recent high incidence of immunosuppressive situations associated with aids, whereas herpes simplex virus type i (hsv) and varicella zoster virus retinitis also occur in immunocompetent individuals [review ]. in our previous study we showed that rather common human pathogenic viruses like respiratory viruses and en-teroviruses or measles virus and adenovirus are also able to infect human rpec [ ]. one of the strategies used by viruses to cause persistent infection so as to evade host immunosurveillance is the interference with the expression or function of cell surface molecules [ , ] . moreover, such mechanisms might influence immune effector cells. we therefore studied the possible influence of viral infection of cultured rpec on the hypothetical mechanisms, especially on mhc antigen and adherence molecule expression. human rpec were isolated from freshly enncleated bulbi from normal donors for corneal transplantation as described previously [ , ] . briefly, the corneoscleral disk was first removed, followed by the lens and vitreous body. the residual eye cup was sectioned with a longitudinal incision toward the optic nerve. repeated rinsing with dulbecco's phosphate-buffered saline (pbs), ca +-and mgz+-free (biochrom, berlin, germany), allowed prompt separation of the remaining vitreous body and neural retina from the retinal pigment epithelium (rpe) layer and detachment of the choroid from the sclera. the rpe adhering to bruch's membrane on the obtained choroidal sheets were washed with pbs and treated three times with . % trypsin edta solution (biochrom) for rain at °c. the isolated cells were centrifuged at g for min, resuspended in cell culture medium rpmi (biochrom), supplemented with % fetal calf serum (biological industries, kibbutz beth haemek, israel). cells were seeded in -cm a tissue culture flasks (becton dickinson, plymouth, uk) for h at °c in a humidified atmosphere with % co . the next day, nonadherent cells were removed and fresh culture media added. all adherent cells contained pigment granulae. confluent cells were trypsinized, washed with pbs and seeded into two tissue culture flasks. after two or three passages, cells were used for virus transfection after seeding on lab-tec chamber slides (nunc, naperville, ill.). rpec were grown in tissue culture flasks for several passages. after seeding, cells quickly came into contact with other rpec and began to proliferate until confluence was achieved after approximately week. in the first two to three passages rpec still contained their typical pigment granulae. for viral infection, cells were transferred onto lab-tec chamber slides and kept under the same culture conditions for approximately week until they were confluent. viral infection was achieved with various viruses on various passages ( and ) of rpec. viruses cmv strain ad (atcc no. -vr) and coxsackievirus b (cvb; atcc no. -vr) were obtained from the american tissue culture collection at rockville, md. hsv type strain wal was described by schroeder et al. [ ] . rpec were cultured on lab-tec chamber slides (nunc) and infected with the various viruses on passages and as described previously [ ] . for infection of rpec, viruses were adjusted to a high multiplicity of infection (m.o.i. > ), which was determined by standard virological methods. cells were kept in culture until cytopathic effects following infection with hsv and cvb were visible or for a maximum of weeks after infection with cmv. productive infection was confirmed by detection of viral antigens cells grown on lab-tec chamber slides were washed twice with pbs and incubated on ice (unfixed) with one of the antibodies diluted in pbs and containing % bovine serum albumin and . % sodium azide for h. after washing thoroughly with pbs, cells were incubated with fitc-conjugated anti-mouse f(ab) igg on ice for min. for double staining, virus-infected cells were permeabilized with % methanol for min at room temperature, then washed and incubated for h with the antibody used for detection of virus infection (see above), followed by a tritc-conjugated rabbit antimouse or anti-human igg for min. extensive washings with pbs were necessary to prevent background staining. cell layers were not allowed to dry out. cells were finally embedded in moviol (hoechst, germany) to reduce fading during observation and examined in a laser scanning microscope (lsm , zeiss, oberkochen, germany). for fitc a -nm argon laser was used for excitation and a bp / filter for emission. for tritc a -nm argon laser was used for excitation and a bp - filter for emission. cells were observed and photographed in transmission scan and in fluorescence scan and presented as a false color overlay micrograph of fitc/ tritc and transmission. infection of human-derived rpec cultures with the rapidly growing viruses cvb and hsv produced typical cytopathic effects such as cell rounding and detachment of cells within and h. infection with the slower growing cmv did not affect all cells visibly. as soon as a cytopathic effect was observed or after a maximum of weeks in cmv-infected cells, viral infection of rpec was confirmed by detection of viral structural proteins with monoclonal or polyclonal antibodies. most of the cells in the culture were infected at the time the experiments were done, as can be seen from the clear nuclear (cmv) and cytoplasmic (cvb) fluorescence (fig. ) . the number of passages of cultured rpec had no influence on the susceptibility of rpecs to virus infection, as we were able to demonstrate in our previous study [ ] . mhc-class i was expressed in abundant amounts by native human rpec in culture, whereas no expression of mhc-class ii was observed (fig. , table ). also, icam- and vcam- were constitutively expressed by rpec, the first to a large extent, the latter weakly. no expression of elam- , icam- or lfa- was observed. after infection with cvb (fig. ) and hsv (not shown), mhc class i expression remained unaltered, and in both cases only a slight increase in fluorescence intensity of icam- was observed. the other cell surface antigens tested remained negative. a very faint immuno- fluorescence indicated borderline expression of vcam- in infected and uninfected cells (table ) . interestingly, infection of rpec with cmv led to a remarkable downregulation of mhc class i antigens from the cell surface (fig. d) , whereas expression of icam- was slightly increased (fig. h ) as shown with double staining. mhc class ii was not upregulated on cultured human bulbus-derived rpec as a direct consequence of virus infection in vitro. the other cell surface molecules tested remained unchanged. retinal pigment epithelium is susceptible to growth of various viruses, but infection of the retina does not necessarily lead to severe destruction, as observed with hsv infection, one of the best-studied viruses in ocular disease [ ] . as shown in animal experiments, mouse hepatitis virus is able to cause only mild inflammatory response but longlasting disease [ , ] . viruses are known to be able to alter "self", thereby inducing an autoimmune response, and presumably a similar mechanism could also play a role in affecting the pigmented layer in humans. autoimmunity to retinal antigens has been implicated in the pathogenesis of endogenous posterior uveitis and retinitis pigmentosa. reid et al. [ ] found that ebv-transformed human lymphocytes from patients with retinitis pigmentosa secreted higher levels of antiretinal antibodies than those from uveitis patients and normal controls, although all groups had low serum titers of antibodies. albeit in the absence of cytopathic effects, persistent viral infection of retinal tissue has been described as leading to cellular dysfunctions [ ] . cmv infection leads to downregulation of mhc class i in human fibroblasts and, as observed, in our experimental design. this seems to favor virus escape from the immune response [ , ] and might support virus persistence in vivo. also, other viruses such as hiv or adenoviruses which are able to induce persistent infections choose this type of strategy [ , , ] . mhc class ii was not upregulated on cultured human bulbus-derived rpec as a direct consequence of virus infection in vitro, not ruling out the possibility that this could occur in vivo due to local production of ifn-y by cmv-specific t-cells. no decrease of adherence molecules was observed in virus-infected rpec. adherence molecules regulating interactions of immune effector cells are known to be involved in retinal inflammation [ , ] . an important member of this class is icam- , which has been shown to mediate lymphocyte binding to rpec [ ] . basal expression of icam- on non-hematopoietic cells is usually rather low, but it can be induced by cytokines such as ifn-y, tumor necrosis factor alpha (tnf-c and interleukin-lbeta (il-i[ ). icam- is also constitutively expressed on unstimulated rpec and can be induced in vivo by various cytokines, most importantly ifn-y [ ] . this mechanism might regulate leukocyte infiltration and might be important for the local immune response of the eye [review ]. lymphocyte binding to rpec is normally icam-l-dependent, but icam-l-independent mechanisms have also been found on stimulated rpec [ ] , suggesting the involvement of further cell surface molecules. other investigators showed that hsv- induced an increase in elam- , icam- and vcam- , whereas coxsackievirus induced an increase in icam- and vcam- in endothelial cells. cytokines such as tnf-c~, il- , il- and il- were shown to effect the expression of adhesion molecules. tnf-c~ also upregulated vcam- and elam- [ ] . however, the adhesion molecules elam- and icam- were not found on rpec, and no induction due to viral infection was observed. as rpec have been shown to function as antigen-presenting cells under certain circumstances [ ] , the modulation of immune regulatory cell surface antigens such as mhc class i or icam- by infectious agents observed in vitro might alter the immune response in vivo. the cell biology of antigen processing and presentation modulation and function of intercellular adhesion molecule- (cd ) on human retinal pigment epithelial cells proliferative vitreoretinopathy: an examination of the involvement of lymphocytes, adhesion molecules and hla-dr antigens human retinal pigment epithelial cells differentially express mhc class ii (hla, dp, dr and dq) antigens in response to in vitro stimulation with lymphokine or purified ifn-gamma interactions between lymphocytes and cells of the blood-retina barrier: mechanisms of t lymphocyte adhesion to human retinal capillary endothelial cells and retinal pigment epithelial cells in vitro retinal pigment epithelial cells modulate lymphocyte function at the blood-retina barrier by autocrine pge and membrane-bound mechanisms cell lines producing human t-cell lymphoma virus type show altered hla expression modulation of mhc antigen expression by viruses and oncogenes cytokinemediated activation of a neuronal retinal resident cell provokes antigen presentation eb-virus transformed human iymphocytes from uveitis and retinitis pigmentosa patients secrete antibodies to retinal antigens murine coronavirus induces an acute and long lasting disease of the retina redistribution and reduction of interphotoreceptor retinoid-binding protein during ocular coronavirus infection down modulation of mhc-i in a cd + t cell line, cem-e , after hiv- infection expression of class i major histocompatibility antigens switched off by highly oncogenic adenovirus in transformed rat cells protection of mice by an apathogenic strain of hsv against lethal infection by a pathogenic strain of hsv type santigen: from gene to autoimmune uveitis molecular mimicry between a uveitopathogenic site of s-antigen and viral peptides. induction of experimental autoimmune uveitis in lewis rats molecular mimicry: uveitis induced in cacaca fascicularis by microbial protein having sequence homology with retinal s-antigen adhesion structures coronavirus (jhm) replication within the retina: analysis of cell tropism in mouse retinal cell cultures monoclonal antibodies against icam- (cd )and lfa- (cdlla/ cd ) inhibit experimental autoimmune uveitis phagocytosis of latex beads is defective in cultured human retinal pigment epithelial cells with persistent rubella virus infection systemic viral infections and their retinal and choroidal manifestations herpes-simplex-virus retinitis. rolle des immunsystems im tierversuch acknowledgement the authors want to thank josef klingenschmid for excellent technical assistance. key: cord- -u eaaj authors: stolf, beatriz s.; smyrnias, ioannis; lopes, lucia r.; vendramin, alcione; goto, hiro; laurindo, francisco r. m.; shah, ajay m.; santos, celio x. c. title: protein disulfide isomerase and host-pathogen interaction date: - - journal: scientificworldjournal doi: . / / sha: doc_id: cord_uid: u eaaj reactive oxygen species (ros) production by immunological cells is known to cause damage to pathogens. increasing evidence accumulated in the last decade has shown, however, that ros (and redox signals) functionally regulate different cellular pathways in the host-pathogen interaction. these especially affect (i) pathogen entry through protein redox switches and redox modification (i.e., intra- and interdisulfide and cysteine oxidation) and (ii) phagocytic ros production via nox family nadph oxidase enzyme and the control of phagolysosome function with key implications for antigen processing. the protein disulfide isomerase (pdi) family of redox chaperones is closely involved in both processes and is also implicated in protein unfolding and trafficking across the endoplasmic reticulum (er) and towards the cytosol, a thiol-based redox locus for antigen processing. here, we summarise examples of the cellular association of host pdi with different pathogens and explore the possible roles of pathogen pdis in infection. a better understanding of these complex regulatory steps will provide insightful information on the redox role and coevolutional biological process, and assist the development of more specific therapeutic strategies in pathogen-mediated infections. host cells have the ability to cope with the progression and severity of infection in response to different types of pathogen. on the other hand, numerous mechanisms have evolved that support the use of the host cell machinery to facilitate pathogen survival and multiplication. such co-evolutionary processes are directly affected by different physicochemical factors within different cell compartments, both in the host and in pathogen. for instance, ph critically affects antigen stability of the influenza virus which modulates endosome acidity that attenuates its own infection [ ] . ros (and reactive nitrogen species) production and the redox state of different cell compartments are also critically involved in cellular hostparasite interaction. among the many redox sensitive proteins that are altered during the course of different infections, protein disulfide isomerase (pdi-) mediated redox switches have been associated with pathogen attachment-internalization, antigen processing in the er/phagosome, and the regulation of ros production by nox family enzymes. thus, pdi emerges as a ubiquitous redox protein that regulates different steps of diverse infection processes. several pathogens also have their own pdi that act as an important virulence factor (table ). other redox modifications directly mediated by ros and especially via nitric oxide (no) generated by inducible nitric oxide synthase (inos), which is abundant in phagocytic cells, have been reviewed elsewhere [ , ] and are not considered in this article. below, the main cellular redox aspects of host and pathogen pdi will be discussed. the ancient pdi is a ubiquitous redox chaperone belonging to the thioredoxin oxireductase super family and can reduce (reaction ), oxidize (reaction ), and catalyse dithiol-disulfide exchange reactions (i.e., isomerase activities, reaction , figure ). such broad range of activities overlaps with the chaperone role of pdi that overall performs a housekeeping function in helping to maintain proteins in a more stable conformation. there are around pdi homologues, and the detailed structure and function of eukaryotic pdis have been covered in recent excellent reviews [ , ] . the classic mammalian pdi ( kda) has several domains ordered as a-b-b -a -c with thioredoxin-like motifs (trp-cys-gly-his-cys) displayed in the a and a domain [ ] [ ] [ ] (figure ). pdi is abundant in the er (∼ . mm) where the relatively oxidizing conditions at basal level (i.e., gsh/gssg ratios ∼ - : ) favours pdi isomerase/oxidase activity, which is primarily involved in client protein redox folding (reaction - , figure ). the oxidizing equivalents for this process are driven mainly by the er thiol-containing oxidase, ero (endoplasmic reticulum oxidase- ), which binds fad and is in turn re-oxidized via electron transfer to oxygen, generating h o in the process [ ] [ ] [ ] [ ] . the h o destiny is elusive, but it can oxidize er-located peroxiredoxin iv (prxiv) that is further reduced by pdi that is oxidized in the process [ ] . this redox circuit is thought to increase total protein folding and thiol oxidation via ero [ ] . however, even in the absence of ero , protein folding still occurs, and it is suggested that other oxidases may compensate for redox demand in the er in some circumstances [ , ] . nevertheless, the pdi-ero -dependent oxidative activity is balanced to cytosolic glutathione levels suggesting a functional redox interplay between these compartments [ ] . pdi reductase activity has been primarily associated to more reducing compartments (i.e., gsh/gssg ratios ∼ - : ), such as those in the vicinity of the plasma membrane [ , ] . pdi redox versatility is mainly governed by the low pka of the proximal cysteine on the active n-terminal a domain. indeed, the lower pka of . renders pdi a much better oxidase than thioredoxin, which has a pka of . and is mainly a reductase in most neutral ph cell compartments. it should also be noted that pdi functions as a chaperone independently of its redox-active domains as especially required for its atpase and ca + activity, although pdi redox motifs still stabilize binding interaction [ , , ] . in the er, pdi is tightly associated with prolyl- hydroxylase (the rate-limiting enzyme for collagen biosynthesis), the sec translocon, and the mhc class i complex (see later). it can be also found as a heterodimer with microsomal triglyceride transfer protein [ , ] . pdi is a soluble homodimer and does not have a transmembrane domain and, similarly to other er chaperones, carries the kdel c-terminal sequence which binds to respective receptors in the cop vesicles that circulate in the er-golgi vicinity, recycling proteins back to er. pdi also undergoes intense intracellular trafficking and is found on the surface of diverse prokaryotic and eukaryotic cells [ ] [ ] [ ] . despite limited knowledge about this traffic, it is possible that pdi exits the er through the translocon sec pore and/or via secretory vesicles [ ] . pdi is thought to attach to lipids, glycans, and integral membrane proteins via electrostatic interactions at the cell plasma membrane [ , ] , where its reductive activity mediates the infection of different pathogens ( figure , discussed later). pdi along with erp have been found in the nuclei in association to dna and affecting transcriptional activity of nf-kb, ap- , and stat [ ] . these transcriptional regulators are key elements in many inflammatory processes, but their functional association to pdi and to different pathogen still elusive. in contrast to many other members of its family, such as thioredoxin itself and erp , pdi is not normally found in the cytosol, where it is likely cleaved by caspase- and - [ ] . protozoans and bacteria have their own pdis ( table ). the function of these pdis in protein folding is poorly understood; however, there are intriguing data correlating pdi expression and the pathogenicity of several parasites, especially obligatory intracellular protozoans. leishmania that leads to distinct types [ ] . if l. chagasi is a subgroup of l. infantum brought to america by european colonist or other specie in its own still a matter of controversy (see discussion in [ ] ). the infection cycle in the vertebrate host and is initiated when leishmania promastigote is injected into the skin by the insect vector. in the host, the promastigote is phagocytised especially by macrophages, and further it is converted into intracellular amastigote. amastigote replicates inside the phagosome within the cell and is liberated after the cell lyses, subsequently infecting other cells resulting in the progression to disease [ , ] . l. amazonensis has at least four pdis, and the use of specific pdi inhibitors substantially affected parasite growth [ ] . in l. major, the increased levels of leishmania pdi (lmpdi) expression and secretion at the parasite surface reflects optimal protein folding balanced to parasite multiplication. importantly, this is correlated to high virulence of the parasite strains [ ] . more recently, the use of lmpdi antigens to generate a vaccine for l. major partially protected balb/c animals and accelerated the cure of different strains of mice [ ] . similarly to leishmania species, other parasites of the trypanosomatid group such as trypanosoma contain several genes predicted to encode for pdis, that can execute n-glycosylation and protein folding in the er [ , ] . although pdi was considered essential for t. brucei survival, pdi activity was not essential for the growth of trypanosomes in vitro [ ] . pdi is also expressed in different species of plasmodium protozoans (table ) , the parasites that cause malaria [ , ] . p. falciparum expresses at least nine different pdis and the pfpdi- has great similarity to the prototype pdi and is expressed during all stages of parasite life cycle. this pdi facilitates the disulfide-dependent conformational folding of eba- protein, an emerging candidate for the development of malaria vaccines [ ] . this is intriguing given that malaria parasites express proteins with high content of cysteine, which are associated to parasite invasion and sequestration in the vertebrate host and transmission into mosquito host [ ] . finally, toxoplasma gondii pdi was identified in host tears, suggesting an extracellular location and adhesion to host cells during the initial phase of infection [ ] . antigen presentation occur through two distinct pathways. antigen presenting cells (apcs; especially macrophages and dendritic cells; dcs) are long-lived cells that capture antigens and subsequently process and present them at the cell surface, where they are recognized by t-lymphocytes. this process provides a long-term adaptive immune response to fungi, bacteria, and parasite. after internalization by the apc, antigens pass through phagosome/lysosome vesicles, where they form complexes with mhc class ii (figure ), which are recognized by helper cd + t lymphocytes (exogenous pathway). in contrast, self cell antigens and virus synthesized within cells (mostly non-apcs) are degraded by the proteasome in the cytosol and nucleus. in successive steps, the antigen is processed, folded, and incorporated into the mhc class i ( figure ) and the complex exposed on the cell surface, and recognized by cytotoxic cd + t lymphocytes (endogenous pathway). these two pathways overlap and some antigens are presented by both mhc class i and ii, in a process called cross-presentation. this has been described in dcs responding to viral infection, transplant rejection, and some autoimmune diseases and cancer. moreover, a wide range of pathogens passing or living in the phagosome such as mycobacterium tuberculosis, salmonella typhimurium, toxoplasma gondii, and especially leishmania spp and trypanosoma cruzi are all crosspresented in association to high levels of cd + t cells [ ] . pdi as part of the er protein folding machinery directly regulates antigen processing of the mhc class i complex [ ] [ ] [ ] [ ] . antigens that are degraded by peptidases and proteasome to shorter peptides in the cytosol and nucleus can be further transported to the er through the tap system, a transmembrane er type of atp-binding cassette (abc) peptide transporter family [ ] . er-located pdi interacts with the peptide-loading complex (pcl) that efficiently promotes peptide assembly with mhc class i molecules and supporting the exit of the peptide-antigen complex from the er [ ] [ ] [ ] . other pcl components include calreticulin, tapasin, erp (another pdi family member), and the tap transporter itself. cells lacking pdi present much less peptide loading to mhc class i and the disulfide bridge between the peptide and mhc groove remains in a reduced redox state [ ] . normally, this interaction is affected by the redox exchange between pdi (predominantly oxidized) and erp (predominantly reduced) [ ] , a condition in which pdi favours the release of peptide-mhc class i from the pcl and the antigen-mhci complex is exited from er [ ] . in fact, pdi-bound peptide facilitates the disassembly of the tapasin-erp complex while the pdi unbound to the complex is unable to interact with tapasin-erp , retaining mhc i molecules in the er [ ] . overall, pdi redox activity modulates the stability of the antigen peptide-mhc class i complex and further determines the transport of the complex to the plasma membrane [ ] [ ] [ ] [ ] . these redox effects may vary according to the type of antigen and some pathogens interfere with this pathway to escape antigen process and evading cd + t-cells recognition. this is the case for us protein from human cytomegalovirus, which enhances pdi degradation via the proteasome [ ] . pdi participation in immune response, however, goes beyond its role in the er protein folding machinery and it acts at other cellular steps of host-pathogen interaction. pdi in the er is also thought to play a role in parasite phagocytosis, and the pdi displayed on the cell surface can mediate the entry of some viral, bacterial, and protozoan. pdi is also implicated in protein unfolding and trafficking of some pathogenic antigens across the endoplasmic reticulum and towards the cytosol by the endoplasmic reticulum-associated degradation system (erad). this is the main pathway where proteins are retrotranslocated from the er to cytosol and further degradated by the proteasome. next, we discuss some examples of the cellular association between host pdi and different pathogens. phagocytosis is the main gate for large microbes to enter into apcs. after binding and attaching to the pathogen, these cells can internalize organisms and large particles even bigger then their own size, which are then phagocytosed in an active process that involves intense membrane remodelling [ ] . proteomics studies accumulated over the last decade revealed the presence of er chaperones in the isolated phagosome, uncovering a process called er-mediated phagocytosis [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . er chaperones were detected in phagosomes of macrophages exposed to different particulate material and pathogens, including latexbeads opsonised or not with immunoglobulin g (igg) or mouse serum (to facilitate entry through the fcr or complement receptors), igg-opsonized erythrocytes, promastigotes of leishmania donovani derived from wild-type cells or cell-surface lpg knockout, among other parasites [ ] . a mix of er and endocytic vesicles in the formation of the parasitophorous vacuoles (pvs) during the uptake of different leishmania spp. was recently shown in macrophages overexpressing er-tagged green fluorescent protein [ ] . the presence of the er proteins sec , bip/grp , and pdi in the phagosome of apcs [ , ] support the idea that the er provides the necessary machinery for antigen translocation from the phagosome to the cytoplasm and thus, possibly converges mhc class i and class ii antigen cross-presentation [ ] (figure ). there are several other complementary hypotheses on how peptides cross from phagosomes to cytoplasm during the cross-presentation process [ , ] . neutrophils are short-lived cells (half-life of - h in the human circulation) and very active in the phagocytosis of large microbes such as bacteria, parasites, and fungus. contrary to apcs, neutrophils only contain restricted amounts of er machinery and are thought to lack the er-mediated phagocytosis process [ ] . whether er proteins functionally operate on phagocytosis-mediated infection has not been well characterised yet. an important work has shown that dictyostelium lacking both er calreticulin and calnexin present altered phagocytic cup formation and substantial decline in phagocytosis [ ] . these two proteins utilise ca, and their disruption per se affects actin filaments and plasma membrane remodelling during phagocytosis [ ] . we recently showed that pdi is critically involved in leishmania parasite infection in vitro [ ] . we showed that phagocytosis of promastigotes (but not amastigotes) of leishmania chagasi was significantly inhibited by macrophage incubation with the thiol/pdi inhibitors dtnb, bacitracin, phenylarsine oxide, and neutralizing pdi antibody in a parasite redox-dependent way [ ] . the phenylarsine response is of particular interest, since this arsenic compound may act similarly to antimonials, widely used in leishmaniasis chemotherapy [ , ] . pdi preferentially affects parasite internalization and the phagocytosis of the promastigote forms is increased when wild-type pdi is overexpressed in macrophages, an effect opposed by pdi knockdown. at later stages of infection (i.e., after h), pdi from promastigote-infected j macrophages was immunoprecipitated and subsequently blotted with an anti-leishmania antibody revealing a parasite band at ∼ kda [ , figure (b), lane ]. subsequent removal and analysis of this band by mass fingerprint spectrometry showed a % match with elongation factor (ef ) of l. major (q q ; data not shown). the incubation of purified bovine pdi (sigma, p ) and parasites did not yield any detectable protein complexes, suggesting that the macrophage milieu may be important to sustain pdi-ef association [ ] . interestingly, leishmania ef has important virulent features and acts as a soluble antigen in lymphocyte stimulation in vitro [ ] and in vivo [ ] . moreover, proteomics studies revealed that ef is secreted during promastigote differentiation into the amastigote stage with potential immunomodulatory proprieties in animal models [ ] . leishmania ef is therefore of particular interest for leishmania therapeutic interventions such as vaccines. although our studies did not address the role of the er in mediating phagocytosis, these data provide compelling evidence for a functional role of er-pdi in a host-parasite interaction. other mechanisms underlining pdi-mediated l. chagasi promastigote phagocytosis involves its association to ros production by phagocyte nadph oxidase and this is discussed next. the nadph oxidase (nox) family of enzymes uses nadph as an electron donor to convert oxygen to superoxide anion (o •− ), a precursor of h o and other powerful oxidants such as hydroxyl radical and peroxynitrite (in the presence of nitric oxide), collectively called ros [ , , ] . each of the seven oxidase family members is characterized by a distinct catalytic subunit (i.e., nox - and duox - ), and has differing requirements for additional protein subunits [ , ] . the prototypic member of the nox family, nox oxidase (or gp phox oxidase), is best known for its role in neutrophil and macrophage phagocytosis. genetic defects in the enzyme are related to chronic granulomatous disease, a condition in which affected children suffer from recurrent severe fungal and bacterial infections due to defective phagocyte function [ ] . each nox isoform forms heterodimers with a lower molecular weight p phox subunit and is predicted to be membrane-bound. nox is normally quiescent and acutely activated by agonists such as pma, lpg, and cytokines in a tightly regulated process in which cytosolic subunits (p phox , p phox , p phox , and rac in the case of macrophages and dendritic cells, or rac in neutrophil) associate with the nox -p phox heterodimer to initiate enzyme activity [ , ] . nox also has electrogenic features [ ] and in apc cells is linked to the regulation of phagosome/lysosome ph and antigen processing [ , ] . usually, phagosome acidity is maintained by a vacuolar atpase (v-atpase) that transports protons from the cytosol into the phagosome lumen, therefore regulating the function of lysosome proteases in the fused phagolysosomes. savina et al. [ ] [ ] [ ] have shown that nox -derived superoxide in the phagosomal vesicle promptly consumes protons maintaining a higher ph ambient in dendritic cells during particle internalization, which favours antigen processing and presentation [ ] [ ] [ ] . opposite results were found in macrophages [ ] [ ] [ ] . the selective role of nox in different phagocytic cells remains to be defined. the jury is out on whether the results shown in macrophages (association of nox to rab a; a member of rab family of gtpases) are related to vesicle traffic molecule assembly and quality, or rather associated to degradation processes [ ] . nox complex protein expression and function is greatly affected by redox compounds, and it is especially regulated by pdi with implications for cell signalling [ ] . the association of pdi to p phox and other nox isoforms in different cell types, especially in vascular cells, has been previously described [ ] [ ] [ ] . a functional and spatial/physical interaction between pdi and the p phox oxidase subunit was shown in macrophages [ ] and more recently between pdi and p phox in neutrophils [ ] . in macrophages, pdi-nox association was correlated to leishmania infection in vitro [ ] . it is well known that during phagocytosis of leishmania, nox is activated and parasite uptake is inhibited by antioxidants such as catalase [ ] . intriguingly, in the course of promastigote infection, some parasites evade that stressful condition and convert themselves into intracellular amastigotes, multiplying and resulting in progression to a disease process. overall, our studies support the view that parasite phagocytosis/infection by macrophages is a redox process mediated by pdi in at least two ways. initially, pdi-nadph oxidase increases ros production generating an oxidizing milieu, which seems to favour promastigote infection. the downstream role of ros generated by pdi-nadph oxidase remains unknown but can be related to the unfolded protein response signalling [ ] or, similar to pdi-ero , to protein folding in the macrophage er compartment with key implications for antigen processing. nevertheless, at later stages of infection, macrophage pdi physically associates with leishmania elongation factor- (as discussed earlier). some viruses envelop their genetic material within a protein-coated capsid in a further lipid membrane layout, for example, influenza virus, baculovirus, hepatitis-c, hiv, and herpes virus. these enveloped particles require successive steps to successfully entry and infect host cells. they usually first attach onto host receptors (and attachment factors), and their membranes fuse to interact with endosome vesicles that traffic the virus toward the endoplasmic reticulum, where it is uncoated. the proteins are finally transported to the cytosol and nucleus [ ] (figure ). there is convincing evidence showing that most viral infections are strongly influenced by changes in the redox environment and that host pdi mediates infection of enveloped viruses [ ] [ ] [ ] [ ] [ ] [ ] . in the course of hiv infection, the virus first binds to attachment factors, for example, mannose binding c-type lectin receptor and intracellular adhesion molecule (icam- ) on the surface of host cd + t cells. the glycoprotein (gp ) subunit of the virus envelope binds to immunoglobulin g of cd + and undergoes conformational changes, allowing the virus to interact with its coreceptors, cxcr or ccr . these interactions favour downstream conversions of gp envelope subunit to a competent fusion conformation. initial studies showed that membrane-impermeable pdi inhibitors and monoclonal antibodies against pdi prevent hiv- infection [ ] . it was then revealed that the domain d of the cd has redox-active disulfide bonds and is regulated by thioredoxin [ ] . using membrane-impermeable reducing agents (especially arsenical-derived compounds) and labelling thiol reagents, it was demonstrated that cd + reactive thiols critically drive hiv entry into cells [ ] . work from another group also revealed that pdi, on the surface of hiv- target cells, reduces disulfide bonds of the recombinant envelope glycoprotein gp (reaction , figure ), a reaction prevented by the usual pdi inhibitors [ ] . intriguingly, pdi silencing in u and hela cells had little impact on hiv infection itself as compared to the effect mediated by general thiol inhibitors [ ] . the reasons for this discrepancy remain to be elucidated and raise the question whether the reductive effect of pdi is coupled to other redox proteins (e.g., thioredoxin or nox's) that could amplify virus-cd redox association in some cells. it is noteworthy that in these later studies, pdi knockdown on the cell surface was not evident as compared to massive loss of most pdis within the er; an observation that supports the idea that pdi in the er has little impact in hiv-mediated infection [ ] . thiol inhibitors also affect viral fusion as that mediated by the fusion (f) protein from the paramyxovirus newcastle disease virus [ ] . the overexpression of pdi family members pfdi and erdj has also been shown to significantly catalyze the reduction of thiols in f protein, facilitating membrane fusion [ ] . there is evidence suggesting a possible association between pdi and infection mediated by some members of the of herpesviridae viruses family [ ] . pdi is also implicated in the attachment of some bacteria from different species of chlamydia [ ] [ ] [ ] . chlamydia is an obligatory intracellular pathogen that causes diverse diseases in humans. the most common species are chlamydia trachomatis, which is sexually transmitted and can cause blindness and infertility, and c. pneumoniae, which affects the respiratory tract. cho cells have impaired endogenous pdi expression due to a defect in truncated mrna processing, thus providing a valuable model to understand the effect of pdi-mediated cell-cell interaction and infection. these cells are very resistant to chlamydia infection showing impaired attachment, an effect restored by ectopic expression of pdi [ ] . similar to hiv infection, the molecular mechanisms most likely include the reductive activity of pdi (reaction , figure ) on the surface of cho cells [ ] . crossing the endoplasmic reticulum (er) membrane is an irreversible process for most proteins. in some cases, however, this flow is reversed and misfolded proteins retained in the er are retrotranslocated to the cytosol via erad to be degraded by the proteasome. this pathway is also exploited by small pathogens, especially non-enveloped viruses and some bacterial toxins, to gain access to the cytosol. in these cases, antigenic particles that reach the er by different means suffer molecular redox rearrangements and binding to pdi allowing them to be transported back to the cytosol or nucleus. well-known examples are infections mediated by polyomaviruses (py) and simian virus (sv ) extensively studied in the field of carcinogenesis. after sv interaction with the gm receptor on the cell surface, the particle enters the host cell through endocytosis and traffics via the caveosome (a particular caveolin containing endosome with neutral ph) towards the er compartment [ , ] . sv -coated pentamers are linked to each other by disulfide bonds between cysteine (c ). further isomerisation in the er is crucial for the viral uncoating process. in vitro cell screening shows that among all er-resident proteins, pdi and erp more specifically regulate sv infection [ ] . pdi silencing substantially decreases sv infection that is also dependent on some redox sensitive cysteines on the viral particle [ ] . pdi cooperation with erassociated erad proteins derlin- and sel l is ca dependent and facilitates sv traffic through erad [ ] . a similar pathway is used by some nonobligatory intracellular bacteria that exert their effect through production of potent endotoxins, such as diphtheria toxin (dt) and cholera toxin (ct). these proteins function similarly to some plant toxins, such as ricin and abrin. conversion into toxic proteins involves cleavage of their interchain disulfide bond, allowing them to traffic into the endocytic pathway within the host cell [ , ] . in humans, ct is derived from the bacterium vibrio cholerae that causes cholera disease and has subunits (a and a ). the protein first attaches to the host cell surface via gm and the subunit a , which contains a kdel sequence, and is transported back to the er (see earlier discussion). there, pdi reduces and unfolds a and a that exit the er via the sec channel into the cytosol. pdi in the reduced state (reaction , figure ) binds to the toxin and subsequent oxidation of pdi, probably via ero α, enables the release of ct toxin [ , ] . the active polypeptide a efficiently modifies a heterotrimeric g protein in the cytosol that leads to massive loss of chlorine and water secretion by intestinal epithelial cells in mammals, resulting in severe diarrhoea. in this article we have reviewed the main cellular aspects of pdi-mediated host pathogen interactions and the pathways that are involved in viral, bacterial (including bacterial toxins), and parasitic infections. a number of cellular mechanisms through which pdi modulates some specific cellular pathways in immune cells have been described, such as redox-sensitive attachment, antigen presentation in the er and exit from it, and association to phagosome and ros production by nadph oxidase (figure ). many of these responses are antigen-specific and the precise mechanisms of action remain to be fully elucidated, especially in the context of redox changes in cross-presentation phenomena. moreover, little is known about the role of pdi in infection per se, as well as how pdi signals to a more integrated cellular response to stress [ ] . pdi global knockout mice are only viable until birth, but partial gene-modified mice and also modified pathogens will help to reveal the significant redox role of pdi and its redox partners. overall, pdi is a key regulator that may propagate or limit the severity of the infection processes, depending on the infectious organism involved. a better understanding of these complex regulatory steps will provide insightful information on the redox role and coevolutional biological process, and assist the development of more specific therapeutic strategies in pathogen-mediated infections. endoplasmic reticulum mhc: major histocompatibility complex h o : hydrogen peroxide erp : a member of pdi family also know as glucose-regulated protein or -kd (grp ) nf-kb: factor nuclear kappa b ap- : activator protein stat- : signal transducer and activator of transcription . cell entry by enveloped viruses: redox considerations for hiv and sars-coronavirus role of nitric oxide synthesis in macrophage antimicrobial activity nitrogen dioxide and carbonate radical anion: two emerging radicals in biology the human pdi family: versatility packed into a single fold 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pdi family: unpredicted non-er locations and functions identification of a disulfide isomerase protein of leishmania major as a putative virulence factor glutathione limits ero -dependent oxidation in the endoplasmic reticulum protein disulfide isomerase is cleaved by caspase- and - during apoptosis tegumentary leishmaniasis as a manifestation of immune reconstitution inflammatory syndrome in patients with aids the molecular basis of leishmania pathogenesis further thoughts on the use of the name leishmania (leishmania) infantum chagasi for the aetiological agent of american visceral leishmaniasis protein disulfide isomerase (pdi) associates with nadph oxidase and is required for phagocytosis of leishmania chagasi promastigotes by macrophages identification and enzymatic activities of four protein disulfide isomerase (pdi) isoforms of leishmania amazonensis comparative evaluation of two vaccine candidates against experimental leishmaniasis due to leishmania major infection in four inbred mouse strains a developmentally regulated gene of trypanosomes encodes a homologue of rat protein-disulfide isomerase and phosphoinositol-phospholipase c characterization of two protein disulfide isomerases from the endocytic pathway of bloodstream forms of trypanosoma brucei cloning of plasmodium falciparum protein disulfide isomerase homologue by affinity purification using the antiplasmodial inhibitor , -bis{ -[n-(cyclohexyl methyl)amino]propyl}piperazine protein disulfide isomerase assisted protein folding in malaria parasites protein disulfide isomerase of toxoplasma gondii is targeted by mucosal iga antibodies in humans oxidative folding and reductive activities of ehpdi, a protein disulfide isomerase from entamoeba histolytica leishmania antigens are presented to cd + t cells by a transporter associated with antigen processing-independent pathway in vitro and in vivo redox regulation facilitates optimal peptide selection by mhc class i during antigen processing molecular 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endoplasmic reticulum are important for phagocytosis molecular mechanisms of antimony resistance in leishmania dual action of antimonial drugs on thiol redox metabolism in the human pathogen leishmania donovani proteomic approach for identification and characterization of novel immunostimulatory proteins from soluble antigens of leishmania donovani promastigotes identification and characterization of t cell-stimulating antigens from leishmania by cd t cell expression cloning approaches for the identification of potential excreted/secreted proteins of leishmania major parasites nadph oxidases in lung biology and pathology: host defense enzymes, and more nox enzymes and the biology of reactive oxygen the superoxide-generating nadph oxidase of human neutrophils is electrogenic and associated with an h + channel the nadph oxidase of professional phagocytes-prototype of the nox electron transport chain systems phagocytosis and antigen presentation in dendritic cells nox controls phagosomal ph to regulate antigen processing during crosspresentation by dendritic cells rab a regulates phagosomal ph and nadph oxidase recruitment to dendritic cell phagosomes nadph oxidase controls phagosomal ph and antigen crosspresentation in human dendritic cells activation of antibacterial autophagy by nadph oxidases novel role of protein disulfide isomerase in the regulation of nadph oxidase activity: pathophysiological implications in vascular diseases regulation of nad(p)h oxidase by associated protein disulfide isomerase in vascular smooth muscle cells protein disulfide isomerase redox-dependent association with p phox: evidence for an organizer role in leukocyte nadph oxidase activation mechanisms and implications of reactive oxygen species generation during the unfolded protein response: roles of endoplasmic reticulum oxidoreductases, mitochondrial electron transport, and nadph oxidase how viruses enter animal cells inhibition of human immunodeficiency virus infection by agents that interfere with thiol-disulfide interchange upon virus-receptor interaction disulfide exchange in domain of cd is required for entry of hiv-i inhibitors of protein-disulfide isomerase prevent cleavage of disulfide bonds in receptor-bound glycoprotein and prevent hiv- entry role of protein disulfide isomerase and other thiol-reactive proteins in hiv- envelope protein-mediated fusion thiol/disulfide exchange is required for membrane fusion directed by the newcastle disease virus fusion protein overexpression of thiol/disulfide isomerases enhances membrane fusion directed by the newcastle disease virus fusion protein the human cytomegalovirus ul immediate-early regulatory protein is an integral membrane n-glycoprotein which traffics through the endoplasmic reticulum and golgi apparatus attachment and entry of chlamydia have distinct requirements for host protein disulfide isomerase chlamydia attachment to mammalian cells requires protein disulfide isomerase protein disulfide isomerase, a component of the estrogen receptor complex, is associated with chlamydia trachomatis serovar e attached to human endometrial epithelial cells simian virus depends on er protein folding and quality control factors for entry into host cells downregulation of protein disulfide isomerase inhibits infection by the mouse polyomavirus cleavage of disulfide bonds in endocytosed macromolecules. a processing not associated with lysosomes or endosomes cell surface sulfhydryls are required for the cytotoxicity of diphtheria toxin but not of ricin in chinese hamster ovary cells unfolded cholera toxin is transferred to the er membrane and released from protein disulfide isomerase upon oxidation by ero the ero α-pdi redox cycle regulates retro-translocation of cholera toxin protein disulfide isomerase and host-pathogen interaction the authors thank daniel c. pimenta for mass spectrometry experiments (centre for applied toxinology cat/cepid, butantan institute). they also thank dr. joão wosniak jr. from vascular biology laboratory, heart institute (incor) for helpful discussion. this paper was supported by auxílio fundação de amparò a pesquisa do estdado de são paulo (fapesp) and conselho nacional de pesquisa (cnpq), instituto do milênio redoxoma; fundação ej zerbini (incor). a. m. shah, s. ioannis and c. x. c. santos are also supported by the british heart foundation and a leducq fondation transatlantic network of excellence award. key: cord- -ej e c d authors: kim, yohan; yewdell, jonathan w.; sette, alessandro; peters, bjoern title: positional bias of mhc class i restricted t-cell epitopes in viral antigens is likely due to a bias in conservation date: - - journal: plos comput biol doi: . /journal.pcbi. sha: doc_id: cord_uid: ej e c d the immune system rapidly responds to intracellular infections by detecting mhc class i restricted t-cell epitopes presented on infected cells. it was originally thought that viral peptides are liberated during constitutive protein turnover, but this conflicts with the observation that viral epitopes are detected within minutes of their synthesis even when their source proteins exhibit half-lives of days. the drips hypothesis proposes that epitopes derive from defective ribosomal products (drips), rather than degradation of mature protein products. one potential source of drips is premature translation termination. if this is a major source of drips, this should be reflected in positional bias towards the n-terminus. by contrast, if downstream initiation is a major source of drips, there should be positional bias towards the c-terminus. here, we systematically assessed positional bias of epitopes in viral antigens, exploiting the large set of data available in the immune epitope database and analysis resource. we show a statistically significant degree of positional skewing among epitopes; epitopes from both ends of antigens tend to be under-represented. centric-skewing correlates with a bias towards class i binding peptides being over-represented in the middle, in parallel with a higher degree of evolutionary conservation. the immune system rapidly detects virus-infected cells through cell-surface presentation of viral peptides to t-cells despite the fact that the half-lives of source proteins are typically orders of magnitude longer than the response time (i.e. days vs. minutes) [ , ] . to explain this paradox, the drip hypothesis proposed that defective ribosomal products, rapidly degraded forms of standard gene products, are a major source of peptides for mhc class i processing pathway [ ] . although the drips hypothesis is well into its teens, surprisingly little is known about the nature of the drips [ ] [ ] [ ] . this is likely due to the low abundance of drips relative to the abundance of folded proteins, which poses a significant challenge to current biochemical/molecular technologies. a central question is what mechanisms dominate in the production of drips [ , ] . one set of mechanisms that leads to the generation of partial protein products is downstream initiation and premature terminations when translating proteins from mrnas [ ] . if such errors are involved in generation of drips, a bias in sampling of regions of mrnas may result. this sampling bias in turn may influence from which regions of proteins epitopes are more often detected, resulting in positional bias of epitopes. for example, a dominance of downstream initiation would result in more epitopes detected from the c-terminal ends; conversely, a dominance of premature termination would result in more epitopes detected from the nterminal regions. to overcome the limitation of the current experimental approaches, we have investigated if a data-driven approach can provide insights into the nature of drips. namely, the availability of a large repository of immune epitopes stored at the immune epitope database (iedb) [ ] makes it possible to indirectly characterize the population of drips by measuring positional bias of viral epitopes. because viruses exploit the translational machinery of the host to synthesize their proteins and are thus relevant in the context of the drips hypothesis, we retrieved all mhc-i restricted t-cell epitopes of viruses for which reference proteomes are available. top viruses based on number of tested peptides are shown in table . for each virus, total number of unique peptides tested, peptides with positive t-cell outcomes (i.e. epitopes), and those with negative outcomes are shown. in the table, vaccinia virus contributed the greatest number of tested peptides, followed by hepatitis c virus, lymphocytic choriomeningitis virus, human herpesvirus / and influenza a virus. after determining positions of viral peptides in their reference antigens and calculating normalized positions, we constructed distributions of normalized positions for positives (i.e. epitopes) and negatives as shown in fig. a and b, respectively. each distribution used bins of equal intervals covering a range [ , ]. distributions plotted using bins showed similar patterns (data not shown). the distributions correspond to probability mass functions, p(x|positive) and p(x|negative), where 'x' represents a binned normalized position (described further in methods section). to show positional bias of epitopes, a probability ratio plot (i.e. p(x|positive)/p(x|negative), see methods for details) is shown in fig. c . by dividing the distribution for positive peptides by the distribution of negative peptides, we implicitly take into account study biases that are otherwise difficult to capture. if positional bias is absent, the corresponding probability ratio plot would show a horizontal line at a probability ratio of . if positional bias is present, regions with lower likelihoods of finding epitopes would show values less than . indeed, in fig. c , the probability ratio plot shows under representation of epitopes at n-and c-termini. furthermore, probability ratio plots generated after cumulative exclusion of data from vaccinia virus and hepatitis c virus, which contributed most in terms of data, resulted in maintaining the overall pattern (supplementary fig. s ) the positional bias of epitopes observed is supported by results of statistical analyses on the corresponding contingency table (supplementary table s ). using a binomial test for each bin, it was determined whether counts of positives significantly deviate from the expected fraction of positives (i.e. (all positives)/(all positives+all negatives) = / ). out of bins, deviated from the expected (two sided; p-value, . ). a possible source of positional bias of epitopes is unequal distributions of amino acids spanning the length of antigens. for instance, the positional bias observed may have been due to hydrophobic residues being preferentially found in middle regions rather than at n-and c-termini. such unevenness would mean that mhc alleles binding peptides with hydrophobic anchor residues would impose positional bias of epitopes. to rule out this possibility, we determined positional bias of predicted binding peptides of viruses to mhc molecules. our prediction strategy uses recently developed peptide:mhc-i binding algorithms, which have achieved high accuracies in benchmarks [ , ] . using -mer peptides generated from a set viral proteins, we made binding predictions to human mhc-i molecules (hla). as a note, hla molecules can be grouped into hla supertypes based on their known overlap of binding specificities [ ] . smm pmbec was used to make binding predictions [ ] . corresponding member alleles for each supertype used for predictions are provided in the supplementary material (table s ) . after grouping predictions based on supertypes, for each supertype, we generated distributions of normalized positions for predicted 'binders' (i.e. those peptides with predicted binding affinities , nm) and 'non-binders' (i.e. those with affinities . nm). probability ratio plots derived from the distributions for hla supertypes are shown in fig. . the lack of divergence from a horizontal line at . indicates absence of positional bias. in the figure, some of the supertypes show slight positional bias. specifically, supertype a favors the middle region of antigens, whereas a favors the c-terminus. a combined plot generated from weighted averaging of probability ratio plots from all supertypes based on frequencies of known mhc restriction data showed absence of positional bias (supplementary fig. s ). to ensure that these observations are not due to the choice of predictive method, we used a different predictive method netmhc [ ] , which is another accurate predictive method, and made the same observations. in addition, scatter plots of predicted binding affinities for the tested peptides and their to defend the host from an infection, the immune system continuously scans cell surfaces for foreign objects. specifically, a virus inside a cell exploits the host to make copies of its proteins; viral proteins are broken up into peptide fragments; and the fragments are displayed on the infected cell's surface, thereby allowing detection and cellkilling. how these peptide fragments for cell-surface presentation are generated remains unknown. an understanding of this step will lead to rational design of vaccines and insights into tumor immunosurveillance and autoimmunity. one possible mechanism is that the peptide fragments come from defective proteins missing either the beginning or end regions, which may result in a bias. here, we analyzed locations of a large set of known viral epitopes, peptide fragments recognized by the immune system, within their proteins. we find that all regions of proteins are represented well by the immune system. however, there is a statistically significant bias in the central regions of proteins, which correlate with a pattern of conservation spanning the length of viral proteins. our results suggest a combined effect of conservation and enhancement of immune responses through repeated exposures in shaping the distribution of known viral epitopes. normalized positions showed no systematic effects (supplementary fig. s ). the presented results largely reflect results reported in [ ] , where mammalian, bacterial, and viral proteomes show lack of influence of mhc class i binding preferences. however, in the same work, a and b supertypes showed bias in signal-peptide regions (i.e. n-terminal). we confirmed that using our prediction strategy, signal-peptide specific biases observed for a and b are present for both human and viral antigens if residue positions are used instead of normalized positions (data not shown). a drip-independent factor that could explain the positional bias of viral epitopes is positional bias of protein conservation. specifically, if ends of proteins are less conserved than the middle region and epitopes tend to be more conserved than non-epitopes, positional bias of epitopes may result. to test this possibility, we calculated conservation scores at the residue-level for proteins of the viruses (see methods for details on calculation of conservation scores). conservation scores could be assigned to , % of proteins. the remaining proteins had an insufficient number of suitably distant homologues to construct reliable conservation scores, and were therefore excluded from this analysis. in fig. , a boxplot showing confidence intervals of means of conservation scores as a function of normalized position is shown. this reveals a pattern of positional bias of conservation, where ends of proteins are less conserved than their cores, similar to the pattern observed for the positional bias of known epitopes in fig. c . for the middle bin, however, we do see a difference. to determine if sequence conservation alone can explain positional bias of epitopes, we first had to determine the relationship between conservation and immune recognition. in fig. a , distributions of conservation scores for positives and negatives are shown. positives tend to be more conserved than negatives (welch's t-test; one-sided; p-value = . ). in fig. b , a plot of probability ratio as a function of conservation score is shown. the plot is a result of calculating ratios of probability masses for positives and negatives shown in fig. a . fig. b , detecting epitopes is less likely as conservation decreases. next, we combined our estimates of conservation bias over the length of a protein with our estimate of correlation between conservation and immune recognition. using conservation as a function of normalized position in fig. as input, we estimated probability ratios as a function of normalized position (fig. c ). its overlay with the probability ratios curve observed earlier (i.e. fig. c ) is shown in fig. d . overall, the probability ratios curve estimated only from the conservation data has a good agreement with the observed one (pearson's correlation r = . ). in addition, for first, second and fourth bins, their confidence intervals between observed positional bias curve and that derived from conservation overlap. thus, the observed positional bias in viral epitopes can be explained by the correlation between conservation and immune recognition alone, consistent with a minor contribution from premature translational termination and downstream initiation. viral epitopes in the context of immunization with peptides display similar level of conservation as nonepitopes there are two possible explanations for the observed correlation between immune recognition and conservation of peptides. one explanation is that the immune recognition machinery has evolved to preferably recognize epitopes that are conserved, as evidenced by an overlap of mhc binding motifs on conserved sequence regions found in [ ] . an alternative explanation is that viral sequences are variable, and responses against epitopes from conserved regions could be higher in individuals that are exposed to multiple variants of a virus over time, as is expected for example in human influenza infections. to determine whether there is an intrinsic enhanced immune recognition for peptides that are conserved in viral species, we retrieved viral epitopes identified in the context of peptide immunization, rather than with viral infection. figure shows distributions of conservation scores for positive and negative peptides, as was done earlier. the two distributions overlap, and their difference in means is not statistically significant (pvalue = . ). thus, we conclude that we cannot detect an intrinsically enhanced immune recognition for peptides that are conserved. this leads us to favor the hypothesis that the observed enhanced immune recognition of conserved viral peptides is due to extrinsic effects such as repeated priming of responses against conserved peptides due to heterologous exposure. by leveraging a large set of experimentally determined epitopes from a wide range of viruses stored in the iedb, we determined positional bias of epitopes in source antigens. the shape of positional bias curve (fig. ) shows significant under-representation of epitopes at n-and c-termini. this could be explained by near equal participation of downstream initiation and premature termination mechanisms in generating drips as a major source of epitopes. our findings, however, point to the conclusion that the central bias of viral epitopes reflects the combined effects of positional bias of epitope sequence conservation and induction of memory cd + t cells by exposure to heterologous boosts. there is a good correlation between positional bias curve estimated only from conservation data with that of the observed position bias curve of epitopes, so we cannot clearly detect (or clearly rule out) partially translated drips transcripts as a major source of viral epitopes. the connection between positional epitope biases with protein conservation is reasonable in the context of boosting effects associated with repeated vaccine administrations. the principle idea behind boosting is that those epitopes already exposed to the immune system tend to dominate in the following exposure [ ] . this repeated exposure is a likely explanation of why mhc-i restricted viral epitopes tend to be more conserved than nonepitopes. providing an additional support to this explanation, kim et al. presented results showing positive correlation between epitope conservation and t-cell response frequency scores, which indicate how often individuals recognize a given peptide from a pathogen [ ] . presumably, this correlation is due to higher likelihood of more conserved epitopes being seen by greater number of individuals. in addition, as more immune epitope data are deposited at the iedb, we expect to see differences in the degree of positional bias for rna versus dna viruses, because rna viruses are more variable. in addition to explanations discussed above, there are a number of recent ones that may be relevant. first, calis et al. have reported correlations between g+c content and potential mhc-i figure . positional biases of predicted binders for hla supertypes. for each supertype, -mer peptide binding predictions were carried out and ratios of probability masses of predicted 'binders' and 'non-binders' were calculated. peptide binding predictions were made for alleles belonging to each supertype, using smm pmbec method. all possible -mer peptides were generated from a set of viral proteins that contain at least one tested peptide from table . relationships between hla molecules and supertypes are provided in [ ] . doi: . /journal.pcbi. .g binders [ ] , where low g+c content indicates pathogenicity. in our hands, we could not detect significant contribution of mhc-i binding affinity preferences to positional bias of epitopes, thereby making g+c content a less likely explanation. in explaining differential conservations between positive and negative peptides, it may be that this is due to viruses selectively mutating t-cell epitopes [ ] [ ] [ ] . the idea is interesting and may be pursued in a future study. our data set however takes protein variability as given and thus cannot be used to delineate its cause/effect relationships. other investigators have also reported epitope conservation for hiv [ , ] and tb [ ] . our results extend their findings to a broad set of viruses, and suggest a possible connection between epitope conservation and boosting through analyses of epitopes determined in the context of immunization with peptides. it remains to be seen whether this boosting effect can be also observed for mhc class ii restricted epitopes as well as in other immunological contexts. regarding the mhc-motif specific biases and conservation, it has been reported that predicted binding affinities of hla molecules positively correlate with conserved regions of a wide range of viruses [ ] , which appears to contradict the results of absence of mhc-specific biases presented here. the absence of bias may be explained by the fact that the correlation between predicted binding affinity and protein conservation reported in [ ] is very small to begin with (spearman's rank correlation of at most , . ), thereby dampening any mhc-specific biases that can be seen. in conclusion, to better understand mechanistic details of antigen processing steps involving drips, positional bias of mhc-i restricted viral t-cell epitopes was measured. our findings indicate that there is indeed such bias in antigens, where epitopes at n-and c-termini are under-represented. although mechanisms associated with translational errors such as downstream initiation and premature termination may contribute to observed positional bias, our data indicate that differential conservation spanning protein length is an alternative explanation. for each virus listed in table , we retrieved mhc-i restricted t-cell epitopes from the immune epitope database (iedb) (http://www.iedb.org), which is the largest publicly available database of epitopes for infectious agents [ ] . to retrieve an appropriate set of epitopes to examine the drips hypothesis, there were a number of important considerations. first, the query should retrieve epitopes derived from proteins newly expressed in a host cell, rather than epitopes recognized after peptide or protein immunization. only for newly synthesized epitopes can defective ribosomal products skew the positional distribution of epitopes in antigens. to meet this requirement, we query the iedb for epitopes identified using assays in which the 'immunogen type' is a whole 'organism' (rather than an individual peptide or antigen). second, we further limit the query to epitopes restricted by mhc class i molecules. third, we limit the query to epitopes with 'virus' as the source organism. we then grouped the epitopes retrieved with the query by viral species. as described below, we want to map all epitopes from one species to a single reference proteome. therefore, we excluded all viruses for which we do not have reference proteomes available, resulting in a total of different viruses. to ensure consistent calculation of positional bias, we mapped all epitopes onto antigens from a single complete reference genome for each species based on sequence similarity rather than using the source antigens listed in the iedb, which are those specified by the author mapping the epitope and are derived from different strains and are of divergent quality. for example, an author may have used truncated versions of an antigen, or epitopes may come from a polyprotein of dengue virus, which later gets cleaved into individual products. consequently, epitope positions can be made relative to the polyprotein or to final cleavage products. to carry out the mapping, we used ncbi's blast with a default setting to search for presence of epitopes in antigens and to retrieve only those hits with exact matches in the reference genome. in addition, we required homology between the originally curated source antigen of the epitope and the antigen in the reference genome using blast searches with an e-value cutoff of . , thereby ensuring meaningful mapping. lastly, we required that there is only a single best match of the epitope in the reference genome to ensure that the position of the epitope in the antigen can be uniquely determined. we did not consider ties because of associated uncertainty in mapping. to derive a measure of epitope position that is independent of protein length, a normalized position, x, is defined as follows: in the equation, 'peptide_start' indicates the position of the first residue from the peptide mapped onto the protein sequence; for each viral protein which also contained at least one tested peptide from table , conservation scores at the residue-level were calculated using rate site [ ] . residue positions were converted into normalized positions and corresponding conservation scores were binned ( bins of equal size). higher conservation score indicates higher degree of conservation. conservation scores were normalized for each protein (i.e. mean = ; sd = ). one thousand bootstrapping over proteins was used to estimate confidence intervals for the means of conservation scores. 'protein_length' and 'peptide_length' are lengths of protein and peptide, respectively. the first residue of a protein has a position of , rather than . our measure of normalized position, x, has the property that if a peptide contains the first residue of the protein (and thus comes from an n-terminal region), then its value is zero; if the peptide contains the last residue, then the value is . . consequently, if all regions of a protein are equally likely to contain epitopes, then one would obtain a uniform distribution of normalized positions in the range [ , ]. this property has been verified using randomly sampled peptide positions and uniformly sampled peptides from a set of proteins, followed by mapping the peptides onto the proteins with blast. deriving probability ratios curve for measuring positional bias of epitopes after mapping peptides with positive (i.e. epitopes) and negative t-cell assay outcomes onto their corresponding antigens, we calculated their normalized positions, x, as described in equation . we then grouped the normalized positions into 'positives' and 'negatives', and binned using bins of equal intervals covering a range [ , ], resulting in probability mass functions (pmfs): p(x|positive) and p(x|negative). the function p(x|positive) gives a probability of observing peptides at binned position x, given that only positives were considered. to indicate positional bias, we calculated ratios of probability masses for positive and negative pmfs: p(x|positive)/p(x|negative). absence of positional bias corresponds to a probability ratio of . for all bins. a probability ratio less than indicates underrepresentation of epitopes while greater than indicates overrepresentation. to determine whether differences in conservation over the normalized position in a protein contribute to positional bias of epitopes, we estimated conservations at the residue level for proteins from the viruses using rate site algorithm [ ] . we chose the algorithm because it was identified as one of the highest performing methods in a recent benchmark to predict a known set of protein catalytic sites [ ] . to estimate conservation, we used a protocol similar to one used by the consurf website [ ] . specifically, a multiple sequence alignment was generated for each protein by running a sequence of psi-blastrcd-hitrmus-cle against the ncbi non-redundant database, using a set of perl scripts retrieved from the consurf website. running the conservation score estimation algorithm on the alignment returned residue position-specific conservation scores. figure s positional bias curves of epitopes after removing varying amounts of data. for reference, the positional bias curve using all data is shown ('all'). the curve labeled 'v' refers to exclusion of data from vaccinia virus. the curve labeled 'v+h' refers to exclusion of data from vaccinia virus and hepatitis c virus. vaccinia virus had the largest amount of immune epitope data, followed by hepatitis c virus. rma/s cells present endogenously synthesized cytosolic proteins to class i-restricted cytotoxic t lymphocytes characterization of rapidly degraded polypeptides in mammalian cells reveals a novel layer of nascent protein quality control commentary: defective ribosomal products (drips): a major source of antigenic peptides for mhc class i molecules? drips solidify: progress in understanding endogenous mhc class i antigen processing innate immune and chemically triggered oxidative stress modifies translational fidelity major histocompatibility class i molecules can present cryptic translation products to t-cells viral alteration of cellular translational machinery increases defective ribosomal products the immune epitope database . a community resource benchmarking predictions of peptide binding to mhc-i molecules evaluation of mhc class i peptide binding prediction servers: applications for vaccine research hla class i supertypes: a revised and updated classification derivation of an amino acid similarity matrix for peptide:mhc binding and its application as a bayesian prior reliable prediction of t-cell epitopes using neural networks with novel sequence representations comparative immunopeptidomics of humans and their pathogens mapping the landscape of host-pathogen coevolution: hla class i binding and its relationship with evolutionary conservation in human and viral proteins jump-starting the immune system: prime-boosting comes of age a synopsis of the existing knowledge of immunoreactivity against hepatitis c virus (hcv): a meta-analysis of the iedb data mhc class i molecules exploit the low g+c content of pathogen genomes for enhanced presentation optimal viral immune surveillance evasion strategies evolution of viral life-cycle in response to cytotoxic t lymphocyte-mediated immunity viruses selectively mutate their cd + t-cell epitopes'Ä îa large-scale immunomic analysis conservation of cytotoxic t lymphocyte (ctl) epitopes as a host strategy to constrain parasite adaptation: evidence from the nef gene of human immunodeficiency virus (hiv- ) clustering patterns of cytotoxic t-lymphocyte epitopes in human immunodeficiency virus type (hiv- ) proteins reveal imprints of immune evasion on hiv- global variation human t cell epitopes of mycobacterium tuberculosis are evolutionarily hyperconserved mapping the landscape of host-pathogen coevolution: hla class i binding and its relationship with evolutionary conservation in human and viral proteins comparison of site-specific rate-inference methods for protein sequences: empirical bayesian methods are superior a comparative study of conservation and variation scores the consurf-db: precalculated evolutionary conservation profiles of protein structures key: cord- -x ardo j authors: pedersen, lasse eggers; harndahl, mikkel; rasmussen, michael; lamberth, kasper; golde, william t.; lund, ole; nielsen, morten; buus, soren title: porcine major histocompatibility complex (mhc) class i molecules and analysis of their peptide-binding specificities date: - - journal: immunogenetics doi: . /s - - - sha: doc_id: cord_uid: x ardo j in all vertebrate animals, cd (+) cytotoxic t lymphocytes (ctls) are controlled by major histocompatibility complex class i (mhc-i) molecules. these are highly polymorphic peptide receptors selecting and presenting endogenously derived epitopes to circulating ctls. the polymorphism of the mhc effectively individualizes the immune response of each member of the species. we have recently developed efficient methods to generate recombinant human mhc-i (also known as human leukocyte antigen class i, hla-i) molecules, accompanying peptide-binding assays and predictors, and hla tetramers for specific ctl staining and manipulation. this has enabled a complete mapping of all hla-i specificities (“the human mhc project”). here, we demonstrate that these approaches can be applied to other species. we systematically transferred domains of the frequently expressed swine mhc-i molecule, sla- * , onto a hla-i molecule (hla-a* : ), thereby generating recombinant human/swine chimeric mhc-i molecules as well as the intact sla- * molecule. biochemical peptide-binding assays and positional scanning combinatorial peptide libraries were used to analyze the peptide-binding motifs of these molecules. a pan-specific predictor of peptide–mhc-i binding, netmhcpan, which was originally developed to cover the binding specificities of all known hla-i molecules, was successfully used to predict the specificities of the sla- * molecule as well as the porcine/human chimeric mhc-i molecules. these data indicate that it is possible to extend the biochemical and bioinformatics tools of the human mhc project to other vertebrate species. electronic supplementary material: the online version of this article (doi: . /s - - - ) contains supplementary material, which is available to authorized users. major histocompatibility complex class i (mhc-i) molecules are found in all vertebrate animals where they play a crucial role in generating specific cellular immune responses against viruses and other intracellular pathogens. they are highly polymorphic proteins that bind - amino acid long peptides derived from the intracellular protein metabolism. the resulting heterotrimeric complexes-consisting of the mhc-i heavy chain, the monomorphic light chain, beta- microglobulin (β m), and specifically bound peptides-are translocated to the cell surface where they displayed as target structures for peptide-specific, mhc-irestricted ctls. if a peptide of foreign origin is detected, the t cells may become activated and kill the infected target cell. mhc-i is extremely polymorphic. in humans, more than , different human leukocyte antigen class i (hla-i) molecules have been registered (as of january ), and this number is currently growing rapidly as more efficient hla typing techniques are employed worldwide. the polymorphism of the mhc-i molecule is concentrated in and around the peptide-binding groove, where it determines the peptide-binding specificity. due to this polymorphism, it is highly unlikely that any two individuals will share the same set of hla-i molecules thereby presenting the same peptides and generating t cell responses of the same specificities-something, that otherwise would give microorganisms a strong evolutionary chance of escape. rather, this polymorphism can be seen as diversifying peptide presentation thereby individualizing t cell responses and reducing the risk that escape variants of microorganisms might evolve. in , we proposed that all human mhc specificities should be mapped ("the human mhc project") as a preamble for the application of mhc information and technologies in humans (buus ) . since then, we have developed large-scale tools that are generally applicable towards this goal: production, analysis, prediction and validation of peptide-mhc interactions (ferre et al. ; harndahl et al. ; hoof et al. ; larsen et al. ; lundegaard et al. ; nielsen et al. nielsen et al. , ostergaard et al. ; pedersen et al. ; stranzl et al. ; stryhn et al. ) , and a "one-pot, read-and-mix" hla-i tetramer technology for specific t cell analysis (leisner et al. ) . here, we demonstrate that many of these tools can be transferred to other vertebrate animals as exemplified by an important livestock animal, the pig. we have successfully generated a recombinant swine leukocyte antigen i (sla-i) protein, sla- * , one of the most common sla molecules of swine (smith et al. ) . using this protein, we have developed the accompanying biochemical peptide-binding assays and demonstrated that the immunoinformatics tools originally developed to cover all hla-i molecules, despite the evolutionary distance, can be applied to sla-i molecules. we suggest that the "human mhc project" can be extended to cover other species of interest. all peptides were purchased from schafer-n, denmark (www.schafer-n.com). briefly, they were synthesized by standard -fluorenylmethyloxycarbonyl (fmoc) chemistry, purified by reversed-phase high-performance liquid chromatography (to at least > % purity, frequently - % purity), validated by mass spectrometry, and quantitated by weight. positional scanning combinatorial peptide libraries (pscpl) peptides were synthesized using standard solidphase fmoc chemistry on -chlorotrityl chloride resins. briefly, an equimolar mixture of of the common fmoc amino acids (excluding cysteine) was prepared for each synthesis and used for coupling in positions, whereas a single type of fmoc amino acid (including cysteine) was used in one position. this position was changed in each synthesis starting with the n-terminus and ending with the c-terminus. in one synthesis, the amino acid pool was used in all nine positions. x denotes the random incorporation of amino acids from the mixture, whereas the single letter amino acid abbreviation is used to denote identity of the fixed amino acid. the peptides in each synthesis were cleaved from the resin in trifluoroacetic acid/ , -ethanedithiol/triisopropylsilane/water : : : v/v/v/v, precipitated in cold diethylether, and extracted with water before desalting on c columns, freeze drying, and weighting. recombinant constructs encoding chimeric and sla- * molecules a synthetic gene encoding a transmembrane-truncated fragment encompassing residues to of human hla-a* : alpha chain followed by a fxa-bsp-hat tag (fxa = factor xa cleavage site comprised of the amino acid sequence iegr, bsp = biotinylation signal peptide, hat = histidine affinity tag for purification purposes; see online resource ) had previously been generated and inserted into the pet expression plasmid (novagen) (ferre et al. ) . synthetic genes encoding the corresponding fragments of the sla- * alpha chain (α α ) and α , respectively, (sullivan et al. ) were purchased from genscript. to exchange domains and generate chimeric human/swine mhc-i gene constructs, a type ii restriction endonuclease-based cloning strategy (seamless® strategene; cat# , revision# a), with modifications, was used. all primers were purchased hplc-purified from eurofins mwg operon (ebersberg, germany), and all pcr amplifications were performed in a dna engine dyad pcr instrument (mj research, mn, usa). all constructs were validated by dna sequencing. the following mhc-i heavy chain constructs were made hhh, hhp, hpp, php, and ppp, where the first, second, and third letter indicates domains α (positions - ), α (positions - ), and α (positions - ), respectively, and h indicates that the domain is of hla-a* : origin, whereas p indicates that it is of sla- * origin. constructs were transformed into dh α cells, cloned, and sequenced (abi prism avant, applied biosystems) . validated constructs of interest were transformed into an escherichia coli production cell line, bl (de ), containing the pacyc expression plasmid (avidity, denver, usa) containing an isopropylβ-d- -thiogalactopyranoside (iptg)-inducible bira gene to express biotin-ligase. this leads to almost complete in vivo biotinylation of the desired product (leisner et al. ). to maintain the pet -derived plasmids, the media was supplemented with kanamycin ( μg/ml) throughout the expression cultures. when appropriate, the media was further supplemented with chloroamphenicol ( μg/ml) to maintain the bira containing pacyc plasmid. e. coli bl (de ) cells transformed with appropriate plasmids were grown for h at °c, and a -ml sample adjusted to od ( ) = was then transferred to a -l fedbatch fermentor (labfors®). to induce protein expression, iptg ( mm) was added at od ( ) ∼ and the culture was continued for an additional h at °c (for in vivo biotinylation of the product, the induction media was further supplemented with biotin (sigma #b , μg/ ml)). samples were analyzed by reducing sodium dodecyl sulfate polyacrylamide gel electrophoresis (sds-page) before and after iptg induction. at the end of the induction culture, protease inhibitor (pmsf, μg/l) was added, and cells were lysed in a cell disrupter (constant cell disruptor systems set at , bar) and the released inclusion bodies were isolated by centrifugation (sorval rc , min, , ×g). the inclusion bodies were washed twice in pbs, . % np- (sigma), and . % deoxycholic acid (sigma) and extracted into urea-tris buffer ( m urea, mm tris, ph . ), and any contaminating dna was precipitated with streptomycin sulfate ( % w/v). the dissolved mhc-i proteins were purified by ni +/ida metal chelating affinity column chromatography followed by q-sepharose ion exchange column chromatography, hydrophobic interaction chromatography, and eventually by superdex- size exclusion chromatography. fractions containing mhc-i heavy chain molecules were identified by a absorbance and sds-page and pooled. throughout purification and storage, the mhc-i heavy chain proteins were dissolved in m urea to keep them denatured. note that the mhc-i heavy chain proteins at no time were exposed to reducing conditions. this allowed purification of highly active pre-oxidized moieties as previously described (ostergaard et al. ) . protein concentrations were determined by bicinchoninic acid assay. the degree of biotinylation (usually > %) was determined by a gel-shift assay (leisner et al. ). the pre-oxidized, denatured proteins were stored at − °c in tris-buffered m urea. recombinant constructs encoding human and porcine beta- microglobulin recombinant human β m was expressed and purified as described elsewhere (ostergaard et al. ) , (ferre et al. ) . using a previously reported e. coli codonoptimized gene encoding human β m as template , a gene encoding porcine β m was generated by multiple rounds of site-directed mutagenesis (quikchange® stratagene, according to the manufacturer's instructions) (online resource ). briefly, the genes encoding human or pig β m were n-terminally fused to a histidine affinity encoding tag (hat) followed by a restriction enzyme encoding tag (fxa), inserted into the pet vector and expressed in inclusion bodies in e. coli. the fusion proteins were extracted into m urea, purified by immobilized metal affinity chromatography (imac), and refolded by dilution. the fusion tags were then removed by fxa restriction protease digestion. the liberated intact and native human or pig β m were purified by imac and gel filtration chromatography, analyzed by sds-page analysis, concentrated, and stored at − °c until use (fig. ). purification and refolding of recombinant porcine β m proteins porcine β m was purified in the same way as human β m (ostergaard et al. ; ferre et al. ) . briefly, the ureadissolved β m protein was purified by ni +/ida metal chelating affinity column chromatography, refolded by drop-wise dilution into an excess refolding buffer under stirring ( mm tris, mm urea, ph . ), and then concentrated (vivaflow, kda). the refolded product was purified by ni +/ida metal chelating affinity column chromatography again (this time in aqueous buffer, i.e., without urea). fractions containing hat-pβ m were iden-tified by sds-page and pooled. removal of the hat tag was performed by cleavage with factor xa restriction protease (fxa) followed by renewed purified by ni +/ ida metal chelating affinity and superdex gel filtration column chromatography, concentrated by spin ultrafiltration ( kda), mixed : with glycerol, and stored at − °c. protein samples were mixed : in sds sample buffer ( % sds, . % glycerol, . % bromophenol blue, . m tris, mm iaa (iodoacetamide)) with or without reducing agent ( -mercaptoethanol) as indicated, boiled for min, spun at , ×g for min, and loaded onto a % or % running gel with a % stacking gel. gels were run at v, ma for min. peptide-mhc class i interaction measured by radioassay and spun column chromatography a hla-a* : -binding peptide, kvfpyalink (nonnatural consensus sequence a con ), was radiolabeled with iodine ( i) using a chloramine-t procedure (hunter and greenwood ) . dose titrations of mhc-i heavy chains (hhh or hhp) were diluted into refolding buffer (tris-maleate-pbs) and mixed with β m (human or porcine) and radiolabeled peptide, and incubated at °c overnight. then binding of radiolabeled peptide to mhc-i was determined in duplicate by sephadex™ g spun column gel chromatography as previously described . mhc bound peptide eluted in the excluded volume, whereas free peptide was retained on the microcolumn. both fractions were counted by gamma spectroscopy, and the fraction peptide bound was calculated as excluded radioactivity divided by total radioactivity. to examine the affinity of the interaction, increasing concentrations of unlabeled competitor peptide were added. when conducted under limiting concentrations of mhc-i molecule, the concentration of competitor peptide needed to effect % inhibition of the interaction, the ic , is an approximation of the affinity of the interaction between mhc-i and the competitor peptide. peptide-mhc class i interaction measured by an enzyme-linked immunosorbent assay peptide-mhc-i interaction was also measured in a modified version of a previously described enzyme-linked immunosorbent assay (elisa) (sylvester-hvid et al. ) . briefly, denatured biotinylated recombinant mhc-i heavy chains were diluted into a renaturation buffer containing β m and graded concentrations of the peptide to be tested and incubated at °c for h allowing equilibrium to be reached. we have previously demonstrated that denatured mhc molecules can de novo fold efficiently, however, only in the presence of appropriate peptide. the concentration of peptide-mhc complexes generated was measured in a quantitative sandwich elisa (using streptavidin as capture layer and the monoclonal anti-β m antibody, bbm , as detection layer) and plotted against the concentration of peptide offered (sylvester-hvid et al. ) . a prefolded, biotinylated flpsdyfpsv/ hla-a* : (kast et al. ) complex was used as standard. because the effective concentration of mhc ( - nm) used in these assays is below the equilibrium dissociation constant (k d ) of most high-affinity peptide-mhc interactions, the peptide concentration, ed , leading to half-saturation of the mhc is a reasonable approximation of the affinity of the interaction. the experimental strategy of pscpl has previously been described (stryhn et al. ) . the construction of the sublibraries and the elisa-driven quantitative measurements of mhc interaction are as given above. briefly, the relative binding (rb) affinity of each pscpl sublibrary was determined as rb (pscpl)=ed (x )/ ed (pscpl) (where ed is the concentration needed to half-saturate a low concentration of mhc-i molecules) and normalized so that the sum of the rb values of the naturally occurring amino acids equals (since peptides with a given amino acid in a given position are times more frequent in the corresponding pscpl sublibrary than in the completely random x library). a rb value above was considered as the corresponding position and amino acid being favored, whereas a rb value below . was considered as being unfavorable (these thresholds represent the % confidence intervals). an anchor position (ap) value was calculated by the equation ∑(rb− ) . a primary anchor position is characterized by one or few amino acids being strongly preferred and many amino acids being unacceptable. we have arbitrarily defined anchor residues as having an ap value above ). the peptide-sla-i* binding activity of each sublibrary was determined using previously published biochemical binding assay (elisa) (sylvester-hvid et al. ) (with the modifications described above). sequences logos describing the predicted binding motif for each mhc molecule were calculated as described by rapin et al. ( ) . in short, the binding affinity for a set of , , random natural mer peptides was predicted using the netmhcpan method, and the % strongest binding peptides were selected for construction of a position-specific scoring matrix (pssm). the pssm was constructed as previously described including pseudo-count correction for low counts. next, sequence logos were generated from the amino acid frequencies identified in the pssm construction. for each position, the frequency of all amino acids is displayed as a stack of letters. the total height of the stack represents the sequence conservation (the information content), while the individual height of the symbols relates to the relative frequency of that particular symbol at that position. letter shown upside-down are underrepresented compared to the background (for details see rapin et al. ( ) ). mhc distance trees were derived from correlations between predicted binding affinities. for each allelic mhc-i molecule, the binding affinity was predicted for a set of , random natural peptides using the netmhcpan method. next, the distance between any two alleles was defined, as d = − pcc, where pcc is the pearson correlation between the subset of peptides within the superset of top % best binding peptides for each allele. in this measure, two molecules that share a similar binding specificity will have a distance close to whereas two molecules with non-overlapping binding specificities would have a distance close to . using bootstrap, such distance trees were generated, and branch bootstrap values and the consensus tree were calculated. we have previously generated highly active, recombinant human mhc-i (hla-i) molecules and accompanying highthroughput assays and bioinformatics prediction resources. here, we transfer the underlying approaches to an important domesticated livestock animal, the pig, and its mhc system, the slas. mhc-i molecules are composed of a unique and highly variable distal peptide-binding platform consisting of the alpha (α ) and alpha (α ) domains of the mhc-i heavy chain (hc) and a much more conserved proximal immunoglobulin-like membrane attaching stalk consisting of the alpha (α ) domain of the hc noncovalently associated with the soluble mhc-i light chain (β m). a priori, the establishment of recombinant sla molecules is complicated by the lack of validated reagents. any failure could therefore be caused either by real technical problems in generating sla molecules, or merely by a lack of information about strong peptide binders to the sla in question. to reduce this uncertainty, we decided to migrate from human to pig mhc-i in a step-wise manner and generate an intermediary chimeric mhc-i molecule composed of a well-known human peptide-binding platform attached to a sla stalk, which might allow us to assess whether we could generate a functional sla stalk consisting of sla- * α hc and pig β m. to this end, we used the α α domains of the hla-a* : molecule, which we expected should be able to bind a known highaffinity hla-a* : -binding peptide (kvfpyalink). this peptide could be i radiolabeled and used in a very robust peptide-binding assay testing whether the human stalk could be replaced with the corresponding sla stalk. once that had been successfully established, the entire sla- * molecule would be constructed and tested. we have previously expressed and purified the extracellular segment spanning positions - of the human hla-a* : in a denatured and pre-oxidized version that rapidly refold and bind appropriate target peptides (ostergaard et al. ; ferre et al. ) . codon-optimized genes encoding the corresponding segments of sla- * (α α ) and sla- * (α ) were constructed as described in the "materials and methods" section and used to replace the hla-a* : gene segment in the above construct generating a new construct allowing for the expression of sla- * . for the generation of hla-a* : /sla- * chimeras, the genes encoding α (spanning positions - ), α (spanning positions - ), and/or α (spanning positions - ) domains of hla-a* : and sla- * were exchanged using seam-less and touch-down cloning strategies. genes encoding the extracellular segments - of the above natural or chimeric mhc-i molecules were c-terminally fused to a biotinylation tag (as indicated for sla- * in online resource ), inserted into pet , and expressed in inclusion bodies in e. coli (fig. shows sds-page of lysates of recombinant e. coli before and h after iptg induction). the fusion proteins were extracted into m urea (without any reducing agents), purified by ion exchange, hydrophobic and gel filtration chromatography (all conducted in m urea, without any reducing agents) (fig. shows sds-page of the purified sla- * after gel filtration), concentrated, and stored in urea at − °c. testing a chimeric molecule consisting of a sla- * stalk and a hla-a* : peptide-binding platform-comparing human versus porcine β m to test the proximal immunoglobulin-like membrane attaching sla stalk, we generated recombinant porcine β m and a chimeric human/porcine mhc-i heavy chain molecule where the α α were derived from the human hla-a* : , and the α was derived from the porcine sla- * . since this construct contains the entire peptide-binding platform of hla-a* : , we reasoned that the binding of the hla-a* : restricted peptide, kvfpyalink, could be used as a functional readout of the refolding, activity, and assembly of the entire chimeric molecule including the porcine sla stalk. for comparison, we tested the supportive capacity of human β m and folding ability of the entirely human hla-a* : . a total of four combinations could therefore be tested: porcine or human β m in combination with either hhp or hhh (where the first letter indicates the origin of the α domain (human hla-a* : or porcine sla- * ), the second letter the origin of the α domain, and the third letter the origin of the α domain). a concentrationtitration of heavy chain was added to a fixed excess concentration ( μm) of β m and a fixed trace concentration ( nm) of radiolabeled peptide. as shown in figs. and , the four combinations gave almost the same heavy chain dose titration with a half-saturation occurring around - nm heavy chain. porcine β m supported folding of the chimeric (hhp) α chain slightly better than it supported folding of the human (hhh) α chain. human β m supported folding of hhp and hhh equally well. thus, a recombinant sla stalk can fold and support peptide binding of the peptide-binding platform. these results also suggest that human β m can support folding and peptide binding of porcine mhc-i heavy chain molecules. using a positional scanning combinatorial peptide library approach to perform an unbiased analysis of the specificity of sla- * and human-pig chimeric mhc class i molecules using human β m to support folding, the recombinant sla- * and human-pig chimeric mhc-i molecule were tested for peptide binding. we have previously described how pscpl can be used to perform an unbiased analysis of mhc-i molecules (stryhn et al. ) . a pscpl consists of sublibraries for each position where one of each of the natural amino acids have been locked and all other positions contain random amino acids. analyzing how much of each pscpl sublibrary is needed to support mhc-i folding (see examples in fig. ) and comparing each sublibrary with a completely random library, the effect of any amino acid in any position can be examined and expressed as a rb value. further, an ap value calculated as the sum of squared deviations of rb values for each position can be used to identify the most prominent anchor position (see "materials and methods" for calculations). thus, the specificity of a nonamer binding mhc-i molecule can be analyzed comprehensively with × + completely random library= sublibraries (stryhn et al. ) . here, this approach was used to perform a complete experimental analysis of sla- * and a limited analysis of the chimeric hpp and php molecules. a nonamer pscpl analysis of sla- * can be seen in table . ap values identified positions , , and (in that order of importance) as the anchor positions of sla- * . in position , the amino acid preferences were dominated by the large and bulky aromatic tyrosine (y), tryptophane (w), and phenylalanine (f), all having rb values above (table ). in the almost equally important position , preferences for negatively charged amino acids glutamic acid (e) and aspartic acid (d) were observed. in the lesser important position , the most preferred amino acids were the hydrophobic amino acids valine (v), isoleucine (i), and leucine (l), followed by the polar amino acids threonine (t) and serine (s). finally, a very limited pscpl analysis was performed for the two chimeric human hla-a* : /porcine sla- * mhc-i molecules, hpp and php (table ) . for both chimeric molecules, it could be demonstrated that position is a strong anchor position. the positively charged amino acids, arginine (r) and lysine (k), were preferred in position of the chimeric hpp molecule, whereas the aromatic amino acid, tyrosine (y), was exclusively preferred in position of the chimeric php molecule. the positively charged amino acids, arginine (r) and lysine (k), were preferred in position of the chimeric hpp molecule similar to the position specificity of the hla-a* : molecule. in contrast, the aromatic amino acid tyrosine (y) was preferred in position of the chimeric php molecule similar to the position specificity of the sla- * molecule. using netmhcpan to predict peptides that bind to sla- * or to human-pig chimeric mhc class i molecules our recently described neural network-driven bioinformatics predictor, netmhcpan (version . ), has been trained on about , peptide-binding data points representing more than different mhc-i molecules (primarily hla-a and hla-b molecules). we have previously shown that netmhcpan is an efficient tool to identify peptides that bind to hla molecules where no prior data exist (nielsen et al. ) and demonstrated that netmhcpan can be extended to mhc-i molecules of other species (hoof et al. ). we applied netmhcpan to our peptide repository of about , peptides, which over the past decade have been selected to scan infectious agents (e.g., sars and influenza, sylvester-hvid et al. ; wang et al. ) , improve coverage of mhc-i specificities (e.g., buus et al. ; christensen et al. ) , etc. we extracted peptides as predicted binders to either the sla- * , the hpp, or the php human/porcine chimeric class i molecules (some of the peptides were predicted to bind to two or even all three of these molecules). all these peptide-mhc-i combinations were tested for binding (table ) ; of peptides bound to the sla- * molecule with an affinity (ic value) better than nm ( with an affinity less than nm); all peptides tested on the php molecule were strong binders with ic values below nm; and of peptides tested on the hpp molecule bound with an affinity better than nm. of the peptide-mhc-i combinations tested, ( %) were found to be good binders, ( %) were average binders, and ( %) did not bind well (table ) . this is in stark contrast to the . % frequency of binders among randomly selected peptides (yewdell and bennink ) . next, the netmhcpan method was used to generate pssms and sequence logos from the corresponding amino acid frequencies as described by nielsen et al. ( ) . for each position, the frequencies of all amino acids were displayed as a stack of letters showing the sequence conservation/information content (the height of the entire a preliminary report of sla- * binding was given in hoof et al. ( ) the normalized relative binding (rb) value indicates whether an amino acid is favored (rb> , bold numbers) or disfavored (rb< . , italic numbers) in a given peptide position. the anchor position (ap) value is given by the equation ∑(rb− ) . the important anchor positions , , and for sla- * are underlined stack) and the relative frequency of amino acids (the height of the individual amino acids). figure shows a specificity tree clustering of the sla- * molecule compared to prevalent representatives of the common hla supertypes that netmhcpan originally intended to cover . by this token, sla- * most closely resembles that of hla-a* : . the limited pscpl analysis of the chimeric mhc-i molecules revealed strong p signals with specificities that seemed to be determined by the origin of the α domain: the hpp chimera showed an hla-a* : -like p specificity, whereas the php chimera showed a sla- * /hla-a* : -like specificity. since the netmhcpan predictor successfully captured these chimeric specificities (see above), we reasoned that the predictor might also be used to perform in silico dissection of these specificities and used the p specificity as an example of such an in silico analysis. the netmhcpan predictor considers a pseudo-sequence consisting of polymorphic positions, which contain residues that are within . Å of the atoms of bound nonamer peptides (nielsen et al. ). of the positions of the pseudosequence, delineates the p binding pocket; however, only of these, positions , , and , differ between sla- * and hla-a* : . to explore the effect of these three residues, we performed in silico experiments where we examined single substitutions y d, g d, and s i (the letter before the position number indicates the sla-a* single letter residue, whereas the letter after indicates the hla-a* : residue) as well as the corresponding triple substitution (ygs-ddi). as described above, pssms were generated for each of the in silico molecules followed by a specificity tree clustering (including sla-a* , hla-a* : , and hla-a* : ). figure shows this tree along with the sequence logo plots showing the predicted binding specificity of each in silico mhc-i molecule. albeit the y d and g d single substitutions showing some of the positively charged p peptide residue preference of hla-a* : , they still clustered with hla-a* : . in contrast, the in silico (ygs-ddi) triple substitution clustered with the hla-a* : . this suggests that the netmhcpan method is capable of defining the residues of the f pocket that determine the specificity of position . we have previously suggested that the specificities of the entire human mhc-i system should be solved ("the human rb and ap values are defined as described in table mhc", buus ; lauemoller et al. ) . however, due to the extreme polymorphism of the mhcs, any attempt to address the specificity of the entire mhc system is a significant experimental undertaking. during the past decade, we have established a series of technologies to support a general solution of human mhc class i and ii specificities. for mhc-i, this includes ( ) a highly efficient e. coli expression system for production of recombinant human and mouse mhc-i molecules (both heavy chain and light chain (β m) molecules) ostergaard et al. ) , ( ) a purification system for obtaining the highly active pre-oxidized mhc-i heavy chain species (ferre et al. ) , ( ) a high-throughput homogenous peptide-mhc-i binding assay for obtaining large data sets on peptide-mhc-i interactions (harndahl et al. ), ( ) a positional scanning combinatorial peptide library approach for a robust and unbiased analysis of the specificity of any mhc-i molecule (stryhn et al. ) , ( ) an immunobioinformatics approach to generate predictors of the peptide-mhc-i interaction, netmhcpan, that allows predictions to be made for any human mhc-i molecules, hla-i, even those that have not yet been covered by existing data set (hoof et al. ; nielsen et al. ) , and finally ( ) we have demonstrated that pre-oxidized mhc-i molecules can be used to generate mhc-i tetramers in a simple "one-pot, mix-and-read" manner (leisner et al. ) . here, we propose that the next goal should be to extend the overall approach to mhc-i molecules of other species of interest. mouse and rats have been extensively studied in the past, but much less reagents and information have accrued for the mhc-i molecules of other species. here, we have used an important livestock animal, the pig, as a model system and demonstrated that it indeed is possible to transfer the original human approach to other species. before attempting to generate a recombinant version of the entire porcine sla- * molecule, we grafted the more conserved membrane-proximal "stalk" (the immunoglobulin-like class i heavy chain α and β m domains) of porcine sla- * onto the peptide-binding domain of hla-a* : generating a chimeric human/ porcine mhc-i molecule. this chimeric molecule retained the peptide-binding specificity of the hla-a* : molecule, and it clearly demonstrated that the recombinant porcine stalk was functional and, by inference, also properly folded. it also suggests that the peptide-binding specificity of the distal domains do not crucially depend upon the identity of the proximal stalk. further, comparing the ability of human and porcine β m to support mhc-i complex formation using either a human or a porcine mhc-i stalk, we demonstrated that every combination (porcine β m/human-α , porcine β m/porcine-α , human β m/human-α , and human β m/porcine-α ) showed al- most the same heavy chain dose titration with identical half-saturations. these results illustrate the ability for porcine and human β m to support complex formation of sla molecules and vice versa and suggest evolutionary that the stalk is quite conserved. next, we generated the entire sla- * heavy chain and succeeded in generating complexes using human β m as the light chain and pcspl as peptide donors. the latter solved the a priori problem of not knowing which peptides would be needed to support proper folding of sla- * , and it did so in an unbiased manner. furthermore, this approach is highly efficient since it readily establishes a complete matrix representing the amino acid preference for each amino acid and each position of a nonamer peptide. the specificity of sla- * shows two primary anchors: one in positions with a preference for aromatic amino acids and another in position with a preference for negatively charged amino acids. in addition, the sla- * features a secondary anchor in position with hydrophobic or polar amino acid preferences. an alternative approach to solve the problem of identifying peptides that support folding of mhc-i molecules of so far unknown specificity is to use our recently developed panspecific predictor, netmhcpan. the successful use of this predictor to initiate peptide-binding studies was recently fig. specificity tree clustering of the sla- * molecule compared to prevalent representatives of the common hla supertypes ). the distance between any two mhc molecules and the consensus tree is calculated as described in "materials and methods". all branch points in the tree have bootstrap values of %. sequence logos of the predicted binding specificity are shown for each molecule. in the logo, acidic amino acids [de] are shown in red, basic amino acids [hkr] in blue, hydrophobic amino acids [acfilmpvw] in black, and neutral amino acids [gnqsty] in green. the axis of the logos indicates in all case positions one through nine of the motif, and the y-axis the information content (see materials and methods) demonstrated for hla-a* . although originally developed to cover all hla-a and hla-b molecules, it has also been shown to extend to mhc-i molecules of other species (hoof et al. ). here, we demonstrate that the netmhcpan predictor is capable of extracting mhc-i sequence information across species and correctly relate this to peptide binding even in the absence of any available data for the specific query mhc-i molecule, i.e., the sla- * as well as the chimeric hpp (hα pα pα ) and php (pα hα pα ) molecules. it is not clear why binding of the php chimera was more efficiently predicted than binding of the hpp chimera. one could speculate that netmhcpan has not captured the effect of the different positions of the pseudo-sequence equally well and not all positions and pockets (and by inference-not all chimeric molecules) are therefore predicted equally well. using the netmhcpan predictor to cluster sla- * and representative molecules of human hla supertypes according to predicted peptide-binding specificities, the sla- * specificity closely resembled that of hla-a* : (iedb, http://www.immuneepitope.org/mhcalleleid/ , accessed march th ). this result was also obvious from an inspection of the pscpl analysis of the sla- * . the pscpl analysis of the p specificity of the sla- * and the two chimeric molecules suggested that the p specificity primarily was determined by the α domain. this contention was further strengthened by a netmhcpan-driven in silico analysis of the residues delineating the f pocket, which interacts with p . this suggests that netmhcpan can be used to design and interpret detailed experiments addressing the structurefunction relationship of peptide-mhc-i interaction. in the case of sla- * , netmhcpan suggests that y , g , and s play a prominent role in defining the p f pocket. whereas the netmhcpan readily captured the p anchor residue of sla- * , it did not capture the p anchor (at least not in the . version). we surmise that this shortcoming is due to insufficient examples of the use of p anchors within the currently available peptide-mhc-i binding data. inspecting the pseudo-sequence of sla- * and hla-a* : vs. hla-a* : suggests that the presence of an arginine in position might fig. comparison of specific in silico mutations of the sla- * molecule and comparison with the two hla molecules: hla-a* : and hla-a* : . the distance between any two mhc molecules and the consensus tree is calculated as described in "materials and methods". all branch points in the tree have bootstrap values of %. the sla- * mutations are indicated as y d, g d, and s i, where the letter before the position number indicates the sla- * single letter residue and the letter after indicates the hla-a* : residue. ygs-ddi is the corresponding triple substitution. sequence logos are calculated and visualized as described in fig. . the axis of the logos indicates in all case positions one through nine of the motif, and the y-axis the information content (see materials and methods) explain the preference for negatively charged amino acid residues in p . future netmhcpan-guided experiments could pointedly address this question, and the resulting data could complement existing data and be used to update and improve the netmhcpan predictor. all in all the two complementary approaches, pscpl and netmhcpan, agreed on the specificity of the sla- * molecule, as well as of the two chimeric mhc-i molecules. thus, the specificity of sla- * appear to be well established. this specificity has successfully been used to search for foot-and-mouth disease virus (fmdv)specific ctl epitopes in fmdv-vaccinated, sla- * positive pigs, and the recombinant sla- * molecules have been used to generate corresponding tetramers and stain pig ctls (patch et al. ) . in conclusion, we here present a set of methods that can be used to generate functional recombinant mhc-i molecules, map their specificities and identify mhc-i-restricted epitopes, and eventually generate peptide-mhc-i tetramers for validation of ctl responses. this suite of methods is not only applicable to humans, but potentially to any species of interest. description and prediction of peptide-mhc binding: the 'human mhc project receptorligand interactions measured by an improved spun column chromatography technique. a high efficiency and high throughput size separation method sensitive quantitative predictions of peptide-mhc binding by a 'query by committee' artificial neural network approach selecting informative data for developing peptide-mhc binding predictors using a query by committee approach purification of correctly oxidized mhc class i heavy-chain molecules under denaturing conditions: a novel strategy exploiting disulfide assisted protein folding high-throughput polymerase chain reaction cleanup in microtiter format peptide binding to hla class i molecules: homogenous, high-throughput screening, and affinity assays netmhcpan, a method for mhc class i binding prediction beyond humans preparation of iodine- labelled human growth hormone of high specific activity role of hla-a motifs in identification of potential ctl epitopes in human papillomavirus type e and e proteins the peptide-binding specificity of hla-a* demonstrates membership of the hla-a supertype an integrative approach to ctl epitope prediction: a combined algorithm integrating mhc class i binding, tap transport efficiency, and proteasomal cleavage predictions identifying cytotoxic t cell epitopes from genomic and proteomic information: "the human mhc project one-pot, mix-and-read peptide-mhc tetramers definition of supertypes for hla molecules using clustering of specificity matrices netmhc- . : accurate web accessible predictions of human, mouse and monkey mhc class i affinities for peptides of length - reliable prediction of t-cell epitopes using neural networks with novel sequence representations improved prediction of mhc class i and class ii epitopes using a novel gibbs sampling approach netmhcpan, a method for quantitative predictions of peptide binding to any hla-a and -b locus protein of known sequence efficient assembly of recombinant major histocompatibility complex class i molecules with preformed disulfide bonds induction of footand-mouth disease virus-specific cytotoxic t cell killing by vaccination the interaction of beta -microglobulin (beta m) with mouse class i major histocompatibility antigens and its ability to support peptide binding. a comparison of human and mouse beta m the mhc motif viewer: a visualization tool for mhc binding motifs peptide binding to the most frequent hla-a class i alleles measured by quantitative molecular binding assays nomenclature for factors of the sla class-i system netctlpan: pan-specific mhc class i pathway epitope predictions peptide binding specificity of major histocompatibility complex class i resolved into an array of apparently independent subspecificities: quantitation by peptide libraries and improved prediction of binding analysis of polymorphism in porcine mhc class i genes: alterations in signals recognized by human cytotoxic lymphocytes establishment of a quantitative elisa capable of determining peptide-mhc class i interaction sars ctl vaccine candidates; hla supertype-, genome-wide scanning and biochemical validation hla class i binding mer peptides from influenza a virus induce cd t cell responses immunodominance in major histocompatibility complex class i-restricted t lymphocyte responses acknowledgments we thank lise lotte bruun nielsen, anne caroline schmiegelow, and iben sara pedersen for their expert experimental support. this work was in part supported by the danish council for independent research, technology and production sciences ( - - ) and by the national institute of health (nih) (hhsn c). key: cord- -plptulfb authors: tilocca, bruno; soggiu, alessio; greco, viviana; piras, cristian; arrigoni, norma; ricchi, matteo; britti, domenico; urbani, andrea; roncada, paola title: immunoinformatic-based prediction of candidate epitopes for the diagnosis and control of paratuberculosis (johne’s disease) date: - - journal: pathogens doi: . /pathogens sha: doc_id: cord_uid: plptulfb paratuberculosis is an infectious disease of ruminants caused by mycobacterium avium subsp. paratuberculosis (map). map is an intracellular pathogen with a possible zoonotic potential since it has been successfully isolated from the intestine and blood of crohn’s disease patients.since no cure is available, after the detection of the disease, animal culling is the sole applicable containment strategy. however, the difficult detection of the disease in its subclinical form, facilitates its spread raising the need for the development of effective diagnosis and vaccination strategies. the prompt identification and isolation of the infected animals in the subclinical stage would prevent the spread of the infection.in the present study, an immunoinformatic approach has been used to investigate the immunogenic properties of map proteins. these proteins were chosen according to a previously published immunoproteomics approach. for each previously-described immunoreactive protein, we predicted the epitopes capable of eliciting an immune response by binding both b-cells and/or class i mhc antigens. the retrieved peptide sequences were analyzed for their specificity and cross-reactivity. the final aim is to employ the discovered peptides sequences as a filtered library useful for early-stage diagnosis and/or to be used in novel multi-subunit or recombinant vaccine formulations. bovine paratuberculosis, also known as johne's disease (jd) is an infectious disease of ruminants caused by mycobacterium avium subsp. paratuberculosis (map). it is characterized by chronic and progressive granulomatous enteritis. the infected animals initially show normal appetite and food consumption, but the intestinal wall thickening and the impaired nutrient absorption cause a reduced feed-conversion rate and a progressive weight loss. milk yield is also negatively affected by the progression of the infection. nevertheless, clinical manifestations do not involve all infected animals [ ] [ ] [ ] ; the subclinical stageof infection can last from to years [ ] and, despite the absence of visible symptoms, animals in this stage can shed map and spread the disease [ , [ ] [ ] [ ] . for these aforementioned reasons, this pathology leads to significantly increased veterinary costs worldwide [ , , ] . the causal agent of jd is map. it is considered a zoonotic pathogen [ ] because of its possible link with crohn's disease. map infection affects animals and there is considerable evidence that might be a co-cause of human crohn's disease [ ] . map isolation from the intestine and blood of crohn's disease patients has extensively documented. more precisely, map presence was found to be seven times higher in crohn's disease patients than what has been found in patients with any other bowel inflammation [ , ] . map also infected animals and crohn's disease patients show similar alterations of the immune system response reinforcing the hypothesis about the analogy between the two [ ] [ ] [ ] [ ] . map is a slow-growing bacterium, commonly acquired via the fecal-oral route by both animals and humans [ ] . despite the pathogenetic mechanism of map, infection has not been fully understood, it has been demonstrated that its acid resistance enables it to survive in the gastric environment, allowing its entrance in the intestinal tract. at the ileal level, map invades the lymphatic system overlying peyer's patches. this stimulates the host's immune response that, besides activating the humoral response, promptly phagocytizes map into macrophages [ ] . as an intracellular pathogen, map can either survive into macrophagic cells or being killed and disassembled to present its antigens to t-lymphocytes [ ] . evidence from multibacillary jd revealed a massive humoral antibody response along with a tendency to suppress the cell-mediated immune response [ , , ] . whereas, a recent comparative study between two groups of cows, one in the sub-clinical and the other in the clinical stage, highlighted an increased t-cell activity in the first group of animals compared to the second one [ ] . studies on cattle at the early stage of map infection revealed an upregulation of class i mhc molecules, suggesting a pivotal role of these molecules in the very beginning of the infective process [ ] ; this is of great interest for both diagnosis and prophylaxis-oriented studies. figure provides an overview of the major immunological mechanisms triggered bymap infections. to date, jd diagnosis relies on both direct (map culture, pcr, microarrays etc.) and indirect (elisa) detection of map from feces, milk and necroscopy-derived tissues. however, all the available diagnostic methods suffer from sensitivity (especially in the sub-clinical phase) that strongly reduce their robustness and efficient applicability on large-scale control programs. the failure to detect the subclinical infection makes it difficult to timely apply the control measures necessary to block the spread of the infection within the same, and to other, herds. a thorough comprehension of the etiopathogenetic mechanisms of map infection and host response would be beneficial for diverse research scenarios, providing guidance for the design of map-specific diagnostic tools capable of jd diagnosis in the subclinical phase. from this perspective, a previous study from our research group [ ] employed an immunoproteomic approach to investigate and select map-specific immunoreactive proteins. here, incubation of map proteome with sera from infected bovines highlighted several possible candidate immunoreactive proteins. these candidates represent a good starting point for an immunoinformatic analysis of their sequences in order to find the best immunoreactive sub-sequences and epitopes. this would provide a library of peptides that might be useful for novel prophylactic strategies and/or for their potential application in the immune-based detection of map. the rapid development of the bioinformatics tools and databases provides the possibility to detect the antigenic/epitopic regions of given protein sequences. this innovative strategy for the in-silicoanalysis is time-and cost-effective compared to the "old-fashioned" laboratory-based approach. recently, carlos et al. [ ] and rana et al. [ ] applied immunoinformatics-based studies to detect class ii mhc epitopes possibly useful for the control of jd in a rapid and cost-effective manner. over the last decade, these computational approaches lead to the achievement of successful epitopes prediction in several research fields as virology [ , ] , bacteriology and cancer research [ ] . in this study, previously-selected immunogenic proteins [ ] were studied via several immunoinformatics approaches aiming at the detection of the most promising peptide sequences useful for diagnostic purposes. the parameters taken into account were affinity for both the humoral antibody binding and the class i mhc molecule binding. we predicted the most suitable peptide sequences and discuss their potential employment in the design of innovative control measures against jd, with a specific focus on the early diagnosis of jd and/or potential use in novel specific vaccine formulations. pathogens , , x for peer review of over the last decade, these computational approaches lead to the achievement of successful epitopes prediction in several research fields as virology [ , ] , bacteriology and cancer research [ ] . in this study, previously-selected immunogenic proteins [ ] were studied via several immunoinformatics approaches aiming at the detection of the most promising peptide sequences useful for diagnostic purposes. the parameters taken into account were affinity for both the humoral antibody binding and the class i mhc molecule binding. we predicted the most suitable peptide sequences and discuss their potential employment in the design of innovative control measures against jd, with a specific focus on the early diagnosis of jd and/or potential use in novel specific vaccine formulations. the peptide sequences of the previously-identified immunoreactive proteins [ ] were used to recall the novel protein identifiers in the ncbinr protein database. because of the continuous evolution of the data repositories and the increasing knowledge on their entries, some protein accession numbers were re-classified into other identifiers. table summarizes the blast-based alignments of the peptides performed to line up the selected proteins to the current identification system. all pblast alignments matched at % with the reference protein kept. the low e-value of each alignment supports the attribution of the immunoreactive proteins to the novel identifiers. the peptide sequences of the previously-identified immunoreactive proteins [ ] were used to recall the novel protein identifiers in the ncbinr protein database. because of the continuous evolution of the data repositories and the increasing knowledge on their entries, some protein accession numbers were re-classified into other identifiers. table summarizes the blast-based alignments of the peptides performed to line up the selected proteins to the current identification system. all pblast alignments matched at % with the reference protein kept. the low e-value of each alignment supports the attribution of the immunoreactive proteins to the novel identifiers. once the updated protein identifiers are inferred, the major immunogenic domains of the selected proteins were predicted through an immunoinformatic approach. prediction of the linear b-epitopes provided a list of epitopes capable of eliciting antibody production (supplementary table s ). all the selected proteins showed relevant epitopes from an immunogenic point of view. a large number of short epitope sequences is predicted for each immunogenic protein; whereas, an average of six candidate epitopes (min -max ) meeting the threshold of a minimal length of aminoacids is predicted for each of the selected immunogenic proteins. figure depicts the ten immunogenic proteins of map along with the relative distribution of the predicted b-epitopes. whole protein calculated immunogenic potential based on the type-b epitopes prediction indicates the "hypothetical protein map_ c" (aas ) as the most immunogenic one. this evidence is supported by its highest number of predicted epitopes and the highest average epitope length ( figure ). on the other hand, the fructose-bisphosphate aldolase (eta ), reported the lowest number of predicted epitopes along with the lowest epitope sequence length. regardless of the number of predictions, candidate epitopes are evenly mapped over the full sequence length of the immunogenic proteins, suggesting a good versatility of the predicted sequences ( figure ). pathogens , , x for peer review of prediction of binding affinity for the diverse class i bolas histocompatibility antigens predicted a high number of peptide sites. the full list of class i mhc epitope prediction is provided in the supplementary table s . epitope prediction from the previously selected immunogenic proteins yielded a total of peptides, each of which scoring a peculiar binding affinity. peptides scoring a binding affinity among the top . % are considered as strong binders (sb); whereas, peptides with a percentile rank comprised between . % and % were labelled as weak binders (wb). sorting all the entries using the "sole" wb and sb resulted in a total of candidate epitopes when considering all the mhc haplotypes for the ten immunogenic proteins (supplementary table s ) . for a better evaluation of the most suitable map epitopes, we focused our attention towards the sequences that are most commonly recognized by the immune system effectors (i.e., bola haplotypes and, in turn, cd + t-cells). figure lists, for each of the tested immunogenic protein, the shared epitopes among the mhc haplotypes. pathogens , , x for peer review of prediction of binding affinity for the diverse class i bolas histocompatibility antigens predicted a high number of peptide sites. the full list of class i mhc epitope prediction is provided in the supplementary table s . epitope prediction from the previously selected immunogenic proteins yielded a total of peptides, each of which scoring a peculiar binding affinity. peptides scoring a binding affinity among the top . % are considered as strong binders (sb); whereas, peptides with a percentile rank comprised between . % and % were labelled as weak binders (wb). sorting all the entries using the "sole" wb and sb resulted in a total of candidate epitopes when considering all the mhc haplotypes for the ten immunogenic proteins (supplementary table s ) . for a better evaluation of the most suitable map epitopes, we focused our attention towards the sequences that are most commonly recognized by the immune system effectors (i.e., bola haplotypes and, in turn, cd + t-cells). figure lists, for each of the tested immunogenic protein, the shared epitopes among the mhc haplotypes. summarizes bolas haplotypes predicted to bind each sequence as means of a color scale that depends on the binding affinity. peptides "amlqdmail" belongs to heat shock protein (caa . ); "aqldlsnal" and "hmlrhqqir" belong to hypothetical protein map_ c (aas . ); "arleppgpl" belong to hypothetical protein map c (aas . ); "gvfnleatl" and "neraveeal" belong to the protein fixa (aas . ); "rlleiepal", "aagqigysl" and "alegvvmel" belong to the malate dehydrogenase protein (p . ); "amqtlpqvl" and "rmaqtgspl" belong to phosphoglucosamine mutase (q s . ); "nqielhpll", "teravsaal", "amdaceasl" and "tqsytplal" belong to uncharacterized oxidoreductase map_ (q vk . ); "emfdlihqm" and "amrkwessm" belong to fructose-bisphosphate aldolase (eta . ); "glyefftpl", "gmigltqal" and "tqmtaaipl" belong to -ketoacyl-acp reductase (ajk . ); "fitwrgipl" and "nqsaiatyl" are peptides of the hypothetical protein ega _ (azp . ). the vast majority of the listed epitopes are classified as sb; while eight of them, belonging to the proteins malate dehydrogenase (p ), uncharacterized oxidoreductase map_ (q vk ) and hypothetical protein ega _ (azp ), are to be considered as wb on the basis of their affinity rank. regardless of the binding affinity, all these sequences are predicted to be commonly bound by a plurality of mhc haplotypes. an average of (min -max ) suitable epitopes are selected for each of the tested protein. such epitopes are predicted to be recognized by five diverse bolas out of the six mhc haplotypes used for the computer-based prediction; except for the immunogenic proteins fixa (aas ) whose epitopes can be bound by four bolas out of six. the bola-hd , bola-jsp. and bola-t c are able to recognize all the selected epitopes sequences . selected class i mhc binding peptides. the heat map displays the selected t-epitopes and summarizes bolas haplotypes predicted to bind each sequence as means of a color scale that depends on the binding affinity. peptides "amlqdmail" belongs to heat shock protein (caa . ); "aqldlsnal" and "hmlrhqqir" belong to hypothetical protein map_ c (aas . ); "arleppgpl" belong to hypothetical protein map c (aas . ); "gvfnleatl" and "neraveeal" belong to the protein fixa (aas . ); "rlleiepal", "aagqigysl" and "alegvvmel" belong to the malate dehydrogenase protein (p . ); "amqtlpqvl" and "rmaqtgspl" belong to phosphoglucosamine mutase (q s . ); "nqielhpll", "teravsaal", "amdaceasl" and "tqsytplal" belong to uncharacterized oxidoreductase map_ (q vk . ); "emfdlihqm" and "amrkwessm" belong to fructose-bisphosphate aldolase (eta . ); "glyefftpl", "gmigltqal" and "tqmtaaipl" belong to -ketoacyl-acp reductase (ajk . ); "fitwrgipl" and "nqsaiatyl" are peptides of the hypothetical protein ega _ (azp . ). the vast majority of the listed epitopes are classified as sb; while eight of them, belonging to the proteins malate dehydrogenase (p ), uncharacterized oxidoreductase map_ (q vk ) and hypothetical protein ega _ (azp ), are to be considered as wb on the basis of their affinity rank. regardless of the binding affinity, all these sequences are predicted to be commonly bound by a plurality of mhc haplotypes. an average of (min -max ) suitable epitopes are selected for each of the tested protein. such epitopes are predicted to be recognized by five diverse bolas out of the six mhc haplotypes used for the computer-based prediction; except for the immunogenic proteins fixa (aas ) whose epitopes can be bound by four bolas out of six. the bola-hd , bola-jsp. and bola-t c are able to recognize all the selected epitopes sequences among the immunogenic proteins. on the other hand, the bola-t a is not showing any binding affinity to the epitope sequences; while bola-d . and bola-t b fail to bind the epitopes of aas protein (figure ). the class i mhc epitopes as of figure are further aligned against both the mycobacteria and cow databases to assess the specificity of the predicted epitope sequences for map. complete list of alignments is available in the supplementary table s . sequence alignment highlighted that the peptide amdaceasl and amrkwessm respectively of the "uncharacterized oxidoreductase map_ " (q vk ) and "fructose-bisphosphate aldolase" (eta ) proteins are the most specific for map. specifically, amdaceasl scores % identity with the map and the mycobacterium aviumcomplex (mac); whereas, hits with other mycobacteria specimens are featured by a lower sequence identity (below %) and a far higher e-value when compared with map and mac hits ( . vs. . , supplementary table s ) . similarly, the peptide amrkwessm scores % sequence identity with map and mac and only less than % of sequence similarity is scored by the alignments with other mycobacteria. the e-value supports the robust alignment against the map and mac (e-value . ) in spite of the other alignments (e-value > ) further supporting the hypothesis on the specificity of this peptide sequence (supplementary table s ). concerning the alignment of the peptides against the cow database, both amdaceasl and amrkwessm did not score relevant matches with any of the cow proteins. several hits were matching with discontinuous sequences of the cow proteins database with high e-values (supplementary table s , topic better commented in the discussion section). the host's immune response to map infection is complex and heterogeneous. debates on the sequelae of immunological events following map infection are currently ongoing. nevertheless, it seems widely accepted that the early stage of the infection elicits an important cell-mediated response. once map is phagocytized, its antigen presentation is accomplished through the loading of the processed antigen onto mhc molecules. the bovine mhc genes complex (i.e., bovine leukocyte antigen, bola) is carried in the chromosome and represent a fundamental component of the bovine immune system that allows the recognition and presentation of a virtually infinite number of antigens [ ] (figure ). such a high versatility relies on diverse factors, including the polygenetic origin of the mhc genes, the codominance of the parental alleles, the polymorphism of the genetic variants and the peptides/proteins splicing [ ] . class i mhc molecules recognize, bind and present peptide antigens from intracellular pathogens to cd + t-lymphocytes [ ] . in this view, class i mhc molecules and cytotoxic t-lymphocytes (ctl) are likely to play a pivotal role in the early stage of the map infection [ ] . thus, of potential interest for early diagnosis-oriented studies and the design of efficient vaccine formulations. class i mhc peptide antigens are to be considered among the main triggers of the cell-mediated responseand their specific immunostimulation would lead to a more efficient prophylactic outcome. nevertheless, a study from rana etal. highlighted an important involvement also of the humoral response to map infection, other than the adaptive immunity mediated by the class ii mhc molecules [ ] . huge efforts have been made to optimize diagnostics for the efficient detection of map by means of both direct and indirect methods [ , , ] . the slow-growing rate of map along with the reduced sensitivity of the culture-based methods raised the need to develop alternative diagnostic strategies. pcr-based diagnosis targeting the multicopy insertion sequence is held the promise of fast detection of map in both environmental and clinical samples. however, the presence of is -like sequences in other bacterial specimens resulted in a reduction of the pcr specificity. this, along with the elevated costs of the reagents, equipment and procedures, precludes the pcr applicability in large-scale programs [ ] . among the indirect methods, elisa-based detection of anti-map antibodies enables faster diagnosis time but still suffer from drawbacks related to sensitivity and specificity. although great improvements have been made in optimizing elisa kits to reduce cross-reactions with environmental mycobacterium strains [ , ] . still, this method suffers from a lack of sensitivity. moreover, the high antigen similarity between map and mycobacterium bovis obstacles the discrimination between bovines infected with tuberculosis and inoculated with live or attenuated paratuberculosis vaccines [ ] . this promotes the seek of molecular target(s) capable of offering a more robust diagnosis. the present work describes a companion study that relies on previously-obtained datasets of our research group [ ] . employing an immunoproteomic approach, we experimentally validated the whole map proteome for its capability of being complexed by the antibodies naturally occurring in sera of infected bovines. ms-based identification of the immunogenic proteins enabled the detection of protein candidates whose protein sequences have been now further investigated for their immunogenic features. we employed an immunoinformatic approach for further focusing on the peptide sequences, potentially involved in the immunostimulation. a key point of the immunoinformatic approach is the prediction of the protein epitope sequences. epitopes prediction can be based on several features such as physical-chemical properties and structural folding of the primary protein sequence [ ] [ ] [ ] . the present investigation is mainly focused on linear epitopes because protein-antibody complexes were selected through two-dimensional electrophoresis ( -de) and western blotting; thus, on linearized proteins [ , ] . however, the application of other mass spectrometry technologies is quickly developing in the field on immunoproteomics [ ] there are still significant limitation to map on a large scale conformational epitopes. as expected from the previous experimental data, all the screened protein sequences showed the capability of being recognized by both b-cells and class i bolas. the comprehension of recognition and binding of map by the host immune cells is still controversial. some studies document a relevant humoral response to map infections. on the other hand, other pieces of evidence describe the importance of the cell-mediated response to control map growth [ ] [ ] [ ] [ ] [ ] . from our perspective and, according to the collected evidence, map-targeted antibodies could play a key role in the specific and sensitive detection of this pathogen in the subclinical stage of the infection. b-cell epitopes prediction highlighted the "hypothetical protein map_ c" (aas ) protein as the most active in stimulating antibody production. this finding is in agreement with our previous study [ ] , where this protein showed a high level of immunoreactivity exclusively against the serum of the map infected animals. to the best of our knowledge, this protein was not described before as an antigen and, according to our dataset on its functional domains, it is possible to hypothesize that it is part of a surface-associated dehydrogenase with oxidoreductase activity involved in pentose phosphate pathway [ , ] . the fructose-bisphosphate aldolase (eta ), instead, is described as less prone to elicit antibody production. this is consistent with its intracytoplasmic localization and with its major role in the central metabolism. despite its cellular localization, several moonlighting properties have been described as part of its multiple functions [ ] [ ] [ ] . interestingly, b-cell epitopes prediction highlighted a homogenous distribution of multiple peptide sequences throughout all the proteins primary sequences (figure ). this suggests the potential usefulness of the selected proteins for a variety of implications where two or more epitopes are needed in a single protein molecule (e.g., sandwich elisa, and other indirect diagnostic tests ensuring higher sensibility) [ , ] . prediction of the class i bolas binding peptides confirmed the immunogenicity of the previously studied proteins. similarly to hlas, bolas are highly polymorphic proteins; thus, including a plurality of bolas while computing the peptide binding affinity would benefit the robustness and reliability of the prediction [ , ] . among the class i mhc epitopes predicted in the present study, the hypothetical protein ega _ (azp ) differs by only one amino-acidic residue from the map membrane protein c (v kre ), whose immunogenic properties have been already demonstrated by both our previous investigation and other studies [ , , ] . it is, indeed, a surface-exposed protein involved in the mechanism of invasion of the epithelial cells [ , ] . its expression is upregulated when the map is exposed to the physicochemical conditions similar to the intestine environment and the specific block of this protein reduces the virulence up to % [ ] . interestingly, this protein is among the entries classified as wb suggesting that more immunogenic properties can also be exploited by the other wb protein besides the others predicted as being sbs. moreover, we specifically focused on the sole peptide sequences whose binding affinity is shared among multiple bolas. in this manner, the most suitable epitope sequences are likely to have a broad recognition in a higher portion of the bovine population [ , ] . epitopes identified with this approach are of potential interest for diverse purposes and studies, including the investigations aimed at elucidating the order of immunological events following the map infection, and shedding light on the controversial aspect of suppression, or not, of the cellular-mediate immune response following map infection [ ] . to prove selected epitopes as suitable candidates for the unbiased diagnosis of map infection, we aligned the peptides sequences against a database comprising the closest taxonomically-related bacteria. such alignment generated a steep reduction of the number of input sequences and returned two peptides suitable for a specific diagnosis of map. these two candidates were not overlapping with other mycobacteria other than mycobacterium avium complex (mac). the described approach resumes the pipeline of an in-silico method, therefore, empirical tests will be required for the definitive assessment of differential diagnosis capability of the selected sequences. the sequence alignment against the host-specific protein (i.e., the publicly available cow proteome) fails to identify significant sequence identities. the only alignment hits observed (supplementary table s ) were not continuously overlapping and showed a low percentage of identity and a high e-value. acknowledged the prediction of linear epitopes, the matching of our candidates with gapped sequences of cow is likely to be of a negligible relevance since regarding amino acid residues that are not laying in a concatenate order. thus, we speculate that the candidate epitopes suggested in this study are of potential value for the design of either multi-subunit or recombinant vaccines to confer protection against the first-time infection of the calves by map. nevertheless, confirmatory experimental trials are warmly encouraged especially to assess the specificity between map infected animals and bovine with tuberculosis [ , ] . although less significant, a certain level of identity hasbeen registered with other mycobacterium strains. however, the discrepancy observed in the sequence alignment might be used as the driving force for the differential diagnosis. at this purpose, application of optimized laboratory protocols expecting high stringency condition might be the key to improve the specificity of the diagnostic methods. finally, empirical evaluation of the synergistic effect of both b-cell epitopes and the class i mhc epitopes are desirable. this will aim at the evaluation of the successful diagnosis of map infection at the subclinical stage and at the potential in elicitation of protective immunity. to conclude, the present study describes an innovative pipeline based on the in-silico survey of selected immunoreactive proteins capable to uncover the immunogenic features of each protein. this pipeline was applied to the detection of a restricted number of peptides potentially useful for the diagnosis of jd at the early subclinical stage. obtained results are as well useful for the implementation of innovative vaccination strategies. the obtained results confirmed the immunogenicity observed experimentally through the immunoproteomic approach applied to the map proteome. this evidence demonstrates, once again, reciprocal support between immunoproteomics and immunoinformatics. nevertheless, empirical confirmations are warmly required to test the provided epitope sequences both in-vitro and ex-vivofor the possible detection of the subclinical phase of the infection and for the efficacy of the eventual vaccinal formulations. such experimental tests might also help with the comprehension of the controversial role of the host immune cell-response underlying behind jd. complementation of the linear epitopes array with other conformational ones is also of importance for befitting efficacy and safety of the deliverables empirical confirmation may serve as further proof of the robustness of the immunoinformatics approaches as key contributors in the study of diverse infectious diseases. this would provide reliable scientific support in a safe, rapid and cost-effective approach. the current study focuses on ten proteins whose immunoreactivity has been experimentally investigated by means of an immunoproteomic approach [ ] . brielfy, the map proteome was incubated with sera from infected animals to screen for proteins with immunoreactive potential. the most promising entries were then subjected to ms-based identification. identifiers of the candidate immunoreactive proteins were queried in the ncbi non-redundant (ncbinr) protein database to retrieve the whole protein sequences and export them as a fasta file. update of the protein accession numbers operated by the reference data repository (i.e., ncbi) required the run of a protein sequence alignment for the attribution of the novel protein identifiers (gi numbers). selected peptide sequences of the immunoreactive proteins were searched against the ncbinr database restricted to mycobacterium avium subsp. paratuberculosis (taxid ) and the best hit was used to transform the former protein accession numbers into the novel ncbi protein gis. the list of proteins employed in this study along with their current gi number is provided in table and supplementary table s . prediction of the protein sequences that are likely to elicit antibody production and/or bind class i mhc proteins was performed through two tools that are commonly employed for the epitope prediction [ ] , namely iedb (http://tools.iedb.org/bcell/) and netmhc (http://www.cbs.dtu.dk/ services/netmhc/), respectively for b-and class i mhc epitopes prediction. bepipred algorithm was chosen for the prediction of linear protein epitopes capable of binding b-cells. this employs a combination of a hidden markov model and a propensity scale method [ ] . each protein residue is scored for its epitope behavior and the sole aminoacid with a score greater than or equal to . was considered as a potential epitope. linear peptide epitopes of the least length of aminoacids were selected for this study. prediction of epitopes binding class i mhc molecules was performed through netmhc prediction tool, using the artificial neural network (ann) algorithm [ ] . the algorithm was set for the prediction of nine-aminoacids long peptides capable of binding the following bola alleles: bola-d . ; bola-hd ; bola-jsp. ; bola-t a; bola-t b; bola-t c. the binding affinity of the peptides wasscored, and a percentile rank wasprovided by computationally comparing the score of each queried peptide sequence against , natural peptides of the same length. peptides scoring a binding affinity up to . % were considered as strong binders (sb); whereas, peptides with a percentile rank comprised between . % and % were labelled as weak binders (wb). all other peptides were discarded [ , , ] . the resulting list of selected peptide epitopes was further quality-checked and filtered. for each of the selected proteins, the epitopes shared among the major number of bola haplotypes were kept (i.e., the most commonly recognized in the bovine population), resulting in a consensus list of epitopes to be further used in the study. a summary of the experimental worklow employed in this study is provided in figure . . schematic representation of the immunoinformatic approach. blue and orange arrows refer to the input and output of the data, respectively. data arising from our previous immunoproteomic study [ ] were used to retrieve the protein sequences of the immunogenic proteins, selected on the basis of their capability of being complexed by the immunoglobulins in the sera of the infected cows. the sequence of the immunogenic proteins is subjected to the epitope prediction through dedicated tools and algorithms. the b-epitope prediction was performed via the iedb prediction tool, that provides a list of candidate b-epitopes. the class i mhc epitopes waspredicted via netmhc. this makes use of the bola haplotypes from the data repository (ncbi) as references for computing the linear peptides capable of being recognized and presented by the diverse bolas. the list of potential t-epitopes is further refined by selecting the most commonly recognized epitopes with a relatively high binding affinity. the refined list of epitopes is further tested for sequence specificity and cross-reactivity by pblast alignment versus the mycobacteria and cow protein database which, in turn, arise from the publicly available data repository (ncbi). the list of epitope sequences was further analyzed through the basic local alignment search tool for protein sequences (pblast) [ ] . this tool implements the pam algorithm to compare protein sequences and calculates the robustness of matches as means of expected values (e-value). . schematic representation of the immunoinformatic approach. blue and orange arrows refer to the input and output of the data, respectively. data arising from our previous immunoproteomic study [ ] were used to retrieve the protein sequences of the immunogenic proteins, selected on the basis of their capability of being complexed by the immunoglobulins in the sera of the infected cows. the sequence of the immunogenic proteins is subjected to the epitope prediction through dedicated tools and algorithms. the b-epitope prediction was performed via the iedb prediction tool, that provides a list of candidate b-epitopes. the class i mhc epitopes waspredicted via netmhc. this makes use of the bola haplotypes from the data repository (ncbi) as references for computing the linear peptides capable of being recognized and presented by the diverse bolas. the list of potential t-epitopes is further refined by selecting the most commonly recognized epitopes with a relatively high binding affinity. the refined list of epitopes is further tested for sequence specificity and cross-reactivity by pblast alignment versus the mycobacteria and cow protein database which, in turn, arise from the publicly available data repository (ncbi). the list of epitope sequences was further analyzed through the basic local alignment search tool for protein sequences (pblast) [ ] . this tool implements the pam algorithm to compare protein sequences and calculates the robustness of matches as means of expected values (e-value). this value describes the statistic of matches occurring "by chance"; thus, it decreases exponentially as the score of the match increases. in the pblast, each epitope sequence has been aligned against both mycobacteria (ncbi taxid ) and cow (ncbi taxid ) protein repertoires to evaluate sequence specificity and cross-reactivity (figure ) , of importance while selecting candidate epitopes to be employed for the effective diagnosis and/or prophylaxis of map. supplementary materials: the following are available online at http://www.mdpi.com/ - / / / /s . table s . b-cell binding protein epitope prediction. the file summarizes the b-epitope prediction results. each sheet of the xls file is relative to one of the ten selected proteins. "position" column is relative to the position of each aminoachid along the protein sequence; "residue" indicates the type of aminoacid; "score" is relative to the epitope propensity attributed by the algorithm and "assignment" rank each aminoacid residue as epitope or not depending on the prefixed settings. table s . class i mhc binding protein epitope prediction. the xls file summarizes the results in a table reporting: the predicted affinity (nm); the percentile rank, and the predicted binding core for all the selected alleles. two additional columns summarize the predictions across alleles: harmonic mean of the %rank calculated over all specified alleles (h_avg_ranks); the number of alleles covered by a given peptide (n_binders). table s . selected peptide epitopes alignment against the mycobacteria and cow databases. the xls file provides a summary of the pblast alignment search. full description of the table columns and the alignment criteria is available at https://blast.ncbi.nlm.nih.gov/blast.cgi?page=proteins. table s . full list of the prototypic peptides alignment. the file summarizes the p-blast alignment of the two selected epitopes against both the mycobacteria and the cow database. full description of the table columns and the alignment criteria is available at https://blast.ncbi.nlm.nih.gov/blast.cgi?page=proteins. the authors declare no conflict of interest. defining resilience to mycobacterial disease: characteristics of survivors of ovine paratuberculosis experimental infection model for johne's disease using a lyophilised, pure culture, seedstock of mycobacterium avium subspecies paratuberculosis gene expression profiles during subclinical mycobacterium avium subspecies paratuberculosis infection in sheep can predict disease outcome control of paratuberculosis: who, why and how. a review of countries paratuberculosis in sheep and goats johne's disease) in cattle and other susceptible species temporal patterns and 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(bola) presented ligands by use of mass-spectrometry-determined ligand and in vitro binding data improved prediction of mhc ii antigen presentation through integration and motif deconvolution of mass spectrometry mhc eluted ligand data production and characterization of monoclonal antibodies against a major membrane protein of mycobacterium avium subsp. paratuberculosis. clin. vaccine immunol a mycobacterium avium subsp. paratuberculosis rela deletion mutant and a kda major membrane protein elicit development of cytotoxic t lymphocytes with ability to kill intracellular bacteria the mycobacterium avium subsp. paratuberculosis kda protein plays a role in invasion of bovine epithelial cells mhc peptidome deconvolution for accurate mhc binding motif characterization and improved t-cell epitope predictions pooshang bagheri, k. in silico rational design of a novel tetra-epitope tetanus vaccine with complete population coverage using developed immunoinformatics and surface epitope mapping approaches new latex bead agglutination assay for differential diagnosis of cattle infected with mycobacterium bovis and mycobacterium avium subsp. paratuberculosis duplex pcr for differential identification of mycobacterium bovis, m. avium, and m. avium subsp. paratuberculosis in formalin-fixed paraffin-embedded tissues from cattle improved method for predicting linear b-cell epitopes gapped sequence alignment using artificial neural networks: application to the mhc class i system in silico identification of epitopes in mycobacterium avium subsp. paratuberculosis proteins that were upregulated under stress conditions protein database searches using compositionally adjusted substitution matrices key: cord- -qrrdi authors: mayer, f; brunner, a title: non-neutral evolution of the major histocompatibility complex class ii gene drb in the sac-winged bat saccopteryx bilineata date: - - journal: heredity (edinb) doi: . /sj.hdy. sha: doc_id: cord_uid: qrrdi the immune genes of the major histocompatibility complex (mhc) are classical examples for high levels of genetic diversity and non-neutral evolution. this is particularly true for the regions containing the antigen-binding sites as, for instance, in the exon of the mhc class ii gene drb. we surveyed, for the first time in the order chiroptera, the genetic diversity within this exon in the sac-winged bat saccopteryx bilineata. we detected alleles among bats, of which were sampled in one population. pairwise comparisons revealed that interallelic sequence differences ranged between and %, although nucleotide substitutions were not evenly distributed along the exon sequence. this was most probably the result of intragenic recombination. high levels of sequence divergence and significantly more nonsynonymous than synonymous substitutions (d(n)/d(s)> ) suggest long-term balancing selection. thus, the data are consistent with the hypothesis that recombination gives rise to new alleles at the drb locus of the sac-winged bat, and these are maintained in the population through balancing selection. in this respect, the sac-winged bat closely resembles other mammalian species. the major histocompatibility complex (mhc) plays a central role in the vertebrate immune system. mhc genes encode two major classes of transmembrane glycoproteins consisting of two chains, a and b. mhc class i genes are expressed in almost all nucleated somatic cells, whereas mhc class ii genes are confined to antigen-presenting cells of the immune system (klein, ) . although this general function of the mhc is conserved among vertebrates, the genetic architecture of the mhc can differ within and between vertebrate classes (recently reviewed in kelley et al., ) . gene duplication occurred frequently, and thus the number of classes i and ii loci can substantially vary between species (kelley et al., ; nei and rooney, ) . in mammals, the genetic diversity at various mhc loci has been studied in several species of all major mammalian orders except for the second largest order chiroptera that comprise more than species worldwide (simmons, ) . bats and flying foxes are of particular immunological interest, because they are hosts of viruses pathogenic to humans including rabies (fooks et al., ; cliquet and picard-meyer, ) , ebola (leroy et al., ) , sars-like viruses (li et al., ) , and nipah and hendra viruses (wong et al., ) . mhc genes are the most variable genes described in vertebrates. up to alleles were described at the human mhc locus hla-b (garrigan and hedrick, ) . highest levels of nucleotide diversity are found in regions containing antigen-binding sites (abs) and patterns of nucleotide substitutions deviate from neutral evolution expectation in almost all studies. some form of balancing selection best explains the enormous diversity of certain mhc genes (hedrick and thomson, ) , and two major mutually nonexclusive mechanisms of pathogen-driven selection have been proposed to explain this pattern. first, the frequency-dependent selection hypothesis or the rare allele advantage (clarke and kirby, ) predicts that rare alleles can have a selective advantage as parasites will adapt to common host alleles. since an advantageous allele will increase in frequency, parasites resistant to this allele will be favoured. negative frequency-dependent selection models of host-parasite coevolution showed that a large number of alleles can be retained (borghans et al., ) . the spatial and rapid temporal variation in frequency of mhc alleles and resistance provides evidence for frequency-dependent selection (hill et al., ; westerdahl et al., ; charbonnel and pemberton, ) . second, the overdominance or heterozygote advantage hypothesis (doherty and zinkernagel, ) assumes that heterozygous individuals have a higher average fitness, because individuals being heterozygous at the antigen-binding region can present a wider array of antigens than homozygous individuals. therefore, heterozyotes are expected to be more resistant against pathogens than homozygotes. an overdominance effect was shown in mice that were coinfected with salmonella enterica and theiler's murine encephalomyelitis virus (mcclelland et al., ) . distinguishing between the frequencydependent selection and the heterozygote advantage hypothesis is difficult, because both models require a correlation between parasites and particular mhc alleles. meanwhile, such relationships were found in several species including many non-model organisms ( table in sommer, ) . the evolutionary ecology of the mhc was recently summarized in several reviews (bernatchez and landry, ; garrigan and hedrick, ; sommer, ; piertney and oliver, ) . we analysed the antigen-binding region of the mhc class ii gene drb of the sac-winged bat saccopteryx bilineata by sequencing cloned pcr products. we selected this neotropical, insectivorous bat species, because completely sampled families consisting of an offspring and both parents were available (heckel and von helversen, ) that allowed testing mendelian inheritance of haplotypes. in this study, we describe the genetic diversity within exon of the mhc class ii gene drb, and discuss the role of recombination and selection in generating and maintaining high levels of genetic diversity in colonies of the sac-winged bat. bats were caught by mist netting at the biological station la selva in costa rica ( n, w, n ¼ ), on barro colorado island (bci) in panama ( n, w, n ¼ ) and at the tiputini biodiversity station (tbs) in the amazonian rainforest of ecuador (b s, w, n ¼ ) between and . animals were sexed, aged and measured before a circular wing biopsy with a diameter of mm was taken from the proximal plagiopatagium: biopsies were stored in % ethanol at room temperature until dna isolation. genetic analysis dna was isolated by a salting out procedure (mü llenbach et al., ) , which yielded about . mg of total genomic dna. the exon of the class ii mhc gene drb was initially amplified by pcr with the primers gh and gh (scharf et al., ) but only small amounts of dna were obtained. the design of two shorter primers er b ( -aaccccgtagttgtgtctgca- ) and er ( -ggatccttcgtgtcccca- ), which have a similar t m value and contain only the core sequences of the primers gh and gh , improved the pcr substantially. the reaction was performed in a total volume of ml, which contained about ng total genomic dna, . % bovine serum albumin, mm of each primer (er b and er ), . mm of each dntp, mm mgcl , . u taq-dna-polymerase (promega corporation, madison, wi, usa, no. m ) and  taq-dna-polymerase buffer (supplied with the enzyme). a pcr was started with incubation at c for min followed by cycles of c for s, c for s, c for . min and finished by min period at c. amplified dna fragments were cleaned with qiaquick pcr purification columns (qiagen, gmbh, hilden, germany) and reconstituted in ml tris-cl buffer ( mm, ph . ). the amplified dna of each individual investigated was cloned using the pgem-t easy kit (promega) and following the manufacturer's protocol. tam -f' extra competent cells (active motif, rixensart, belgium) were used in transformation. positive clones were picked and transferred into a ml pcr mix as described above. if a dna fragment of the expected length was amplified, the same clone was picked again, transferred into ml lb medium containing mg/ml ampicillin and incubated at c for h. dna was extracted using the qiaprep miniprep kit (qiagen) following the recommendations of the manufacturer. six to clones with an insert of the drb fragment were sequenced from each individual on a dna sequencer (li-cor) using the thermosequenase dyenamic direct sequencing kit (amersham biosciences, piscataway, nj, usa). the probability of missing one of two gene copies among six clones is . ¼ . and decreases to po . with or more clones. we excluded sequence types from the analysis, if they were found only in one clone to account for nucleotide substitutions or recombination during the pcr or cloning. the inheritance of sequence types was studied in juvenile bats with known mothers and/or fathers from a large colony at the biological station la selva. they were assigned to their parents by behavioural observations and genotyping of the juveniles and their putative parents at microsatellite loci as described in heckel and von helversen ( ) . allele frequencies were calculated only from adult males and females, which were sampled in costa rica. using these allele frequencies, the heterozygosity at a given amino-acid site was calculated (hedrick et al., ) . hardy-weinberg exact tests were performed with the program genepop (web version . ) (raymond and rousset, ) using the default values (markov chain: batches and iterations per batch). the software package mega (kumar et al., ) allowed the calculation of the relative rate of nonsynonymous and synonymous substitutions according to nei and gojobori ( ) by applying the correction of jukes and cantor ( ) for multiple hits. the program arelequin . (schneider et al., ) was used to perform the ewens-watterson homozygosity test of neutrality. the computer program geneconv (sawyer, ) was used to test for statistical evidence of gene conversion (sawyer, ) . we used the default settings that do not allow mismatches. global and pairwise comparisons were statistically evaluated with permutation runs. p-values were corrected for multiple comparisons. all other statistical tests were performed with spss . for mac osx. we detected sequence types ( figure ) by sequencing - clones from each of the individuals studied. except for two individuals that had three different sequence types, only one or two sequence types were found per individual. the sequence type b was detected only in one and b in another individual mhc diversity in the sac-winged bat f mayer and a brunner (two out of nine clones in each individual), although two different a sequence types were found in both individuals. the three sequence types indicate the existence of at least two drb loci in the genome of the sac-winged bat. a and b sequence types differ in length by bp. all sequence types contained an open reading frame without stop codons ( figure ) and showed high similarity to the exon amino-acid sequence of the mammalian drb locus. inheritance of drb sequence types was studied in single parent-offspring pairs and complete families that consisted of a juvenile and both parents. among all individuals, only a sequence types were found. the genotypes of all juveniles matched to the genotypes of their parents as expected under mendelian inheritance. according to the differences in length between a and b sequence types and the mendelian inheritance of a sequence types, we assume that all a sequence types are coded by a single locus and refer to them as alleles of the drb locus of s. bilineata. the genotypes of adult individuals from the la selva population did not deviate from hardy-weinberg expectation (p ¼ . ). it remains unclear, whether the two b sequence types originate from one or two loci. alignment of all polymorphic nucleotide sites among all sequence types found. boxes mark identical sequence fragments among drb alleles that are statistically supported by permutation tests implemented in the software geneconv (sawyer, ) . a shared fragment between the sequence types a and b is underlined. only significantly shared fragments of global comparisons are shown. b sequence types are characterized by a bp fragment that lacks in a sequence types. the complete dna sequences are available at genbank (accession nos. ef -ef ). , , , , , , , , , , , , , , , , and are assumed to be antigen-binding sites (brown et al., ) and are labelled with an asterisk. mhc diversity in the sac-winged bat variation at the drb locus; evidence of recombination/ gene conversion ten alleles were detected within the main sample of individuals from the la selva population. allele a was most frequent and occurred at a frequency of . % among adult bats (figure ) . the alleles a , a , a and a were detected in only one individual. observed heterozygosity ( . ) was lower than expected heterozygosity ( . ). the hardy-weinberg exact test on heterozygote deficiency (genepop) revealed an almost significant result (p ¼ . ). within the panama sample of three individuals, allele a was also most common (four copies) and a single copy of allele a and a new allele a were found. in contrast, allele a was most common in three animals from ecuador (three copies). the remaining alleles were a (two copies) and a (one copy). the dna sequence consists of base pairs of which ( . %) were polymorphic. on average, alleles differed by . nucleotides ( . %). alleles a and a differed by only six nucleotides ( . %). the most distinct alleles were a and a , which differed by nucleotides ( . %). pairwise dna sequence differences between the alleles are given in table . allele a , which was only found in individuals from panama, was not more distinct to alleles from costa rica than alleles from costa rica to each other. several alleles share different sequence fragments of identical sequence (figure ). these shared fragments were significantly similar when the global comparison procedures and permutation test implemented in the software geneconv (sawyer, ) was applied. thus, some alleles are partly characterized by different arrangements of these fragments that is likely due to gene conversion. the two most common alleles (a and a ) are distinct at . % of all nucleotide sites and do not share an obvious sequence fragment. the two b sequence types do not share a sequence fragment with each other. only the sequence type b has a short sequence fragment in common with the a allele. in contrast, most drb alleles (that is a sequence types) are linked by shared sequence fragments. this corroborates our view that b sequence types are not alleles of the drb locus. evidence for balancing selection drb alleles differed in ( %) to ( %) of all amino-acid sites studied. up to five different amino acids were found at a single codon position and ( . %) amino-acid sites were variable (figure ). in humans, amino-acid positions, located within the investigated sequence region, are documented to be important in the abs using x-ray crystallography (brown et al., ) . most variation was concentrated at these abss. the proportion of variable positions was significantly higher at abs than at nonantigen-binding sites (non-abss) ( of ( %) and of ( %), respectively, w ¼ . , po . ). using the observed allele frequencies in the costa rica sample, we calculated the heterozygosity for each amino-acid position (figure ) . figure average heterozygosity for individual amino-acid positions calculated from the allele frequencies in the costa rica sample. asterisks indicate positions, which are assumed to be antigenbinding sites in humans (according to brown et al., ) . mhc diversity in the sac-winged bat the highest heterozygosity was found at position ( . ). an average heterozygosity of . was observed at abs, which was significantly higher than the average heterozygosity at non-abss ( . , u ¼ , p ¼ . ). the maximum of five different amino-acid substitutions was only found at two abs (positions and , figure ). higher divergence among amino-acid sequences than among dna sequences, and the concentration of aminoacid variation at abs indicate the existence of balancing selection. additional evidence comes from the comparison of the relative rate of nonsynonymous (d n ) and synonymous (d s ) substitutions at the antigen (abs) and the nonantigen-binding amino-acid sites. at abs, nonsynonymous substitutions (d n ¼ . . ) were on average . times more frequent (z ¼ À . , n ¼ , po . ) than synonymous substitutions (d s ¼ . . ). the corresponding ratio d n /d s at non-abss was reverse ( . ) and also both substitution according to the overdominance hypothesis and the negative frequency-dependent selection hypothesis, balancing selection should lead to a more even distribution of allele frequencies than under neutral expectation, which can be tested by the commonly used ewens-watterson homozygosity test of neutrality. we did not find a significant deviation from neutral expectation (p ¼ . ) by applying this test to our costa rica sample. the first analysis of a mhc gene in a bat species revealed concordant diversity patterns as in species of other mammalian orders. in the genome of the sac-winged bat s. bilineata, we found evidence for two gene copies of the mhc class ii gene drb, which is involved in antigen presentation. cloning and sequencing pcr products amplified by mhc class ii b-specific primers produced a maximum of three haplotypes per individual. three haplotypes were found only in two out of individuals ( %). only in these two individuals, one haplotype differed from all the other haplotypes detected by a bp insertion and thus likely originated from another (second) locus. the occasional occurrence of three haplotypes can have two reasons. either duplicated drb loci exist only in some individuals or the primers we used amplify preferably only one locus. at the moment, we cannot distinguish between both hypotheses. although we used drb exon -specific primers, which were also successfully applied in other mammalian orders, including primates and rodents (gyllensten et al., ; schad et al., ; froeschke and sommer, ) , we cannot rule out the amplification failure of a locus due to mutations in a primer-binding site. in % of all bats studied, we amplified one or two haplotypes. we regarded them as alleles from one locus (drb ). this assumption is supported by the mendelian inheritance of alleles that was tested in parentoffspring pairs and by the observation that genotype frequencies matched hardy-weinberg expectations. allelic diversity a total of alleles was found within a small geographic area of the biological station la selva in costa rica. alleles differed substantially in frequency. although allele a occurred at a frequency of %, the frequencies of the remaining alleles were % or less. we did not find evidence of an amplification bias among alleles, which could explain such frequency differences. among heterozygous individuals, clones containing the allele a were not more frequent than clones containing another allele (wilcoxon signed-rank test, z ¼ À . , p ¼ . ). the number of drb alleles varies substantially among mammalian species ranging from one or two in some small and inbred populations (ruminants: mikko et al., ; canids: aguilar et al., ; rodents: sommer and tichy, ; pinnipeds: weber et al., ) to alleles at the human hla-drb locus (garrigan and hedrick, ) . this enormous variation has to be treated with caution because the number of alleles detected also depends on the number of individuals sampled and population coverage. the highest numbers of drb alleles are described from intensively studied species like humans ( alleles, garrigan and hedrick, ) or sheep ( alleles, konnai et al., ) . in the sac-winged bat, the number of drb alleles increased to by analysing additional six individuals from panama and ecuador. therefore, the allele number found in the sacwinged bat is within the range of other mammalian species, although it ranges at the lower end of an outbred population (for example, mikko et al., ) . recombination between alleles (intragenic recombination) or between loci (intergenic recombination) can rapidly generate allelic diversity at mhc class ii genes (andersson and mikko, ) . our study provides further evidence for the significance of recombination in generating allelic diversity of mhc class ii genes. several drb alleles of the sac-winged bat shared different sequence motifs with different alleles (figure ). in contrast, the two b sequence types, which are likely encoded by another locus, did not share long-sequence fragments with drb alleles. this suggests that intragenic recombination of the drb locus and less intergenic recombination plays an important role in generating allelic diversity at the drb locus of the sac-winged bat. despite the lack of clear evidence for intergenic recombination we are not able to rule it out, because we might have missed to amplify paralog drb loci possibly involved in intergenic recombination. methodological artefacts, like recombination during pcr, can be ruled out because recombined alleles were independently detected in several individuals. several recent studies on genetic diversity of mhc class ii genes documented intragenic recombination (human hla-dpb : zangenberg et al., ; deer mouse eb: richman et al., ; chamois drb: schaschl et al., ; stickleback mhc class iib genes: reusch and langefors, ) . usually only short-sequence motifs are shared among alleles. in contrast, shared sequence fragments are rather long in the sac-winged bat. a similar pattern of shared sequence fragments among alleles was observed by zangenberg et al. ( ) , who studied the rate of evolution within the exon of the human hla-dpb locus by screening pools of sperm. the similarity between this sperm analysis study and our study suggests that some recombination events among drb alleles in the sac-winged bat occurred rather recently. this is further supported by the lack of nucleotide substitutions between some dna sequence fragments that are shared by two alleles (figure ). one of the strongest arguments for balancing selection comes from a higher rate of nonsynonymous nucleotide substitutions than synonymous nucleotide substitutions (garrigan and hedrick, ) . the comparison of nonsynonymous to synonymous nucleotide substitutions (d n /d s ) revealed contrasting results between sites involved in abs and non-abs. at abs, nonsynonymous mutations were . times more frequent than synonymous mutations, whereas this was not the case for non-abs. in addition, abss were more variable than non-abss. the proportion of polymorph abs was significantly higher than that of non-abs; and at the protein level, amino-acid sites involved in antigen binding showed higher levels of heterozygosity than nonantigen-binding amino acid sites. the d n /d s test is most commonly used in mhc studies to infer a possible role of balancing selection. in of studies, the rate of nonsynonymous substitutions exceeded the rate of synonymous nucleotide substitutions (d n /d s ) (bernatchez and landry, ) , and thus our study on the sac-winged bat is in line with these studies. whether balancing selection occurred over the history of the species and whether it still acts in contemporary populations can be tested by different approaches (garrigan and hedrick, ) . the nucleotide sequence of the alleles differed substantially (mean . %). this is in accordance with balancing selection in the past, which can maintain particular haplotype lineages for a long evolutionary period. therefore, despite the important role of balancing selection for maintaining allelic diversity for a long evolutionary time, its contemporary role remains unclear. our data do not support ongoing balancing selection at least not within our local population of sac-winged bats from the biological station la selva. we neither found evidence for heterozygote excess nor did the ewens-watterson test reveal a deviation of heterozygosity from a neutral equilibrium distribution. instead the hardy-weinberg-exact test on heterozygote deficiency revealed an almost significant result due to an increased proportion of homozygotes of the most common allele. this trend is in contrast to the expectation of ongoing balancing selection due to a heterozygote advantage (doherty and zinkernagel, ) . however, detection of selection from heterozygote excess has low power. small deme size itself leads to heterozygote excess (pudovkin et al., ; luikart and cornuet, ; balloux, ) and cryptic population structure could cause homozygote excess. thus, one would need a comparative study with other markers to see if observed excess was itself in excess of a heterozygote or homozygote excess at other loci. despite the diversity of divergent alleles, the observed and expected heterozygosity was very low ( . and . , respectively). such low levels of heterozygosity are known only from populations of small size or with a bottleneck in the past. they are characterized by a small number of alleles with allele frequencies not exceeding % in polymorphic populations (sena et al., ; aguilar et al., ; amills et al., ; weber et al., ) . in contrast, in the sac-winged bat, a singlefrequent allele (a ) occurred at a frequency of % that caused the low heterozygosity. the nine remaining alleles had a frequency of % or less. such a high frequency of a single allele can be the result of genetic drift and is unexpected if heterozygotes (doherty and zinkernagel, ) or rare alleles (clarke and kirby, ) have an advantage. we observed more homozygotes for the common allele than expected, although this result was not significant. an excess of a particular homozygous genotype is expected, if individuals with this genotype have higher fitness, for example, by having a higher chance of survival. one possibility is pathogen-driven selection that was recently shown for a number of wild populations of several species. particular alleles were significantly increased in frequency in individuals, infected by a particular pathogen compared to uninfected individuals (reviewed in bernatchez and landry, ; piertney and oliver, ) . for example, the frequency of one allele differed by the factor of more than two between nematode infected and uninfected striped mice rhabdomys pumilio (froeschke and sommer, ) . the high frequency of the a allele in the sac-winged bat could be a result of positive selection by an unknown pathogen that invaded our study population. this remains purely speculative at the moment, as we do not have information on the infection of sac-winged bats by parasites. even when positive selection occurs, allelic diversity can be maintained due to immigrant individuals from other populations, if these populations experience different selection pressures. the analysis of bats from other localities and of neutrally evolving sequence regions close to the drb locus will give some further insights (garrigan and hedrick, ; charlesworth, huang mm, arnheim n, erlich h ( ) . new hla-dpb alleles generated by interallelic gene conversion detected by analysis of sperm. nat genet : - . mhc diversity in the sac-winged bat high mhc diversity maintained by balancing selection in an otherwise genetically monomorphic mammal low diversity in the major histocompatibility complex class ii drb gene of the spanish ibex, capra pyrenaica generation of mhc class ii diversity by intra-and intergenic recombination heterozygote excess in small populations and the heterozygote-excess effective population size mhc studies in nonmodel vertebrates: what have we learned about natural selection in years? mhc polymorphism under host-pathogen coevolution three-dimensional structure of the human class ii histocompatibility antigen hla-dr a long-term genetic survey of an ungulate population reveals balancing selection acting on mhc through spatial and temporal fluctuations in selection balancing selection and its effects on sequences in nearby genome regions maintenance of histocompatibility polymorphisms rabies and rabies-related viruses: a modern perspective on an ancient disease enhanced immunological surveillance in mice heterozygous at the h- gene complex european bat lyssaviruses: an emerging zoonosis mhc class ii drb variability and parasite load in the striped mouse (rhabdomys pumilio) in the southern kalahari perspective: detecting adaptive molecular polymorphism: lessons from the mhc genetic diversity at class ii drb loci of the primate mhc genetic mating system and the significance of harem associations in the bat saccopteryx bilineata evidence for balancing selection at hla heterozygosity at individual amino acid sites: extremely high levels for hla-a and -b genes human leukocyte antigens and natural selection by malaria evolution of protein molecules comparative genomics of major histocompatibility complexes the natural history of the major histocompatibility complex sequences and diversity of new ovar-drb alleles from three breeds of sheep mega : integrated software for molecular evolutionary genetics analysis and sequence alignment fruit bats as reservoirs of ebola virus bats are natural reservoirs of sars-like coronaviruses estimating the effective number of breeders from heterozygote excess in progeny major histocompatibility complex heterozygote superiority during coinfection monomorphism and polymorphism at mhc drb loci in domestic and wild ruminants an efficient saltchloroform extraction of dna from blood and tissues simple methods for estimating the numbers of synonymous and nonsynonymous nucleotide substitutions concerted and birth-and-death evolution of multigene families the evolutionary ecology of the major histocompatibility complex on the potential for estimating the effective number of breeders from heterozygote-excess in progeny genepop (version . ): population genetics software for exact tests and ecumenicism inter-and intralocus recombination drive mhc class iib gene diversification in a teleost, the three-spined stickleback gasterosteus aculeatus relative roles of mutation and recombination in generating allelic polymorphism at an mhc class ii locus in peromyscus maniculatus statistical tests for detecting gene conversion mhc variability of a small lemur in the littoral forest fragments of southeastern madagascar sequence analysis of the hla-dr beta and hla-dq beta loci from three pemphigus vulgaris patients recombination and the origin of sequence diversity in the drb mhc class ii locus in chamois (rupicapra spp) arlequin: a software for population genetics data analysis. genetics and biometry lab polymorphisms in mhc-dra and -drb alleles of water buffalo (bubalus bubalis) reveal different features from cattle dr alleles mammal species of the world: a taxonomic and geographic reference the importance of immune gene variability (mhc) in evolutionary ecology and conservation major histocompatibility complex (mhc) class ii polymorphism and paternity in the monogamous hypogeomys antimena, the endangered, largest endemic malagasy rodent christian voigt and gerald heckel participated in field work. we are grateful for discussions and suggestions by gerald heckel, otto von helversen, martina nagy, simone sommer, jana ustinova and christian voigt. two anonymous referees provided important suggestions. the research was financially supported by the deutsche forschungsgemeinschaft (dfg) and the deutscher akademischer austauschdienst (daad). key: cord- -v h yro authors: han, ki-cheol; park, daechan; ju, shinyeong; lee, young eun; heo, sun-hee; kim, young-ae; lee, ji eun; lee, yuna; park, kyong hwa; park, se-ho; lee, hee jin; lee, cheolju; jang, mihue title: streamlined selection of cancer antigens for vaccine development through integrative multi-omics and high-content cell imaging date: - - journal: sci rep doi: . /s - - -z sha: doc_id: cord_uid: v h yro identification of tumor antigens that induce cytotoxic t lymphocytes (ctls) is crucial for cancer-vaccine development. despite their predictive ability, current algorithmic approaches and human leukocyte antigen (hla)-peptidomic analysis allow limited selectivity. here, we optimized a method to rapidly screen and identify highly immunogenic epitopes that trigger ctl responses. we used a combined application of this method involving immune-specific signature analysis and hla-associated peptidomics using samples from six patients with triple-negative breast cancer (tnbc) in order to select immunogenic hla epitopes for in vitro testing. additionally, we applied high-throughput imaging at the single-cell level in order to confirm the immunoreactivity of the selected peptides. the results indicated that this method enabled identification of promising ctl peptides capable of inducing antitumor immunity. this platform combining high-resolution computational analysis, hla-peptidomics, and high-throughput immunogenicity testing allowed rapid and robust identification of highly immunogenic epitopes and represents a powerful technique for cancer-vaccine development. . scheme of the rapid high-throughput approach for discovering natural ctl epitopes. preselected til-resident tnbc tumors underwent hla-peptidomic analysis to identify hla-bound peptides. integrated wts data revealed a higher priority to select promising hla-peptides via high-resolution bioinformatics analysis, showing immune-cell-specific signatures and tcr-repertoire diversity in tumors. combined ngs analysis and the use of predictive algorithms for mhc-binding affinity enabled selection of highly immunogenic hla-peptide candidates. analysis of ifnγ-producing cd + t cell response using the highcontent imaging system in a -well format at the single-cell level for discovery of immunogenic hla epitopes eliciting a ctl response. filtrating lymphocytes) play a significant role in tumor-sites. additionally, large amounts of tils correlate with improved tumor survival ; therefore, we preselected til-resident tnbc tissues for histologic analysis to identify potentially promising cancer epitopes ( fig. a and supplementary fig. s ) and scored til density by measuring the proportion of the stromal area infiltrated by lymphocytes, as previously described . to select highly immunogenic hla epitopes, we analyzed the intratumoral heterogeneity of the tcr repertoires in til-resident cancers from six patients with tnbc (fig. b,c) . the tcr repertoires comprise somatic recombination of the tcrα and β chains, allowing the specificity of each t cell clone to be determined by rearrangement of the v, d, and j segments of the tcrβ chain during generation of the highly variable complementary determining region , . to evaluate tcr diversity of tils, we assembled cdr sequences using the sequence reads of rna-seq data. a unique cdr sequence of tcrα and tcrβ, respectively, was defined as a clone, and the number of clonotypes represents the number of unique clones per sample after normalization with the corresponding rna-seq depth (fig. b) . the t cell clonal fraction was defined as the frequency of the top % of tcrα or tcrβ clones among total tcr clones (fig. c) . the top most abundant tcrα and tcrβ sequences in each patient are shown in supplementary fig. s , and expression of the three hla-class i genes (hla-a/b/c) is shown in fig. d . we found a linear positive correlation between the number of tcrβ clonotypes and the expression of mhc-class i genes according to pearson's correlation coefficient (r = . ) (fig. e) . til immunoprofiles and immune-specific signatures. several recent studies demonstrated the strong relationship between significant overall survival (os) and cancer patients harboring a high number of cd + t cells and a low number of foxp + t cells . in particular, the abundance of regulatory t (treg) cells and macrophages correlated with worse outcomes, whereas the abundance of intratumoral cd + t cells and cd + t-helper (th) cells correlated with better prognosis . additionally, immune-specific signatures in tils are of potential clinical significance; therefore, we estimated the distribution of til types in each patient according to wts data using cibersort computational analysis (fig. and supplementary fig. s ). the relative proportion of each infiltrated immune cell was evaluated in each patient by quantifying immune composition from bulk-tissue gene-expression profiles, as enrichment of cd /cd ro and th cells are considered positive prognostic factors . the results indicated that cd + t cells were highly infiltrated in both patients tnbc# and tnbc# , whereas a large proportion of treg cells was observed in patients tnbc# and tnbc# (fig. a-c) . patient tnbc# displayed a highly enriched frequency of cd + t cells, as well as treg cells, suggesting increased accumulation of tils. interestingly, we found a significantly increased proportion of cd + t cells relative to treg cells in patient tnbc# as compared with that observed in other patients (fig. d) , suggesting the emergence the relationship between the number of tcrβ clonotypes and the expression of mhc class i genes. pearson's correlation was calculated between two groups. of promising tumor-associated antigens. on the other hand, we found elevated levels of immune-suppressive macrophages in patients tnbc# , tnbc# , and tnbc# , which is predictive of a negative outcome (fig. e ). to identify naturally existing mhc class i -restricted ligands, we used an immunoproteomic approach involving tissue from six patients with tnbcs and an mhc-І antibody specific for the hla-a, b, and c molecules ( fig. and supplementary fig. s ). after immunoprecipitation, high-resolution lc-ms/ms analysis identified and quantified the hla peptide sequences with a % false discovery rate (fdr) (supplementary fig. s ) . notably, the number of eluted peptides from each of the six patients were substantially different ( supplementary fig. s ), although > % of the hla peptides analyzed by lc-ms/ ms showed typical properties associated with epitope length. as expected, most of the peptides were nine amino acids long, with only a few having to amino acids, suggesting a high level of consistency ( fig. a and supplementary fig. s ). clustering of the -mer hla peptides showed predominant enrichment of residues at peptide positions and and consistent with the anchor motifs required by the binding groove of each hla molecule (fig. b and supplementary fig. s ). additionally, we found high numbers of cd + t and cd + th cells infiltrating into the tumor sites of patient tnbc# and relative to treg cells, with patient tnbc# showing a -fold higher number of cd + t cells as compared with that in patient tnbc# and accompanied by the lowest expression of mhc class i genes, suggesting a higher accumulation of antigen-specific ctls in patient tnbc# (supplementary figs. s ,s ). a total of peptides were identified from the tissue of patient tnbc# along with elevated expression of hla genes, whereas only five peptides were received from tissue from patient tnbc# and all showing the lowest expression of mhc class i genes ( fig. c and supplementary fig. s ). moreover, we observed a positive correlation between the number of eluted peptides relative to input lysate and the expression of mhc class i genes (fig. c) . we then performed kyoto encyclopedia of genes and genomes (kegg) pathway enrichment analysis to investigate the genes associated with the hla-binding peptides in patient tnbc# and the homotypic hla-a* : allele (fig. d) . interestingly, the high-count genes (n > ) were significantly enriched in kegg pathways related to cancer, protein processing in the endoplasmic reticulum (er), viral carcinogenesis, and www.nature.com/scientificreports www.nature.com/scientificreports/ antigen processing and presentation. numerous cancer-related genes overexpressed in cancer tissues contribute to cancer-specific or associated epitopes , and hla epitopes require proteasomal digestion and translocation into the er to bind mhc class-І molecules . it would be expected that the expression of genes encoding machinery responsible for antigen processing would be elevated under these circumstances. these results suggested that the eluted hla peptides identified were naturally presented by hla molecules. we further investigated the levels of the eluted peptides based on rna-seq analysis of corresponding mrna from the same sample (fig. e ,f). compared with normal breast tissues, of peptides corresponding to proteins from the same sample showed elevated abundances in cancer tissues accompanied by significant differences in mrna expression (≥ log fold change). subsequent in silico prediction of the hla-binding affinities to the hla peptides and calculation of their respective binding affinity to specific alleles (predicted ic ) yielded a list of the top highest ranking peptides derived from patient tnbc# (table ) . a rapid imaging-based screening method to determine antigen-specific t cell response at the single-cell level. to determine whether the experimentally identified peptides can functionally elicit an immune response, we evaluated cytokine production by the cd + t cells. currently, intracellular cytokine staining (ics)-based detection methods for monitoring ex vivo ifnγ response show low throughput relative to the number of candidate antigens being tested. moreover, an individual antigen test requires large amounts of immune cells . therefore, we developed an efficient and comprehensive screening system to test ctl response based on a high-content, high-throughput imaging approach ( supplementary fig. s ). this fluorescence-imaging-based screening system allows the use of lower numbers of viable cells up-scaled performance , . development of a -well format capable of screening mixed populations of t cells for their response against large number of www.nature.com/scientificreports www.nature.com/scientificreports/ peptides enables a cost-effective approach to phenotype analysis. notably, cancer-associated antigens are highly attractive targets for determining their efficacy in triggering a t cell response; however, numerous clinical trials targeting taas for vaccine development have failed to demonstrate clinical efficacy due to immune self-tolerance. to identify highly immunogenic peptides incapable of eliciting self-tolerance, we tested the antigen-specific t cell response in pbmcs from a healthy donor. monitoring ex vivo ifnγ-producing pbmc reactivity using our fluorescence-labelled cell-based screening system ( fig. a -c, supplementary figs. s ,s ) revealed significant ifnγ responses to two individual epitopes (eif a -p and tcp -p) in cd + t cells labelled with a fitc conjugated anti-human cd antibody (fig. b) . additionally, treatment with the hla-a* : -specific epitopes allowed detection of apc-conjugated ifnγ released from cd + t cells (fig. a,b) , with pma and ionomycin co-treatment used to trigger t cell activation as a positive control. similarly, pbmcs from two of the three healthy donors were reactive against same two epitopes (fig. c) . to further analyze the peptide-induced cd + t cells, we generated hla-a* : tetramers targeting specific peptide-reactive cd + t cells. facs analysis revealed that . % and . % of t cells were targeted by eif a -p and tcp -p, respectively, and detectable on day of ex vivo t cell expansion (fig. d) . these findings suggested the efficacy of our method to screen highly immunogenic ctl epitopes using an imaging system on the detection of intracellular ifnγ levels following peptide stimulation. the two genes associated with the peptide epitopes, the translation initiation factor eif a and tcp , a member of the chaperonin-containing complex tcp -containing ring complex (tric), are involved in tumor proliferation and survival. eif a controls translation initiation and is a critical checkpoint protein involved in cell proliferation and tumorigenesis . additionally, tcp , as a tric member, is involved in tumor survival and growth and an oncogene driver . moreover, these two genes are significantly overexpressed in tumor tissues according to wts data and data from the genotype-tissue expression project public proteomic database, suggesting their potential as therapeutic targets. these results identified two epitopes derived from eif a and tcp as potential promising immunogenic antigens to boost t cell response. patients with tnbc, which is defined by the absence of estrogen receptor, progesterone receptor, and human epidermal growth factor receptor , have a higher tendency for recurrence at ~ years after diagnosis , . there are currently no known therapeutic targets for tbnc patients due to the molecular heterogeneity of the disease . recent accumulation of massive and comprehensive bioinformatics data allowed identification of potential therapeutic targets associated with clinical survival . interestingly, a tnbc subtype characterized by high levels of immune genes involved in t cell function, immune response, and antigen processing, was found to be associated with favorable prognosis, suggesting a close correlation between immune-gene signatures and better clinical outcomes . therefore, it is possible that tils controlling clinical cancer progression represent key factors for preselection of tumors prior to hla-immunopeptidomics. in this study, til-resident tissues were pre-selected comprehensively to investigate a diversity of tcr repertoires and immune profile as predictors of clinical outcome. we then found a positive correlation between tcr diversity, reflecting clonal composition, and the expression of mhc-class І molecules, suggesting that active tumor-antigen presentation promotes the generation of antigen-specific tils. additionally, immune-specific signature analysis can discriminate specific immune-cell types in each patient and thus enhance the efficiency of selective hla-peptidomic approaches. notably, the expressional comparison of cd + t cells relative to immune-suppressive treg cells is extremely crucial to select the high antigenicity of antigens reflecting the therapeutic efficacy. thus, the extreme increase in the number of cd + t cells relative to that of treg cells in patient tnbc# presented a major analytical reason for further in vitro t cell response testing. furthermore, our results indicated a positive correlation between the number of peptides identified via hla-peptidomics and the amount of hla molecules expressed on the surface of cancer cells. it is also possible that the observed difference in the number of eluted peptides might have been influenced by hla-expression levels resulting from active induction of antigen presentation on mhc molecules, which subsequently elicited a strong immune response. thus, these findings suggested that several factors should be considered for successful hla-peptidomic approaches influenced by tcr diversity and elevated expression of hla genes. www.nature.com/scientificreports www.nature.com/scientificreports/ additionally, we showed that integrating hla-peptidomics with imaging-based immunogenicity screening is applicable for the discovery of highly immunogenic ctl epitopes. characterization of antigen-specific cellular immune response is essential to confirm vaccine-related effects specific to a cancer antigen. currently, there are www.nature.com/scientificreports www.nature.com/scientificreports/ only few assays (e.g., the enzyme-linked immunosorbent spot assay) capable of quantifying t cell responses , , and the choice of which assay to use depends on the experimental scale, cost, equipment, reproducibility, and required detection sensitivity. a high-throughput imaging system provides an optimal platform for highly sensitive and quantitative analysis of individual t cells at the single-cell level. moreover, ics-based cytokine detection allows identification of specific cell subpopulations, even when using a small number of cells. the immunopeptidome approach serves massive hla-associated peptides as the collection of cancer epitopes; however, there are obstacles to rapidly determining the optimal set of promising epitopes by testing an enormous pool of peptide candidates in a single measurement. in this study, the hla-peptidomics approach combined with comprehensive analysis of immune-specific signatures and tcr repertories showed high selectivity to determine the immunogenic t-cell epitopes. sequentially, the high-content imaging system allowed high-resolution analysis for t cell reactivity. despite the need for discovery of tumor-derived antigens for effective cancer vaccine development, selection of antigens that elicit robust immune response remains challenging. here, we report a smart strategy for streamlined selection of cancer antigens in vaccine development. through integrative multi-omics and high-content cell imaging, we identified highly immunogenic epitopes from patients with tnbc. identification of potential vaccine epitopes coupled with immune-specific signature analysis, hla-peptidomics, and single-cell-based immunogenicity testing offers a discriminative and powerful tool for cancer-vaccine development. analysis of high-throughput sequencing. dna and rna were simultaneously extracted from cryo-pulverized tnbc tissue powder using an allprep kit (qiagen, hilden, germany), and libraries for whole-exome sequencing and total rna sequencing, respectively, were prepared using truseq library prep kits (illumina, san diego, ca, usa). the libraries were sequenced using the hiseq platform (illumina), and raw data were mapped onto hg using the bwa mem algorithm (http://bio-bwa.sourceforge.net/). variant calling was performed using the genome analysis toolkit (https://software.broadinstitute.org/gatk/) as previously described . a custom proteogenomic search database was generated for the variants using the proteomics tool quilts (http://www.fenyolab.org/tools/tools.html). for wts data, contaminating adapters and low-quality bases were removed with trimmomatic , and the trimmed data were mapped onto hg using star (version . . a) . gene expression, including that of hla genes, was calculated by rsem (version . . ) , and hla presentation on multiple cancer cells was evaluated using the expression atlas (https://www.proteinatlas.org/humanproteome/ tissue/cancer) . for identification and quantification of tils from wts data, we used mixcr (v. . . ; https:// mixcr.readthedocs.io/en/master/) with the alignment parameter -p rna-seq. . hla typing at -digit resolution was performed using the hlascan method and wes data (synthekabio, korea). purification of hla-class i peptides. the lc-ms/ms analysis was performed as previously described . hla-class i peptides were purified from tnbc tissues, and frozen tissues were pulverized with a cp cry-oprep automated dry pulverizer (covaris, woburn, ma, usa), followed by incubation at °c for h with lysis buffer containing . % sodium deoxycholate, . mm iodoacetamide, mm edta, mm pmsf, % octyl-β-d-glucopyranoside (sigma-aldrich, st. louis, mo, usa), and a protease-inhibitor cocktail (roche, mannheim, germany) in phosphate-buffered saline (pbs). the lysates were cleared by centrifugation for min at , g and °c. hla-class i molecules were purified using the w / monoclonal antibody bound to amino-link beads (thermo scientific, waltham, ma, usa), as previously described . the anti-hla-class i antibody was purchased from abcam (cambridge, uk). hla-peptide complexes were eluted from the affinity column using five column volumes of . n acetic acid. the eluted hla-class i proteins and the released peptides were loaded on sep-pak tc columns (waters, milford, ma, usa), and the peptide fraction was eluted with % acetonitrile in . % trifluoroacetic acid, followed by drying by vacuum centrifugation. the lc-ms/ms analysis was performed as previously described . dried peptide samples were reconstituted in µl of . % formic acid, and an aliquot containing ~ μl was injected from a cooled ( °c) autosampler into a reversed-phase magic c aq column ( cm × μm (packed in-house); michrom bioresources, auburn, ca, usa) on an eksigent nanolc d system at a flow rate of nl/min. prior to use, the column was equilibrated with % buffer a ( . % formic acid in water) and % scientific reports | ( ) : | https://doi.org/ . /s - - -z www.nature.com/scientificreports www.nature.com/scientificreports/ buffer b ( . % formic acid in acetonitrile). the peptides were eluted with a linear gradient from % to % buffer b over min and % to % buffer b over min, followed by an organic wash and aqueous re-equilibration at a flow rate of nl/min, with a total run time of min. the high-performance liquid chromatography system was coupled to an ltq-orbitrap xl mass spectrometer (thermo fisher scientific, bremen, germany) operated in data-dependent acquisition mode. full scans (m/z - ) were acquired at a resolution of , using an automatic gain-control (agc) target value of e and a maximum ion-injection time of ms. tandem mass spectra were generated for up to precursors by collision-induced dissociation in the ion-trap using a normalized collision energy of %. the dynamic exclusion was set to s, and fragment ions were detected at a normal scan mode using an agc target value of e and a maximum ion-injection time of ms. source ionization parameters were as follows: spray voltage, . kv; and capillary temperature, °c. data analysis of the hla-class i peptidome. ms data were analyzed using maxquant software (v. . . . ) against the uniprot database (april , ; https://www.uniprot.org/), and personalized human database from patient-derived wes data. n-terminal acetylation and methionine oxidation were set as variable modifications, enzyme specificity was set as "unspecific, " and fdrs for peptides and proteins were set at . and , respectively. possible sequence matches were restricted to eight to amino acids, a maximum peptide mass of da, and a maximum charge state of three. main search peptide tolerance was set at , and the box for "use ms centroids" was checked. hits to the reverse database and contaminants were removed from the "peptide. txt" output file produced by maxquant. peptides were subjected to hla-class i binding and immunogenicity prediction analyses using netmhc (http://www.cbs.dtu.dk/services/netmhc/) and the mhc i immunogenicity portion of the iedb analysis resource (http://tools.iedb.org/immunogenicity/), respectively. the logo-plots were constructed using a seq. logo method . pbmcs isolated from three hla-a* : -positive healthy donors. t cell responses to treatment with individual peptides were monitored after days of in vitro culture, as previously described . briefly, pbmcs were cultured in rpmi- supplemented with l-glutamine, non-essential amino acids, hepes, β-mercaptoethanol, sodium pyruvate, penicillin/streptomycin (gibco; thermo fisher scientific), and % human ab serum (gibco), with a total of × pbmcs used in each well. for antigen-specific t cell expansion, individual peptides ( µg/ml) were incubated with pbmcs in the presence of interleukin (il)- ( ng/ml; peprotech, rocky hill, nj, usa) for days. each peptide with > % purity was synthesized by synpeptide (shanghai, china). for non-specific t cell expansion, t cells were stimulated using a t cell activation/expansion kit (miltenyi biotec, bergisch gladbach, germany), and cells were cultured every days by replacing the medium with fresh half-medium containing il- ( ng/ml) and il- ( ng/ml; peprotech). on day , for ex vivo intracellular ifnγ detection, each peptide ( µg/ml) or phytohemagglutinin (pma) ( ng/ml; sigma-aldrich) plus ionomycin ( µg/ml; sigma-aldrich) for the positive control was administered in the presence of brefeldin a ( : ; biolegend, san diego, ca, usa) for overnight incubation according to the ics protocol. after intracellular fixation using bd cytofix/cytoperm fixation/permeabilization kit (bd biosciences, san jose, ca, usa), cells were stained with antibodies against surface markers and ifnγ at °c for either h or overnight. for visualization of ifnγ-producing t cells, a fluorescein isothiocyanate (fitc)-conjugated anti-human cd α antibody (r&d systems, minneapolis, mn, usa), an alexa -conjuated anti-human cd antibody (biolegend), and an allophycocyanin (apc)-conjugated anti-human ifnγ antibody (biolegend) were specifically labeled for cd + t, cd + t, and intracellular ifnγ capture, respectively. cells were then washed with pbs buffer, and high-throughput imaging was performed using the operetta cls high-content analysis system equipped with harmony software (perkinelmer, waltham, ma, usa). to generate the p/mhc tetramer, a biotinylated hla-a* : monomer complexed with each peptide was obtained from immunomax co., ltd. (seoul, korea). peptides with > % purity were synthesized by synpeptide (china), and their sequences are provided in table . to generate an apc-labeled p/mhc complex tetramer, p/mhc complex monomers were tetramerized in the presence of apc-conjugated streptavidin (bd biosciences). t cells were then stained with ng/µl of the p/mhc tetramer in fluorescence-activated cell sorting (facs) buffer (biolegend) for min at room temperature, and after washing, immunofluorescence was detected by facs. t cells were gated according to the populations of cells labeled with the fitc-conjugated anti-human cd antibody (biolegend). neoantigens in cancer immunotherapy vaccines for established cancer: overcoming the challenges posed by immune evasion translating tumor antigens into cancer vaccines antigen-specific vaccines for cancer treatment targeting neoantigens to augment antitumour immunity neoantigens generated by individual mutations and their role in cancer immunity and immunotherapy a unique tumor 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agonist, gla, via inflammatory caspases, il- , and ifn-gamma assessment of antigen-specific cellular immunogenicity using intracellular cytokine staining, elispot, and culture supernatants the genome analysis toolkit: a mapreduce framework for analyzing next-generation dna sequencing data trimmomatic: a flexible trimmer for illumina sequence data star: ultrafast universal rna-seq aligner rsem: accurate transcript quantification from rna-seq data with or without a reference genome expression atlas update-an integrated database of gene and protein expression in humans, animals and plants mixcr: software for comprehensive adaptive immunity profiling comprehensive analysis of human protein n-termini enables assessment of various protein forms soluble plasma hla peptidome as a potential source for cancer biomarkers maxquant enables high peptide identification rates, individualized p.p.b.-range mass accuracies and proteomewide protein quantification seq logo: a method for construction and visualization of amino acid binding motifs and sequence profiles including sequence weighting, pseudo counts and two-sided representation of amino acid enrichment and depletion we would like to express my special appreciation and thank dr. supplementary information is available for this paper at https://doi.org/ . /s - - -z.correspondence and requests for materials should be addressed to k.-c.h., c.l. or m.j.reprints and permissions information is available at www.nature.com/reprints.publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons license, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons license, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this license, visit http://creativecommons.org/licenses/by/ . /. key: cord- -p hhd ed authors: Şahar, esra atalay; can, hüseyin; İz, sultan gülçe; döşkaya, aysu değirmenci; kalantari-dehaghi, mina; deveci, remziye; gürüz, adnan yüksel; döşkaya, mert title: development of a hexavalent recombinant protein vaccine adjuvanted with montanide isa v and determination of its protective efficacy against acute toxoplasmosis date: - - journal: bmc infect dis doi: . /s - - - sha: doc_id: cord_uid: p hhd ed background: toxoplasma gondii is an obligate intracellular parasite that can infect almost all warm-blooded animals, avian species and humans. toxoplasmosis is asymptomatic in healthy individuals, whereas it may lead to death in immune suppressed or deficient patients. a vaccine against t. gondii is required to prevent consequences of the infection. the aim of this study is to generate a multivalent recombinant protein vaccine against t. gondii. methods: previously discovered antigenic proteins of t gondii were evaluated by their expression level in e. coli and by comprehensive bioinformatics analyses to determine antigenic epitopes. based on these analyses, six vaccine candidate proteins were selected to generate a hexavalent recombinant protein vaccine adjuvanted with montanide isa v. humoral and cellular immune responses were determined by flow cytometry and elisa. vaccinated mice were challenged with t. gondii ankara strain tachyzoites. results: in mice vaccinated with hexavalent vaccine, strong total igg (p < . ) and igg a (p < . ) responses were induced compared to controls, the ratio of cd (+) and cd (+) t lymphocytes secreting ifn-γ increased, and significantly higher extracellular ifn-γ secretion was achieved compared to the controls (p < . ). the survival time of the vaccinated mice increased to . ± . days which was significantly higher than controls (p < . ). conclusions: altogether, these results show that the hexavalent vaccine which is developed for the first time against t. gondii induced strong and balanced th and th immune responses as well as conferred significant protection against challenge with lethal toxoplasmosis in murine model. toxoplasma gondii is an obligate intracellular parasite that causes toxoplasmosis in all warm blooded animals and humans. it is reported that one third of the world's population is estimated to be infected with t. gondii. t. gondii usually causes an asymptomatic infection in healthy people, but can be life-threating in immunecompromised patients (organ transplant recipients, acquired immunodeficiency syndrome patients, and cancer patients). congenital toxoplasmosis may cause abortion, neonatal death or foetal abnormalities in the foetus [ ] . farm animals such as sheep, goats, and pigs have also been shown to be susceptible to t. gondii infection. toxoplasmosis infection in farm animals causes significant economic losses as a result of prenatal death, abortion, and neonatal death. moreover, t. gondii is linked to mental disorders and may affect human behaviour, personality, and other phenotypic traits [ ] . cats and other felines are the definitive hosts of t. gondii [ ] . in the life cycle of t. gondii the main infective forms are tissue cysts (containing bradyzoites) and oocysts (containing sporozoites). the infection sources for humans are consumption of vegetables, fruits or water contaminated with faeces of infected cats containing oocysts and raw or undercooked meat contaminated with tissue cysts [ ] . for these reasons, development of a safe and protective vaccine against t. gondii infection that can be used in animals and humans has utmost importance. recombinant protein vaccines are safe and efficient, and have a great potential for prevention or eradication of diseases. one of the most important issues during a recombinant protein vaccine development is the selection of the vaccine candidate antigen(s) [ ] . antigens to be used in vaccine formulations against toxoplasmosis should actively induced strong immune response, produce long-lasting immunity, and be antigenic in each stage of the parasite [ ] . in our study group's previous studies, we used protein microarray containing candidate exon products of t. gondii to screen well-characterized sera from acute and chronic toxoplasmosis human cases and murine model infected orally with oocysts or tissue cysts. during these studies, screening human sera prioritized antigenic proteins and screening these antigens with murine sera prioritized proteins based on their immunogenicity [ ] [ ] [ ] . in this study, we selected six proteins based on their antigenic epitopes using bioinformatics and protein expression level in e. coli. thereafter, we developed a hexavalent recombinant protein vaccine adjuvanted with montanide isa v and administered to a murine model to determine its immunogenicity and protection efficiency against lethal toxoplasmosis. animals - week old female healthy swiss outbred mice were obtained from the bornova veterinary control institute animal production facility and used during the experiments. animals were housed under standard and suitable conditions. specifically rooms had ambient temperature and humidity, adequate light cycle, and diet was specific for each animal type. all animals were checked for humane endpoints every day such as rapid weight loss more than~ % of gross body weight, inability to assess water or food, or loss of skin elasticity indicative of dehydration. in any of these circumstances, we pre-euthanized the animals with ketamine hydrochloride ( mg/kg) and % xylazine ( mg/kg) and then euthanized with cervical dislocation. based on the data obtained from previous studies [ ] [ ] [ ] , proteins were selected based on their immunogenicity in humans and murine sera (table ). in order to determine the vaccine candidate antigens, expression levels of these proteins were analysed using western blot and moreover, a comprehensive bioinformatics analyses was performed. the gene sequences of the different genes were accessible from the toxoplasma genomic resource (http:// www.toxodb.org/toxo/). the plasmids expressing these recombinant proteins were constructed as previously described [ ] [ ] [ ] . thereafter, the plasmids were cloned into chemically competent escherichia coli (e. coli) bl star (de ) plyss cells according to the manufacturer's protocol (invitrogen, usa). e. coli bl star (de ) plyss cells containing different plasmids were incubated at °c with shaking at rpm for - h until the optical density (od ) reached an absorbance of , ng/μl. then, recombinant protein expression was induced with . mm iptg (isopropyl-d-thiogalactopyranoside) and the bacterial cultures were incubated for h, °c with shaking at rpm. next, cell cultures were harvested by centrifugation at ×g for min. the pellets were homogenized with lysis buffer [ . % triton x- , mm tris-cl and . m nacl (ph: . )] and were freeze-thawed times. the homogenates were incubated on a rotator at room temperature for min and then were centrifuged at . ×g for min. the supernatants were incubated with ml ni-nta superflow beads (qiagen, usa) for min with shaking at rpm. at the end of incubation, the suspension was centrifuged at rpm for min and the supernatant was discarded. then, the ni-nta beads were washed with mm tris-cl and . m nacl and mm imidazole (ph: . ) for min with shaking at rpm. thereafter, the suspension was centrifuged at rpm for min and the supernatant was discarded. next, ni-nta beads were incubated with mm tris-cl and . m nacl and . m imidazole (ph: . ) for min with shaking at rpm. finally, the suspension was centrifuged at rpm for min and supernatants were analysed with western blot as described below to determine the expression level of recombinant proteins. the most abundantly expressed recombinant proteins were selected for vaccine development. the protective immune response against toxoplasmosis is conferred by mainly by cellular immune response and through humoral immune response [ , ] . in addition, the expression of recombinant proteins of t. gondii will be performed in e. coli. t. gondii is a eukaryotic obligate intracellular parasite. protein post-translational modifications are common events in most eukaryotes, such as glycosylation. glycosylation of peptide effects protein immunogenicity and major histocompatibility complex (mhc) binding [ ] . thus, apart from the determination of the expression levels, proteins will analysed by bioinformatics tools to determine the presence of antigenic epitopes as well as glycosylation sites. at the end of the bioinformatics analyses, we aimed to select the proteins that have cellular and humoral immune response inducing epitopes with the least glycosylation sites. since t. gondii is an intracellular parasite, immunemediated protection against toxoplasmosis is mainly conferred by interaction between cd + t cells and mhc class i and cd + t cells and mhc class ii [ ] [ ] [ ] [ ] . antigenic sites recognized by cd + t cells and cd + are peptides containing to and amino acids associated with the mhc class i (mhc-i) and mhc-ii molecules, respectively [ ] [ ] [ ] . predicted epitopes of cd + t cells table the plasmids encoding selected t. gondii antigenic proteins toxodb a name plasmid name toxodb a name plasmid name tgme _ _ pa tgme _ _ pb tgme _ _ pb tgme _ _ pc tgme _ _ pc tgme _ _ pd tgme _ _ pd tgme _ _ pe tgme _ _ pe tgme _ _ pf tgme _ _ pf tgme _ _ pg tgme _ _ pg tgme _ _ ph tgme _ _ ph tgme _ _ pa tgme _ _ pa tgme _ _ pb tgme _ _ pb tgme _ _ pc tgme _ _ pc tgme _ _ pd tgme _ _ pd tgme _ _ pe tgme _ _ pe tgme _ _ pf tgme _ _ pf tgme _ _ pg tgme _ _ pg tgme _ _ ph [ ] [ ] [ ] [ ] [ ] [ ] . the potential linear b-cell epitopes of antigenic proteins were also analysed by support vector machine (svm) which has been utilized by combining the tri-peptide similarity and propensity scores (svmtrip; http://sysbio.unl.edu/ svmtrip/prediction.php) [ ] . in many eukaryotic pathogens, proteins may have nand o-linked glycosylation sites through post translational modification which may affect their interaction with their host organisms. protein glycosylation is common in t. gondii as it is in other eukaryotes [ ] . in this study, e. coli was used as the expression system and we aimed to select the vaccine candidate proteins among the recombinant proteins with least glycosylation sites in predicted antigenic epitopes. the potential n-glycosylation and oglycosylation sites of antigenic proteins were analysed using netnglyc . server (http://www.cbs.dtu.dk/services/netnglyc/) netoglyc . server (http://www.cbs. dtu.dk/services/netoglyc/) [ ] . according to protein expression levels and bioinformatics results, proteins were [ph (tgme _ _ ), pa (tgme _ _ ), pe (tgme _ _ ), pd (tgme _ _ ), pe (tgme _ _ ), ph (tgme _ _ )] were selected as vaccine candidate. thereafter, e. coli bl star (de ) plyss cells containing the ph , pa , pe , pd , pe , and ph stocks were inoculated into individual ml lb broth medium supplemented with ampicillin ( μg/ml) and incubated overnight at °c with rpm shaking. next day, the overnight cultures were inoculated into the bioreactor (bioflo , new brunswick, usa) containing . l enrichment medium supplemented with ampicillin ( μg/ml). the dissolved oxygen and ph levels were maintained at - and . ± . with vigorous mixing ( rpm) at °c until od reached . . then, the cell cultures were induced at a final concentration of . mm iptg and incubated for h at °c. the cells were centrifuged at ×g for min and the pellet was resuspended with ml pre-chilled loading buffer ( mm tris-cl, . m nacl, ph . ) and homogenized with a blender for s (waring, usa). then, the homogenized cells were disrupted twice using a microfluidizer processor (microfluidics m- l pneumatic, usa) at a low temperature under internal pressure of , psi and centrifuged at , g for ½ h at °c. the homogenates were centrifuged at , ×g for min at °c. the clarified supernatants were filtered using . μm pore filter (corning, usa). the filtered samples were purified with akta fast protein liquid chromatography (fplc) system, controlled by uni-corn™ software (ge health, usa), using ml hitrap ni + chelating hp column (ge health, usa). approximately ml filtered supernatant was loaded to the hitrap ni + chelating hp column. after binding, the column was washed with buffers containing increasing concentrations of imidazole ( mm, mm, mm). the recombinant proteins (rh , ra , re , rd , re , and rh ) were eluted by raising the imidazole concentration to . m. purity and identity of the purified proteins were analysed by % (sodium dodecyl sulfatepolyacrylamide gel electrophoresis) sds-page and western blot analysis and concentrated with vivaspin (sartorius, germany). the proteins were further purified by fplc on a superdex / - gl [ - , molecular weight cut-off (mwco)] column (ge health, usa) to remove excess endotoxin. the subsequent protein fractions were pooled, concentrated and quantitated by bradford method (pierce, usa). to observe the expression levels, purity and immunoreactivity, proteins were separated by % sodium dodecyl sulfate-polyacrylamide gel (sds-page). the separated proteins were transferred to polyvinylidene difluoride (pvdf) transfer membrane (immobilon-p, millipore, ma), blocked by . % non-fat dry milk for h at room temperature. the membranes were probed with a / dilution of monoclonal anti-polyhistidine antibody (sigma-aldrich, usa) for . h at room temperature. then the membranes were probed with a / dilution of alkaline phosphatase-conjugated goat anti-mouse igg (h + l) antibody (sigma-aldrich, usa) for h at room temperature. the blot was developed with diethanolamine buffer ( % diethanolamine, m hcl ph: . , the amount of endotoxin in the purified vaccine candidate recombinant proteins were determined with limulus ameobocyte lysate (lal) gel-clot test using pyrotell single test vials according to the manufacturer's protocol (cape cod inc., usa). the test sensitivity was . endotoxin units (eu)/ml. e. coli o :h control standard endotoxin (cape cod inc., usa) was used for positive control and lal reagent water was used for negative control. the concentration of endotoxin-depleted, purified recombinant protein samples were calculated with bradford method (pierce, usa) and stored at − °c until use. vaccination and t. gondii challenge infection - weeks old female swiss webster outbred mice were randomly divided into four groups, each group consisting of eleven mice (n: ). mice were vaccinated intraperitoneally (i.p.) twice at weeks intervals. vaccines and control groups as shown in table . the first group was immunized with hexavalent ( antigens) recombinant proteins adjuvanted with montanide isa v (seppic, france) prepared according to the manufacturer's protocols. montanide isa v was used as adjuvant due to its efficiency in inducing both humoral and cellular immune response. three groups were considered as control; one control group was administered only with hexavalent recombinant proteins without montanide isa v adjuvant. the adjuvant control group was inoculated with only montanide isa v [ μl montanide isa v adjuvant + μl phosphate buffered saline (pbs)] and the last control group was inoculated only pbs ( μl pbs). tail bleeds were performed weeks after each vaccination. nine weeks after first vaccination, eight mice from all four groups were challenged intraperitoneally with × tachyzoites of t. gondii local strain called ankara [ ] . thereafter, the infected mice were observed for the symptoms of toxoplasmosis such as loss of fur brightness and appetite and survival times were recorded on a daily basis. detection of total igg and igg subclass antibody response using rec-elisa determination of t. gondii specific total igg, igg and igg a antibodies in vaccinated mice were performed by recombinant enzyme-linked immunosorbent assay (rec-elisa) as described [ , ] . in brief, each well of microplates (nunc, usa) were coated with μl of recombinant proteins solution (containing μg/ml of each rh , ra , re , rd , re and rh in × pbs) and incubated overnight at °c. next day, plates were washed times with μl pbs-t ( . % tween in pbs) and then blocked with % nonfat dry milk containing . % pbs-t for h at room temperature. mice sera were diluted to / with blocking buffer supplemented with e. coli lysate at a final concentration of mg/ml protein to block anti-e. coli antibodies and incubated for min at °c. then, the mouse sera were added to the wells in duplicate and incubated for h at °c with gentle shaking. after three washes with μl pbs-t, the plates were incubated with μl of anti-mouse igg to evaluated cytokine production, three mice from per group were euthanized weeks after the prime vaccination and their spleens were removed. single-cell suspensions of splenocytes were prepared as previously described [ , ] . aliquots of × viable splenocytes in growth medium [ × rpmi supplemented with % fcs (nbcs, hyclone, thermo fisher scientific, usa), mm l-glutamine (gibco, invitrogen,usa) penicillin ( u/ml) and streptomycin ( μg/ml) (sigma-aldrich, usa), . mm non-essential amino acid and thereafter, the cells were permeabilized and labelled with pe conjugated rat anti-mouse ifn-γ (bd biosciences, usa) or pe conjugated rat anti-mouse il- antibodies (bd biosciences, usa) according to the manufacturer's recommendations. antibodies were diluted in perm/wash solution (bd biosciences, usa) for intracellular staining. all antibodies were used at a final concentration of . μg/ cells. t cell populations of cells, gated with cd + positive expression, were analysed to quantify: the percentage of rh , ra , re , rd , re and rh proteins specific cd + t lymphocytes secreting ifn-γ and cd + t lymphocytes that secreted il- and ifn-γ using facs diva software (bd biosciences, usa). all data were obtained on a bd facsaria flow cytometer (facsaria; bd biosciences, usa). data obtained during the experiments were processed using microsoft excel software and prism . program (graphpad, san diego, ca). a two-tailed unpaired t-test with % confidence interval was used to determine the significance between the vaccination groups. kaplan-meier survival curves were constructed to illustrate protection from lethal toxoplasmosis. humoral and cellular immune responses and survival time were expressed as mean ± standard deviation (s.d.). the expression of recombinant proteins were induced with . mm iptg when growing cells reached an absorbance of . ng/μl at od nm. the cell cultures were harvested h after induction, homogenized with lysis buffer and purified by ni-nta beads. then, the recombinant protein expression levels were assessed by western blotting and rh (tgme _ _ ), ra (tgme _ _ ), re (tgme _ _ ), rd (tgme _ _ ), re (tgme _ _ ), and rh (tgme _ _ ) had detectable bands at . kda, . kda, . kda, . kda, . kda, and . kda, respectively. bioinformatic analyses to predict mhc-i, mhc-ii, and b cell epitopes of the recombinant proteins using immune epitope database and analysis resource (iedb) and svmtrip showed that there were , , , , , and predicted mhc-i epitopes (fig. a) ( table ) , , , , , , and predicted mhc-ii epitopes (fig. b) (table ) and , , , , , and predicted b-cell epitopes (fig. c) (table ) in re , rd , rh , re , ra and rh , respectively. the n-glycosylation and o-glycosylation sites of antigenic proteins were predicted using netnglyc . server and netoglyc . server. moreover, n and oglycosylation sites on the predicted mhc-i, mhc-ii, and b-cell epitopes were also analysed ( table ) . the results showed that rh , re , rd , re , ra , and rh have ( . %), ( . %), ( . %), ( . %), ( . %), and ( . %) o-glycosylation sites, respectively (fig. a) ( table ) . regarding n-glycosylation, rd , ra , re and rh proteins have ( . %), ( . %), ( . %), ( . %) sites respectively, while rh and re proteins didn't have n-glycosylation site (fig. b) (table ) . next, we next focused on the potential glycosylation sites inside predicted mhc-i, mhc-ii, and b cell epitopes of re , rd , rh , re , ra , and rh . the results showed that in the mhc-i epitopes, re and rh have only one n-glycosylation site, re , re , rh , and rd have , , and o-glycosylation sites, respectively (table ). in mhc-ii epitopes, re and rh have and one n-glycosylation site, re , rd , rh , re , and rh have , , , , and o-glycosylation sites, respectively (table ) . on the other hand, b-cell epitopes of re , rd , rh , re , and ra have , , , and oglycosylation sites but did not contain any n-linked glycosylation site (table ) . based on the protein expression levels and mhc-i, mhc-ii, and b cell epitope prediction results, rh , ra , re , rd , re , and rh were grown in big batches of lb using a bioreactor, purified in on a hitrap ni + chelating column and then polished on a gel filtration column to remove excess endotoxin. the purity of rh , ra , re , rd , re and rh were assessed by sds-page and western blotting as shown in fig. a and b. the purified rh , ra , re , rd , re and rh had apparent molecular weight of approximately . kda, . kda, . kda, . kda, . kda, and . kda, respectively. some of the proteins gave several bands possibly due to multimerization of recombinant protein, degradation or stalling of protein synthesis in e. coli. to determine total igg, igg and igg a antibodies against rh , ra , re , rd , re , and rh proteins in adjuvanted and control group mice serum samples, rec-elisa was performed. according to the results, total igg response detected at day was significantly higher in sera of mice administered with hexavalent recombinant protein mixture (+) montanide isa v vaccine and the control group administered with only hexavalent recombinant protein mixture compared to the pre-vaccination sera (p < . , ***). in control groups administered with pbs or montanide isa v didn't induce a significant total igg response. significantly high levels of total igg immune response was detected in the hexavalent recombinant protein mixture (+) montanide isa v when compared with the mice administered with hexavalent recombinant protein mixture (p = . , **) (fig. ) . to assess the polarization of igg /igg a which is a preliminary marker of whether vaccine induced the th or th immune response, the igg and igg a response was analysed by rec-elisa. the polarization of igg and igg a response is shown in fig. . hexavalent recombinant protein mixture (+) montanide isa v vaccine induced significantly high levels of igg and igg a at day compared to the controls groups (p < . , **). overall, in mice administered with hexavalent recombinant protein mixture (+) montanide isa v, igg a and igg levels showed a strong and balanced immune response. in control groups, pbs or montanide isa v didn't induce a significant igg or igg a response. (fig. ) . single-cell suspensions of splenocytes obtained from mice administered with hexavalent recombinant protein mixture (+) montanide isa v and controls were stimulated with purified rh , ra , re , rd , re , and rh proteins. concentration of extracellular cytokine il- and ifn-γ were determined using elisa. according to the results, ifn-γ level was significantly higher in mice vaccinated with the hexavalent recombinant protein mixture (+) montanide isa v compared to mice vaccinated with only montanide isa v (p = . ,***) or pbs (p = . ,***) (fig. a) . on the other side, il- level was also significantly high in mice vaccinated with the hexavalent recombinant protein mixture (+) montanide isa v compared to control groups administered with pbs (p = . , *) or hexavalent recombinant protein mixture (p = . , *) (fig. b) . flow cytometry analysis was used to determine the ratio of cd + t lymphocytes secreting ifn-γ and cd + secreting ifn-γ and il- in vaccinated and control groups. protection against intracellular parasite like t. gondii is primarily achieved by cd + t lymphocytes secreting ifn-γ. for this reason, cd + t cell response is important for an ideal vaccine against toxoplasmosis [ , , ] . t cell population has been gated with cd + staining. in mice vaccinated with hexavalent recombinant protein mixture (+) montanide isa v vaccine, the ratio of cd + t lymphocytes secreting ifn-γ increased , . and . times compared to control groups vaccinated with pbs, montanide isa v, and only hexavalent recombinant protein mixture (fig. a) . besides, the ratio of cd + t lymphocytes secreting ifn-γ increased . and . times in mice vaccinated with hexavalent recombinant protein mixture (+) montanide isa v compared to control groups vaccinated with pbs, montanide isa v, and only hexavalent recombinant protein mixture (fig. b) . on the other hand, the ratio of cd + t lymphocytes secreting il- in mice vaccinated with hexavalent recombinant protein mixture (+) montanide isa v increased . , . , and . times compared to control groups vaccinated with pbs, montanide isa v, and only hexavalent recombinant protein mixture (fig. c) . cd + t cells from all experimental groups proliferated to comparable ratios in response to cona (data not shown). protection against lethal toxoplasmosis in mice vaccinated with hexavalent recombinant protein mixture (+) montanide isa v was determined using t. gondii ankara strain tachyzoites. eight from each the results are summarized in fig. . recently, our study group has discovered some t. gondii proteins that can be used as vaccine candidate against toxoplasmosis using in silico and immunoscreening approaches based on protein microarrays [ , ] . we selected the antigens from this screening approach because antigens selected without a screening approach ended with disappointing clinical results such as the malaria vaccine rts,s which is a hybrid protein particle designed in s. the major surface glycoprotein (msg) containing hybrid protein was formulated in a multi-component adjuvant (as ) and showed % protection in east african children in [ ] . for these reasons, development of a multivalant recombinant protein vaccine using some of these discovered proteins was the aim of this study. for this purpose, we used the data from protein microarray screening and prioritized proteins based on their immunogenicity. to further analyse these proteins for their availability for vaccine development against toxoplasmosis, we conducted small scale protein expression experiments in conjunction with bioinformatics analyses. according to protein expression levels proteins were suitable to be used in the vaccine development. among them, rh (tgme _ _ ) is a dnak family protein, ra (tgme _ _ ) is rop , re (tgme _ _ ) is a sag-related sequence srs a, rd (tgme _ _ ) is an fig. extracellular a ifn-γ and b il- levels elicited by hexavalent recombinant protein mixture (+) montanide isa v and control groups. the red bars represent ifn-γ response and the green bars, il- response. each bar represents the mean ± sd value of ifn-γ and il- responses of mice from each group. each bar represents the mean ± sd value of ifn-γ and il- responses of mice from each group. single cell suspensions were stimulated with rh , ra , re , rd , re , and rh recombinant proteins with a final concentration of μg/ml. in figure, *** represent p ≤ . and * represent p ≤ . ubiquitin carboxyl-terminal hydrolase, re (tgme _ _ ) is a hypothetical protein, and rh (tgme _ _ ) is a plectin. the protection against t. gondii infection is dependent on cd + t-cytotoxic lymphocytes which play a significant role in cell mediated protection as well as b cells which is important in humoral immune response [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . for this reason, mhc-i, mhc-ii, and b-cell epitopes of the vaccine candidate proteins were predicted by bioinformatics. svmtrip online service was used to analyse the b-cell epitopes of re , rd , rh , re , ra , and rh proteins. as shown in fig. c , the presence of b-cell epitopes on the vaccine candidate proteins suggests that they have a strong potential to act as a bcell antigen. we also used the online service iedb to analyse mhc-i and mhc-ii epitopes of re , rd , rh , re , ra , and rh proteins and found mhc-i and mhc-ii epitopes on the vaccine candidate proteins ( fig. a and b) . protein glycosylation has great importance in terms of protein stability, three-dimensional structure, surface expression, activity, and antigenicity [ ] . previously, it was shown that t. gondii contains n-linked glycosylated proteins and o-linked glycosylated proteins by lectin-probed western blot analysis. moreover, it is reported that n and o-linked glycosylated proteins are found throughout the secretory pathway of the t. gondii and n-linked glycosylation of proteins is essential for the survival of parasite [ ] . for this reason, nand o-linked glycosylation sites of antigenic proteins, which may be candidates for vaccination, was predicted by bioinformatics. the results suggest that the vaccine candidate proteins contain n-linked glycosylation sites except for the rh and re proteins (fig. b) . o-linked glycosylation sites are found on re , rd , rh , re , ra , and rh proteins (fig. a) . at this stage, we further examined the n-and olinked glycosylation sites in b cell, mhc-i, and mhc-ii epitopes of the vaccine candidate antigens by bioinformatics. the results demonstrate that b-cell epitopes of vaccine candidate proteins were not containing n-linked glycosylation sites. b-cell epitopes of re , rd , rh , re , ra were o-glycosylated ( table ). the n-linked glycosylation site in the mhc-i epitopes were only found in the re and rh proteins and the o-linked glycosylation is detected in the re , re , rh , and rd proteins ( table ). the n-linked glycosylation site in the mhc-ii epitopes were similarly found in the re and rh proteins and the o-linked glycosylation is detected in the re , rd , rh , re , and rh proteins (table ). these results show that the vaccine candidate proteins and their epitopes are glycosylated at various ratios and their antigenicity is high. thereafter, we developed a hexavalent recombinant protein protein vaccine adjuvanted with montanide isa v which has shown to induce strong cellular and humoral immune response. after vaccination of swiss webster outbred mice, strong total igg, igg and igg a responses were detected in mice administered with hexavalent recombinant protein mixture (+) montanide isa v compared to control groups vaccinated with only montanide isa v or pbs (p < . ) indicating strong and balanced th and th responses (figs. and ) . the production of extracellular ifn-γ was significantly higher in mice vaccinated with the hexavalent recombinant protein mixture (+) montanide isa v compared to mice vaccinated with only montanide isa v or pbs (p < . ) (fig. a) . flow cytometry results were also in compatible with extracellular elisa in which hexavalent recombinant protein mixture (+) montanide isa v showed increment in ratio of cd + and cd + t lymphocytes secreting ifn-γ ( fig. a and b) . cd + t lymphocytes secreting il- were also increased according to flow cytometry results as well as there was an increase in extracellular il- secretion according to elisa (figs. b and c) . flow cytometry results, specifically cd + t lymphocytes secreting il- cell ratio is bigger than cd + t lymphocytes secreting ifn-γ which contradict with elisa results in which ifn-γ was higher than il- secretion. this discrepancy can interpreted as ifn-γ levels detected by elisa can be related to cd + t lymphocytes secreting and macrophages other than cd + t lymphocytes. on the other side, the main protective cells against toxoplasmosis are conferred by cd + t lymphocytes secreting ifn-γ which has increased with recombinant protein mixture (+) montanide isa v compared to controls. the protective efficacy of hexavalent recombinant protein mixture (+) montanide isa v against lethal toxoplasmosis was evaluated by infecting mice intraperitoneally with t. gondii ankara strain tachyzoites. t. gondii ankara strain is africa genotype and causes death in mice in approximately - days [ ] . challenging study showed that survival was prolonged from to days which was observed in control group mice administered with only montanide isa vand pbs to more than days in two mice vaccinated with hexavalent recombinant protein mixture (+) montanide isa v (fig. ) . in this study, montanide isa v was selected as an adjuvant due to its efficiency in inducing both humoral and cellular immune responses. montanide isa v was used as adjuvant in previous studies against bovine herpesvirus , boophilus microplus, foot and mouth disease virus (fmdv), and leishmania major [ ] [ ] [ ] [ ] . in the vaccine trial against fmdv, boophilus microplus, and leishmania major, montanide isa v induced significant levels of protective cytokine production and/ or antibody response. during the vaccine trial with recombinant glycoprotein d of bovine herpesvirus , a mixed th /th response was elicited [ ] . in this study, hexavalent recombinant protein mixture (+) montanide isa v showed strong and balanced th and th responses. overall, the th part of the immune response elicited by hexavalent recombinant protein mixture (+) montanide isa v induced significant levels of cd + and cd + t lymphocytes secreting ifn-γ and conferred significant protection in swiss outbred mice challenged with lethal dose of t. gondii ankara strain tachyzoites. in literature, multivalent recombinant protein vaccines have been developed against toxoplasmosis. in these studies, surface related antigens rsag , rsag , rsag , rsrs , rp , rsrs , and rsrs ; dense granule proteins rgra , rgra , rgra , rgra , rgra , and rgra ; rhoptry proteins rrop , rrop , and rrop as well as rtgpi- , rmag and rbag have been used [ , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . in all of the recombinant protein or dna vaccine studies against toxoplasmosis, murine models are being used to determine the immunity and protection conferred by vaccinations. michima et al., vaccinated mice with a mixture of rsag , rsag , rsag , rsrs , and rp proteins adjuvanted with freund and achieved % survival up to days after i.p. challenging with t. gondii beverley strain bradyzoites [ ] . golkar et al., vaccinated mice with a mixture of rgra and rgra proteins adjuvanted with monophosphoryl lipid a and achieved . % decrease in brain cyst formation after i.p. challenging with t. gondii pru strain cysts [ ] . in one study, rrop , rgra , rgra proteins and cholera toxin were administered through intranasal route to mice and oral challenge with veg cysts resulted in . % decrease in brain cysts compared to controls [ ] . in a study that used antigenic epitopes of sag , gra , and mag proteins adjuvanted with freund decreased the brain cysts formation by % [ ] . dziadek et al., , evaluated rrop , rrop , rgra , and rsag in three vaccine formulations adjuvanted with incomplete freund administered subcutaneously to mice and challenge with t. gondii dx cysts results with to % decrease in parasite burden [ ] . in another study, a synthetic peptide, generated using b-cell and two t-cell epitopes derived from sag , gra , and gra antigens was adjuvanted with freund and challenge with gjs tachyzoites increased survival time [ ] . in other study, mice were vaccinated with the mixture of rrop and rsag adjuvanted with freund and challenged with the t. gondii rh strain. the results showed that t. gondiispecific igg antibodies levels and lymphocyte proliferative responses are increased in vaccine group and conferred more efficient protection compared to the control groups [ ] . sun et al., vaccinated mice with a mixture of rbag , rsrs , and rsrs proteins adjuvanted with freund or recombinant mindin. the results showed that vaccine using mindin as an adjuvant efficiently stimulated humoral and cellular responses, including antigen-specific igg and igg a, as well as lymphocyte proliferation. also the improved protection against t. gondii infection was observed in the mindin adjuvanted vaccine group compared with the other controls [ ] . in another study, t-and b-cells epitopes of ama , ron , and ron proteins was used to develop multivalent peptide vaccine formulations. the iga levels were increased in the mice immunized with single rron , while the igg levels were higher in the mice immunized with rama (+) rron. significant level of ifn-γ was detected in mice immunized with rama (+) rron . infection with t. gondii is naturally occurs through the oral route by water or food contaminated with tissue cysts (containing bradyzoites) or oocysts (containing sporozoites). interestingly, in this study, the mice were challenged orally with × tachyzoites of t. gondii rh strain. it was reported that the control mice died within days. the mice immunized with a + r achieved % survival rates. the mice immunized with ama , a + r + r , and ron achieved , , and % survival rates, respectively [ ] . another vaccine trial using t-and b-cells epitopes of sag , gra , gra , and rop proteins resulted with higher levels of igg and igg a subclass titters, significant production of ifn-γ, percentage of t lymphocyte subsets and longer survival times against intraperitoneal challenge with t. gondii rh strain tachyzoites [ ] . rgra and rbag were used in a multivalent recombinant protein vaccine adjuvanted with alum. significant increase in igg, igg a subclass titters as well as significant increment in ratio of ifn-γ secreting cd + and cd + t lymphocyte subsets were achieved. mice were challenged orally with - t. gondii pru strain tissue cysts and the amount of tissue cysts in vaccinated group decreased . % compared to control groups. in sum, multistage and multivalent rbag and rgra vaccine increased immune response but induced partial protection against toxoplasmosis [ ] . picchio et al., vaccinated mice with rtgpi- , rrop , and rgra proteins ajuvanted with alum intradermally or with cpg-odn intranasally and mice were orally challenged with the t. gondii me tissue cyst. p + r + g vaccine formulations induced significant decreases in the number of cysts per brain compared to the control group. according to the levels of igg and igg a subclasses p + r + g vaccine groups showed a mixed th /th immunity [ ] . overall, in the present study a hexavalent recombinant protein vaccine adjuvanted with montanide isa was first time developed and administered to mice to protect against lethal toxoplamosis. moreover, the immunogenic and protective efficiency of rrop , dnak family protein, srs a, ubiquitin carboxyl-terminal hydrolase, and plectin was first time tested in an animal model. in addition, montanide isa v was first time used as an adjuvant in mice model against toxoplasmosis. apart from these, multiplexing recombinant proteins induced strong and balanced th and th immune responses and improved protection against toxoplasmosis and thus showing the the importance of using multivalant recombinant protein vaccines in future vaccine development studies against ruminants, cats or humans. development of toxoplasma gondii vaccine: a global challenge is toxoplasma gondii infection related to brain and behavior impairments in humans? evidence from a population-representative birth cohort partial protective effect of intranasal immunization with recombinant toxoplasma gondii rhoptry protein against toxoplasmosis in mice current progress toward vaccines against toxoplasma gondii new approaches in vaccine development discovery of new toxoplasma gondii antigenic proteins using a high throughput protein microarray approach screening sera of murine model infected orally with oocysts and tissue cysts identification of potential serodiagnostic and subunit vaccines antigens by antibody profiling of toxoplasmosis cases in turkey identification of toxoplasma gondii 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vaccination with sag and srs evaluation of protective effect of recombinant dense granule antigens gra and gra formulated in monophosphoryl lipid a (mpl) adjuvant against toxoplasma chronic infection in mice toxoplasma gondii: evaluation of an intranasal vaccine using recombinant proteins against brain cyst formation in balb/c mice toxoplasma gondii: chimeric dr fimbriae as a recombinant vaccine against toxoplasmosis evaluation of three recombinant multi-antigenic vaccines composed of surface and secretory antigens of toxoplasma gondii in murine models of experimental toxoplasmosis increased survival time in mice vaccinated with a branched lysine multiple antigenic peptide containing b-and t-cell epitopes from t gondii antigens the virulence-related rhoptry protein (rop ) of toxoplasma gondii is a novel vaccine candidate against toxoplasmosis in mice the extracellular matrix protein mindin as a novel adjuvant elicits stronger immune responses for rbag , rsrs and rsrs antigens of toxoplasma gondii in balb/c mice protective immunity induced by peptides of ama , ron and ron containing t-and b-cell epitopes via an intranasal route against toxoplasmosis in mice toxoplasma gondii: vaccination with a dna vaccine encoding t-and b-cell epitopes of sag , gra , gra and rop elicits protection against acute toxoplasmosis in mice vaccine potential of antigen cocktails composed of recombinant toxoplasma gondii tgpi- , rop and gra proteins against chronic toxoplasmosis in c h mice publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations not applicable. authors' contributions eaŞ, hc, ayg, md conceived the study and participated in its design. eaŞ, hc, sgİ, add, mkd, md participated in in vitro and in vivo studies. eaŞ, hc, rd, ayg, md helped in discussion of results. eaŞ and md performed the statistical analyses. eaŞ, hc, ayg, md interpreted the results and drafted the manuscript. all authors read and approved the final manuscript. this study was supported by the scientific and technological research council of turkey (tubitak) grant s to ayg. the funding bodies played no role in the design of the study and collection, analysis, and interpretation of data and in writing the manuscript. the dataset analyzed during the current study is available from the corresponding author on reasonable request. the animal study was performed under the instructions and approval of the institutional animal care and use committee (iacuc) of ege university for animal ethical norms (permit number: - ). not applicable. the authors declare that they have no competing interests. key: cord- -qhtj pef authors: dash, raju; das, rasel; junaid, md; akash, md forhad chowdhury; islam, ashekul; hosen, sm zahid title: in silico-based vaccine design against ebola virus glycoprotein date: - - journal: adv appl bioinform chem doi: . /aabc.s sha: doc_id: cord_uid: qhtj pef ebola virus (ebov) is one of the lethal viruses, causing more than epidemic outbreaks to date. despite having available molecular knowledge of this virus, no definite vaccine or other remedial agents have been developed yet for the management and avoidance of ebov infections in humans. disclosing this, the present study described an epitope-based peptide vaccine against ebov, using a combination of b-cell and t-cell epitope predictions, followed by molecular docking and molecular dynamics simulation approach. here, protein sequences of all glycoproteins of ebov were collected and examined via in silico methods to determine the most immunogenic protein. from the identified antigenic protein, the peptide region ranging from to and the sequence hkegaffly from the positions of – were considered the most potential b-cell and t-cell epitopes, correspondingly. moreover, this peptide (hkegaffly) interacted with hla-a* : with the highest binding energy and stability, and also a good conservancy of . % with maximum population coverage. the results imply that the designed epitopes could manifest vigorous enduring defensive immunity against ebov. ebola virus (ebov) is an antisense-strand rna virus from the filoviridae family, and it is structurally filamentous. although the initial discovery of ebov was in , till now more than epidemics have been reported from africa, mostly with the zaire species (http://who.int/mediacentre/factsheets/fs /en/). [ ] [ ] [ ] the genome of ebov enciphers the seven structural proteins, ie, nucleoprotein (np), viral structural proteins (vp , vp , vp , and vp ), glycoprotein (gp), and rna-dependent rna polymerase (l). among these, three different versions of glycoprotein are transcribed by the gp gene. [ ] [ ] [ ] [ ] both attachment protein (gp ) and entry/fusion protein (gp ) are expressed from the full length of the gp chain, which are synthesized from messenger rnas (mrnas), containing an additional nontemplated adenosine. the soluble gp (sgp) is synthesized from the unedited rna transcript. on the contrary, small soluble gp (ssgp) is translated during this process by adding two additional adenosine residues. the gps are expressed virally on the virion surface, which plays a crucial role in the catalysis of membrane fusion and amalgamation to host cells. as a result, it is considered not only a crucial component for vaccines but also an essential target for developing inhibitors and antibodies of attachment and fusion. [ ] [ ] [ ] protein sequence retrieval, evaluation analysis, and antigenic protein identification all available sequences of the gp of ebov were extracted from the uniprot database. after that, multiple sequence alignment was performed by using the clustalw tool, and a phylogenetic tree was assembled by mega . software. and then, vaxijen v . was used to predict most efficient antigenic protein from the available protein sequences. top scored eiptope subjected to ns md simulation **rmsf **rmsd **hydrogen bond occupency analysis secquence, having highest vaxijen score prediction of b cell epitope, using-**t cell epitope prediction by proteasomal c terminal cleavage, tap transport efficiency and mhc class binding **epitopes with ic value less than for their binding to mhc class molecule from iedb analysis along with binding to highest number of alleles in both analyses were chosen **epitope conservancy analysis **population coverage analysis **kolaskar and tongaonkar antigenicity scale **emini surface accessibility prediction **karplus and schulz flexibility prediction **bepipred linear epitope prediction **chou and fasman beta turn prediction vaxijen analysis with a threshold score of > . secquence, having highest vaxijen score vaxijen analysis with a threshold score of > . in silico-based vaccine design against ebov gp t-cell epitope identification and conservancy analysis t-cell identification was done using the netctl . server, setting thresholds at . , . , and . for sensitivity and accuracy. mhc-i binding of the identified epitopes and epitope conservancy were then calculated using tools from the immune epitope database (iedb). [ ] [ ] [ ] these tools calculate the half maximal inhibitory concentration (ic ) value of epitope binding to human leukocyte antigen (hla) molecules using the stabilized matrix base method. , the restriction for epitope identification was set to mhc-i supertypes. prior to the run, all the alleles were considered, and the length of the peptides was set at . . the population coverage tool from iedb was applied to determine the population coverage for every single epitope by selecting hla alleles of the corresponding epitope. allergenicity of the predicted epitope was calculated using allerhunter, which can predict both nonallergens and allergens with a high level of accuracy , by comparing the input sequence with the sequence of known allergen. molecular simulation analysis of hla allele interaction design of the three-dimensional structure of epitope and hla protein the three-dimensional structures of all the five epitopes were predicted by a pep-fold web-based server. for each sequence, this server predicted the five most provable structures, the best of which, having the lowest energy model, was chosen for further analysis. to validate the binding of identified epitope and hla molecule, we considered the homology modeling as there is no relevant structure available in the protein data bank. we selected homology modeling using the most popular online protein fold recognition server, phyre , to generate the three-dimensional structure of hla-a* : (accession id: am ). then, modrefiner was used to minimize and correct the hypothetical structure. the validation of the predicted structure was done using procheck, verify d, errat, prove, and qmean. molecular docking analysis was performed using autodock vina, by considering hla molecule as a protein and identified epitopes as ligands. first, we used the protein preparation wizard of ucsf chimera to prepare the hypothetical protein for docking analysis by adding hydrogens and gasteiger-marsili charges. the prepared file was then converted into pdbqt format. the parameters used for the docking simulation were set to default. the size of the grid box in autodock vina was kept at . , . , and . , respectively, for x, y, and z. the energy range was kept at , according to the default setting. autodock vina was implemented via the shell script offered by autodock vina developers. docking results were observed by negative score in kcal/mol, as binding affinity of ligands. binding energy estimation and molecular dynamics (md) simulation the binding free energy of hla-epitope complexes were calculated by using mm (charmm) -generalized born surface area (gbsa) and poisson-boltzmann surface area (pbsa) protocols, implemented in accelrys discovery studio . . using implicit solvent models of gbsa and pbsa, the binding free energy (Δg bind ) for each epitope was calculated by maintaining salt concentration of . m. default value was set for conformational entropy and ligand minimization. the distance cutoff value was set to . Å. the binding energy was calculated by using following equation: the entire dynamics simulation study for the hlaepitope complex was accomplished in yasara dynamics software. prior to simulation, the complex was cleaned and optimized the hydrogen bond network. after that, a cubic simulation cell was created with a periodic boundary condition, and the atoms of the complex were typed using the amber force field. the pka (acid dissociation constant) values of protein titratable amino acids were calculated and solvated the simulation box using the transferable intermolecular potential points (tip p) water model (density: . g/l - ). the system consistent with atoms was energy minimized using the steepest gradient approach ( cycles) followed by simulated annealing method. restrained and unrestrained all-atom molecular dynamics simulation were performed in solvent using the pme method to describe long-range electrostatic interactions at a cut off distance of Å at physiological conditions ( k, ph . , . % nacl). a multiple time step algorithm together with a simulation time step interval of . fs was chosen. molecular dynamics simulations of ns long were performed at constant temperature using a berendsen thermostat and constant submit your manuscript | www.dovepress.com dash et al pressure. the md trajectories were saved every ps for analysis. the trajectories generated from the simulation were analyzed for the stability by various evaluative measures viz. rmsd, rmsf (rms fluctuations), and initial and final protein backbone comparisons using yasara structure built in macros and vmd software. to detect b-cell epitope, various tools from iedb were used to identify the b-cell antigenicity, together with the emini surface accessibility prediction, kolaskar and tongaonkar antigenicity scale, karplus and schulz flexibility prediction, and bepipred linear epitope prediction analysis. since antigenic parts of a protein belong to the beta turn regions, the chou and fasman beta turn prediction tool was also used. a total of gp sequences from the different variants of ebov were collected from the uniprotkb database. multiple sequence alignment analysis was then performed, and a phylogenetic tree (figure ) was constructed thereby. using the unweighted pair-group method with arithmetic mean, a phylogram was constructed using the bootstrap with , replications in mega . from the multiple sequence alignment analysis, it is clearly seen that protein sequences that isolated from various strains were having a close relationship. also, from the multiple comparison result, the selected sequences of ebov of the same subtype have %- % similarity. this result also confers the possibilities of mutation in glycoprotein of all strains, which demonstrates a good agreement with the results from veljkovic et al. antigenic protein prediction protein sequences in this study were considered to screen out using vaxijen web server for the identification of potent antigenic protein. as a corollary, uniprotkb id: q ymg was identified as the most potent antigenic protein having a maximum total prediction score of . . here the threshold of . is considered as the potent antigenicity. this sequence was used for further analysis. on the basis of the high combinatorial score, the five best epitopes were predicted by the netctl server from the selected protein sequence in a preselected environment. the identified epitopes are represented in table . in combination with several methods such as proteasomal cleavage/transporter associated with antigen processing (tap)/mhc-i combined predictor, mhc-i processing of the netctl server calculates an overall score for each peptide's intrinsic potential from a protein for the designing of t-cell epitope. peptides with a higher score represent higher processing capabilities. the five t-cell epitopes were subjected to mhc-i binding prediction, using the stabilized matrix base method. the epitopes that elicited higher affinity (ic < nm) were subjected to afterward analysis (table ) . notably, proteins are transformed into peptides by proteasome complex, which cleaved the peptide bonds. by combining with class i mhc molecules, these peptides were deported to the cell membrane, where they were introduced to t helper cells. as shown in table furthermore, this epitope retained the highest conservancy of . %, according to the iedb conservancy analysis, as tabulated in table . as population coverage in vaccine design generally plays a crucial role, it was calculated in this study. the cumulative percentage of population coverage was obtained for the predicted epitope hkegaffly. as shown in table , the population coverage for east africa was found to be . %; in west and north africa, it was . % and . %, respectively; and for central africa it was observed to be . %. the population coverage was recorded at . % for the east asian region, which was a major hotspot for viral infection. for north america, the population coverage was found to be . %. in current vaccine design pipeline, allergenicity is considered the most prominent barrier in vaccine designing, since most vaccines convert the immune system into an "allergic" reaction by inducting type t helper cells and immunoglobulin e. that is why we predicted allergenicity of the selected epitope by the allerhunter web server, where the probability is > . . the epitope hkegaffly was scored . (sensitivity = . %, specificity = . %), and was thus considered a nonallergen, according to the food and agriculture organization/world health organization evaluation system of allergenicity prediction. dash et al protein model having > % of the residues in the core and allowed regions can be considered a high-quality model. the hypothetical model was further analyzed using errat and verify d. for a good model, structure should retain an errat score > . , against which the model in this study obtained an errat score of . . verify d graph indicates that . % of residues of this model had an averaged d- d score of . , which is good. along with the qmean analysis, the protein model in our interest resulted in a z-score of − . , and the total score was . . this value denotes a higher quality of the model, where the acceptable score ranges between and ( figure b ). on the basis of the results obtained from the aforementioned structural validation programs, the model ( figure c ) showed much reliability and was considered for further study. molecular docking simulation revealed that the proposed epitopes bound in the cleft of the hla-a* : ( figure s ), where the highest binding affinity was − . kcal/mol (table s , observed for the hkegaffly epitope). the chimera program was used to visualize the interactions of docked hla-a-epitope complexes, as shown in figures and s . then, binding energy calculation was carried out to understand the binding of hla with epitopes. here the binding free energies of mm-gbsa and mm-pbsa are approximate free energies of binding, so a more negative value denotes stronger binding. from mm-gbsa analysis, the highest binding free energy was observed for hla-a* : with epitope (hkegaffly) of - . kj/mol (table s ). on the contrary, the lowest binding free energy was obtained for atedpssgy epitope, i.e. - . kj/mol. in contrast of mm-gbsa, the hkegaffly epitope was also resulted the highest binding energy of - . kj/mol, while the lowest binding free energy was seen for tedpss-gyy epitope, - . kj/mol. since hkegaffly epitope obtained the highest docking affinity and binding free energy, its complex subjected for molecular dynamics simulation. table interaction, binding, and conservancy of identified t-cell epitopes validation of predicted t-cell epitope as described in the "materials and methods" section, the hypothetical structure of hla-a* : protein was generated using the homology technique. the structure was then analyzed through various web-based protein validation software. as shown in figure , the ramachandran plot generated by the procheck server showed that about . % of the residues of protein are located in the most favored region, as against % in the outlier region and . % in the generously allowed region. it should be noted that the and remained stable in the range from . Å to Å. in case of epitope, similar rmsd pattern was observed, where the order of magnitude was seen to fluctuate in some range. the average energy of the simulation was - . kj/mol; the average coulombic charge and van der waals interactions was - . kj/mol, . kj/mol, respectively. we also calculated the contribution of each residue for both hla and epitope in the simulation, in terms of rmsf and rmsd. as seen in figure a , highest rmsd was observed the ns md simulation of hla-epitope (hla-a* : -hkegaffly) complex was carried out using amber force field, following the energy minimization protocol. the stability of the hla-epitope complex by means of rmsd was calculated and rendered in figure a . from the results, it is revealed that the hla molecule was stabilized after ns simulation and tended to remain in plateau phase thereafter for rest of the period. the rmsd value of hla was observed to grow up quickly from . for arg residue at the position of in hla, while lowest rmsd observed for cys . however, this residue was also resulted highest rmsf value of . Å, while the rests of the residues were in lowest fluctuation. in case of epitope, the histidine residue at the first position and the tyrosine residue in th position were seen to be very much flexible, as these residues were resulted with highest rmsd and rmsf ( figure b ). in the meanwhile, we calculated the number of hydrogen bond formed between the epitope and hla molecule during the simulation. the results represented in figure b , showed that hydrogen bond at initial stage was , and the range decreased to . during the simulation, the number of hydrogen bond was at a range of - , in silico-based vaccine design against ebov gp potentiality to express the b-cell response. furthermore, the surface accessibility of the protein was also analyzed using the emini surface accessibility prediction methods, since a potent b-cell epitope should be accessible through the surface. as shown in figure s and table , higher accessibility was found in regions - and - amino acid residues. figure s represents the β-turns region identified by chou and fasman β-turn methods. according to the result, the region from to (in the region of - and - ) is regarded as β-turns as well as hydrophilic in nature. these are two properties required to be a potent b-cell epitope. experimentally, antigenicity is related to the protein flexibility. that is why we implemented the karplus and schulz flexibility prediction method, where it was evident that the regions of - and - were regarded as the most flexible ( figure s ). finally, based on the hidden markov model, the bepipred linear epitope prediction tool was utilized to predict linear b-cell epitopes. the predicted result is rendered and tabulated in figure s and table . hence, by comparing the foregoing results, the peptide sequences ranging from to are which indicates the strong binding of epitope-hla complex. hence, all analyses lead to the conclusion that hkegaffly is one of the most prominent t-cell epitopes for gp based designing of vaccine. for the identification of potential b-cell epitopes, amino acid scale base methods have been used in this study. consistent with this protocol, we used diverse investigation processes for the calculation of an incessant b-cell epitope. according to the analysis of kolaskar and tongaonkar's antigenicity prediction method, the average antigenicity was . , while . and . were the maximum and minimum, respectively. the kolaskar and tongaonkar antigenicity prediction uses a semiempirical method to predict antigenicity on the basis of physicochemical properties of the residues in a protein and their diversity in experimentally known epitopes, where values > . were considered to denote a potential antigen. as summarized in table and figure s [ ] [ ] [ ] [ ] [ ] however, the information representing the population coverage in the worldwide are still limited. in such case, computational based epitiope screening is very much efficient in context of hla class i molecules, and also much safe, high specificity and cost effective. therefore, this study incorporated various immunoinformatics and molecular modelling tools to identify potential epitopes present in ebov gps. initially, a set of glycoprotein sequences from the different strains of ebov has been subjected to perform multiple sequence alignment. previous gp sequences analysis of different strains of each ebov species revealed a high degree of sequence similarity, , and thereby, it is believed that targeting gp from old strain could provide strong and cross reactive immunity against the new strain and previous outbreaks in . interestingly, in our molecular analysis, we have found ~ - % conservation for the amino acid sequences of different strains within the species, which confers the degree of f lss ngvatdvpsatkrwgfrsgvppkvvnyeagewae kkpdgseclpaapdgirg vsgtgpca tf kdf plrepvnatedpssgyys tgfgtne ytsgkrsntt peidtti tgilqlpr tsfflwviilfqrtfsiplgvihnstlqvsdvdklvcrdk lrsvgln atdvpsa rsgvppkvvny gseclpaap fprcryvhkvsgtgpcagdfafh egafflydrlastviyr aegvvaflilpqa fsshplrep sgyysttiryq lfevdnltyvqlesr tpqfllqlnet iwkvnpe able to provoke the immune response as b-cell epitope for gp-based designing of vaccine. in recent trends, the primary focus of vaccine development is very much rely on gps, as they are involved in cell attachment, fusion and entry as well as assist in invasion; and thus plays the role of pathogenesis of disease. the central role in silico-based vaccine design against ebov gp similarity and support the previous analysis. from this set of gps, the most antigenic protein sequence was determined by vaxijen server. based on auto cross covariance (acc), the vaxijen server transform the protein sequence into uniform vectors of physicochemical properties of proteins. with % sensitive, % accuracy and specificity, the l -cv (leave one -out cross validation) was used to identify antigenicity of protein for viral species. the resultant antigenic protein (vaxijen score ≥ . ) was then subjected for various immunoinformatics analysis, followed by iedb web server. at the beginning five potent -mer epitopes have been predicted from netctl . server and selected for further study. using the threshold of . , the netctl . server predicts maximum number of epitopes without compromising the specificity or sensitivity levels, covering all mhc class i supertypes. the five most potent epitopes are represented in table , and the scores are the predicted mhc class i affinities in the form of -logic and ic value. for mhc-i binding prediction, peptides with ic values < nm are considered high affinity, < nm for intermediate affinity, and < nm for low affinity. therefore, we selected maximum alleles having binding affinity < nm. it is advocated that t-cell epitope binding to specific multiple hla supertypes are termed as promiscuous in vaccine design, since they effectively increase the coverage of higher proportions of human populations. , according to the results, both hkegaffly and lfevdnlty bind to the highest number of alleles. however, hkegaffly represents highest conservancy and was hence considered as epitope of choice. we also validated each epitope by molecular docking simulation and mm-gbsa/mm-pbsa studies with hla-a* : protein, as it was found common in the results from mhc-i binding interaction analysis. prior of docking simulation, the three dimensional structure of hla molecule was prepared by using the phyre , followed by intensive mood. as a result, residues ( %) of hla-a* : modelled at > % accuracy. in these study, bck_chain a, crystal structure of hla-a* showed the highest similarity of % ( figure s ). the selected model has been chosen from the twenty models generated by phyre , on the basis of similarity and confidence level. phyre is one of the best protein prediction servers that allows remote fold reorganization and homology detection. using hidden markov model (hmm), this server predicts the structure of given protein sequence by constructing backbone, loop modelling and adding side chains. however in intensive mode, additional ab initio approach is used for reconstruction of missing region, backbone and side chain. in docking simulation, among the other epitopes, hkegaffly obtained highest binding affinity (table s , supplementary material). in addition, mm-pbsa and mm-gbsa techniques are frequently to re-rank docking poses from molecular docking study, as they achieve a much better performance than docking scoring functions. nevertheless, the success rate of the absolute binding free energy prediction strongly depends on the systems. thence, we used both of these solvation models to predict binding energy more accurately, where the results from mm-pbsa examine the accuracy and reliability of the results from mm-gbsa. results of binding energy calculation are shown in table s . the relative magnitude of binding free energy obtained from gb methods is found to be consistent with those calculated using pb method, despite of the differences in the absolute value of salvation energy. as a corollary, these results also demonstrated the consistence with relative stabilities of hla-epitope complexes. in previous published reports regarding in silico epitope identification of ebov, [ ] [ ] [ ] [ ] the studies are limited to sequence-based scoring function techniques and some extend to docking simulation. these techniques have certain limitations, though these are very useful. in docking simulation of peptide and protein, it faces problem like peptide's flexibility. , whereas, energy based approach like molecular mechanics and interaction energy scoring can add valuable information to sequence based results. , therefore, we have performed md simulation study of ns long to enhance the predictive power of the peptide affinity calculations to mhc molecule. in molecular dynamics simulation, both epitope and hla protein were seen to achieve equilibration, while different fluctuations of rmsd were seen by the time evolution. higher rmsd values of epitope indicate the flexibility in binding with hla molecule, during the simulation. these results were further confirmed by the analysis of per residue contributions in dynamics simulation by means of rmsf and rmsd. low values of rmsf indicate the core region of the hla protein was stable, while high values of rmsd demonstrated the motion of the protein during the simulation. in like manner, the rmsd and rmsf profiles of eptiope confirm the synergic conformation changes to accommodate the binding pocket of hla. the hydrogen bond occupancy analysis between the hlaepitope further confirmed the stability of the complex during the simulation. overall, these results evidently demonstrate that both hla and epitope have remarkable conformation changes to facilitate the binding and formed stable complex in thermodynamic environment (figure ). submit your manuscript | www.dovepress.com dash et al it is one of important factors in vaccine design that the distribution of hla varies according to the diverse ethnic groups and geographic regions around the world. therefore, wide range of population coverage must be considered during the designing of an effective design. according to the results from population coverage analysis, the epitope hkegaffly showed wide range of population coverage in different regions of the world (table ) , where the highest coverage was observed central africa; one of the most ebov infected areas. this result indicates that it will specifically bind with the prevalent hla molecules in the target population, where the vaccine will be employed. in other aspects, the b-cell epitope stimulates minimal immune unity, which is very much strong enough to elicit a potent humoral immune response, causing no harmful side effects to human body. thereby, we are also calculated and found that the sequences ranging from - as a b-cell epitope, by taking consideration of amino acid property, hydrophilicity, accessibility, flexibility, turns, exposed surface, polarity and antigenic propensity. this study could provide a solid base for vaccine design. in recent years, most vaccines have been developed based on b-cell immunity; however, the current strategy relies mostly on t-cell epitope owing to long-lasting immunity. both b-cell and t-cell epitopes are offered in this study for stimulating immunity in several ways. the resulting peptides showed b-cell and t-cell selectivity, better conservancy, population coverage, and significant interaction with mhc- allele with good affinity. above all, the predicted epitopes are anticipated to offer long-term and high protective immunity against ebov. the authors report no conflicts of interest in this work. computational biophysics; chemoinformatics and drug design; in silico adme/tox prediction. the manuscript management system is completely online and includes a very quick and fair peer-review system, which is all easy to use. visit http://www.dovepress.com/testimonials. php to read real quotes from published authors. dash et al ebola, marburg and disease ebola viral disease: what should be done to combat the epidemic in ? treatment of ebola virus disease ebola viral disease outbreak -west africa genome structure of ebola virus subtype reston: differences among ebola subtypes genomic rna editing and its impact on ebola virus adaptation during serial passages in cell culture and infection of guinea pigs ebola virus rna editing depends on the primary editing site sequence and an upstream secondary structure deep sequencing identifies noncanonical editing of ebola and marburg virus rnas in infected cells ebolavirus delta-peptide immunoadhesins inhibit marburgvirus and ebolavirus cell entry the multiple roles of sgp in ebola pathogenesis ebolavirus glycoprotein structure and mechanism of entry structure of the ebola virus glycoprotein bound to an antibody from a human survivor prediction and identification of mouse cytotoxic t lymphocyte epitopes in ebola virus glycoproteins a highly conserved wdypkcdra epitope in the rna directed rna polymerase of human coronaviruses can be used as epitope-based universal vaccine design more than one reason to rethink the use of peptides in vaccine design immunogenicity and safety of a novel therapeutic hepatitis c virus (hcv) peptide vaccine: a randomized, placebo controlled trial for dose optimization in healthy subjects conserved epitopes of influenza a virus inducing protective immunity and their prospects for universal vaccine development approaching rational epitope vaccine design for hepatitis c virus with meta-server and multivalent scaffolding construction and immunological evaluation of multivalent hepatitis b virus (hbv) core virus-like particles carrying hbv and hcv epitopes in silico-based vaccine design against ebov gp uniprot: the universal protein knowledgebase clustal w and clustal x version . vaxijen: a server for prediction of protective antigens, tumour antigens and subunit vaccines large-scale validation of methods for cytotoxic t-lymphocyte epitope prediction development of an epitope conservancy analysis tool to facilitate the design of epitope-based diagnostics and vaccines sensitive quantitative predictions of peptide-mhc binding by a 'query by committee' artificial neural network approach generating quantitative models describing the sequence specificity of biological processes with the stabilized matrix method modeling the mhc class i pathway by combining predictions of proteasomal cleavage, tap transport and mhc class i binding allerhunter: a svm-pairwise system for assessment of allergenicity and allergic cross-reactivity in proteins combining pairwise sequence similarity and support vector machines for detecting remote protein evolutionary and structural relationships pep-fold: an updated de novo structure prediction server for both linear and disulfide bonded cyclic peptides protein structure prediction on the web: a case study using the phyre server identification and characterization of three novel hla alleles, hla-a* , hla-a* and hla-dqb * improving the physical realism and structural accuracy of protein models by a two-step atomic-level energy minimization aqua and procheck-nmr: programs for checking the quality of protein structures solved by nmr verify d: assessment of protein models with three-dimensional profiles verification of protein structures: patterns of nonbonded atomic interactions deviations from standard atomic volumes as a quality measure for protein crystal structures qmean: a comprehensive scoring function for model quality assessment autodock vina: improving the speed and accuracy of docking with a new scoring function, efficient optimization, and multithreading ucsf chimera--a visualization system for exploratory research and analysis rotamer libraries in the st century charmm general force field (cgenff): a force field for drug-like molecules compatible with the charmm all-atom additive biological force fields assignment of protonation states in proteins and ligands: combining pka prediction with hydrogen bonding network optimization lipid : the amber lipid force field fast empirical pka prediction by ewald summation new ways to boost molecular dynamics simulations induction of hepatitis a virus-neutralizing antibody by a virus-specific synthetic peptide a semi-empirical method for prediction of antigenic determinants on protein antigens prediction of chain flexibility in proteins improved method for predicting linear b-cell epitopes structural evidence for induced fit as a mechanism for antibody-antigen recognition empirical predictions of protein conformation mega : molecular evolutionary genetics analysis version . in silico analysis suggests interaction between ebola virus and the extracellular matrix identification of novel potential vaccine candidates against tuberculosis based on reverse vaccinology vaccination and allergic disease: a birth cohort study computational analysis and binding site identification of type iii secretion system atpase from pseudomonas aeruginosa a method to identify protein sequences that fold into a known three-dimensional structure rational design, synthesis, and biological evaluation of -azaindole derivatives as potent focused multi-targeted kinase inhibitors turns in peptides and proteins antigenic determinants in proteins coincide with surface regions accessible to large probes (antibody domains) efficacy and effectiveness of an rvsv-vectored vaccine expressing ebola surface glycoprotein: interim results from the guinea ring vaccination clusterrandomised trial an adenovirus vaccine expressing ebola virus variant makona glycoprotein is efficacious in guinea pigs and nonhuman primates potent neutralizing monoclonal antibodies against ebola virus infection mechanism of binding to ebola virus glycoprotein by the zmapp, zmab, and mb- cocktail antibodies safety and immunogenicity of novel 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is assisted by bound water molecules key: cord- -pkm fq authors: deseke, malte; prinz, immo title: ligand recognition by the γδ tcr and discrimination between homeostasis and stress conditions date: - - journal: cell mol immunol doi: . /s - - -y sha: doc_id: cord_uid: pkm fq t lymphocytes comprise cells expressing either an αβ or a γδ tcr. the riddle how αβ tcrs are triggered by specific peptides presented in the context of mhc was elucidated some time ago. in contrast, the mechanisms that underlie antigen recognition by γδ tcrs are still baffling the scientific community. it is clear that activation of γδ tcrs does not necessarily depend on mhc antigen presentation. to date, diverse and largely host-cell-derived molecules have been identified as cognate antigens for the γδ tcr. however, for most γδ tcrs, the activating ligand is still unknown and many open questions with regard to physiological relevance and generalizable concepts remain. especially the question of how γδ t cells can distinguish homeostatic from stress conditions via their tcr remains largely unresolved. recent discoveries in the field might have paved the way towards a better understanding of antigen recognition by the γδ tcr and have made it conceivable to revise the current knowledge and contextualize the new findings. t cells are divided into αβ and γδ t cells based on the expression of their respective t-cell receptor (tcr). with a frequency of . - % of all t cells in human peripheral blood γδ t cells are the smaller subset although higher abundances can be observed in peripheral tissues. , nevertheless, this enigmatic immune cell subset is conserved among almost all jawed vertebrates, which indicates their importance for the immune system. they are able to recognize a plethora of infections such as by mycobacteria, plasmodium, , or cytomegalovirus (cmv) and induce potent infection containing reactions, such as granzyme and cytokine release. , besides, γδ t cells were also shown to be effective in inducing antitumor responses. [ ] [ ] [ ] besides the different immune receptors expressed by γδ t cells like nk cell receptors and ncrs, their tcr is one of the main cellsurface molecules that is involved in the recognition of pathological conditions. this assumption is supported by blocking experiments , and by the clonal expansion of specific γδ tcrs upon infections. , like its αβ counterpart, the γδ tcr is composed of two chains, named gamma and delta, whose diversity is generated by the recombination of variable (v), diversity (d, only in δ-chain) and joining (j) fragments. with some exceptions in sharks, somatic hypermutation as in immunoglobulin genes does not occur in tcrs so that the recombined region, which makes up the complementarity defining region (cdr ), comprises most of the receptor's diversity. the recognition of antigens, however, seems to be entirely different when compared between αβ and γδ tcrs. most αβ tcrs bind to major histocompatibility complexes (mhc) i or ii presenting small peptide fragments derived from pathogens or tumor specific proteins. together with co-receptor engagement of cd or cd and co-stimulation through cd , this elicits t-cell activation. in contrast, the γδ tcr does not require mhc-mediated antigen presentation and no general requirement for co-receptor interaction has been identified so far. this has led to the notion that the mere binding of the γδ tcr to its cognate antigen is sufficient for activation. moreover, γδ tcrs have the ability for both innate and adaptive ligand recognition via either germline-encoded regions of the receptor, reminiscent of prrs or adaptive antigen binding via the cdrs. although central for understanding γδ t-cell biology and their application in therapeutic approaches, the identification of γδ tcr antigens has been proven challenging for several reasons. first, since no general restricting molecule could be identified for the γδ tcr, the antigens could be virtually any molecule present on cell surfaces or in the surrounding extracellular space. this becomes particularly problematic if not only proteins, which are already very diverse, but also carbohydrates, lipids and nucleic acids could be recognized or at least involved in the recognition because this further increases the complexity of the question and is technically demanding to address. second, the affinity of tcrs to their antigens is typically low being mostly within the range of - µm. therefore, classical methods of protein biochemistry cannot be applied. alternative methods like the generation of blocking antibodies, genetic approaches or tetramer staining with known tcell antigens is on the one hand side tedious and labor-intensive or on the other hand requires a priori knowledge of possible candidates, which introduces a bias. , third, it is difficult to assess whether the recognition of certain antigens by γδ tcrs can be generalized, because a number of known antigens are bound solely by particular clones that have been identified in individuals. moreover, findings in the mouse system can in most cases not be translated into the human system and vice versa because the tcr sequences and subsets in mice and humans differ substantially. this also complicates the assessment of physiological relevance of many human γδ tcr ligands since they can be identified only in vitro without the possibility to test their functionality in transgenic animals. despite all these obstacles, several antigens for the γδ tcr have been identified since the discovery of γδ t cells (see table ). due to recent progress made in this field, we aim to summarize in this review the current knowledge about adaptive recognition of mhclike molecules, the concept of immunoglobulin-like antigen recognition as well as innate recognition of phosphoantigens and butyrophilins by the γδ tcr. moreover, we speculate on how the γδ tcr might be able to discriminate between physiological and pathological conditions. the paradigm of γδ t cells not being restricted to mhc is based to a large extent on the observation that γδ t cells develop normally in β -microglobulin knockout mice whereas αβ t cells are missing due to missing positive selection in the thymus. in contrast, experiments with mice expressing another t /t -reactive γδ tcr (kn ) and an mhc class i-reactive γδ tcr showed that the development and maturation of γδ t cells were impaired in a β -microglobulin knockout background. as the β -microglobulin knockout does not completely eliminate surface expression of t /t , a mouse model with a more specific knockout of t and t was generated and led to the conclusion that the antigen is important for the development of γδ t cells. however, even the complete absence of the respective antigen failed to abolish the generation of at least some t -reactive γδ t cells, which might be explained by a certain plasticity of the γδ tcr for different ligands. thus, γδ t cells can develop without the presence of their cognate antigen but their functional maturation is heavily impaired. moreover, nonexpanded clones reactive to mhc or mhc-like molecules likely make up only a small part of the total γδ tcr repertoire and their disappearance might not be detectable in β -microglobulin knockout mice. therefore, clones reactive to mhc or mhc-like molecules likely coexist next to those recognizing non-mhc molecules. examples of γδ tcr-ligands that are classical mhc molecules comprise murine mhc class ii molecule i-e and class i molecule h- , both found to activate cytotoxic γδ t-cell clones derived from athymic mice. in humans, hla- , hla-b , and hla-a were specifically activating γδ t-cell clones derived from healthy individuals that have been expanded in culture. all these identified interactions have in common that they are independent of peptide presentation by the mhc, as their activation also occurred in cell lines with peptide-loading defects. these γδ t-cell clones were therefore qualified as alloreactive. the fact that some of them are able to cross-react with some subtype of the same mhc indicates that they might even be binding to less polymorphic parts of the mhc molecule. additionally, a further alloreactive vγ vδ + tcr has recently been found to recognize hla-a * : on cancer cells. it was shown to be dependent on peptide loading of the hla complex but not the presentation of a specific peptide. given the increased stability of peptidepresenting mhc molecules on cell surfaces, the authors reasoned that the random peptide presentation might be rather required for target stabilization than for specific antigen presentation. in contrast to this, vδ + γδ t cells derived in vitro from human hematopoietic stem and progenitor cell (hspc) were reactive to mhc hla-a -restricted peptide presentation of the melanoma antigen mart- . however, the resolved structure of the interacting proteins suggests that binding of the respective γδ tcrs to mart- presenting mhc is less peptide-centric as compared to the interaction with a mart- -specific αβ tcr. hence, one might speculate that mart- -mhc-specific activation of some γδ tcr is still different from classical αβ tcr mhcrestriction and that mart- could also be a specific stabilizer for the mhc that is required for proper detection by the respective γδ tcrs. besides classical mhc recognition some γδ tcrs are reactive towards mhc class ib or mhc-related proteins in mice and humans. the lipid-antigen-presenting molecules cd -c and cd -d are amongst the best studied examples of this group. , [ ] [ ] [ ] [ ] [ ] human γδ tcrs recognizing cd -molecules are vδ + or vδ + and they can react to several presented phospho-and glycolipids. , however, unloaded cd -d was also able to bind to vδ + γδ tcrs albeit with lower affinity than if presenting lipids, which would be in-line with the mentioned alloreactivity observed in mhc-reactive γδ tcrs. another mhc-ib molecule that is a putative ligand for the γδ tcr is murine qa- b presenting an artificial glutamine-tyrosine polypeptide. in addition, in vivo expansion of γδ iels in response to salmonella infection was dependent on peptide loading of qa- b . yet, unambiguous evidence that physiological peptides bound to qa- b are specifically recognized by γδ tcrs does not exist and a mere stabilizing function as in the case of human hla * : cannot be excluded. other functional interactions of γδ tcrs with mhc-like molecules do not require the presentation of antigens as is the case for mhc-related protein (mr- ), endothelial protein c receptor (epcr), mhc class i-related chain a or b (mica/ micb), , - ul -binding protein (ulbp ) and t /t in mice. [ ] [ ] [ ] [ ] the reasons are that either the reactive γδ tcrs are binding independently of the presented antigen (mr- ), no further molecules are presented (epcr) or the antigen-binding cleft of the respective ligand is truncated, which precludes the loading of antigen (t /t ). thus, overall γδ tcr recognition of classical mhc or mhc-like molecules seems to be independent of the presentation of foreign antigens, which is in contrast to αβ tcr antigen binding. reactivity of γδ tcrs to mhc or mhc-like molecules is largely dependent on the cdrs with a substantial focus on the cdr δ in most cases (t /t , cd -d, mart- hla-a ) and the tcr-chains are commonly composed of vδ − -or vγ − vδ + sequences. furthermore, reactive tcrs were usually derived from particular private clones (epcr, hla * : ) that were not shared between individuals or were of low abundance in peripheral blood (mr- , cd , t /t ). however, γδ tcr repertoire analysis revealed that clones of the vδ − -or vγ − vδ + subsets can undergo rapid and sustained clonal expansion in response to e.g., cmv infection , and mart- -hla-a reactive γδ t cells could be expanded from pbmcs in vitro. these features of mhc-and mhc-like-reactive γδ tcrs are reminiscent of the adaptive responses observed in αβ t cells, hence this type of antigen recognition in γδ tcrs was termed adaptive as has been reviewed by willcox & willcox as well as davey et al. , as a consequence, it is often difficult to judge whether the ligand-specificities observed are a general phenomenon that is particularly relevant, since most of the interactions were identified in cell culture systems in vitro and, so far, evidence for physiological relevance is still rare. on the other hand, also in αβ t cells the amount of particular antigen-specific clones is low prior to expansion and it is conceivable that antigennaïve but potentially reactive γδ t cells present at low frequencies would expand upon antigen exposure. in fact, the epcr-reactive les clone (vγ vδ + ) made up about % of the entire t-cell ligand recognition by the γδ tcr and discrimination between homeostasis. . . m deseke and i prinz peptides of mycobacterial and mammalian origin recognized [ ] [ ] [ ] ligand recognition by the γδ tcr and discrimination between homeostasis. . . m deseke and i prinz repertoire in a cmv-positive transplanted patient. in addition to the low abundance of naïve γδ t cells, it is possible that other mhc-or mhc-like reactive γδ tcrs escaped the detection by tetramer staining as in the case of cd -d or mr- because the affinity for their cognate antigen was too low for flow cytometry approaches. concerning the methodology employed for the identification of the so far investigated mhc molecules as γδ tcr ligands, it has been criticized that it relied to a large extent on previous knowledge and techniques from αβ tcrs and the detection of mhc or mhc-like molecules as γδ tcr ligands might thus not appear very surprising. despite this technical bias in many studies published in the past, the identification of hla * : as an antigen for the alloreactive vγ vδ + γδ tcr by kierkels et al. indicates that also approaches without preconceived ideas of putative antigen candidates can reveal mhc or mhc-like molecules as γδ tcr ligands. the question to what extent the reactivity to mhc molecules can be generalized awaits further investigation and unbiased identification of reactive γδ tcrs. adaptive or adaptive like antigen recognition by γδ tcrs is by no means limited to mhc-or mhc-like molecules as shows the wide and diverse range of cell surface or soluble molecules reported to be γδ tcr antigens. these include the cell stress-induced annexin a and ephrin receptor a (epha ), which were recognized by vδ − γδ t-cell clones in a tcr-dependent manner on cells that were either transformed, cmv-infected or exposed to abiotic stressors, e.g., heat. the human dna mismatch repair protein muts-homologue (hmsh ) is found at the cell surface of malignant cells and induces target cell killing dependent on vδ + γδ tcrs. furthermore, histidyl trna synthetase , is recognized by a vγ vδ + tcr identified in a polymyositis patient although it remains elusive how this target can be reached by the γδ tcr since no cell-surface exposition has been shown to date. in both cases the γδ tcrs were vγ − vδ + . foreign antigens derived from pathogenic or nonpathogenic organisms that have been reported to activate γδ tcrs comprise herpex simplex virus glycoprotein i (hsv-gi) recognized by a murine vγ vδ + tcr, bacterial superantigens such as sea, oxys, and dx - and even the algal protein phycoerythrin (pe), which is a prototype b-cell antigen but can also be bound by range of different murine, ruminant as well as human γδ tcrs. similar observations were made with the haptens cy and np. however, it is very unlikely that pe or haptens represent actual antigens under physiological conditions. nevertheless, these studies underline the plasticity of γδ tcr target recognition and it might be used in experimental models. strikingly, not only entire proteins but also small peptide fragments can be detected by γδ tcr without the requirement for presentation by other cells or molecules. examples are the insulin peptide b: - in mice, , peptides derived from mycobacterial and mammalian heat shock proteins in mice and humans [ ] [ ] [ ] and from the lysteria monocytogenes protein listeriolysin o in humans. other peptides, however, do not bind the tcr directly but seem to require presentation on target cells such as those derived from tetanus toxin , and from immunoglobulin λ-chain from b-cell lymphoma. , recognition of these peptides, whether presented or not, was found to be γδ tcr dependent but neither were direct interactions between γδ tcr and peptides shown nor exists evidence for their physiological relevance. recently, murine γδ nkt cells with a vγ vδ . + tcr were found to be activated constitutively when cultured in vitro. the reaction was tcr-dependent and driven by polyanions present on the treated plastic ware used for culturing the cells. although both chains were required for the reactivity, the vγ -chain seemed to have a slightly higher importance with only secondary relevance of the cdr . interestingly, many of the aforementioned peptidespecificities of γδ tcrs mice are mediated via vγ + tcrs. ligand recognition by the γδ tcr and discrimination between homeostasis. . . m deseke and i prinz although the peptides described are not necessarily polyanionic, reactivity for short polymeric sequences by this group of tcrs might be a common feature that is, however, probably not cdr dependent. together with the different mhc and mhc-like molecules, these examples of recognized molecules illustrate the high diversity of ligands for the γδ tcr that is in contrast to αβ tcrs, which can recognize a wide range of peptides but all in the context of the less polymorphic mhc class i and ii molecules. furthermore, comparison of the cdr lengths between the different adaptive immune receptors revealed that overall γδ tcrs resemble more immunoglobulins with a shorter cdr γ and a longer cdr δ, which is in contrast to cdr α and cdr β that have comparable lengths. although recognition of antigen by vdj-recombined adaptive immune receptors depends on more factors than the mere cdr length, this points to an antigen-binding mode of γδ tcrs that is substantially different from αβ tcrs. together with the great variety of γδ tcr antigens this has led to the concept of a rather immunoglobulin-like (ig-like) recognition of antigens by γδ tcrs. in accordance with this, similar to immunoglobulins, γδ tcrs seem to be able to recognize structural as well as sequence epitopes. moreover, the fact that many different γδ tcrs can be specific for the same target molecule as e.g., in the case of pe, cd -d, or mr- is reminiscent of polyclonal antibodies with the same target specificities but different binding modes. despite these striking similarities between γδ tcrs and immunoglobulins with regard to their antigen recognition properties, one should always bear in mind that fundamental differences exist. the affinity of most γδ tcrs for their antigens is low in contrast to high-affinity antibodies, which has important considerations for technical applications, such as flow cytometry or co-immunoprecipitation. due to the high density of γδ tcr molecules on the cell surface in physiological contexts and similarly high expression of the respective ligands, the high avidity of this interaction probably circumvents the single molecule lowaffinity binding. to what extent a possible ig-like antigen recognition by γδ tcrs plays a role in vivo remains a matter of debate. in humans the largest subset of γδ t cells in peripheral blood express a semi-invariant tcr composed of a restricted vγ jp rearrangement together with a more diverse vδ chain. this subset is also found in other species, e.g., in non-human primates , or alpaca but not in rodents. , vγ vδ + tcrs recognize small non-proteogenic phosphorylated molecules termed phosphoantigens (p-ags) in an mhc-independent way. the most potent p-ag is (e)- -hydroxy- -methyl-but- -enyl pyrophosphate (hmbpp), an intermediate of the prokaryotic non-mevalonate pathway of isoprenoid biosynthesis. isopentenyl pyrophosphate (ipp) and dimethylallyl pyrophosphate (dmapp) are present in both prokaryotes and eukaryotes. however, their efficiency to activate vγ vδ + tcrs is lower compared to hmbpp. ipp and dmapp can accumulate inside eukaryotic cells in response to stress situations e.g., infection or malignant transformation whereas hmbpp is produced directly by pathogens such as grampositive bacteria, plasmodium or toxoplasma gondii. besides the naturally occurring p-ags, synthetic aminobisphosphonates as alendronate, zoledronate or pamidronate can activate vγ vδ + tcrs by inhibiting the enzyme farnesyl pyrophosphate synthase in the mevalonate pathway of eukaryotic isoprenoid biosynthesis leading to an intracellular accumulation of ipp. , vγ vδ + t cells can therefore be expanded for cancer immunotherapy by administration of these aminobisphosphonate drugs as has been reviewed by morita et al. and legut et al. the recognition of ubiquitous microbial or stress signals by γδ tcrs is reminiscent of the pamp-detection by pattern recognition receptors, which is supported by the semi-invariant v-usage of these γδ tcrs and rather polyclonal expansions in response to p-ags. therefore, vγ vδ + tcr-mediated recognition of p-ags has been termed innate-like. despite the requirement for p-ags for the activation of vγ vδ + tcrs, these small phosphorylated moieties are not the cognate antigens binding to γδ tcrs and cell to cell contact is necessary. especially, the protein butyrophilin a (btn a ) has been shown to be essential for γδ tcr-mediated p-ag-recognition. , btn a belongs as the other butyrophilins to the b receptor family-like proteins and consists of two extracellular ig-like domains, transmembrane and juxtamembrane domains and an intracellular b . domain. after the discovery of btn a as a central mediator of p-ag reactivity, two different models of interaction with p-ags were proposed. vavassori et al. first showed evidence for an antigen presentation by btn a and a direct interaction between p-ag presenting btn a and the vγ vδ + tcr. in contrast, sandstrom et al. made the observation that the intracellular b . domain of btn a binds the p-ags in a pocket with basic residues and no direct interaction between the extracellular igv-domain of btn a and the vγ vδ + tcr was detected. subsequent mutagenesis experiments with btn a intra-and extracellular domains revealed that the second model of intracellular p-ag binding held true. moreover, rodent cells expressing human btn a are not able to stimulate vγ vδ + tcrs upon p-ag exposure indicating that other mechanisms and molecules are probably involved. thus, the current concept of btn a -mediated activation of vγ vδ + tcrs implies that intracellular p-ag binding to the b . domain leads to conformational changes that translate to the extracellular domain of btn a in order to be sensed indirectly by vγ vδ + tcrs. inline with this hypothesis, nmr-studies of the b . domain and the juxtamembrane domain of btn a revealed conformational changes upon p-ag binding. [ ] [ ] [ ] furthermore, the cytoskeletal adaptor protein periplakin as well as the gtpase rhob were reported to interact with the b . domain and thereby assist to organize btn a membrane organization influencing vγ vδ + tcr activation. , however, btn a alone is not sufficient for p-ag recognition by vγ vδ + tcrs as the btn a isoforms btn a and btn a were shown to be required by the use of knock down and knockout cell lines. , in contrast to the observation of btn a homodimers, btn a and btn a seem also to be able to form heterodimers, which enable the correct btn a localization to the cell membrane and complete functionality in terms of p-ag reactivity. interestingly, expression of btn a alone can lead to vγ vδ + tcr activation in response to p-ag, albeit with much lower efficiency. to which extent btn a homo-or heterodimers play important functions in p-ag recognition by vγ vδ + tcrs or whether interconversion between both structural arrangements can occur is still to be elucidated. although the evidence for btn a as the p-ag sensing molecule and its influence on vγ vδ + tcr activation is compelling, no direct interaction with the γδ tcr has been established so far. rodent cell lines expressing transgenic human btn a were shown to require human chromosome for inducing functional p-ag reactivity in vγ vδ + tcrs. hence, another component encoded on this chromosome appeared to be essential to induce p-ag responses. recently, btn a has been identified by two different approaches to be this enigmatic "factor x" and to be a direct ligand for vγ vδ + tcrs. rigau et al. employed tetramerized soluble tcr staining of target cells and a genome-wide crispr screening to identify candidates for vγ vδ + tcr ligands. at the same time, karunakaran et al. generated radiation hybrids of chinese hamster ovarian (cho) cells containing human chromosome and screening for abrogation of p-ag reactivity led to the identification of the gene encoding btn a . both publications suggested binding of the ligand recognition by the γδ tcr and discrimination between homeostasis. . . vγ -chain to btn a via germline-encoded regions and without major involvement of the cdrs with an affinity of around - µm. btn a itself seems to form homodimers linked by disulfide bridges and it associates with btn a on the cell surface as has been shown by co-immunoprecipitation and fret. interestingly, not only the extracellular igv domains of btn a and btn a but also their intracellular regions seem to be at least in close proximity although only the b . domain of btn a is able to bind p-ag as shown by isothermal calorimetry (itc). , the domains of the vγ -chain involved in interaction with btn a were determined by mutagenesis of the γδ tcr and btn a and molecular modeling in silico. the data suggest that interaction occurs between the c, c', f, and g β strands (cfg interface) of btn a and residues of the germline-encoded hypervariable region (hv ) as well as cdr γ. the results from the mutagenesis study conducted by rigau et al. were not entirely consistent with these observations. based on their mutagenesis experiments, they concluded that the outer face of the abed βsheet is important for the interaction with btn a . however, in both cases the interaction interface of the vγ -chain with btn a was germline-encoded and a central glutamic acid residue at position was considered to be relevant by both groups. further investigation might be required to completely solve this discrepancy. a still unsolved question concerning btn-mediated p-agreactivity is how the cdr γ and cdr δ, which are both reported to be required for a vγ vδ + tcr response to p-ag, are involved in this process. mutations in the cdr γ and the cdr δ led furthermore to the abrogation of p-ag reactivity but not the binding of the vγ -chain to btn a . this indicated that binding to btn a is required but not sufficient for a vγ vδ + tcr-mediated response. thus, it has been speculated that at least a second interaction is necessary. whether this is mediated by btn a as proposed by rigau et al. or if a yet completely unknown ligand is involved as suggested by karunakaran et al., remains to be defined. the role of butyrophilins in γδ t-cell biology exceeds their implication in p-ag sensing and the activation of vγ vδ + tcrs. other proteins with structural similarity to the b receptor superfamily such as skint- and butyrophilin-like (btnl) and in mice as well as btnl and in humans were shown to be relevant for the development and possibly for the tissue homing and homeostasis of certain γδ t-cell subsets as has been reviewed recently by haday and vantourout. murine detcs bearing a canonical vγ vδ + tcr require skint- expression, since mice with a mutation in the skint- gene leading to a premature insertion of a stop-codon lack this skin-resident γδ t-cell subset. , it is in particular the homing to the skin as well as the phenotype of these vγ vδ + t cells that is affected rather than their differentiation in general. this effect was additionally shown to be γδ tcr-dependent, which makes skint- a putative ligand. , mutagenesis of the membrane distal domain of skint- and nmr-studies suggest a putative receptor-interaction surface but a direct interaction with the γδ tcr has not been shown so far. the case of btnl/btnl proteins in mice and humans seems to be, however, much clearer. btnl and form heterodimers and are required for the development of vγ + iels in the gut in a γδ tcrdependent manner. likewise, human intestinal vγ + tcrs can be activated by the co-expressed btnl-homologues btnl and btnl . , tcr-dependent responsiveness to btnls seems thus to be evolutionarily conserved. recently, direct binding as well as the mode of interaction of btnl/btnl proteins and the respective γδ tcrs were revealed. , the interaction was mediated via the germline-encoded γ-chain hv with the involvement of some cdr residues and the cfg-domain of btnl and btnl , reminiscent of superantigen binding to αβ tcrs. other cdrs and the entire δ-chain were not involved but are available for clonally specific ligand binding of e.g. cd -d or epcr. this indicates that γδ tcrs are intrinsically able to combine clonal adaptive reactivity and nonclonal innate responsiveness to common ligands. the physiological role of btnlresponsiveness by γδ tcrs beyond its implication in γδ t-cell development remains to be elucidated. it has, however, been discussed, that it may serve as a signal of normality, keeping the cells ready to respond to cognate antigens under stress conditions. , whether germline-encoded recognition of b family-like proteins via the γ-chain hv extends also to other γδ tcrs and represents a general principle is unclear. the tissue-specific expression of certain γ-chains would reflect such a broadly applicable mechanism. moreover, the recently investigated interaction mode between btn a and the public vγ jp-chain follows the same principle as between btnl/btnl proteins and γδ tcrs. in both cases, the hv was described as the major mediator of the interaction, suggesting that a binding mode via germlineencoded domains might describe a more general feature of butyrophilin binding by γδ tcrs. hallmarks of antigen recognition by αβ tcrs are the recognition of pathogen-derived peptides presented by mhc molecules and additional regulation via co-receptors, which allow all together the fine-tuned discrimination of foreign from self. ligands of γδ tcrs, however, are representing a wide range of largely host-cellderived molecules, which are believed to be signals of cellular stress (see table ). hence, the question arises of how γδ t cells are able to discriminate normal homeostatic conditions from pathological ones. especially the direct ig-like binding of the antigen harbors the potential for autoimmune reactions and thus molecular mechanisms to circumvent this problem must exist (fig. ) . one option to regulate activation of the γδ tcr is to induce tissue-specific antigen expression upon stress conditions (fig. a) . examples of antigens differentially expressed at the cell surface are mica/micb, annexin a and epha in humans, which were upregulated upon cmv-infections, hypoxia, heat shock and metabolic reprogramming. in mice, the expression of γδ tcr ligands t and t was described to be upregulated upon antigenic activation of αβ t cells. furthermore, some in vitro studies suggest that the yet unknown antigens for the respective investigated γδ tcrs in malaria and listeria-infection can already be recognized on target cells prior to any stress stimulus and that the γδ tcr reactivity was increased upon e.g., infection. , besides a transcriptional and translational upregulation of antigen expression, differential subcellular localization of otherwise intracellular γδ tcr ligands might regulate activation. this has been suggested for hmsh , an otherwise nuclear protein involved in dna damage repair. although this theory of differential expression of γδ tcr ligands seems quite compelling, it does not sufficiently explain all observations made with γδ tcrs investigated for their recognition and binding behavior. in some cases, the expression of the recognized antigen on certain target cells does not induce the respective γδ tcr, e.g., in the case of epcr, epha and the mhc-molecules i-e and hla-a * : . interestingly, although identified as the antigen of a cmv-reactive γδ tcr, the expression of the epcr is unchanged upon cmv infection. put together, these observations indicate that either binding of the γδ tcr to its cognate antigen is not always sufficient for its activation and that additional signals or molecular changes in the antigen itself are necessary. ligand recognition by the γδ tcr and discrimination between homeostasis. . . m deseke and i prinz fig. mechanisms for the discrimination of health and stress conditions via the γδ tcr. a the putative γδ tcr ligand might be differentially expressed depending on the stress level of the cell. in the case of stress-induced antigens such as annexin a or mica/micb this would mean an upregulation whereas btnl molecules might be downregulated allowing for γδ tcr activation via the cdr . b co-stimulatory molecules such as cd or jaml might be required for full activation of the γδ tcr and their upregulation might be triggered by stress conditions. c changes in the conformation of the ligand might increase the accessibility of a particular γδ tcr binding domain. btn a for example undergoes conformational changes upon p-ag binding. d multimerization or monomerization of the respective ligand can be triggers for γδ tcr as in the case of the hlamolecule a* : . e glycosylation patterns are modified upon infections or tumor development. these changes in post-translational modifications might lead to different outcomes of γδ tcr interaction with the same ligand with different glycan residues on the extracellular domain ligand recognition by the γδ tcr and discrimination between homeostasis. . . like αβ t cells which rely on co-receptor and co-stimulatory signals to regulate their response via e.g., cd /cd and cd , γδ tcrs might also require certain co-receptors for full activation (fig. b) . butyrophilins and the butyrophilin-like molecules btnl/ btnl might represent candidates for such a regulatory function. as they are binding to germline-encoded regions in the vγ-chain distinct of the cdr , they could exert co-receptor functions in addition to the clonotype-specific antigens. however, binding of btnl to human les tcr (vγ vδ ) was reported to inhibit the interaction with epcr via the cdr , indicating that simultaneous interaction with btnls and antigen is impossible. therefore, btnls might not function as classical co-receptors acting in parallel to tcr signaling but rather confer signals of normality before the actual γδ tcr activation. upon stress conditions such as an infection, btnls could be downregulated allowing for the subsequent recognition of stress-induced ligands (fig. a) . inline with this is the observation that the btnl expression is altered upon intestinal inflammation and colon cancer in humans and mice. , evidence for this regulatory role of btnls in γδ tcr activation is however still missing and it remains therefore purely hypothetical. a wide range of co-stimulatory molecules required for full tcrdependent γδ t-cell activation have been described. these comprise amongst others cd , , cd , , cd in the p-ag-dependent stimulation of vγ vδ + γδ t cells and jaml in the activation of murine detcs. besides, cd α has been shown to increase the reactivity to cancer cells of some vδ + tcrs , , and in cmv-positive bone marrow grafts the frequency of cd + vδ + γδ t cells was increased, indicative of a potential costimulatory role of cd for vδ + tcrs. the exact contribution of all these proposed co-stimulatory molecules to γδ t-cell-mediated immune responses remains to a large extent unclear as they are often only expressed by certain γδ t-cell subsets and functional in vivo studies are missing. even more controversial is the role of the natural-killer group , member d (nkg d) receptor that was reported to enhance tcrmediated immune responses of γδ t cells. , , interestingly, nkg d-ligands mica and micb are also recognized by vδ + tcrs and γδ t cells expressing both receptors seem to require signals from both receptors to get activated. like other nk-receptors, nkg d is able to activate γδ t cells without involving the γδ tcr. it is thus difficult to properly dissect whether the role of nkg d can be defined as co-stimulatory or whether γδ tcr and nkg d act sequentially as suggested by ribot et al. besides differential expression and co-stimulation by other surface molecules, intramolecular mechanisms might be involved in the regulation of γδ tcr enabling the distinction of homeostatic and stress conditions. these include e.g., conformational changes that increase the accessibility of domains required for proper γδ tcr activation (fig. c) or post-translational modifications such as glycosylation (fig. e) . the p-ag-mediated conformational changes in btn a mentioned previously are an example for this. , although no direct binding of the vγ vδ + tcr to btn a has been observed so far, this conformational change is required for p-ag-mediated activation either via btn a interacting with the cdrs of the tcr or indirectly via a yet unknown molecule. conformational differences of γδ tcr ligands might also be caused by multimerization as it is the case for the mhcmolecule hla * : recognized by the alloreactive vγ vδ + tcr (fig. d) . being present as homodimers under regular conditions, malignant transformation seems to induce changes in the spatial organization of hla * : at the cell surface so that it occurs as monomers. this restructuring is essential for γδ tcr recognition as fixation of the dimeric form of hla * : led to the abrogation of tcr-mediated activation. the influence of antigen glycosylation on γδ tcr activation has so far not been investigated in detail but some publications suggest that they are relevant for ligand recognition. in fact, changes in surface glycosylation patterns are known to occur in infected or transformed cells. , it is thus conceivable that immune receptors recognizing mostly glycosylated cell-surface molecules like γδ tcrs are to some extent reacting to these changes (fig. e) . as the investigation of carbohydrates and their effects is difficult with conventional molecular biology techniques, data on the influence of glycosylation on γδ tcr binding and activation is still quite sparse. for example, the recognition of hla i-e by the murine γδ tcr lbk is influenced by changes in the nglycosylation at position of its α-chain. furthermore, recognition of glycosphingolipids by a subset of murine γδ t cells has been reported to be dependent on specific carbohydrate residues but to what extent the γδ tcr is involved in this process has not unambiguously been shown. , more recently, a study with soluble γδ tcrs from a patient with lyme arthritis revealed that binding of the investigated receptors is sensitive to cell-surface digestion of glycans. although the actual ligand for the respective γδ tcr was not identified, this hints to an essential role of carbohydrates in the binding mechanism to the target cells. whether the γδ tcrs in all these studies directly interacts with the respective carbohydrates and whether the influence thereof can be generalized to other γδ tcrs requires further investigation. finally, recent data on mr- -reactive γδ tcrs suggest that also the binding mode of the γδ tcr to its cognate antigen could be of relevance for the degree of activation induced by this interaction. some of the investigated human vδ + tcrs were interacting with the α -domain on the side of mr- while others were binding from the top to the actual antigen-presenting cleft of mr- . the γδ tcrs also differed in their capacity to induce intracellular signal transduction. binding to the α -domain led to erk-phosphorylation but no upregulation of the activation marker cd , whereas interaction with the top-side of mr- induced full activation of the employed reporter cells. although these controversial binding modes where observed in γδ t cells ex vivo from several individuals it is unclear to what extent this applies also to physiological conditions and whether it is a singular or more general phenomenon. during the past decades, research on γδ tcr ligands revealed a plethora of diverse molecules that are recognized by either unique specific γδ tcrs or by omni-present tcrs with canonical γδ rearrangements such as vγ vδ . although some general patterns like involvement of butyrophilins and btnls or the recognition of mhc-like molecules can be observed, an overarching concept of what is driving the activation of γδ tcrs is still missing. thus, in order to broaden our understanding of γδ tcr specificities and their implications in health and disease, unbiased and large-scale screening approaches for further ligands will be required. for αβ tcrs, a multitude of these methods is already established as recently reviewed by gerber et al. a prominent example is the expression of mhc-αβ tcr hybrids (mcr) on the surface of a reporter target cell line, which led to the expression of a fluorescent reporter gene construct upon functional αβ tcr binding. to γδ tcrs however, these approaches cannot be applied since no general restrictive molecule such as the mhc exists or has at least not been identified yet. therefore, the emerging possibilities of crispr-cas technology might be better applicable in this context. with the appropriate readout systems at hand, such as target cell killing or binding of tcr-tetramers, genome-wide crispr libraries might allow for a negative selection of those cells where the target for the γδ tcr is not present anymore. to get a better understanding of γδ tcr activation, the mere identification of ligands on its own will, however, not be sufficient. especially for host-cell-derived ligands for the γδ tcr, which might be directly recognized without further processing, it is essential to understand the mechanisms by which a discrimination between normal and stress conditions can take place. besides the differential expression of the respective ligand, co-stimulatory effects by other molecules, multimerization, conformational changes and differential glycosylation of the putative ligand should be considered and investigated more in this context. this should help to reconcile so far contradicting observations in γδ tcr research and help to better understand ligand recognition by the γδ tcr. expression of the gamma-delta t-cell receptor on intestinal cd + intraepithelial lymphocytes homing of a gamma delta thymocyte subset with homogeneous t-cell receptors to mucosal epithelia selection by two powerful antigens may account for the presence of the major population of human peripheral γ/δ t cells preferentially expanding vγ + γδ t cells are associated with protective immunity against plasmodium infection in mice a macrophage colony-stimulating-factor-producing γδ t cell subset prevents malarial parasitemic recurrence implication of gammadelta t cells in the human immune response to cytomegalovirus shared reactivity of vδ neg γδ t cells against cytomegalovirusinfected cells and tumor intestinal epithelial cells effector functions and control of human γδ t-cell activation harnessing γδ t cells in anticancer immunotherapy recognition of stress-induced mhc molecules by intestinal epithelial γδ t cells translating gammadelta (γδ) t cells and their receptors into cancer cell therapies cd -restricted recognition of exogenous and self-lipid antigens by duodenal γδ + t lymphocytes the nkg d ligand ulbp binds to tcrγ /δ and induces cytotoxicity to tumor cells through both tcrγδ and nkg d human γδ t cells are quickly reconstituted after stem-cell transplantation and show adaptive clonal expansion in response to viral infection clonal selection in the human vδ t cell repertoire indicates γδ tcr-dependent adaptive immune surveillance somatic hypermutation of t cell receptor α chain contributes to selection in nurse shark thymus the innate biologies of adaptive antigen receptors γδ tcr ligands: the quest to solve a -millionyear-old mystery γδ t cell responses: how many ligands will it take till we know? semin most gamma delta t cells develop normally in the absence of mhc class ii molecules most γδ t cells develop normally in β -microglobulin-deficient mice structure and specificity of a class ii mhc alloreactive γδ t cell receptor heterodimer identification of a tumor-specific allo-hla-restricted gdtcr cd d-lipid antigen recognition by the γδ tcr cytomegalovirus and tumor stress surveillance by binding of a human γδ t cell antigen receptor to endothelial protein c receptor positive selection is not required for thymic maturation of transgenic γδ t cells blockade of transgenic gamma delta t cell development in beta -microglobulin deficient mice requirement for positive selection of γδ receptor-bearing t cells role of a selecting ligand in shaping the murine γδ-tcr repertoire structure and specificity of t cell receptor γ/δ on major histocompatibility complex antigen-specific cd +, cd −, cd − t lymphocytes specificity of human t lymphocytes expressing a γ/δ t cell antigen receptor. recognition of a polymorphic determinant of hla class i molecules by a γ/δ clone identification of a novel hla-b subtype by restriction analysis of a cytotoxic gamma delta t cell clone cytotoxic activity and lymphokine production of t cell receptor (tcr)-αβ+ and tcr-γδ+ cytotoxic t lymphocyte (ctl) clones recognizing hla-a and hla-a mutants. recognition of tcr-γδ+ ctl clones is affected by mutations at positions and generation and molecular recognition of melanomaassociated antigen-specific human γδ t cells self-recognition of cd by γ/δ t cells: implications for innate immunity recognition of pollen-derived phosphatidyl-ethanolamine by human cd d-restricted γδ t cells cardiolipin binds to cd d and stimulates cd d-restricted γδ t cells in the normal murine repertoire molecular analysis of lipid-reactive vδ γδ t cells identified by cd c tetramers crystal structure of vδ t cell receptor in complex with cd d-sulfatide shows mhc-like recognition of a self-lipid by human γδ t cells cutting edge: cd d restriction and th /th /th cytokine secretion by human vδ t cells qa- restricted recognition of foreign antigen by a γδ t-cell hybridoma infection-induced expansion of a mhc class ib-dependent intestinal intraepithelial γδ t cell subset a class of γδ t cell receptors recognize the underside of the antigen-presenting molecule mr cell stress-regulated human major histocompatibility complex class i gene expressed in gastrointestinal epithelium t cell antigen receptor engagement and specificity in the recognition of stress-inducible mhc class i-related chains by human epithelial γδ t cells crystal structure of a t-cell receptor specific for the human mhc class i homolog mica recognition of the product of a novel mhc tl region gene ( b) by a mouse γδ t cell receptor the nature of major histocompatibility complex recognition by γδ t cells a population of murine γδ t cells that recognize an inducible mhc class ib molecule crystal structure of a γδ t cell receptor ligand t : a truncated mhc-like fold recasting human vδ lymphocytes in an adaptive role vδ +t cells--two subsets for the price of one sensing of cell stress by human γδ tcr-dependent recognition of annexin a γδ t-cell conference : close encounters for the fifth time ectopically expressed human tumor biomarker muts homologue is a novel endogenous ligand that is recognized by human γδ t cells to induce innate anti-tumor/virus immunity antigen recognition properties of a vγ . vδ -t-cell receptor from a rare variant of polymyositis target specificity of an autoreactive pathogenic human γδ-t cell receptor in myositis hsv- glycoprotein i-reactive tcr gamma delta cells directly recognize the peptide backbone in a conformationally dependent manner specific recognition of staphylococcal enterotoxin a by human t cells bearing receptors with the vγ region a novel strategy to screen bacillus calmette-guérin protein antigen recognized by γδ tcr identification of a new tuberculosis antigen recognized by γσ t cell receptor γδ t cells recognize a microbial encoded b cell antigen to initiate a rapid antigen-specific interleukin- response gamma delta t cells recognize haptens and mount a haptenspecific response gamma delta t cell receptors confer autonomous responsiveness to the insulin-peptide b: - γδ t cells recognize the insulin b: - peptide antigen when it is dimerized through thiol oxidation isolation of cd -cd -mycobacteria-reactive t lymphocyte clones from rheumatoid arthritis synovial fluid recognition of a peptide antigen by heat shock-reactive γδ t lymphocytes heat shock protein hsp -reactive γδ cells: a large, diversified t-lymphocyte subset with highly focused specificity human t-cell recognition of listeria monocytogenes: recognition of listeriolysin o by tcrαβ+ and tcrγδ+ t cells human tcr-γ+/δ+, cd + t lymphocytes recognize tetanus toxoid in an mhc-restricted fashion dual antigenic recognition by cloned human γδ t cells cytotoxic t lymphocytes specific for self tumor immunoglobulin express t cell receptor δ chain gamma delta t cell recognition of tumor ig peptide recognition of synthetic polyanionic ligands underlies "spontaneous" reactivity of vγ γδtcrs cdr length in antigen-specific immune receptors cells: t cells with b-cell-like recognition properties γδ t cells: first line of defense and beyond restricted diversity of vγ -jp rearrangements in unstimulated human γ/δ t lymphocytes evolution of the v, d, and j gene segments used in the primate t-cell receptor reveals a dichotomy of conservation and diversity conservation of nonpeptide antigen recognition by rhesus monkey vγ vδ t cells vγ and vδ t cell antigen receptor genes and butyrophilin (btn ) emerged with placental mammals and are concomitantly preserved in selected species like alpaca (vicugna pacos) alpaca (vicugna pacos), the first nonprimate species with a phosphoantigen-reactive vγ vδ t cell subset nonpeptide antigens, presentation mechanisms, and immunological memory of human vγ vδ t cells: discriminating friend from foe through the recognition of prenyl pyrophosphate antigens early innate responses to pathogens: pattern recognition by unconventional human t-cells human t cell receptor γδ cells recognize endogenous mevalonate metabolites in tumor cells alkylamines cause vγ vδ t-cell activation and proliferation by inhibiting the mevalonate pathway the promise of γδ t cells and the γδ t cell receptor for cancer immunotherapy human γδ tcr repertoires in health and disease direct presentation of nonpeptide prenyl pryophosphate antigens to human gamma delta t cells key implication of cd /butyrophilin- (btn a) in cellular stress sensing by a major human γ δ t cell subset the molecular basis for modulation of human vγ vδ t cell responses by cd /butyrophilin- (btn a)-specific antibodies regulation of immunity by butyrophilins butyrophilin a binds phosphorylated antigens and stimulates human γδ t cells the intracellular b . domain of butyrophilin a binds phosphoantigens to mediate activation of human vγ vδ t cells sensor function for butyrophilin a in prenyl pyrophosphate stimulation of human vγ vδ t cells the butyrophilin a intracellular domain undergoes a conformational change involving the juxtamembrane region phosphoantigen-induced conformational change of butyrophilin a (btn a ) and its implication on vγ vδ t cell activation the juxtamembrane domain of butyrophilin btn a controls phosphoantigen-mediated activation of human vγ vδ t cells activation of human γδ t cells by cytosolic interactions of btn a with soluble phosphoantigens and the cytoskeletal adaptor periplakin rhob mediates phosphoantigen recognition by vγ vδ t cell receptor heteromeric interactions regulate butyrophilin (btn) and btn-like molecules governing γδ t cell biology vγ vδ tcr-activation by phosphorylated antigens requires butyrophilin a (btn a ) and additional genes on human chromosome butyrophilin a is essential for phosphoantigen reactivity by gd t cells butyrophilin- a directly binds germline-encoded regions of the vγ vδ tcr and is essential for phosphoantigen sensing vγ vδ t cell receptor recognition of prenyl pyrophosphates is dependent on all cdrs skint , the prototype of a newly identified immunoglobulin superfamily gene cluster, positively selects epidermal γδ t cells skint- is a highly specific, unique selecting component for epidermal t cells skint- identifies a common molecular mechanism for the development of interferon-γ-secreting versus interleukin- -secreting γδ t cells positive selection of dendritic epidermal γδ t cell precursors in the fetal thymus determines expression of skin-homing receptors selection of the cutaneous intraepithelial γδ+ t cell repertoire by a thymic stromal determinant characterization of a putative receptor binding surface on skint- , a critical determinant of dendritic epidermal t cell selection epithelia use butyrophilin-like molecules to shape organ-specific γδ t cell compartments the γδtcr combines innate immunity with adaptive immunity by utilizing spatially distinct regions for agonist selection and antigen responsiveness butyrophilin-like directly binds a human vγ + t cell receptor using a modality distinct from clonally-restricted antigen ligand recognition by the γδ tcr and discrimination between homeostasis structure of the superantigen staphylococcal enterotoxin b in complex with tcr and peptide-mhc demonstrates absence of tcr-peptide contacts antigen recognition by gammadelta t cells recognition of listeria infection by germline elements of the vγ . vδ . tcr activation of tcr vδ + and vδ − vδ − γδ t cells upon controlled infection with plasmodium falciparum in tanzanian volunteers the specificity of a weak gamma delta tcr interaction can be modulated by the glycosylation of the ligand altered expression of butyrophilin (btn) and btnlike (btnl) genes in intestinal inflammation and colon cancer chronic inflammation permanently reshapes tissue-resident immunity in celiac disease searching for 'signal ': costimulation requirements of γδ t cells human t cell clones with γ δ and α β receptors are differently stimulated by monoclonal antibodies to cd selective activation of γ/δ t cell clones by single anti-cd antibodies functional expression of cd on t cell antigen receptor γ/δ-bearing t lymphocytes cellular and molecular basis of human γδ t cell activation. role of accessory molecules in alloactivation cd -cd interactions provide survival and proliferative signals that regulate t cell receptor-driven activation of human γδ peripheral blood lymphocytes the junctional adhesion molecule jaml is a costimulatory receptor for epithelial t cell activation γδt cells elicited by cmv reactivation after allo-sct crossrecognize cmv and leukemia cd + γδ t cells are more frequent in cmv seropositive bone marrow grafts and display phenotype of an adaptive immune response mica engagement by human vγ vδ t cells enhances their antigen-dependent effector function nkg d costimulates human vγ vδ t cell antitumor cytotoxicity through protein kinase cθ-dependent modulation of early tcr-induced calcium and transduction signals activation of vγ vδ t cells by nkg d infection with listeria monocytogenes impairs sialic acid addition to host cell glycoproteins mucin antibodies-new tools in diagnosis and therapy of cancer immunization with glycosylated kb-binding peptides generates carbohydrate-specific, unrestricted cytotoxic t cells crystal structure of an mhc class i presented glycopeptide that generates carbohydrate-specific ctl detection of cell surface ligands for human synovial γδ t cells identification of antigenic targets deciphering cd + t cell specificity using novel mhc-tcr chimeric receptors which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons license, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons license, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder competing interests: the authors declare no competing interests. key: cord- - lmj op authors: ada, gordon title: overview of vaccines and vaccination date: journal: mol biotechnol doi: . /mb: : : sha: doc_id: cord_uid: lmj op of the -plus known infectious agents pathogenic for humans, there are now more than vaccines against mainly viral and bacterial infections and these greatly minimize subsequent disease and prevent death after exposure to those agents. this article describes the nature of the vaccines, from live attenuated agents to subunits, their efficacy and safety, and the kind of the immune responses generated by those vaccines, which are so effective. to date, all licensed vaccines generate especially specific antibodies, which attach to the infectious agent and therefore can very largely prevent infection. these vaccines have been so effective in developed countries in preventing mortality after a subsequent infection that attempts are being made to develop vaccines against many of the remaining infectious agents. many of the latter are difficult to manipulate; they can cause persisting infections or show great antigenic variation. a range of new approaches to improve selected immune responses, such as immunization with dna or chimeric live vectors, viral or bacterial, are under intense scrutiny, as well as genomic analysis of the agent. vaccines are designed as a prophylactic measure to induce a lasting immune response so that on subsequent exposure to the particular infectious agent, the extent of infection is reduced to such an extent that disease does not occur ( ). there is also increasing interest in designing vaccines that may be effective as a therapeutic measure, immunotherapy. there are two contrasting types of infectious processes. some organisms, including all viruses and some bacteria, are obligate intracellular infectious agents, as they only replicate inside a susceptible cell. some parasites, such as plasmodia, have intracellular phases as part of their life cycle. in contrast, many bacteria and parasites replicate extracellularly. the immune response required to control the different patterns of infections may therefore differ. in the case of an acute infection, exposure of a naïve individual to a sublethal dose of the infectious agent may cause disease, but the immune response generated will clear the infection within a period of days or weeks. death occurs if the infecting dose is so high that the immune response is qualitatively or quantitatively insufficient to prevent continuing replication of the agent so that the host is overwhelmed. in contrast, many infections persist for months or years if the process of infection by the agent results in the evasion or subversion of what would normally be an effective immune control reaction. most of the vaccines registered for use in developed countries, and discussed briefly in the next section, are designed to prevent acute human infections. . one approach, pioneered by edward jenner, is to use a virus that is a natural pathogen in another mammalian host as the basis of a vaccine in humans. examples of this approach are the use of cowpox and parainfluenza viruses in humans, and the turkey human virus in chickens. more recently, the use of avipox viruses such as fowlpox and canarypox, which undergo an abortive infection in humans, is being used in humans as vectors of dna coding for antigens of other infectious agents for which vaccines are not yet available ( ). . the measles, mumps, rubella, and yellow fever vaccines are typical of the second approach. the wild-type viruses are extensively passaged in tissue culture/animal hosts until an acceptable balance is reached between loss of virulence and retention of immunogenicity in humans. . type polio virus is a naturally occurring attenuated strain that has been highly successful. more recently, rotavirus strains of low virulence have been recovered from children's nurseries during epidemics ( ). . a fourth approach has been to select mutants that will grow at low temperatures but very poorly above °c ( ). the cold-adapted strains of influenza virus grow at °c and have mutations in four of the internal viral genes ( ). such strains were first described in the late s and have since been used successfully in russia, have undergone extensive clinical trials in the united states ( ) and the vaccine has now been licensed, but not for young children and the elderly. in contrast to these successes, bacillus calmette-guerin (bcg) for the control of tuberculosis was for many years the only example of a live attenuated bacterial vaccine. although still widely used in the world health organization (who) expanded programme of immunization (epi) for infants, it has given highly variable results in adult human trials. in general, it has proven more difficult to make highly effective attenuated bacterial vaccines, but with increasing examples of antibiotic resistance occurring, there is now a greater effort. a general approach is to selectively delete or inactivate one or more genes ( ). salmonella strain ty a has a faulty galactose metabolism, and strains with other deletions are being made. the most recent addition to this category is a vibrio cholerae vaccine. a new approach is to sequence the bacterial genome to establish the properties and hence the probable location of different proteins in the bacteria, and this has now been done for many different bacteria ( ). genetic modification can be useful for complex viruses. thus, open-reading frames, including six genes involved in nucleotide metabolism, have been selectively deleted from the copenhagen strain of vaccinia virus. the product, nyvac, has low virulence but has retained immunogenicity ( ). the same approach has been used with simian immunodeficiency virus (siv) but with limited success. it now seems very unlikely that a live, attenuated strain of human immunodeficiency virus (hiv) will ever become available. live, attenuated agent vaccines have the potential to stimulate strong humoral and cell-mediated immune responses that can be highly effective in preventing or clearing a later infection in most recipients. viruses and bacteria can be treated to destroy their infectivity (inactivation) and the product used with varying efficacy as a vaccine ( table ) . compared to attenuated preparations, inactivated preparations must be given in larger doses and sometimes administered more frequently. the vi-molecular biotechnology volume , ral vaccines are generally effective in preventing disease. the lower efficacy of the influenza viral vaccine is partly due to the difficulty of closely matching the specificity of the virus strains that are circulating when the vaccine finally becomes available. this is due to the continuing antigenic drift that characterizes this virus ( ). the only bacterial vaccine of this nature widely used is the whole cell pertussis vaccine, which is quite effective ( table ) , but has been replaced in some developed counties by the subunit (acellular) vaccine to avoid the adverse effects attributed to the whole cell vaccine ( ). inactivated whole vaccines should induce many of the desirable immune responses, particularly infectivity-neutralizing antibodies. generally, they do not induce a class i major histocompatibility complex (mhc)-restricted cytotoxic t-cell response, which is the major response required to clear intracellular infections by viruses, and by some bacteria and parasites. class ii mhc neutralizing antibodies are adequately induced. the generation of antibodies that prevent infections by both intra-and extracellular microorgan- isms has been regarded as the prime requirement of a vaccine. the epitopes recognized by such antibodies are usually restricted to one of a few proteins or carbohydrate that is exposed at the external surface of the microorganisms. isolation (or synthesis) of such components formed the basis of the first viral and bacterial subunit vaccines. the influenza virus vaccine was composed of the two surface protein antigens, the hemagglutinin and neuraminidase, and the hepatitis b virus vaccine of the surface antigen, hbsag. encapsulated bacteria have a coating of polysaccharides that is not recognized by the immune system of very young children (< yr old) and only moderately well by older individuals (mainly an immunoglobulin[ig] m response), therefore polysaccharide vaccines needed to be improved. in , it was found ( ) that immunizing with a polysaccharide/protein conjugate gave a much stronger antipolysaccharide response (mainly iggs, because t cells were now involved). much later, we now have three such conjugate vaccines ( table ) that are highly immunogenic and these are saving especially many young lives ( ). more will become available. the two bacterial toxoids, tetanus and diphtheria, represent a special situation in which the primary requirement was neutralization of the toxin secreted by the invading bacteria. whereas toxoids have traditionally been made by treatment of the toxins with chemicals, it is now being achieved by genetic manipulation ( ) . hbsag exists in the blood of hepatitis b virus (hbv)-infected people and infected blood was the source of antigen for the first vaccines. production of the antigen in dna-transfected yeast cells initiated the era of genetically engineered vaccines ( , ) . a second genetically engineered subunit preparation from borrelia burgdorferi to control lyme disease later became available in the mid- s but was withdrawn from sale in because of poor sales. all available data concerning the efficacy and safety of candidate vaccines are reviewed by regulatory authorities before registration ( ). at that stage, potential safety hazards, which occur at a frequency of about / doses, should have . this advice has been accepted by several other developed countries such as australia. after successful vaccination campaigns that greatly reduced disease outbreaks, the low levels of undesirable side effects after vaccination gained some notoriety. the evidence bearing on causality and specific adverse health outcomes following vaccination against some childhood viral and bacterial diseases, mainly in the united states, has been evaluated by an expert committee of the institute of medicine (iom) ( ). the possibility of adverse neurological effects was of particular concern, and evidence for these as well as several immunological reactions, such as anaphylaxis and delayed type hypersensitivity, was examined in detail. in the majority of cases, there was insufficient evidence to support a causal relationship, and where the data were more persuasive, the risk was considered to be extraordinarily low. measles has provided an interesting example of vaccine safety. the experience of the who epi shows that the vaccine is very safe ( ). although natural measles infection induces a state of immunosuppression, even immunocompromised children rarely show this effect after vaccination ( ) . in developing countries, the epi schedule is to give the vaccine at mo of age. this delay is meant to allow a sufficient drop in the level of maternally derived antibody so that the vaccine can take. in some infants, this decay occurs by mo, resulting in many deaths from measles infection in the ensuing to mo, "high-titer" vaccines were therefore developed that could be given at mo of age. trials in several countries showed the apparent safety and efficacy of the new vaccine, but after who authorized its wider usage, some young girls in disadvantaged countries died, leading to the withdrawal of the vaccine ( ). one possibility is that the high titer vaccine caused a degree of immunosuppression sufficient to allow infections by other infectious agents. another example is the rotavirus vaccine that was registered for use in the united states in . it was withdrawn in after administration to . million children because of an unacceptable level-about one case per , recipients in some areas-of the condition intussusception ( ). this was surprising because the disease itself does not cause this condition. fortunately, other preparations are well advanced as the need for such a vaccine is great especially in developing countries. it is particularly difficult to attribute causality to the onset of diseases that may occur many months after vaccination. when such claims are made, national authorities or who establish expert committees to review the evidence. there have been claims-rarely in the medical literature but often by antivaccination groups-that a vaccine can cause sudden infant death syndrome (sids), multiple sclerosis, autism, asthma, or a specific allergy. there is no sound medical, scientific, or epidemiological evidence to support these claims. for example, at least different investigations have found no evidence that inflammatory bowel disease and autism occur as a result of measles, mumps, and rubella (mmr) vaccination ( , ) . this claim was apparently made by the antivaccination lobby simply because the increase in austism in the united states late last century coincided with the introduction of a second mmr vaccination. many countries keep yearly records of diseases incidence and the cdc in atlanta have kept records from as early as . table compares the incidence of cases of some common childhood infectious diseases during a major epidemic before the vaccine was licensed, with levels in and , some years after the introduction of the vaccine. most show a very high level of efficacy. pertussis, the cause of whooping cough, is an exception in showing an increase in cases recently and this may lead to the introduction of an additional dose of the vaccine. the crowning achievement of a vaccination program would be to eradicate some infectious diseases. jenner ( ) had proposed that his new vaccination procedure could be used to eradicate smallpox, but a century and a half passed before this suggestion was taken seriously. having achieved control of smallpox infections in their country, the russians in proposed to the world health assembly (wha) the global eradication of that disease. a -yr unfunded, voluntary program was initiated by who. great progress was achieved in developed countries but in , there were million deaths and million disease cases in developing countries. in , a -yr funded (usd $ million) program was begun and despite many difficulties, such as vaccine shortages and wars, the last case was treated in . eradication was declared to the wha in ( ). three senior investigators, f. fenner, d. a. henderson, and i. arita shared the japan prize for this achievement. would it be possible to repeat this outstanding success with another infectious disease? poliomyelitis and measles were two considered. both ful-filled the necessary requirements but not the desirable properties ( table ) . the former was chosen as there was already some success in limiting transmission of the disease in developed countries. the global polio eradication initiative was spearheaded by who, rotary international, the united nations children's fund (unicef), and cdc in , and joined later by other groups. there were many difficulties. the infection persisted in some individuals for many months and at one stage, one of the three vaccine virus strains mutated into a virulent form. repeated vaccinations became necessary so that the deadline had to be progressively extended until to . in , the virus was still endemic (less than cases) in six countries, but especially in nigeria. in kano province, a religious group refused vaccination because of concerns that the vaccine was contaminated with hiv and other factors that could affect the fertility of their children. by the time (mid- ) such contamination claims were shown to be wrong and the concern abated, infectious cases had occurred in previously infection-free countries in west and central africa. re-vaccination of children under the age of yr in countries in this region will begin in late and extend into , provided an ongoing funding gap of usd $ million is overcome ( ). the reason for such funding is that for every case of infection found, up to million children in surrounding areas may need to be re-vaccinated. having been at the edge of successful eradication, it would be a disaster if the extra funding was not made available. as of early october , this vaccination program has begun. for yr until the measles vaccine was introduced in , the yearly incidence of infections in the united states was never less than , cases, with epidemics occurring every to yr. the maximum incidence of reported cases was , in ( table ) . introduction of the measles vaccine in led to a reduction in incidence of cases by > . %. a -yr epidemic of , cases began in and led to the introduction of a second vaccine dose for mainly chil-molecular biotechnology volume , dren about to go to school. in , the cdc concluded that measles was no longer an endemic disease in the united states. in , it was established that all cases had been brought into the country by visitors from specified countries. in , ministers of health from north and south america met and resolved to eliminate measles from all the western hemisphere by . the pan american health organization (paho) under ciro de quadros contributed greatly with catch-up and follow-up campaigns with the result that for mo in , transmission of measles in the whole of the americas was prevented. in , the paho strategy was successfully extended to seven south african countries ( ). although a major difficulty to prevent transmission of measles is the high level of vaccination coverage necessary ( %), the interruption of indigenous measles transmission was also achieved in cuba from , and in england and wales from . some countries such as japan, italy, france, and germany do not consider this a national priority ( ). but there is still hope that one day, a global eradication program will be announced: "it is not a dream to imagine a world free of measles by year " ( ). this may depend on the final result of the poliomyelitis eradication campaign. and new vaccines vaccines are not currently available for more than infectious agents pathogenic for humans. vaccine development is well advanced for some of these and is in progress for the most of the remainder. table contains a short list of agents for which improved vaccines are desirable, and a longer list of those agents that should have priority in vaccine development plans. although the current measles vaccine is highly effective, a vaccine that could be administered well before mo would save many lives. in the new category, the "terrible trio," hiv, m ycobacterium tuberculosis, and malaria, would be at the top of many lists. they are major killers and despite great efforts over many years, effective vaccines are some way off. fortunately in subtropical developed countries, and with effective drugs, each agent is largely controlled. the advantages of this approach include the fact that the anti-idiotype should mimic both carbohydrate-based and peptide-based epitopes, and the conformation of the epitopes in question. the potential advantages of the former point have dis- vaccines ( , ) . the use of this technology may be largely restricted to very special situations, such as identifying the nature of the epitope recognized by very rare antibodies that neutralize a wide spectrum of hiv- primary isolates ( ). the receptors on t cells (lymphocytes) recognize on the surface of the antigen-presenting cell (apc) or infected target cell, a complex composed of a peptide from a protein in the apc or infected cell attached to a mhc antigen. simi-larly, the ig receptors on b cells may recognize a pattern on an antigen often formed by a linear peptide sequence. therefore, it could be advantageous to link such linear sequences to form a particular antigen. the sequences may contain either b-cell epitopes or t-cell determinants, or often both (see subheading . .). sequences containing b-cell epitopes may either be conjugated to carrier proteins, which act as a source of t-helper cell determinants, or linked in different ways to achieve particular tertiary configurations. some of the obvious advantages of this approach are that the final product contains the critical components of the antigen and avoids other sequences that may mimic host antigen sequences, and thus potentially induce an autoimmune response. multiple antigenic peptide systems (maps) are more immunogenic than individual sequences ( ), and the immunogenicity of important "cryptic" sequences may sometimes be enhanced by the deletion of other segments ( ). new methods of synthesis offer the possibility of more closely mimicking the conformational patterns in the original protein. this approach is likely to be applicable, especially for some bacterial and parasitic vaccines. however, the first peptide-based candidate vaccine, despite giving encouraging results in an early small trial, gave disappointing results in an efficacy trial in malaria endemic regions ( ). a preparation composed of polymers of linked peptides from group a streptococcus, which was effective in a mouse model ( ), is currently undergoing clinical trials. this is now a well-established procedure. three cell types have been used: ( ) prokaryotes (bacteria), ( ) lower eukaryotes, mainly yeast; and ( ) mammalian cells, either primary cells (e.g., monkey kidney), cell strains (with a finite replicating ability), or cell lines (immortalized cells such as chinese hamster ovary cells [cho]). each has its own advantages. as a general rule, other bacterial proteins should preferably be made in transfected bacterial cells, and human viral an- molecular biotechnology volume , tigens, especially glycoproteins, in mammalian cells, because of the substantial differences in properties, such as post-translational modifications in different cell types. table lists the viruses and bacteria mostly used for this purpose. of the viruses, the greatest experience has been with vaccinia and its derivatives such as the highly attenuated, modified vaccinia virus ankara. these have a wide host range, possess many different promoters, and can accommodate dna coding for up to averagesized proteins. the avipox viruses, canary and fowlpox, undergo abortive infection in mammals, making them very safe to use as vectors ( ). adenovirus ( ), polioviruses ( ), and salmonella ( ) are mainly used for delivery by a mucosal route, although vaccinia and bcg have been administered both orally and intranasally. making such chimeric vectors has also been an effective way to evaluate the potential role of different cytokines in immune processes. inserting dna coding for a particular cytokine as well as that for the foreign antigens results in the synthesis and secretion of the cytokine so its maximum effect should be displayed. thus, interleukin (il)- and il- have been shown to have dominant effects in inducing a humoral or cell-mediated immune response, respectively. incorporation of dna coding for il- into the genome of ectrome-lia virus greatly increased the virulence of this virus in otherwise resistant mice, and even if the latter had been immunized to increase resistance before challenge ( ). the most fascinating and exciting of the recent approaches to vaccine development has been the injection of plasmids containing the dna coding for antigens of interest, either directly into muscle cells or as dna-coated tiny gold beads into the skin, using a "gene gun" ( , ) . in the latter case, some coated beads bind to the toll-like receptor (tlr) on langerhan's (dendritic) cells, and during passage of the cells to the draining lymph nodes ( h), the expressed foreign protein is processed. appropriate peptide sequences attach to mhc molecules and the resulting complex is expressed at the cell surface. these complexes are recognized by naïve, immunocompetent t cells in the node and this stimulates activation of the cells. a basically similar process occurs after muscle injection. in lower primates, this procedure primarily induces a type- t-cell response, resulting in both an antibody-and cell-mediated immune response, with both cd + and cd + effector t cells. in many respects, this is similar to the response following an acute infection. one advantage is that the response to dna occurs in the presence of specific antibody to the encoded antigen. our knowledge of the properties of lymphocytes-the cell type of major importance in vaccine development-has increased enormously in recent years ( ). the major role of b lymphocytes is the production of antibodies of different isotypes and of course, specificity. the other class of lymphocytes, t cells (so-called because they mature in the thymus), consist of two main types. one, with the surface marker cd , exists in two subclasses, the th- and th- cells (h = helper activity). the major role of th- cells is to help b cells differentiate, replicate, and in the form of plasma cells, secrete antibodies of a defined specificity, and of different subclasses, igg, ige, and iga. furthermore, there are different subclasses of igg. this is facilitated by the secretion of different cytokines (interleukins), which are listed in table . th- cells also have a small but important role in helping b cells produce antibody of various igg subtypes, but the overall pattern of cytokine secretion is markedly different. factors, such as interferon (ifn)-γ, tumor necrosis factor (tnf)-α, and tnf-β, have several functions, such as antiviral activity and up-regulation of components (e.g., mhc antigens on other cells). with macrophages, this can lead to their activation and if infected, greater susceptibility to recognition by t cells. th- cells can also mediate (through cytokine secretion) delayed-type hypersensitivity (dth) responses that may have a protective role in some infections. it was recognized that during an infection by a pathogenic microorganism, the immune response may be suppressed to limit immunopathology, and this has recently been shown to be due to a particular cd + t cell, now called a regulatory cell (treg). it is not expected that these cells would be activated during vaccination by a live attenuated vaccine. a second type of t cell has the cell-surface marker cd , and its pattern of cytokine secretion is similar to that of cd + th- cells, although they generally mediate dth responses rather poorly. as primary effector cells in vivo, cd + t cells recognize and lyse cells infected by a virus or by intracellular bacteria and some parasites, hence the name cytotoxic t lymphocytes (ctls). an important aspect of this arm of the immune re- molecular biotechnology volume , sponse is that susceptibility of the infected cell to lysis occurs shortly after infection, and many hours before infectious progeny is produced, thus giving a "window of time" for the effector cell to find and destroy the infected cell ( ). both cd + and cd + t cells recognize on the surface of the apc a complex between a mhc molecule and a peptide from the foreign antigen. in the first case, the peptide (average length, amino acids), which is derived from antigen being degraded in the lysosomes, complexes with class ii mhc antigens. in the second case, the peptide (average length, amino acids) is derived from newly synthesized antigen in the cytoplasm, and binds to class i mhc molecules. class ii mhc antigens are expressed mainly on cells that can act as apcs. in contrast, nearly all cells in the body may become infected and express class i mhc molecules. thus, the role of ctls has been described as performing a continuous molecular audit of the body ( ). table ascribes particular roles to specific antibody and to the t-cell subsets. some general conclusions regarding adaptive immune responses are: . specific antibody is the major mechanism for preventing or greatly limiting an infection; . ctls are the major mechanism for controlling and finally clearing most acute intracellular infections ( during an acute model infection such as murine influenza, the sequence of the appearance in the infected lung of adaptive responses is: first, cd + th cells, then cd + ctls, and finally antibody-secreting cells (ascs). the ctls are largely responsible for virus clearance and it has been thought that the subsequent decline in ctl activity that occurs shortly after infectious virus can no longer be recovered ( ), and this is because of the short half-life of these cells. the two dominant cytokines are il- , which favors a th- response ( ), and il- , which favors a th- response. the production of il- , which results in the enhancement of the antibody response, may curtail the life of the ctls. the finding that if il- is "artificially" induced very early after infection, ctl production is substantially suppressed ( ), supports this notion. one important point is that a large pool of memory ctls has already been formed by the time that ctl activity usually disappears. these memory cells persist, and are rapidly activated if the host is exposed to the same or a closely related infectious agent at a later time. it is now recognized that, as well as affecting the magnitude and persistence of immune responses to noninfectious preparations, adjuvants can also greatly influence the type of immune response ( ). some adjuvants, such as alum and cholera toxin and its b subunit, favor cd + th- responses. water-in-oil emulsions, such as freund's complete and incomplete adjuvant and lipopolysaccharide, favor a th- response. a variety of delivery systems is available for the induction of ctl responses ( ). although new technologies are making it more likely that attempts to develop vaccines to an in-creasing number of infectious agents will be successful, many other factors can influence the final outcome. some of these are: . the simpler the agent, the greater the chance that important protective antigens will be identified. . the occurrence of great antigenic diversity in the pathogen can be a major hurdle, especially in the case of rna viruses because escape mutants (antigenic drift) may readily occur. . integration of dna/cdna into the host cell genome is likely to lead to lifelong infection, which is difficult for a vaccine to prevent/overcome. . if a sublethal natural infection does not lead to protection from a second infection, it becomes necessary to understand the pathogenesis of the infection and how the normal protective responses (antibody, cell-mediated) are subverted or evaded. . the ready availability of an inexpensive animal model that mimics the human disease can be very helpful, but is rare. most early studies are done with mice but it is becoming increasingly common to then work with lower primates before initiating clinical studies. vaccine delivery is a major cost component in vaccination programs. combining vaccines so that three or more can be administered simultaneously results in considerable savings, therefore there are determined efforts to add other vaccines to longtime successful combinations (diphtheria, acellular pertussis, tetanus [dapt] and mmr) such as dapt-hepatitis b-h aemophilus influenzae type b. there must be compatibility and no interference by one component on the other. there is the risk of antigenic competition that occurs at the t-cell level, and the likelihood of such interference is difficult to predict. however, in the case of mixtures of protein/carbohydrate conjugates, using the same carrier protein should decrease this risk. the use of the same vector (e.g., the same poxvirus) in mixtures of chimeric constructs should minimize this difficulty. vaccination with dna should offer the same advantage. some antigens to be used in vaccines, mainly subunits, and many antibodies can be produced in transgenic plants. initially it was thought that simply "eating the fruit of the plant" would achieve satisfactory vaccination (i.e., edible vaccines) and that the technique would be particularly suitable for use in developing countries ( ). but the idea has evolved more recently into obtaining licensed products under strict biological regulations and subject to the same safeguards as products produced by conventional techniques ( ). in the long run, such products may be less expensive than conventional products, but not by a very large margin. most recently, although different antigens have been produced in this way, there are still substantial regulatory hurdles to be overcome in the use of these products ( ). in an effort to limit vaccine administration by needles, progress is being made in inducing immunization by direct application of the antigen, with a strong adjuvant such as cholera toxin, to prewashed skin, using a patch. with this technique, called transcutaneous immunization, the antigen rapidly contacts and is taken up by dendritic (langerhan's) cells in the epidermis and later, on arrival at draining lymph nodes, the processed antigen is presented to t cells ( ). using a gene gun is an efficient way of introducing dna coding for different antigens into the epidermis and directly into langerhan's cells and sometimes into the cell's nucleus. tiny gold beads are coated with the dna and this becomes an efficient way to induce an immune response ( ). in contrast with the time-honored approach of giving several doses of the same preparation of an antigen to obtain a stronger and longer immune response, the concept arose of priming with one formulation of an antigen and boosting with a different formulation. immunization of naïve volunteers with an hivgp / vaccinia virus construct followed by boosting with a recombinant gp preparation gave higher anti-gp antibody titers compared to using either preparation for both priming and boosting ( ). it was then shown that molecular biotechnology volume , mice immunized with a chimeric dna preparation and later boosted with chimeric fowlpox virus both expressing influenza hemagglutinin (ha), gave anti-ha titers up to -fold higher than those found after two injections of the same preparation ( ). this approach has been vigorously pursued to induce high and persistent ctl responses to hiv- , siv, ebola virus, m. tuberculosis, and plasmodia antigens with positive results in mice or monkeys ( ). two phase clinical trials using preventive aids vaccine candidates, one by an australian consortium supported by the national institutes of health, washington, and the other, an oxford-nairobi-uganda partnership supported by the international aids vaccine initiative, new york, were recently completed. both used chimeric dna followed by a chimeric live poxvirus vector to generate strong cell-mediated immunity (cmi) responses (ctls), but unfortunately they gave disappointing results ( ). the reason is not clear but at least temporally, the results cast a shadow over this technology in humans. studies are proceeding using different chimeric live vectors for priming and boosting. whole genome sequencing of complex microbes such as bacteria and parasites is poised to revolutionize the way different components are chosen to form a vaccine ( ).this enables the characterization of potential candidate proteins that might be recognized by infectivity-neutralizing antibodies, and which ones may provide important t-cell determinants. in one recent example, mice immunized with with of proteins from steptococcus pneumoniae that had been identified from the dna sequence as having appropriate structural characteristics, were protected from disease when later challenged with this organism ( ). infectious agents in his book, guns, germs and steel ( ), jared diamond retraces the history of civilization over the past , yr. (this should be of special interest to those younger than yr old.) because of the exposure, of especially europeans, to domesticated and other animals for most of this period, some of the animal infectious agents had evolved over many years to become essentially human infectious agents. these include smallpox, influenza, tuberculosis, malaria, plague, measles, and cholera. many infected people died, but the population survived and expanded. but when they traveled abroad, some of the group might be infected. beginning with columbus's voyage in , shortly followed by cortes' invasion of the aztec empire, the european invasion of north america, and in the late eighteenth century, the british invasion of australia, the mortality rate of indigenous peoples exposed for the first time to these agents was often as high as % and occasionally much higher. within the first yr, the indigenous populations in these regions had decreased by at least %, due mainly to the introduced infections. at the beginning of the twentieth century, in well-off countries, families were large because it was expected that several children in one family could die from childhood infections. parents were especially proud if they reached the biblical goal of three score years and ten. but why was the mortality rate so high? the nobel prize in physiology or medicine was awarded to peter doherty and rolf zinkernagel for research they had done in the early s at the australian national university. based on their experimental findings, they predicted that ctls could distinguish between infected and normal cells because the former expressed at the cell surface a complex between a mhc molecule and a protein (later shown to be a peptide) from the infecting virus ( ). thirteen years later, this interpretation was shown to be correct by xray crystallography ( ). the human chromosome contains loci, each containing dna coding for a particular mhc antigen. at conception, the fetus receives such genes from each parent giving a total of . dna at six loci codes for class i mhc molecules and at the other loci, the dna codes for class ii mhc molecules. yet on a population basis, there are many, sometimes > dna molecules coding for different mhc antigen specificities, anyone of which can occupy the same loci in different individuals. thus, individual humans possess only a small sample of the total number of mhc specificities. one result is that it was highly unlikely that all people would have the best protective response to all infections. person a may make a strongly protective response against infection x, but a weakly protective response against infection y. person b may make the opposite responses. the resistance or susceptibility of different inbred strains of mice to different pathogens supports this view. thus, c bl mice are relatively resistant to ectromelia (mousepox) infection, whereas balbc mice are susceptible, with a high mortality rate. the protection afforded by the vaccination programs, especially death from childhood infections in nonindigenous populations in developed countries, has dramatically changed the previous family pattern. at present, the family size is generally much smaller as parents expect all children to survive, and many adults are living much longer, well into their s or s. there are many known infectious agents for which vaccines are not yet available, but the biggest danger is emerging infectious diseases. a recent article on this topic lists about a dozen such agents ( ). table lists some of these agents where there is substantial evidence for their animal origin, showing them as zoonoses or vector-borne diseases. many induce a high mortality rate in humans. some such as sars (severe acute respiratory syndrome) has spread around the world. there were three influenza virus pandemics in the past century, the "spanish flu" causing million deaths due to the special properties of the hemagglutinin gene of the virus ( ). the appearance of a/h n in hong kong in and in neighboring countries in , and possibly the first indication (new york times, sept , ) that it may have recently spread from one person to another, has heightened concern. the maximum opportunity for a pandemic occurs when the bird virus infects a person currently infected with a human strain, allowing the different particles to enter the same cells and while replicating, to exchange genes. the hiv- , which causes aids, is also a very serious hazard as there have now been million infections worldwide. it jumped from non-human primates about yr ago, probably by the consumption of infected "bushmeat" and this form of transmission has occurred six more times since. in richer countries, it is controlled by a complex mixture of drugs. in the absence of treatment, the average time to death is yr but can be as long as yr, so it is difficult to know the exact death rate, but it is > %. there is a small group of long-term nonprogressers whose immune system keeps the hiv virus levels very low. in some african countries, which have about a % incidence of hiv infection, the average lifespan has dropped from yr to about yr. despite intensive efforts, it has so far not been possible to develop an effective vaccine. other infections listed in table have mortality rates varying from about % (sars) to between % and % (ebola and marburg hemorrhagic fevers). there is a number of infections called reemerging diseases. these are infections that were reasonably localized but have suddenly spread to other regions. although originally confined to one region, one example, west nile virus, is causing epidemics of encephalitis in the united states and russia due to migratory birds, and an abundance of both vector mosquitoes and susceptible local bird species in those two countries. as the population of the world grows and there is increasingly frequent interaction between mankind and wildlife, it is inevitable that new diseases and re-emerging diseases will continue to occur. efforts to rapidly identify, investigate, and control such outbreaks will continue to be coordinated by who, ably assisted by designated authorities in different countries such as the cdc in the united states. although special situations such as hiv/aids can be kept under control in rich countries by a cocktail of drugs, the basic global need is to be able to develop appropriate vaccines. this must continue to be a top priority if we and our descendants are to live fruitful lives vaccines advances in immunology: vaccines and vaccination rotavirus vaccine temperature sensitive mutant vaccines influenza vaccine-live current status of live, attenuated influenza virus in the us regulation of host immune responses by modification of salmonella virulence genes the use of complete genome sequences in vaccine design nyvac, a highly attenuated strain of vaccinia virus cocirculation and divergence of human influenza viruses pertussis vaccine chemoimmunologial studies on conjugated carbohydrate proteins. . immunological specificity of synthetic sugar-protein antigen preparation of polysaccharide-conjugate vaccines genetic detoxification of bacterial toxins vaccine perspectives from the vantage of hepatitis b the cellular basis for the lack of antibody responses to hepatitis b vaccine in humans assuring the quality and safety of vaccines. regulatory expectations for licensing and batch release vaccine protocols an epidemiological and clinical evaluation of guillain-barre syndrome reported in association with the administration of swine influenza virus vaccine acute encephalopathy followed by permanent brain injury or death associated with further attenuated measles vaccines vaccines today adverse events associated with childhood vaccination. evidence bearing on causality. institute of medicine indications and contraindications for vaccines used in the expanded programme of immunization increased mortality following high titer measles vaccines: too much of a good thing intussusception among infants given an oral rotavirus vaccine measles, mumps, rubella immunization, autism, and inflammatory bowel disease: an update. communicable disease intelligence mmr vaccine: the continuing saga smallpox and its eradication. world health organization new polio cases confirmed in guinea, mali and sudan. cases reported as kano, nigeria, resumes immunizations is global measles eradication feasible? perspectives for the elimination/eradication of diseases with vaccines crystal structure of a neutralizing human igg against hiv- : a template for vaccine design synthetic peptide vaccine design: synthesis and properties of a high density multiple antigenic peptide system efficacy trial of a malaria vaccine spf in gambian infants towards a vaccine for rheumatic fever: identification of a conserved target epitope on m protein of group a streptococci new multi-determinant strategy for group a streptococcal vaccine designed for the australian aboriginal population live viral vectors: vaccinia virus live viral vectors: construction of a replication-deficient recombinant adenovirus academic development of attenuated salmonella strains that express heterologous antigens expression of mouse interleukin- by a recombinant ectromelia virus suppresses cytolytic lymphocyte responses and overcomes genetic resistance to mousepox molecular medicine; dna vaccines dna vaccination: an update the immune system cytotoxic t cells recognise very early, minor changes in ectromelia-infected target cells unravelling the mysteries of molecular audit: mhc class i restriction the immunology of vaccination the role of antibody in controlling and/or clearing virus infections expression of cytokines by recombinant vaccinia viruses: a model for studying cytokines in virus infections in vivo the immune response to antigens; the immunological principles of vaccination the adjuvant effect of il- in a vaccine against leishmaniasis major interleukin mediates down regulation of antiviral cytokine expression and cytotoxic t lymphocyte responses and exacerbates vaccinia virus infection in vivo adjuvant formulations for experimental vaccines comparison of numerous delivery systems for the induction of cytotoxic t lymphocytes by immunization plant biotechnology: new procedures and applications oral vaccines derived from transgenic plants edible vaccines not ready for main course transcutaneous immunization: a human vaccine delivery strategy using a patch the powderjet particle-mediated epidermal delivery of dna vaccines. a new technology augmentation of human immunodeficiency virus type- neutralizing antibody by priming with gp recombinant vaccinia and boosting with rgp in vaccinia-naive adults generation of enhanced immune responses by consecutive immunization with dna and recombinant fowlpox virus hiv dodges one-two punch the use of complete genome sequences in vaccine design use of a whole genome approach to identify vaccine molecules affording protection against streptococcus pneumoniae infection guns, germs and steel. a short history of everybody for the past , years. vintage a biological role for the major histocompatibiliy antigens the foreign antigen-binding site and t cell recognition regions of class histocompatibility antigens the challenge of emerging and re-emerging infectious diseases enhanced virulence of influenza a viruses with the haemagglutinin of the pandemic virus key: cord- -emmoeuqd authors: cooper, joanne c.; dealtry, gillian b.; ahmed, mohamed abdelrahman; arck, petra c.; klapp, burghard f.; blois, sandra m.; fernández, nelson title: an impaired breeding phenotype in mice with a genetic deletion of beta- microglobulin and diminished mhc class i expression: role in reproductive fitness( ) date: - - journal: biol reprod doi: . /biolreprod. . sha: doc_id: cord_uid: emmoeuqd beta- microglobulin (b m) plays a pivotal role in the biology of mammals, including its association with major histocompatibility complex (mhc) class i gene products. the latter molecules have been shown to affect reproduction in both mice and humans, although the exact mechanism is still unknown. here we report the results of a longitudinal study of the reproductive performance of a genetically modified b m deficient mouse strain with low mhc class i expression. our data show that this mouse strain has an impaired reproductive performance. however, the mice superovulate well and show a normal estrous cycle. breeding studies from crosses between the transgenic mice and the wild-type parental strain show that b m deficient mice have a significantly lower frequency of mating than the control b m(+/+) mice. in addition, the litter size and weaning success of b m deficient mice were lower than the control. perinatal lethality of the b m deficient offspring was also inflicted by cannibalism of the young pups by the b m deficient female. the impaired breeding phenotype (ibp) can be reversed by reintroducing the b m gene in f heterozygous b m(+/−) animals; thus the presence of b m confers a normal breeding pattern. the acquisition of an impaired breeding phenotype (ibp) as a result of the knockout of b m directly implicates b m in the reproductive cycle of mice and raises the possibility of an effect of b m on the reproduction of other mammals. beta- microglobulin (b m) is the product of a single copy, low-polymorphic gene, which encodes a dalton nonglycosylated protein belonging to the immunoglobulin superfamily. the mouse b m gene is di-allelic, encoding two proteins that differ at a single amino acid residue [ ] . b m exists as a soluble product in serum, and, since its gene lacks a transmembrane coding exon, it is released into solution from a variety of cells. the b m protein forms physical associations with a number of molecules; these include major histocompatibility complex (mhc) class i and cluster of differentiation (cd ) [ ] . b m also associates with the fc receptor of the placenta, where it allows trans-placental transport of igg from the mother to the foetus [ ] , and associates with the fc receptor of neonatal gut epithelium for transport of igg in the gut epithelium [ ] . the major histocompatibility complex-related fc receptor (fcgrt) is also implicated in igg homeostasis in the adult [ ] . in addition, it participates in the regulation of albumin [ ] and in hfe (iron transport) [ ] . the b m protein plays a pivotal role in the biosynthesis of mhc class i molecules, controlling assembly, peptide binding, and cell-surface expression [ ] . the majority of the fully assembled mature mhc class i molecules exist as an assembled complex with b m, with the exception of free class i heavy chains of the h- b haplotype [ ] . thus b m is an essential molecule in the process of antigen presentation in the adaptive immune system. one dramatic effect of the deletion of b m in homozygous knockout mice is that cell-surface and cytoplasmic expression of mhc class i molecules are significantly reduced [ ] , leading to a reduction of the cd À cd þ subpopulation of mature t cells [ , ] . as a consequence, adult mice are susceptible to some parasitic diseases such as trypanosoma cruzi infections [ ] , but not leishmaniasis [ ] or blood-stage malarial infections [ ] . these mice also have delayed viral clearance and increased mortality after theiler virus infection [ ] ; and are susceptible to mouse hepatitis virus with the infected mice being more susceptible to acute hepatitis and encephalitis [ ] . in rheovirus infection, b m À/À mice showed enhanced intestinal mucosal and systemic immune responses by elevated igg and iga [ ] . however, quite remarkably, in the absence of infections, a b m À/À strain has a healthy phenotypic appearance, develops well into adulthood and has a normal life span, despite a deficiency in iron metabolism [ ] . of critical importance is the finding that b m knockout mice are distinguishable by scent from identical mice with an intact b m gene [ ] , thus supporting the notion that mhc products are important in providing olfactory recognition and odortypes in rodents and humans [ ] [ ] [ ] [ ] [ ] [ ] . in the course of experiments designed to generate mouse embryos for gene transcription studies and immuno-detection of mhc products [ ] [ ] , we consistently observed an impaired reproductive capacity in the b m deficient mice. we report this observation here, which we have termed impaired breeding phenotype (ibp), and discuss the biological implications of such a defect. all the mice in this study were of the c bl/ genetic background. our transgenic founding colony consisted of females and males, all homozygous b m À/À on the c bl/ background [ ] . mice from this colony, the wild-type parental strain (b m þ/þ c bl/ ) and heterozygous offspring of crosses between these two strains were used. the frequency of mating was defined by the appearance of postcopulatory vaginal plugs after overnight pairing of single b m À/À , b m þ/þ , or b m À/þ males with groups of up to three females of b m À/À , b m À/þ , or wild-type b m þ/þ genotypes. breeding data were collected over a period of / years from age-matched crosses generated between b m À/À b m À/À ( pairs of mice; breeding period: - weeks of age); b m þ/þ b m þ/þ ( pairs of mice; breeding period: - weeks); b m þ/þ b m À/À ( pairs of mice; breeding period: - weeks); b m À/À b m þ/þ ( pairs of mice; breeding period: - weeks) and b m þ/À b m þ/À ( pairs of mice; breeding period: - weeks). once a mouse was seen to be pregnant, the male was removed from the breeding pair and the female was housed alone until her pups were weaned. thus stress due to overcrowding or the presence of the male was avoided. all procedures described within were reviewed and approved by the institutional animal care and use committees of the university of essex and were performed in accordance with the guiding principles for the care and use of laboratory animals. in order to avoid shifts in the circadian rhythm and hence the estrous cycle, all mice were bred and maintained in a temperature-, humidity-, and lightcontrolled room (lights-on to h) and housed in identical cages (university of essex, biological services unit). mice were allowed food (laboratory diet rm [e], sds ltd., witham, essex) and water ad libitum. our mouse colony was free of mouse hepatitis virus. this behavioral characteristic was defined as a defect in the normal care of the newborn pups, reflected in visible physical damage to the pups and detachment of the mother from the newborn litter. adult ( - weeks of age) females were stimulated with iu ecg (folligon, intervet uk ltd., milton keynes, uk) and iu hcg intraperitoneal (pregnyl, organon laboratories ltd., cambridge, uk) at h, h apart, to induce superovulation. the control group consisted of naturally cycling females of the same strain (without stimulation). the estrous cycle was studied using cells removed by gentle mechanical disruption following vaginal washes, stained with hematoxylin/eosin solution and analyzed by light microscopy. samples were classified as follows: proestrus, nucleated epithelial cells with occasional leukocytes and small degenerative nuclei cells; estrus, numerous cells from squamous epithelium and small epithelial cells; metestrus, numerous leukocytes and small cornified epithelial cells, and diestrus, mucus and nucleated cells. data are expressed as means and sem. statistical significance between groups was tested using chi-square test for homogeneity and mann-whitney utest for two proportions. significance was set at p . (*), p . (**), p . (***). a longitudinal study designed to quantify the reproductive fitness of the b m deficient mouse was conducted. due to a steady depletion of breeding stocks of the transgenic mice, we began to outbreed the transgenic b m deficient c bl/ mice with wild-type c bl/ mice to produce b m heterozygous stocks. the f litters from these mating were then crossed to b m þ/þ female b m þ/þ male b m À/À female b m À/À male * *** b m À/À female b m þ/þ male *** b m þ/þ female b m À/À male *** b m À/þ female b m À/þ male * a values are presented as mean sem. * p , . and ***p , . versus wild-type females analyzed by student t-test. percentage of natural deaths occurring before weaning is indicated for number of mating crosses (n) is shown. percentage of natural deaths occurring with b m À/À genotype was comparable to the b m þ/þ wild-type c bl/ . percentage natural deaths fell for the b m þ/þ b m À/À and the b m þ/À b m þ/À mating. results are expressed as mean sem. chisquare test was used to determine differences between the groups. **p . ; and ***p . . replenish the b m negative mice. the data show that the b m deficient mice have a lower number of litters born than the control group of c bl/ mice (p , . ). the total number of pups born to b m deficient mice was significantly lower than the number born to the control mice (p , . ) ( table ) . analysis of postnatal mortality as determined by natural death (not cannibalized animals) (fig. ) , revealed that the b m homozygous deficient mice (b m À/À ) had a similar proportion of postnatal natural deaths ( . %) to the c bl/ b m þ/þ homozygous control mice ( . %), although the control group had a higher total number of pups born. interestingly, overall postnatal natural death declined when a b m þ/þ female was crossed with a b m À/À male ( . %) or a b m À/À female was crossed with a b m þ/þ male ( . %) (p , . ). this decline was even greater when the heterozygous b m þ/À mouse strain was crossed with identical b m þ/À mice ( . %) (p , . ), suggesting that crossing mice heterozygous for b m confers a higher rate of survival of the offspring. since the progeny of this cross will include genotypes of b m þ/þ , b m þ/À , or b m À/À , this finding suggests that the maternal genotype may be an important determining factor rather than the embryo genotype. we then examined the postnatal mortality rate by cannibalism (fig. ) and observed that pups born from homozygous b m À/À parents exhibited a significantly higher rate of death than pups born to the b m þ/þ mice ( . % and . % of total pups born respectively [p , . ]). the presence of the b m gene appears to confer a higher survival rate since the homozygous b m þ/þ female mice crossed with the homozygous b m À/À males exhibited a low cannibalism death rate ( . %). the reverse mating combination that is homozygous b m À/À female mice crossed with the homozygous b m þ/þ males also exhibited a low cannibalism death rate ( . %). similarly, the mating of b m þ/À heterozygous individuals showed a lower death rate by cannibalism ( . % [p , . ]). the impact of the aforementioned variables on the weaning success of the b m À/À deficient mice as measured by the survival rate of the pups to adulthood was examined (fig. ) . a measure of association between the survival rate and the mating genotypes revealed a high statistical significance (p , . ). mice born to a cross between heterozygous b m þ/À females and b m þ/À males exhibited the highest survival rate. similar levels of significance were observed between the rate of mortality (natural death plus cannibalism) and the type of mating (p , . ). pups born to b m À/À parents showed the poorest survival rate. in addition, the b m À/À mice showed a very significant disproportionate increase in postnatal mortality due to cannibalism when compared with the mice carrying the b m gene (p , . ). fig. . cannibalistic behavior by the mother is increased on a b m À/À background. percentage of pups cannibalized by the mother is indicated for all four mating crosses b m À/À b m À/À ( . %), b m þ/þ b m þ/þ ( . %), b m þ/þ b m À/À ( . %) b m À/À b m þ/þ ( . %) and b m þ/À b m þ/À ( . %). number of mating crosses (n) is indicated. incidence of cannibalism falls as b m is reintroduced into the genotype of the mice. results are expressed as mean sem. chi-square test was used to determine differences between the groups. ***p . . weaned percentage is illustrated for all four mating crosses; b m À/À b m À/À ( . %), b m þ/þ b m þ/þ ( . %), b m þ/þ b m À/À ( . %), b m À/À b m þ/þ ( . %) and b m þ/À b m þ/À ( . %). number of mating crosses (n) is indicated. due to a high degree of cannibalism in the b m À/À -deficient mice, the weaned success is lower compared with the b m þ/þ wild-type mice. incidence of natural deaths and cannibalism falls for the b m þ/þ b m À/À and b m b m þ/À mating crosses, and hence weaning success increases. results are expressed as mean sem. chisquare test was used to determine differences between the groups. ***p . . cooper et al. mating frequency was significantly reduced in the b m deficient dams compared with the b m þ/þ wild-type mice. a reduction of the percentage of mating on night to night on b m deficient ( , p , . ) versus the b m wild-type mice ( ) was observed. i. nterestingly, the capacity of the b m-deficient mouse strain to respond to superovulation with the hormone hcg and ecg was similar to other mouse strains ( table ) . moreover, there was no difference in the estrous cycle analysis between b m deficient and wild-type female mice (fig. ) . these results suggest that the b m À/À mice have an intact and responding corpus luteum and therefore a normal ovulation pattern. the possibility of resorption of the entire litter as a cause of poor breeding was ruled out, since mating of a b m À/À female, detected by the presence of vaginal plugs, normally led to a successful pregnancy and the birth of a litter. in this study we have shown that the b m knockout mouse has an impaired breeding phenotype (ibp). the results implicate the absence of b m, and consequently diminished expression of mhc class i in the altered reproductive performance of this mouse strain. the female b m À/À mice also exhibited a high level of cannibalism towards the newborn pups, suggestive of an alteration of the maternal nurturing behavior and the mechanism that control progeny recognition by the mother. the cannibalistic behavior of the b m À/À mice extends observations on the relationship of offspring recognition by the mother, and communal nesting and communal nursing in mice [ ] . b m is found in the serum and body fluids, both in a free form and associated with mhc class i heavy chain. previous studies have shown that soluble mhc class i molecules can act as pheromones, influencing mating signals within rodent colonies [ ] [ ] [ ] [ ] [ ] . therefore, a possible mechanism operating in the b m-deficient mice is the absence of secreted mhc class i products, and thus the absence of mating signals resulting in impaired breeding. this mechanism might also render the female unable to recognize the offspring as her own, leading to cannibalistic behavior. it has been postulated that mhc genes act as kin-recognition markers to increase relatedness among communal nesting partners [ , , ] . mice can recognize one another by individual characteristic body odors that reflect their genetic constitution at the mhc [ ] and mothers are able to recognize syngeneic pups from other pups differing only in mhc [ ] . pup lethality, a form of cannibalism affecting female nurturing behavior, has also been reported as a result of fosb gene deficiency [ ] ; although in a different context, the phenotypic effect bears some similarity to the effect of the b m deficiency. in humans, there is also data suggesting that mhc genes are linked to olfactory receptors and act as odor recognition cues in mating choices [ ] [ ] [ ] [ ] [ ] [ ] . the homozygous b m À/À and heterozygous b m þ/À females produce the same number of oocytes following superovulation as the control b m þ/þ females, suggesting that they should produce as many offspring per mating as their wild-type counterparts. however, litter sizes are smaller indicating that a lack of b m impairs embryo development. the b m À/À mice have a severely diminished expression of mhc class i products both in the adult tissues and throughout development. a reasonable postulate is that b m and, in turn, mhc deficiency are implicated in the poor breeding capacity of the b m deficient mice by affecting mating, embryo development, and maternal behavior. the ibp phenomenon can be rescued by introducing the b m gene back into the genome of the mice. breeding experiments in which c bl/ b m À/À transgenic mice were crossed with the c bl/ b m þ/þ parental mouse strain restored the reproductive pattern of the heterozygous progeny to normality. these observations are consistent with the notion that mhc disassortative mating preferences, driven probably by pathogens, favor mhc heterozygosity [ ] . the impact of variations in the maternal uterine genotype and maternal cytoplasmic contributions to the oocyte has been controlled by use of mice of identical c bl/ genetic background in all crosses. the only genetic variable is the presence or absence of functional b m allele(s). in previous studies, we have shown that c bl/ (h -d b ) derived mhc class i products are synthesized at the one-cell embryo stage, arguing for a requirement of mhc in development [ ] . this finding was confirmed in another mouse strain; arcellana-panlilio and schultsz showed that in the cd- swiss albino mice, h- k q products were also expressed at all stages of preimplantation development [ ] . the exact mechanism for a role of mhc in early development is still unclear. it may involve stage-specific antigen synthesis to allow differentiation and cell division, similar to the role of differentiation molecules involved in the ontogeny of lymphocytes [ ] . mhc-mediated transmembrane signaling pathways, particularly in the case of h -q products, which have glycosidylinositolphosphate, anchored transmembrane domains [ ] might also be a signal mechanism operating in mammalian embryogenesis. we have postulated that a background level of mhc class i is required for successful mouse development and for protection against maternal large granular cells and natural killer cells (nk) [ ] , as it has become established that in the adult, nk effector cell recognition involves targeting cells with diminished mhc expression [ ] . conversely, it has also been shown that overexpression of mhc class i products has a detrimental effect on development. indeed, jaffe and colleagues have shown that embryos overexpressing h- d d antigen under the control of the human beta-actin promoter, when introduced into pseudopregnant syngeneic foster females fail to develop past the mid-point of gestation; while control embryos that overexpressed an irrelevant protein developed normally [ ] . the above observations suggest that a homeostatic level of mhc might be protective and influence normal development and viability early in life. it is possible that in the b m À/À mouse strain the lack of b m impairs the synthesis and cell-surface expression of mhc class i, thus altering the normal balance of mhc expression during early stages of development. b m normally associates with the fc receptor of the placenta [ ] , therefore, in the pregnant female b m À/À mice, the fc receptor may have a reduced capacity to transport igg molecules across the placenta to the developing embryo, affecting development and reducing litter size. taken together these results indicate that the b m deficient mouse strain has an impaired reproductive pattern, that we have termed impaired breeding phenotype (ibp). the acquisition of ibp as a result of selective genetic deficiency of b m directly implicates the b m protein in the reproductive cycle of mice and raises the possibility of an effect of b m in the reproduction in humans. the natural history of the major histocompatibility complex expression of murine cd on gastrointestinal epithelium a major histocompatibility complex class i-like fc receptor cloned from human placenta: possible role in transfer of immunoglobulin g from mother to fetus an fc receptor structurally related to mhc class i antigens the mhc class i-like igg receptor controls perinatal igg transport, igg homeostasis, and fate of igg-fc-coupled drugs the major histocompatibility complexrelated fc receptor for igg (fcrn) binds albumin and prolongs its lifespan a novel mhc class i-like gene is mutated in patients with hereditary haemochromatosis early events in the assembly of mhc class i antigens b -microglobulin is not required for cell surface expression of the murine class i histocompatibility antigen h- d b or a truncated h- d b normal development of mice deficient in beta- -m, mhc class i proteins, and cd þ t cells beta- -microglobulin deficient mice lack cd À cd þ cytolytic t cells the cd þ repertoire in b -microglobulin deficient mice is biased towards reactivity against selfmajor histocompatibility class i susceptibility of b -microglobulin-deficient mice to trypanosoma cruzi infection targeted activation of cd cells and infection of b -microglobulindeficient mice fail to confirm a primary protective role for cd cells in experimental leishmaniasis resolution of blood-stage malarial infections in cd þ cell deficient b -mÀ/À mice theiler's virus infection of b -microglobulin-deficient mice cellular reservoirs for coronavirus infection of the brain in beta( )-microglobulin knockout mice enhanced mucosal and systemic immune responses to intestinal reovirus infection in beta- -microglobulin-deficient mice b -m knockout mice develop parenchymal iron overload: a putative role for class i genes of the major histocompatibility complex in iron metabolism effect of b -m gene disruption on mhc-determined odortypes kin recognition and the major histocompatibility complex-an integrative review mhc-mediated fetal odourtypes expressed by pregnant females influence male associative behaviour parentprogeny recognition as a function of mhc odortype identity communal nesting patterns in mice implicate mhc genes in kin recognition discrimination of odour differences in mhc-deficient mice origin, functions and chemistry of h- regulated odorants how do major histocompatibility complex genes influence odour and mating preferences? discrimination of odortypes determined by the major histocompatibility complex among outbred mice fetal h- odortypes are evident in the urine of pregnant female mice chemosensory recognition of mouse major histocompatibility types by another species the major histocompatibility complex and the chemosensory recognition of individuality in rats mating patterns in seminatural populations of mice influenced by mhc genotype volatile signals of the major histocompatibility complex in male mouse urine allogeneic recombinant soluble mhc class i molecules modify urinary odor cues in rats mhc antigens in urine as olfactory recognition cues kin recognition and the major histocompatibility complex: an integrative review fetal odortypes are evident in the urine of pregnant female mice olfactory individuality distinctive urinary odors governed by the major histocompatibility locus of the mouse a distinctive change in odortype determined by h- d- mutation odortypes determined by the major histocompatibility complex in germ-free mice chemosensory recognition of phenotypes determined by the tla and h- k regions of chromosome- of the mouse expression of urinary h- odortypes by infant mice olfactory receptor-like genes are located in the human major histocompatibility complex evidence suggesting that the odortypes of pregnant-women are a compound of maternal and fetal odortypes mhc-linked olfactory receptor loci exhibit polymorphism and contribute to extended hla/or-haplotypes hla and mate selection: no evidence in south amerindians hla and mate choice in humans paternally inherited hla alleles are associated with women's choice of male odor a highly sensitive rt-pcr system for analysing gene expression in embryos and single cells expression and assembly of mhc class i products and associated molecules during pre-implantation development communal nesting and communal nursing in house mice, mus musculus domesticus a defect in nurturing in mice lacking the immediate early gene fosb the role of infectious disease, inbreeding and mating preferences in maintaining mhc genetic diversity-an experimental test preimplantation mouse embryos express mhc class i genes before the first cleavage division temporal and spatial expression of major histocompatibility complex class i h- k in the early mouse embryo a critical review of the role of the major histocompatibility complex in fertilization, preimplantation development and feto-maternal interactions a single gene encodes soluble and membrane-bound forms of the major histocompatibility qa- antigen: anchoring of the product by a phospholipid tail embryonic development and the major histocompatibility complex rejection of class i mhc deficient hemopoietic cells by irradiated mhc-matched mice distinct patterns of expression of mhc class i and b microglobulin transcripts at early stages of mouse development we would like to thank dr. geoffrey butcher (babraham, cambridge) and dr. richard d. jurd (university of essex, england) for reading the manuscript and for their advice and helpful suggestions. key: cord- -avkgldnp authors: perlman, stanley; wu, gregory f. title: selection of and evasion from cytotoxic t cell responses in the central nervous system date: - - journal: adv virus res doi: . /s - ( ) - sha: doc_id: cord_uid: avkgldnp cytotoxic cd t lymphocytes (ctls) are critical for the clearance of noncytopathic viruses from infected cells. this chapter discusses one mechanism used by viruses to persist—namely, the selection of a variant virus in which changes in the sequence of a ctl epitope abrogate recognition. the unique features of cytotoxic cd t cell function in the central nervous system (cns) are discussed. the role of ctl escape mutants in the viral evasion of the immune system and subsequent disease progression in non-cns infections are summarized. the immune response in the cns is similar to the response in extraneural tissue, but several aspects of the activation of the immune response, cellular trafficking, and antigen presentation are unique to the cns. although the cns has classically been considered a site of immune privilege, surveillance of the normal cns by circulating, activated lymphocytes occurs, with a limited number of lymphocytes being present in the normal cns at any given time. in mice infected with mouse hepatitis virus and in some humans persistently infected with human immunodeficiency virus type , hepatitis b virus or hepatitis c virus, ctl escape mutants play an important role in virus amplification and disease progression. the immune response in the cns is similar to the response in extraneural tissue, but several aspects of activation of the immune response, cellular trafficking, and antigen presentation are unique to the cns (reviewed in lassmann, ; lassmann et al., ; matyszak, ; williams and hickey, ) ). the function of cytotoxic cd t cells in the inflamed cns and the major histocompatibility complex (mhc) class i antigen expression in this tissue are most pertinent for the topic of this review and will be preferentially discussed in this section. although the cns has classically been considered a site of immune privilege, surveillance of the normal cns by circulating, activated lymphocytes occurs, with a limited number of lymphocytes being present in the normal cns at any given time (hickey, hsu, and kimura, ) . however, considerable data suggest that the immune response in the cns is initiated not in the parenchyma but, rather, in draining lymph nodes located in the deep cervical tissue (cserr and knopf, ) . dendritic cells, critical for antigen presentation, are virtually absent from the normal cns, suggesting that virus antigen must spread to the draining lymph node tissue before activation can occur (hart and fabre, ; matyszak, ; matyszak and perry, ) . in support of this contention, influenza virus inoculated directly into the cns parenchyma does not induce an immune response until virus spreads to the cerebrospinal fluid (csf) and, soon thereafter, to the draining lymph nodes (stevenson et al., a; stevenson et al., b) . once the immune response has been initiated, breakdown of the blood-brain barrier, presence of plasma proteins, and recruitment of leukocytes characterize the cns inflammatory response. notably, cells with the phenotype of dendritic cells are detected as part of the cellular infiltrate in inflammatory processes and are believed to contribute to sustaining the immune response during chronic inflammation (matyszak, ; matyszak and perry, ) . within the cns, cd and cd t cell effector functions are virtually the same as in peripheral sites of inflammation. cd t cells are responsible for the majority of cytotoxic activity and exhibit three effector mechanisms: perforin/granzyme-mediated killing; fas/fas ligand-mediated killing; and cytokine secretion (liu, young, and young, ; smyth and trapani, ) . in one well-documented example, perforin is critical for the cd t cell response to lcmv, both peripherally and in the cns (kagi et al., ) . fas/fasl interactions have not been documented to be critical for cd t cell effector function in the cns, but fasl expression by infiltrating cd t cells has been impli-cated in oligodendrocyte death in inflammatory settings such as experimental allergic encephalomyelitis (eae) and multiple sclerosis (ms) (d'souza et al., ; sabelko et al., ; waldner et al., ) . secretion of interferon-~/(ifn-~/) and of other cytokines are of critical importance in orchestrating multiple components of the cellular and humoral immune responses. furthermore, in some cases, cytokines are capable of directly inhibiting viral replication. for example, ifn-• appears to be critical for virus clearance from infected oligodendrocytes in mice infected with mouse hepatitis virus (parra et al., ) . in another example, genetic disruption of ifn-~ or its receptor inhibits virus clearance in strains of mice normally resistant to infection with theiler's murine encephalomyelitis virus (tmev) (fiette et al., ) . ctls also release chemokines through granule exocytosis, thereby sustaining the immune response by contributing to the influx of other inflammatory cells (reviewed in baggiolini, dewald, and moser, ; luster, ) . the influx of both antigen-specific and nonspecific cells may contribute to virus clearance but also has the potential for increasing damage to nearby uninfected cells (bystander damage). this mechanism has been postulated to be significant in the pathogenesis of several cns diseases with inflammatory components, including demyelination in animals with virus-induced or autoimmune demyelination (houtman and fleming, b; lassmann, ) . one major difference between the cns and extraneural tissue is found in mhc class i antigen expression. in extraneural tissue, mhc class i antigen is expressed constitutively by most cells. however, in the uninflamed cns, constitutive expression of mhc class i and ii antigen is infrequent and is confined to a subset of endothelial cells (mhc class i) and macrophages/microglia (mhc class ii) (reviewed in lampson, ; shrikant and benveniste, ) . expression of these molecules by astrocytes, oligodendrocytes, or neurons is not believed to occur in the normal cns. however, this conclusion may need to be modified since, in a recent study, the coordinated expression of mhc class i antigen (heavy chain and ~ -microglobulin) and of a t cell receptor component (cd ~) by neurons during development was postulated to be important for synapse formation (corriveau, huh, and shatz, ) . mhc class i and class ii expression are upregulated in the cns in multiple pathological settings with infectious or noninfectious etiologies (also reviewed in lampson, ; shrikant and benveniste, ) . as discussed above, mhc class i expression by antigen-presenting cells resident in the cns is not involved in initiation of the ctl response but is likely to be critical for its perpetuation. expression of mhc class i molecules by infected cells is also required for recognition and lysis by activated antigen-specific cd t cells. the most convincing data show that mhc class i and class ii antigens are upregulated on microglia/macrophages in inflammatory diseases of the cns, including ms and tmev-and mhv-induced demyelinating encephalomyelitis. t cell costimulatory molecules such as b - and b - are also upregulated on these cells and, in some cases, activated microglia/macrophages isolated from the cns can directly stimulate antigen-specific t cells proliferation (bo et al., ; matyszak, ; pope et al., ; xue et al., ) . mhc class i and class ii expression by astrocytes and oligodendrocytes has been demonstrated in vitro, although, in some cases, only after exposure to proinflammatory cytokines, such as interferon- (lampson, ; shrikant and benveniste, ) . whether astrocytes and oligodendrocytes upregulate mhc class i and class ii expression in the inflamed cns remains quite controversial, with expression of these molecules being reported in some studies (lampson, ; shrikant and benveniste, ) . in most recent studies, mhc antigen expression by astrocytes and oligodendrocytes in inflamed tissue was not detected (e.g., pope et al., ; xue et al., ) , but these negative conclusions must be tempered because a biologically significant level of expression might not be detectable with presently available assays. mhc class i antigen upregulation on neurons has not been reported in pathological conditions, although a recent set of elegant studies demonstrated the expression of these molecules following inhibition of electrical activity (neumann et al., ; neumann et al., ) . if verified in the infected cns, these results would suggest that only neurons sufficiently damaged by infection with a virus or another infectious agent would be recognized by antigen-specific ctls. furthermore, this mechanism would minimize destruction of infected, but still functionally active, neurons. although there is no agreement on the specifics, these studies clearly demonstrate that mhc class i and ii antigens are expressed on cns-derived cells in vitro and in vivo during inflammatory processes. as a consequence, they provide a framework for interpreting experiments that describe the selection of ctl escape mutants in the cns of virus-infected mice. occurring outside the cns the first experimental evidence for virus escape from ctl recognition by mutation of a cd t cell epitope was provided by pircher et al., ( ) . in mice that were transgenic for a t cell receptor (tcr) specific for lymphocytic choriomeningitis virus (lcmv), infection with lcmv resulted in the rapid appearance of virus mutated in the target ctl epitope. infection of tcr transgenic mice, but not of immunocompe-tent b mice, with mutant lcmv resulted in a delay in the kinetics of virus clearance. similar results were obtained when wild-type mice were infected with lcmv variants in which some or all of the appropriate ctl epitopes were mutated (lewicki et al., ; moskophidis and zinkernagel, ) . notably, lcmv escape mutants are not generated in immunocompetent mice after infection with wild-type virus. in these mice, ctls recognizing multiple epitopes are detected, suggesting that an immune response directed against several epitopes precluded the selection of ctl escape mutants, at least in settings in which virus clearance was relatively efficient. a subsequent study suggested that ctl escape mutants may emerge and become the predominant virus in human populations in which a single hla allele is overrepresented, and in which an immunodominant epitope is restricted by that allele. the strain of epstein-barr virus (ebv) circulating in humans residing in the coastal regions of papua new guinea contains a mutation in an immunodominant cd t cell epitope that diminished binding to the hla-all molecule (de campos-lima et al., ) . hla-all is present at a high frequency in this population, suggesting that the selection of virus encoding this mutation was immune-driven. however, this conclusion may not be warranted since the same mutation is found in virus circulating in the highland populations of papua new guinea, even though the hla-all allele is not overrepresented in that group (burrows et al., ) . the studies of lcmv-infected mice suggested that efficient virus clearance precluded the selection of ctl escape mutants. conversely, less efficient virus clearance would be predicted to facilitate the emergence of these mutants. therefore, the potential contribution of ctl mutants to pathogenesis was examined next in several persistent infections, including human and nonhuman primates chronically infected with the human immunodeficiency virus (hiv- ), hepatitis b virus (hbv), and hepatitis c virus. the emergence of ctl escape mutants during the course of persistence was documented in each of these infections (franco et al., ; mcmichael and phillips, ; weiner et al., ) . the most convincing data suggesting a role for these variants in disease progression comes from studies in which the emergence of ctl escape mutants are documented early during the infection. selection at early times postinfection is consistent with a role in delaying the elimination of virus from the infected host. in one study, borrow et al. ( ) described an hiv-infected patient in which the initial ctl response was directed at a single hla b -restricted epitope. early in the course of the infection, a mutation in a presumptive anchor residue was detected, leading to a loss of recognition by the patient's ctls. shortly thereafter, the ctl response spread to addi-stanley perlmanandgregoryewu tional, presumably less immunodominant, epitopes. these responses were able to control the virus for a short period, but were, ultimately, inadequate because the patient exhibited a rapid rate of disease progression with a fatal outcome. in another study, mutations resulting in ctl escape were identified in the nefgene in a patient during primary hiv infection. these mutations abrogated recognition by nef-specific ctls harvested from the patient, suggesting that their selection was also immune-driven and that it contributed to virus persistence (price et al., ) . in still another study, ctl escape mutants were identified in hivinfected patients several years after the primary infection, coincident with increased virus replication and decreased cd t cell counts . it is uncertain, in these cases, whether ctl escape caused disease progression or whether it was a consequence of increasingly ineffectual control of virus replication by other components of the immune system, such as antibodies and cd t cells. ctl escape mutants that emerge only after immune surveillance has diminished may still contribute to disease progression in hiv-infected patients, even if they are not the primary cause for the loss of control by the immune system. ctl escape mutants were also selected in an hiv-infected individual treated with ctls directed against a single epitope (koenig et al., ) , thereby showing that, as in lcmvinfected tcr transgenic mice, a strong, monospecific ctl response facilitated their appearance. ctl escape variants are occasionally detected in patients chronically infected with hbv, usually in the context of a narrowly focused, strong immune response (bertoletti et al., a; bertoletti et al., b) . a more common finding in patients infected chronically with hbv is the lack of a significant ctl response (rehermann et al., ) , suggesting that variation in hbv-specific t cell epitopes is important in only a minority of patients. similarly, ctl escape mutants were detected in a chimpanzee chronically infected with hepatitis c virus (weiner et al., ) , but persistence appears to be associated, most commonly, with a weak ctl response (rehermann et al., ) . mutations in an epitope or in its flanking sequences can result in escape from surveillance by cd t cells, by affecting any one of several steps along the pathway used to generate peptides for presentation by mhc class i molecules. antigen processing involves proteolytic cleav-age by proteosomes in the cytoplasm and transport of the resulting peptides to the endoplasmic reticulum for binding to the mhc class i molecule. mutations in sequences flanking a ctl epitope may affect proteolytic cleavage or transport into the endoplasmic reticulum. only a few examples of mutations affecting either of these processing steps have been described (eisenlohr, yewdell, and bennink, ; ossendorp et al., ) . in one example, comparison of the sequence of an immunodominant ctl epitope encoded by two related murine leukemia virus families (akv/mcf and friend/moloney/rauscher [fmr] ) showed that a single amino acid change present in the fmr strains abrogated cd t cell recognition (ossendorp et al., ) . the mutation did not affect binding of peptide to the mhc class i molecule or immunogenicity of a peptide corresponding to the variant epitope. rather, the loss of recognition occurred only after endogenous processing of antigen and resulted from alterations in proteosomal processing, which were shown using purified s proteosomes in vitro. these conclusions assume that processing in vitro mimics what occurs in the cell and that other differences in the amino acid sequence between the two viruses did not contribute to the observed differences. mutations in sequences flanking a ctl epitope are difficult to detect, and therefore may be more important in ctl escape than is appreciated at present. more commonly, mutations in ctl epitopes that affect binding to the mhc molecule, or affect recognition by the tcr, have been identified. some of the mutations described above, in hiv- infected individuals, diminish binding to the mhc class i molecule or result in increased rates of dissociation from the mhc molecule (reviewed in mcmichael and phillips, ) . mutations that impair binding to the mhc molecule are the simplest to interpret and, depending on the diminution of binding, will completely abrogate recognition by epitopespecific cd t cells. mutations that affected binding to tcrs were first reported in studies describing selection of lcmv ctl escape mutants in mice transgenic for an lcmv-specific tcr (pircher et al., ) . peptides mutated in residues that directly contact the tcr have also been described in virus isolated from hiv-infected patients . these mutations may result in partial or complete loss of recognition by autologous ctls. during the course of these studies on viral variation, mutations were identified in viral rna and dna isolated from patients persistently infected with hiv- or hbv that not only diminished recognition by epitope-specific cd t cells, but also inhibited recognition of wild-type epitope if both epitopes were present in the infected cell (bertoletti et al., b; klenerman et al., ) . the mechanism of this phenomenon (tcr antagonism) is not completely understood, but is believed to involve delivery of an altered signal to the cd t cell by the mutated peptide/mhc class i complex, resulting in nonresponsiveness or diminished responsiveness to the target agonist peptide (sette et al., ) . although tcr antagonism has only been demonstrated by using ctl clones, it has also been documented to occur when variant and wild-type epitopes are presented by different cells, a scenario that presumably mimics the situation in the infected host (meier et al., ) . another mechanism similar to antagonism is interference with ctl priming (plebanski et al., ) . in this case, a variant epitope does not inhibit ctl effector function but, rather, the generation of activated antigen-specific ctls from naive precursor cd t cells. this mechanism, identified in humans and mice infected with malaria, has not yet been described in any viral disease. one of the best examples of a pathological setting in which ctl escape mutants contribute to virus persistence and the development of disease is that of mice infected with the neurotropic coronavirus, mouse hepatitis virus--strain jhm (mhv-jhm). selection of escape mutants was not anticipated because immune selective pressure in the cns, a site that is relatively protected from immune surveillance, might be predicted to be less than in the periphery. neurotropic coronaviruses, including mhv-jhm, the a strain of mhv (mhv-a ) and mhv- , cause acute and chronic infections of the cns in susceptible mice and rats (reviewed in houtman and fleming, b; lane and buchmeier, ; stohlman, bergmann, and perlman, ) . mhv-jhm is highly neurovirulent and infection of susceptible mice results in widespread infection of neurons with a rapidly fatal outcome. however, more generally interesting are those model systems in which mhv-jhm and mhv-a are induced to cause either acute or chronic demyelination. general strategies for modifying the acute infection have been recently reviewed (perlman, ; stohlman, bergmann, and perlman, ) , and two general approaches will be briefly described here. in the first, mice are infected with attenuated virus. attenuated virus was cloned from pools of virus harvested from suckling mouse brain (stohlman et al., ) . alternatively, viral mutants were selected by chemical mutagenesis or by treatment with neutralizing antibody directed against the surface (s) glycoprotein (dalziel et al., ; fleming et al., ) . in each case, the variant virus causes acute encephalitis in only a small percentage of mice but is able to persist and cause demyelination in most survivors. in mice infected with these variants, infectious virus is, in general, cleared by - weeks after inoculation. clinical symptoms are most evident within the same time frame. mice that survive the infection slowly recover, although evidence of ongoing demyelination can be detected for several months after the acute infection has resolved. in the second approach, mice are infected with wild-type mhv-jhm and protected from the acute encephalitis by administration of anti-mhv antibody or mhv-specific cd or cd t cells (buchmeier et al., ; stohlman et al., ; yamaguchi et al., ) . this intervention also results in decreased infection of neurons, but does not prevent infection of cells in the white matter or demyelination. virus clearance is enhanced by treatment with protective antibodies or t cells in some studies (buchmeier et al., ; yamaguchi et al., ) , but not all . in a variation of the latter approach, suckling c bl/ (b ) mice are protected from acute encephalitis with nursing by dams previously immunized to mhv-jhm . the suckling mice are protected from acute encephalitis and remain asymptomatic for - weeks. at that time, a variable percentage ( - %) develop histological evidence of demyelination and clinical signs of hind-limb paralysis. immunization of the dams may be accomplished either by active immunization with infectious virus , or by passive infusion of neutralizing antibody (pewe, xue, and perlman, ) , and with similar outcomes. the temporal course of this disease is very different from that observed in other models of mhv-jhm-induced demyelination, because mice are asymptomatic for several weeks before developing disease, and because infectious virus is not cleared. in a series of reports, pewe et al showed that escape from the cytotoxic cd t cell response was a major factor in the disease progression in these mice (pewe et al., ; pewe, xue, and perlman, ; pewe, xue, and perlman, ) . ctl escape mutations were detected in all mice that developed clinical disease several weeks postinfection in this model. in b mice, two cd t cell epitopes, encompassing residues - (s- - ) (h- db-restricted) and - (s- - (h- kb-restricted), of the s glycoprotein are recognized (bergmann et al., ; castro and perlman, ) . these two epitopes are located within a region of the protein that is prone to both mutation and deletion (termed the "hypervariable region") (banner, keck, and lai, ; stanley perlman and gregory f. wu parker, gallagher, and buchmeier, ) . the location of the epitopes within a region that can tolerate mutation, without loss of function, most likely contriljutes to the selection of ctl escape mutants. mutations have only been detected in epitope s- - , the immunodominant epitope of the two , and not in epitope s- - . mutations are not detected in regions of the genome flanking epitope s- - or in most mhv-specific cd t cell epitopes. the one possible exception is that a mutation is detected in a cd epitope encompassing residues - of the s glycoprotein (s- - ) in several mice, although wild-type epitope is also detected in the same animals. the significance of this mutation is not known, because it does not appear to decrease recognition by cd t cells specific for the epitope . mutations have been detected in amino acids at positions - of epitope s- - , with only a single variant generally being detected in any individual infected mouse (pewe, xue, and perlman, ) . a summary of mutations detected thus far is shown in fig. . based on the crystallographic structure of the h- d b molecule and on mutagenesis studies (hudrisier et al., ; young et al., ) , some of these mutations are predicted to affect binding to the mhc class i molecule, whereas others inhibit binding to the tcr of the cd t lymphocyte. mutations in epitope s- - are selected within - days postinfection, a time at which virus is not cleared in other mhv infections (houtman and fleming, a; lin et al., ) . detection of ctl escape mutants at a time before virus clearance is expected to be complete is consistent with a role for these mutants in virus persistence. notably, epitope s- - -specific ctl activity can be detected in the cns as early as days postinfection, prior to the detection of ctl escape mutants (unpublished observations). mutations are also not detected in infected mice with severe combined immunodeficiency (scid mice), suggesting that their selection is cd t cell-driven (pewe, xue, and perlman, ) . in this model, mice that do not develop hind-limb paralysis by - days postinfection remain asymptomatic. mutations are only occasionally detected in asymptomatic mice at - days postinfection suggesting that escape from cd t cell surveillance correlates well with virus amplification in b mice but is not sufficient for the development of clinical disease. as discussed above, the role of ctl escape mutants in viral pathogenesis is controversial. these mutants are not expected to be relevant to pathogenesis if the ctl response is directed at several ctl epitopes, as is believed to occur in many human infections. it has also been postulated that, even if a single epitope is immunodominant, a t cell summary of mutations detected in epitope s- - in virus isolated from mice with hind-limb paralysis. a panel of peptides resulting from single nucleotide changes in the sequence of the wild-type epitope were tested in cytotoxicity assays. only those changes resulting in a -fold decrement in activity are included in the figure. the amino acids listed above the wild-type sequence have been detected in virus isolates, whereas those listed below the wild-type sequence (underlined) have not yet been detected. the l to p change in position results in only a -fold decrease in recognition but has been detected in a minority of cdna clones in single mice with hind-limb paralysis (marked with parentheses). *mhc (m) or tcr (t) contact-based on published reports (hudrisier et al., ; hudrisier et al., ; young et al., ) . response that is comprised of many cd t cell clonotypes would preclude ctl escape, because a single mutation would affect recognition by only a subset of the epitope-specific t cells (ishikawa et al., ) . to determine the biological significance of the mutations that were detected in mhv-infected mice, several additional experiments were performed. first, a panel of variant epitopes were analyzed in cytotoxicity assays, using either lymphocytes derived from the cns of mice with acute encephalitis directly ex vivo or bulk populations of splenocytes cultured in vitro for weeks with peptide s- - pewe et al., ) . this panel comprised all the peptides resulting from single nucleotide changes in epitope positions - and . these assays showed that nearly all of the mutations that were stanley perlman and gregory f. wu selected in vivo resulted in a complete or substantial loss of recognition by either population of lymphocytes. several additional mutations also resulted in a nearly complete loss of recognition by these lymphocytes, but have not yet been detected in vivo (fig. ) strikingly, some mutations in position , a secondary anchor for binding to the mhc molecule, resulted in loss of recognition, but were never selected in infected mice. whether this apparent selectivity reflects a sampling error, or is in fact biologically driven (i.e., results in detrimental changes in rna or protein structure), remains to be determined. the biological significance of these mutations was also addressed by infecting maternal antibody-protected suckling c bl/ mice with virus mutated in epitope s- - . mice infected with this virus do not recognize epitope s- - but still respond to epitope s- - . variant virus-infected mice have significantly greater mortality and morbidity when compared to mice infected with wild-type virus (pewe, xue, and perlman, ) . these results suggest that the cd t cell response to epitope s- - is an important component of the host response to mhv-jhm, and support the idea that escape from this response is beneficial to the virus and results in increased virus replication and disease progression. these results suggest that the ctl response to epitope s- - is monospecific, because single mutations in residues important for binding to either mhc antigen or the tcr result in substantial loss of activity in cytotoxicity assays. the monospecificity of the response is consistent with a monoclonal or an oligoclonal response to the epitope. to directly assess the diversity of the t cell response, tcr v~ element usage and the complexity of the complementarity determining region (cdr ) were determined. the tcr is a heterodimer consisting of an a and a ~ chain. the great diversity in the t cell response results from the large number of different v, d, and j elements in the germ line, coupled with imprecise joining at the v-d and d-j junctions ([ chains) or v-j junction (a chain). the cdr encompasses the junctional region of the a and chains and makes direct contact with the mhc/peptide complex. analysis of a monoclonal or oligoclonal t cell response should reveal usage of only one or a small number of different v~ and va elements and minimal heterogeneity of the cdr . sequence analyses of the cdr were facilitated by the use of soluble mhc/peptide tetramers specific for epitope s- - (tetramer s- ). soluble mhc class i/tetramers were first described by altman et al., and have been shown, in several subsequent studies, to detect antigen-specific t cells with high sensitivity and specificity (e.g., altman et al., ; flynn et al., ; murali-krishna et al., ) . epitope s- - -specific cd t cells were isolated directly from the cns of mice with acute encephalitis by sorting with a fluorescentactivated cell sorter (facs) after staining with anti-cd antibody and tetramer s- . the tetramer s- -positive t cells were analyzed initially for v~ usage. the results showed usage of a large number of different v[~ elements by cd t cells responding to the epitope, with preferential usage of some v~ elements occurring, specifically v~ and v[~ . analysis of individual cdr sequences from a subpopulation of tetramer s- -positive cells, those expressing the v~ element, revealed substantial heterogeneity. the cdr sequences were shown to fit, approximately, a logarithmic distribution (fisher, corbet, and williams, ; taylor, kempton, and woiwod, ) . from this distribution, it was possible to calculate that approximately - epitope s- - -specific cd t cell clonotypes were present in the cns of a mouse with acute encephalitis. similar measurements using lymphocytes harvested from the cns of mice with chronic demyelination revealed that approximately - different clonotypes were present in these animals. previous studies suggested that either , (butz and bevan, ) or (bousso et al., ) precursor cd t cells for any single epitope were present in an individual mouse. our calculation of the number of t cells recognizing epitope s- - agrees more closely with the lower estimate. notably, none of the wild-type epitope s- - rna, or little of it, is present in the cns of mice with chronic demyelination, but cd t cells specific for the epitope are still detectable. whether this represents stimulation by residual wild-type antigen (which may be cleared more slowly than viral rna (knopfet al., ; zhang et al., ) , or by variant sequence remains to be determined. furthermore, many of the [~ chain cdr sequences were detected in more than one mouse. in the case of mice with chronic demyelination, very few cdr sequences were unique to a single animal. thus, unlike other experimental systems (bousso et al., ) , the repertoire of cd t cell tcrs recognizing epitope s- - is not unique to each naive animal but is, in large part, common among all c bl/ mice. this lack of variability may facilitate the emergence of ctl escape mutants in a large fraction of infected mice. these results suggest that ctl escape mutants are critical in virus persistence and in the development of clinical disease in mice infected with mhv-jhm. these results also show that ctl escape mutants are selected in the presence of a polyclonal, but monospecific, cd t cell response. as already mentioned, the importance of ctl escape stanley perlman and gregory f. wu mutants in other infections is much less certain, suggesting that the following features, present in the mhv-jhm-infected cns, must predispose to the selection of these mutants: . ctl escape mutants are observed in mhv-infected rodents only when b mice are inoculated at the suckling stage with virulent wildtype virus and protected from acute disease by nursing by immunized dams. inoculation with virulent mhv-jhm and use of b mice are essential for disease to develop. chronic demyelination does not develop if suckling mice are inoculated with the less neurovirulent a strain of mhv (unpublished observations). in most other models of mhv persistence, older mice are inoculated with attenuated strains. persistence is manifested by the presence of viral rna and ongoing demyelination in the absence of infectious virus (adami et al., ; houtman and fleming, a; rowe et al., ) . ctl escape mutants have not been detected in these models, possibly because clearance of infectious virus is so rapid . a normal immune system is also required, however, because even when clearance is delayed in mice infected with attenuated virus, as occurs in immunodeficient mice, ctl escape mutants are not commonly selected . . infection with wild-type virus at the suckling stage ( - days old) is also crucial in this model. three-week-old b mice inoculated with wild-type virus and protected from acute encephalitis by passive administration of small amounts of neutralizing antibody are protected from acute encephalitis, but they do not develop hind-limb paralysis at later times (sun and perlman, ) . demyelination can be detected in the spinal cords of these mice at months postinfection. these mice have not formally been assessed for the presence of ctl escape mutations at late times after infection, but they clearly do not develop a clinical disease consistent with the selection of ctl escape mutants. these results suggest that inoculation of mice with virulent virus at the suckling stage is essential for the selection of ctl escape mutants or, as a minimum, for their clinical manifestation. this requirement for inoculation of suckling mice may reflect the immaturity of the immune system. a consequence of infecting mice at days of life may be suboptimal clearance of virus. consistent with this possibility, after inoculation at the suckling stage, low levels of infectious virus can be detected at days postinfection in balb/c mice (unpublished observations) . balb/c mice never develop evidence of hind-limb paralysis or other clinical disease at any time and develop completely normally (castro et al., ; . . another consequence of inoculation of mice with an immature immune system may be an aberrantly polarized immune response. a striking feature of the immune response in most c bl/ mice infected with mhv at the suckling stage is the lack of an appreciable antibody response, when they are assayed by elisa or in neutralizing assays (jacobsen and perlman, ; . the cd and cd t cell response in maternal antibody-protected mice that develop chronic demyelination is proinflammatory and readily detected (castro et al., ; . this strongly polarized, cell-mediated immune response in the presence of a defective humoral response may also predispose to the selection of ctl escape mutants. in support of this, balb/c mice inoculated at the suckling stage in the maternal antibody model mount a significant antibody response and do not develop hind-limb paralysis (castro et al., ; . . mhv infection of congenic b .a( r) (kbddl d) mice results in the development of hind-limb paralysis, albeit at a lower frequency than in either b or c bl/ mice (castro et al., ) . two ctl epitopes, s- - and an la-restricted epitope encompassing residues - of the nucleocapsid (n) protein (n- - ) , are recognized in these mice. the immunodominant epitope recognized in b mice, epitope s- - , is not recognized in b .a( r) mice since these mice do not encode the h- d b allele. virus isolated from b .a( r) mice with hind-limb paralysis was evaluated for the presence of mutations in epitopes s- - and n- - , and none were detected. the development of hind-limb paralysis in b .a( r) mice, in the absence of mutations in either ctl epitope, suggests that a factor expressed on the c bl/ background (decreased production of antibody?) may contribute to the increased amount of virus replication observed in these mice. the outgrowth of ctl escape mutants in b mice, in conjunction with this other putative factor, results in a much higher rate of clinical disease than is observed in b .a( r) mice in which ctl escape mutants are not selected. . a ctl response directed at a single immunodominant epitope enhances the likelihood that ctl escape mutants will be selected. in b mice, two mhv-specific epitopes are recognized. cytotoxicity assays using cns-derived lymphocytes from mice with acute encephalitis, directly ex vivo (castro and perlman, ) , and limiting dilution assays using splenocytes harvested from mice intraperitoneally immunized with mhv , suggested, in both cases, that the two epitopes were recognized by similar numbers of cd t cells. more recent measurements, using mhc class i peptide/tetramers to detect antigen-specific cells or methods to detect interferon-~ production after stimulation with peptide, confirmed stanley perlman and gregory f. wu these results (unpublished observations). however, much more peptide s- - is required to sensitize target cells for lysis. thus, the ctl response in these mice may be functionally directed at a single epitope. conversely, a ctl response to a single immunodominant epitope is not sufficient for ctl escape mutants to be selected. the ctl response in balb/c mice is directed at a single epitope (n- - ), yet these mice do not develop hind-limb paralysis in the maternal antibody-protection model (castro et al., ) , and ctl escape mutants are not selected (unpublished observations). the ability of the virus to tolerate mutations in the part of the protein that contains the target cd t cell epitope is also important. epitope s- - is in a region of the s protein prone to deletion and mutation, while the balb/c epitope is located in a highly conserved region of the n protein. as mentioned above, mutations in this epitope are not detected in b .a( r) mice, even though the background of this strain may be favorable for the selection of ctl escape mutants. . mhv-jhm is neurotropic and the infection caused by this agent is confined, for the most part, to the cns. as discussed above, the processes of initiation of an inflammatory response, trafficking to the site of inflammation, and antigen presentation within the cns differ in several aspects from similar processes occurring at extraneural sites. in particular, mhc class i expression is minimal in the normal cns, and while clearly upregulated within the infected cns, determining the cellular sites of expression remains an area of active research. therefore, while it is speculative at present, it is formally possible that infection within the cns results in a modified immune response that increases the likelihood that ctl escape mutants will be selected during the course of an infection. what can be concluded from these studies about the role of ctl escape mutants in the pathogenesis of viral infections in the cns? the following conclusions may also be applicable for ctl escape mutants arising in extraneural tissue. first, ctl escape mutants are not commonly selected in most infections. one explanation for this is that the selection of ctl escape mutants is uncommon if the cd t cell response is directed against several epitopes. however, a cd t cell response directed against one epitope or a few, has been observed in several human and experimental infections (cole, hogg, and woodland, ; flynn et al., ; lehner et al., ; moss et al., ; murali-krishna et al., ; wallace et al., ) --suggesting that an immunodominant response is not unusual. nevertheless, ctl escape mutants are not detected in most experimental settings even when only one epitope is immunodominant, or a few are. second, the recognition of an epitope by a diverse population of cd t cell clonotypes does not prevent emergence of these variants. what does appear to be true, however, is that the diversity of the response matters less than whether the different cd t cell clonotypes recognize the same or different parts of the mhc/peptide complex. a response narrowly focused on one part of the complex will facilitate the selection of ctl escape mutants. the ctl response to epitope s- - is very sensitive to changes in residues and pewe et al., ) , two residues important for binding to the tcr (young et al., ) . strongly focused recognition of central residues of an immunodominant h- kb-restricted epitope, which is recognized in sendai virus-infected b mice, has also been reported (cole, hogg, and woodland, ) . third, in mice infected with mhv and in some humans persistently infected with hiv- , hbv, or hepatitis c virus, ctl escape mutants play an important role in virus amplification and disease progression. in most cases, however, it is likely that there would not have been sufficient time for selection of ctl escape mutants if virus clearance were rapid and complete. thus, selection of ctl escape mutants in the absence of any other factor is probably not sufficient to prevent rapid virus clearance from an infected host. in mhv-infected b mice, their selection appears to be facilitated by a suboptimal humoral immune response. this deficient antibody response may be a consequence of infection of suckling mice in the presence of maternally derived antibody. maternal immunization has been shown to impair immune responses in other infections (wang et al., ) , although it has never been shown to affect selectively humoral, and not cellular, immunity. fourth, an interesting aspect of this model system is the number of questions that it raises about the dynamics of mhv growth and cellular tropism in the cns. unlike the situation in mice chronically infected with tmev, in which macrophages are the main reservoir for virus (lipton, twaddle, and jelachich, ) , no single type of cns cell has been identified as the predominant target in mice persistently infected with mhv. the selection of ctl escape mutants in the cns of mhv-infected mice demonstrates that mhv infection of a cell expressing mhc class i antigen is a critical part of the pathogenic process. astrocytes, macrophages/microglia, and oligodendrocytes are all infected in these animals xue et al., ) . of all of these cells, only macrophages/microglia express detectable levels of mhc class i antigen in mhv-infected mice with chronic demyelination (xue et al., ) . one interpretation of these results is that macrophages/microglia are in fact the primary site of productive virus infection, with replication in oligodendrocytes or astrocytes being of secondary importance or abortive. infection of the latter two cells might be crucial for the development of clinical disease, but might be less important for propagation of the virus. alternatively, astrocytes or oligodendrocytes might express mhc class i antigen, albeit at levels below the detection limits of assays presently available. escape from cd t cell surveillance might occur in these cells, thereby resulting, simultaneously, in increased virus replication and disease 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and from the national multiple sclerosis society. g.f.w. was supported in part by a national research service award from the nih. key: cord- -vu b a authors: panahi, heidar ali; bolhassani, azam; javadi, gholamreza; noormohammadi, zahra title: a comprehensive in silico analysis for identification of therapeutic epitopes in hpv , , and oncoproteins date: - - journal: plos one doi: . /journal.pone. sha: doc_id: cord_uid: vu b a human papillomaviruses (hpvs) are a group of circular double-stranded dna viruses, showing severe tropism to mucosal tissues. a subset of hpvs, especially hpv and , are the primary etiological cause for several epithelial cell malignancies, causing about . % of all cancers worldwide. due to the high prevalence and mortality, hpv-associated cancers have remained as a significant health problem in human society, making an urgent need to develop an effective therapeutic vaccine against them. achieving this goal is primarily dependent on the identification of efficient tumor-associated epitopes, inducing a robust cell-mediated immune response. previous information has shown that e , e , and e early proteins are responsible for the induction and maintenance of hpv-associated cancers. therefore, the prediction of major histocompatibility complex (mhc) class i t cell epitopes of hpv , , and oncoproteins was targeted in this study. for this purpose, a two-step plan was designed to identify the most probable cd + t cell epitopes. in the first step, mhc-i and ii binding, mhc-i processing, mhc-i population coverage and mhc-i immunogenicity prediction analyses, and in the second step, mhc-i and ii protein-peptide docking, epitope conservation, and cross-reactivity with host antigens’ analyses were carried out successively by different tools. finally, we introduced five probable cd + t cell epitopes for each oncoprotein of the hpv genotypes ( epitopes in total), which obtained better scores by an integrated approach. these predicted epitopes are valuable candidates for in vitro or in vivo therapeutic vaccine studies against the hpv-associated cancers. additionally, this two-step plan that each step includes several analyses to find appropriate epitopes provides a rational basis for dna- or peptide-based vaccine development. hpvs are a large branch of the papillomaviridae family, grouped in different genera (alpha-, nu-/mu-, beta-and gamma-papillomaviruses), with more than genotypes [ ] [ ] [ ] [ ] . the classification of papillomaviruses (pvs) has been based on l gene sequence. they are clinically divided into two groups: low-risk hpvs, like hpv and , which cause benign lesions (warts and benign papillomas), and high-risk hpvs (hrhpvs), like hpv and , which are a a a a a response. however, the addition of cd + t cell epitopes can significantly augment its strength and duration [ , , ] . cd + ctls commonly recognize intracellular-originated peptides presented by mhc-i molecules. they accommodate peptides with - residues; the ideal length is residues. while cd + helper t lymphocytes (htls) commonly recognize extracellular-originated peptides presented by mhc-ii molecules. they accommodate peptides with - residues or even more; the ideal length is [ ] [ ] [ ] [ ] [ ] . the strength of the interaction between a t cell receptor and a peptide-mhc complex (pmhc), depends on the presented peptide and the mhc structure [ , ] . the binding of a peptide to mhc-i molecule is the most selective stage in the way of peptide presentation [ ] . bioinformatics tools can predict the potential immunogenic epitopes from thousands of epitopes in a short time [ ] . generally, the algorithms of these tools range from ones programmed to determine peptide-mhc molecule binding data to those based on structural similarity, molecular modeling, and molecular docking [ ] . peptides that bind to a specific mhc molecule have sequence similarity. therefore, peptide sequence patterns have been used to predict their binding to mhc molecules [ ] . in recent years, the accuracy of these methods has increased strikingly, and more than % of natural epitopes have been recognized at a high specificity of % [ ] . this improvement in performance was achieved by the expanding experimental binding data, available in the immune epitope database (iedb) and analysis resource (http://www.iedb.org/), and by the improvement of machine-learning algorithms [ ] . regarding the fundamental importance of epitope prediction in vaccine development, we investigated the best potential cd + t cell epitopes from the e , e , and e oncoproteins of four prevalent hrhpv genotypes ( , , and ) in the world and iran [ ] , as shown in a two-step plan was designed to identify the most probable cd + t cell epitopes (fig ) . for the first step, mhc-i and ii binding, mhc-i processing, mhc-i population coverage and mhc-i immunogenicity prediction analyses, and for the second step, mhc-i and ii protein-peptide docking, epitope conservation, and cross-reactivity with host antigens analyses were considered. the second step analyses were performed only for the selected peptides in the first step. in jan , in order of priority, the refseq, reviewed or unreviewed sequences of hrhpv oncoproteins (e , e , and e ) were retrieved from the national center for biotechnology information database (ncbi) (http://www.ncbi.nlm.nih.gov/) and uniprotkb/swiss-prot database (http://www.uniprot.org/). the isoform sequences of hpv , , , and oncoproteins were retrieved from hpv t cell antigen database (http://cvc.dfci.harvard.edu/hpv/ html/search.php). all the sequences are accessible in supporting information (s file). binding of epitopes to mhc-i molecules is an essential step for antigen presentation to ctls. herein, it was predicted by four online servers, as illustrated in table . the hla supertypes and frequently occurring hla-i alleles provided by the servers were included in the analysis. however, when an allele (e.g., hla-b � : ) was not provided, but its allele group (i.e., hla-b � ) was available, we used the allele group instead of the allele. the used human and mouse alleles, or allele groups are provided in supporting information (s table) . currently, eight prediction methods are available in the iedb mhc-i binding prediction tool, i.e., iedb recommended [ ] , consensus [ ] , netmhcpan [ , ] , artificial neural network (ann) [ , ] , smm with a peptide-mhc binding energy covariance matrix (smmpmbec) [ ] , stabilized matrix method (smm) [ ] , comblib_sidney [ ] , pickpocket [ ] , netmhccons [ ] and netmhcstabpan [ ] . the iedb-recommended and consensus are not independent methods; they use ann, smm and comblib_sidney methods to generate a representative index for each predicted pmhc; the median of percentile ranks (prs) or binding scores obtained from the used methods is reported as a representative pr or consensus score in the iedb-recommended or consensus method respectively. the pr is calculated by comparing the half maximal inhibitory concentration (ic ) of subjected peptide against a group of random peptides from swiss-prot database. the ic value, expressed as nanomolar, shows binding affinity. the lower ic or pr means higher binding affinity. as a rough guideline, peptides with ic values < nm are considered as high affinity, - nm intermediate affinity and more than - nm low affinity. no known t cell epitope has got an ic value > nm to date [ ] . in this study, iedb recommended method was used. the outputs for each pmhc in this method consisted of a median pr, a method-specific ic , and a method-specific pr. predictions were made against frequently occurring human mhc-i alleles (including hla supertypes) and mhc-i mouse alleles. epitope length was set on , , , and mer. peptides with median pr < . are applied for the analysis. netmhcpan mhc-i binding prediction. netmhcpan server predicts binding of peptides to the known mhc molecules using anns method. it is trained on a combination of naturally eluted ligands ( human and mouse mhc-i alleles) and binding affinity data ( mhc molecules from human, mouse, cattle, primates, and swine). besides, the user can perform a prediction against any custom mhc-i molecule by uploading its full-length sequence [ ] . in this study, predictions were performed for , , , and mer peptides against frequently occurring human mhc-i alleles and mhc-i mouse alleles. pr thresholds for strong and weak binders were set on . and . , respectively. peptides with pr < . were applied for the analysis. rankpep mhc-i binding prediction. rankpep predicts binder peptides of a given protein sequence or sequence alignments to mhc-i and ii molecules. the algorithm of rankpep based on the comparison of sequence similarities, using position-specific scoring matrices (pssms) method. it employs profiles of a group of aligned peptides recognized to bind to a specific mhc molecule and creates a consensus sequence by determining the most common residue for each position. then, it allocates an optimal score to the consensus sequence, compares the score of the subjected peptide with the optimal score, and gives the peptide a percentile optimal value for comparison. finally, it highlights strong binders in red [ , ] . herein, the prediction was made against frequently occurring hla-i and h -i alleles. the server did not provide all common lengths of epitopes for all the mhc alleles. thus, the used alleles and their provided epitope lengths are shown together, as given in supporting information (s table) . syfpeithi mhc-i binding prediction. syfpeithi (http://www.syfpeithi.de/ -home. htm/) is a database of over published and verified peptide sequences of human, mouse, and other organisms, known as natural binders of mhc-i and ii molecules. when syf-peithi analyzes a peptide for binding prediction against a specific mhc-i allele, its scoring system evaluates every residue of the query and gives it an arbitrary value between and , according to whether it is an anchor, auxiliary anchor, or preferred residue. it allocates the value to those residues which slightly preferred in that particular position, to the ideal anchor residues, and - to - to those residues which exhibit an adverse effect on the binding ability. the sum of these values is the score of the peptide. the maximal score could vary between different mhc alleles [ , ] . herein, the prediction was made against frequently occurring hla-i alleles and h -i alleles. epitope length was set on , , , and mer. every predicted pmhc which got a score less than % of the reference sequence score was excluded from the analysis. the allele-specific reference sequence was selected from rankpep's consensus sequence [ ] , or our syfpeithi predicted epitopes, whichever got the highest score in syfpeihi server. the reference sequences, their sources, and their scores are given in supporting information (s table) . recognition of high immunogenic cd + t cell epitopes was the primary aim of this study. therefore, all predictions were primarily made against epitopes with - residue length. however, it was valuable to determine that which mer mhc-i epitope is the core peptide of the mhc-ii epitope(s) too. the core peptide lies on the mhc-ii molecule grooves, and play the central role in constructing pmhc. with this strategy, the short minimal predicted epitopes could be used in designing of synthetic long peptides (slps), resulting in peptide loading to both mhc-i and ii molecules. iedb mhc-ii binding prediction. in this study, the mhc-ii binding prediction was made by iedb mhc-ii binding predictor (http://tools.iedb.org/mhcii/) [ , , ] . iedb possess seven prediction methods for mhc-ii binding prediction: iedb-recommended, consensus [ ] , netmhciipan [ ] , nn-align [ ] , smm-align [ ] , combinatorial libraries [ ] and sturniolo's method [ ] . herein, the iedb-recommended method was used, and all peptides with pr< . were selected for the analysis. the prediction was made against human alleles (iedb reference set) and three mouse alleles, given in supporting information (s table) . the server has fundamentally set the epitope length on mer. each iedb-recommended method participated in the prediction process offered a core peptide ( mer) for each predicted epitope ( mer). we associated the mer mhc-ii core peptides with the mer mhc-i predicted epitopes to determine that which mhc-i epitope is the core peptide of the mhc-ii epitope(s) too. mhc-i t cell epitope processing predictions of e , e , and e oncoproteins are made by the iedb combined predictor (http://tools.iedb.org/processing/). this tool combines predictors of three main steps of mhc-i antigen presentation pathway (proteasomal processing, transporter associated with antigen processing (tap) transport, and mhc-i binding) and calculates a total processing score for each predicted epitope. it allows the user to choose a method from ann, smm, smmpmbec, comblib_sidney , netmhcpan, netmhccons and pickpocket methods for the binding prediction. in the current update ( ), the iedb team has changed the choice of the recommended prediction method for the processing tool to be netmhcpan . rather than a consensus, since the processing tools requiring an ic value, which the consensus method does not provide. furthermore, netmhcpan . has provided all mhc alleles and has performed the predictions very well in recent comparisons [ ] . there are two types of proteasomes, the housekeeping types which are expressed instinctively, and immuno types which are provoked by ifn-γ secretion. the immunoproteasomes are believed to improve the efficiency of antigen presentation [ , ] . in this study, the immunoproteasome option was selected. the program outputs for every predicted epitope consisted of proteasome score, tap score, mhc score, processing score (proteasome + tap score), total score (proteasome + tap + mhc score), and mhc-i ic . the tap scoring system calculates a-log (ic ) value for the binding of a peptide (or n-terminal of its precursors) to the tap molecules. the higher tap score, the higher transport rate. [ , , ] . herein, the analysis was made against the human and mouse mhc-i alleles used later in the iedb binding prediction, with the iedb-recommended method and other default settings of the program. epitopes with ic < nm for hla-i alleles and < nm for h -i alleles were included in the analysis. several factors could clarify the difference between epitope and non-epitope peptides; an essential factor is epitope immunogenicity, i.e., it could be recognized by t cells. some amino acids, particularly those with large and aromatic side chains (especially tryptophan, phenylalanine, and isoleucine), are associated with immunogenicity. moreover, the positions p - of a peptide are more critical for immunogenicity [ ] . in this study, the mhc-i immunogenicity of all predicted epitopes was determined by the iedb web server (http://tools.iedb.org/immunogenicity/) [ ] . this tool uses the properties of amino acids and their locations to predict the immunogenicity of a pmhc. the default option was selected to specify which positions of the query peptide to be masked from the analysis, because it masked positions which are also suggested for the most frequent human mhc-i allele, hla-a � : . iedb population coverage prediction tool (http://tools.iedb.org/population/) [ ] is used to predict the hla-i population coverage of all - mer predicted epitopes in the first step. this tool can accept a target population by two query levels: ) area-country-ethnicity and ) ethnicity alone. it can integrate allele frequency information retrieved from the allele frequency net database (afnd) (http://www.allelefrequencies.net/default.asp) [ ] . iedb also accepts custom populations with allele frequencies defined by users. since, hla-i and hla-ii t cell epitopes elicit immune responses from two different t cell populations (ctl and htl, respectively), the server provided three different population coverage modes: ) hla-i lonely, ) hla-ii lonely, and ) hla-i and hla-ii together. herein, the mhc-i promiscuous predicted epitopes and their binding hla-i alleles (ic < nm or pr< . ) were entered as inputs for the analysis against the world population. the primary aim of molecular docking is the prediction of the binding site of a ligand at a protein receptor surface, and then docking and modeling the ligand into the recognized site. in this study, the binding ability of the first step selected peptides to human and mouse mhc molecules, was analyzed by cabs-dock (http://biocomp.chem.uw.edu.pl/cabsdock/) server. the server uses a multistage procedure that involves multiple programs, with the cα-cβ-side chain (cabs) model at its heart. the detailed information about these stages is given in supporting information (s file) [ , ] . also, fig shows the pipeline of cabs-dock protocol [ ] . cabs-dock gets the d structure of the receptor and the sequence of the peptide as obligatory inputs. furthermore, there are some non-obligatory inputs as recommendations which could improve outputs. in this study, duplicate dockings for each peptide ( dockings in total) were done against the most significant human/mouse mhc-i and ii molecules which had at least one well-structured protein data bank (pdb) file in the rcsb protein data bank (https://www.rcsb.org/), as shown in table . these pdb files are in the complex with their peptidic ligand and some x-ray crystallography solution molecules (heteroatoms). thus, these excess molecules, as well as redundant mhc molecules were removed before executing docking process. since, the binding site of epitopes on the mhc molecules was well-known previously, the unlikely regions to bind masked before the analysis. cabs-dock returns ten representative models (medoids) as the best-simulated models and ranks them by cluster density (cd). cluster density is equal to the number of elements in a cluster divided by their average ligand root mean square deviation (rmsd). the higher cd value implies greater accuracy. ligand rmsd value shows the differentiation measure between cluster elements. as a guideline; rmsd < . Å means high accuracy; rmsd � . and � . Å means medium accuracy and rmsd > . Å means low accuracy [ ] . herein, the rmsd and cd of the best-simulated models were selected for the analysis. the best model, which has the highest cd value, is not necessarily the top-ranked model, because, in some cases, peptides were not attached to their binding site properly. thus, these malformed models were excluded from the analysis. it is important to note that, due to the different frequency of mhc alleles in human populations, the equal cd value of different mhc alleles, don't have equal value regarding population coverage. thus, to involve the effect of population coverage, the cd value of every model was multiplied by its allele population coverage (divided by hundreds for more facility) to obtain a weighted index. then, the sum of all hla-i or ii weighted indexes of each peptide was calculated to get a total docking score (tds), used as a score to compare the candidate peptides. it is the first time that the tds has been formulated and used for this purpose. this formula is also applicable to the similar docking scores obtained from other servers. the use of highly conserved epitopes in a vaccine formulation reduces the risk of tumor immune escape and provides broader protection against different virus strains or genotypes. thus, the conserved areas are preferred to use in therapeutic vaccines, if they are appropriate epitopes. herein, the epitope conservancy analyses for the first step selected peptides were done in three levels: . inter-isoform conservancy: the percent of conservancy between all isoforms of each e , e , or e oncoprotein. . inter-type conservancy: the percent of conservancy between hpv and (alpha-papillomavirus ), as well as between hpv and (alpha-papillomavirus ). . inter-hrhpv conservancy: complete ( %) conservancy between all hrhpvs (hpv , , , , , , , , , , , and ). the selected peptides in the first step were analyzed to find inter-strains and inter-types conservancy percentages by iedb tool, conservation across antigens, (http://tools.iedb.org/ conservancy/). the inter-hrhpvs conservancy analysis was done by the iedb and expasy clustalw servers (https://embnet.vital-it.ch/software/clustalw.html). cross-reactivity with host antigens can cause adverse immune responses. therefore, the selected peptides in the first step were checked for similarities with the mouse and human proteomes by the ncbi blastp tool (https://blast.ncbi.nlm.nih.gov/blast.cgi). regarding the studies, different peptides usually get different scores/ranks in different analyses. this inconsistency indicates that these results needed to be analyzed with an integrated approach. indeed, integrated approach is more practical and efficient in such conditions in comparison with analysis by analysis filtering approach, in which those epitopes are chosen for the next analysis that have gotten an acceptable score in the previous analysis. herein, the integrated approach was applied in both steps of epitope selection. since the ultimate goal of the discovery of therapeutic epitopes is to use them in human vaccines, only the scores/ranks of human alleles were used to rank epitopes in some studies. however, investigators usually test therapeutic vaccines on mouse species in preclinical trials, thus in the current study, the binding status of the predicted epitopes to mouse mhc-i alleles was also studied by several binding predictors and molecular docking, as well. as stated above, ctl-mediated responses play a crucial role in killing the malignant cells. besides, the binding of epitopes to mhc-i molecules is the most selective step for antigen presentation to ctls. therefore, in the first step, the selection was made primarily by the comparison of obtained mhc-i binding, processing and immunogenicity scores/ranks, and population coverage percentages. however, the mhc-ii binding ranks were actually of secondary importance to the selection process as an added advantage. additionally, the population coverage has a dual application. first, it determines the coverage of a given peptide in the target population. second, it is the best index for summarizing and evaluating of the hla-i binding predictions too, since it is calculated from the results of hla-i binding prediction analyses. in the first step, ten peptides (tables - ) from each hpv genotype oncoprotein ( peptides in total), which got better results in the first step analyses were selected for the second step analyses, including protein-peptide molecular docking, epitope conservation, and crossreactivity with host antigens. the individual detailed results of the mhc-i and ii binding (s file), mhc-i immunogenicity (s file) and mhc-i population coverage (s file) predictions, as well as, mhc-i and ii molecular docking (s file) and epitope conservation (s file) analyses are given in supporting information, as excel files. indeed, cabs-dock returns ten representative medoids as the best-simulated models and ranks them by cluster density (cd). cluster density is derived from two factors (the number of elements in a cluster and their average ligand rmsd) that is an advantage for this server. in the second step, five peptides out of ten selected peptides in the first step (tables - ) , which got better results in all analyses of both steps, were selected as the final-predicted epitopes. none of the final predicted epitopes showed more than % sequence similarity with mouse and human proteomes. high prevalence and mortality of oncogenic infectious pathogens such as hpv and helicobacter pylori have caused serious problems for humans. currently, people who are infected with hrhpvs but show normal cytology or precancerous lesions do not have any treatment option, causing the disease progress toward invasive carcinoma in some cases. unfortunately, no fda-approved immunotherapy exists for pre-existing hpv infections or their related cancers to date. immunotherapy of hpv-associated cancers by dna or peptide-based vaccines, depends on the recognition of highly immunogenic epitopes, inducing robust and specific immune responses, particularly cell-mediated responses against the malignant cells. the primary aim of this study was the prediction of cd + t cell epitope from the e , e and e oncoproteins, using a comprehensive two-step selection plan. these proteins chose because they play a pivotal role in the cell transformation, immune evasion, and maintenance of malignancy, as well as, their permanent expression (e and e ) by the malignant cells [ ] [ ] [ ] . expression of e oncoprotein occurs in the early phase of hpv infection. evidence indicates that e play a prominent role in the genesis of hpv-associated cancers, but is not essential for cancer progression [ ] , since when hpv genome integrates into the host genome, it usually results in the disruption of e , e , and e genes. therefore, targeting e protein provides an opportunity for treatment of hpv infections and preventing the precancerous lesions from the progression to established carcinomas [ , ] . some genotypes of hrhpvs are more involved in the genesis of epithelial tissue malignancies [ ] . thus, in this study, hrhpv , , and were targeted due to their high prevalence in the hpv-associated cancers, especially cervical carcinoma. there are several limitations for epitope prediction: ) the major drawback of peptidebased vaccines is low immunogenicity [ , ] . many studies have focused on enhancing immunogenicity using immune stimulating agents or adjuvants to avoid this problem. another solution is the use of agonist epitopes [ ] . epitope immunogenicity is a crucial factor in vaccine development. however, many of known natural epitopes when are analyzed in silico by iedb mhc-i immunogenicity predictor, do not obtain a high score. therefore, in this study, epitope selection was based on the integrated approach, in which one analysis does not play an important role alone. ) there are certain drawbacks associated with the function of each method invented for the mhc-peptide binding prediction [ ] . for this reason, several predictors and a molecular docking program were used to augment the prediction accuracy. ) some web tools have been developed for mhc-ii epitope prediction. since mhc-ii groove can bind to peptides with variable lengths, and different peptides have the different number of residues between their n-terminus and first anchor [ ], the exact assignment of mhc-ii core peptide would be a difficult problem which reduces the success rate of these prediction tools. therefore, most mhc-ii prediction tools did not usually make epitope predictions as accurately noted for mhc-i molecules [ , ] . in cancer immunotherapy, the ctl-mediated responses play the central role in eradication of malignant cells, and the binding of epitopes to mhc-i molecules is an essential step for antigen presentation to ctls. thus, in this study, predicted epitopes were primarily selected by their mhc-i binding and processing scores. however, the mhc-ii binding scores were actually of secondary importance to the epitope selection process as an extra advantage. additionally, there are several other essential determinants which significantly affect the outcomes, such as antigen processing, immunogenicity, population coverage, conservancy and cross-reactivity with host antigens. vaccine development requires a comprehensive approach to cover all these effectual elements, covered in this study. the primary aim of molecular docking is the recognition of binding site of a ligand at a protein receptor surface, and docking and modeling the ligand into this recognized site. in this study, cabs-dock server was used for molecular docking analyses. cabs-dock has several main advantages: ) the method does not require any data about the peptide structure and its binding site. ) during docking process, peptide conformation is entirely flexible. ) it is possible to apply dynamic conformational changes in the receptor structure and ) to exclude some receptor regions from the docking search, leading to the more efficient search in the vicinity of the binding site at a sensible time. [ , ] . in comparison with protein-ligand (small molecules) docking, protein-peptide docking analysis is more problematic, since significant conformational changes occur during the process. as a general rule, how much the length of the query peptide to be longer, there are more torsions and conformational flexibilities. additionally, in comparison to protein-protein interactions, protein-peptide dockings are more transient, and their binding affinities are notably weaker [ ] . these factors make structural predictions of long peptides very challenging. therefore, in this study, mer peptides were preferred for selection compared to other possible lengths. they are also preferred by all mhc-i molecules as epitope and by mhc-ii molecules as the core peptide of epitopes. moreover, expansion of or mer ctl epitopes to longer peptides may create a practical alternative, containing both cd + htl and cd + ctl epitopes; especially, when cd + htl epitopes, covering ctl epitopes, are not recognized [ ] . [ , , [ ] [ ] [ ] [ ] [ ] [ ] . however, the prediction of t cell epitopes inducing strong responses has remained a big challenge. for therapeutic hpv vaccines, many candidates have been designed to trigger the activation of ctls or htls, mostly by targeting two major hpv oncoproteins, e or e [ ] , and in a few studies, e oncoprotein [ , ] . as well as, several clinical trials have been launched for immunotherapy of hpv-associated cancers [ ], although, they have not been so immunogenic, to induce a sufficient cellular immunity and eradicate malignant cells completely. some studies have suggested that the use of e and e slps, containing both cd + htl and cd + ctl epitopes, led to more potency and durability of cd + t cell reactivity in vivo, in comparison with the minimal ctl epitopes [ , ] . in , as pioneers in hpv epitope studies, feltkamp et al. recognized the hpv -e sequence rahynivtf as an mhc-i epitope that can provoke ctl-mediated responses and eradicates established hpv l -induced tumor cells in mice [ , ] . this sequence is the first hpv -e predicted epitope in our study as well. in , kumar et al. studied hpv -e oncoprotein to predict the candidate t-cell and bcell epitopes [ ] . they have screened potent epitopes for mhc-i molecules according to pr and the immunogenicity score, using iedb mhc-i binding and immunogenicity predictors. they found a mer potent epitope, safrcfivyiifvy, having the lowest pr and the highest immunogenicity score, i.e., . and . , respectively. notably, our second hpv -e predicted epitope, safrcfivy, is the n-terminal part of safrcfivyiifvy, and our first predicted epitope, flihtharf, is the c-terminal part of the third epitope of their study, vyiplflihtharf. in , tsang et al. scanned the hpv -e and e oncoproteins for the match peptides with the consensus motif of hla-a binding peptides [ ] . the bimas algorithm [ ] was employed to rank probable binding peptides according to the predicted one-half-time dissociation of pmhcs. three potential ctl predicted epitopes of the e protein (klpqlctel, kiseyr-hyc, and qqynkplcdl) and three of the e protein (ymldlqpet, tlheymldl, and rtledllmgt) were selected. they showed the immunogenicity of these peptides was enhanced when their agonist epitopes were used. the klpqlctel and tlheymldl sequences are the seventh and the fifth predicted epitopes of hpv -e and hpv -e in our study, respectively. experimental evidences about hrhpv-derived epitopes in literatures are mostly limited to e and e oncoproteins of hpv and . among our first-step predicted epitopes: fllcfcvll and yiifvyipl from the e -derived epitopes [ ] , fafrdlcivy [ ] , cyslygttl [ ] , vydfafrdl [ , ] , kfyskisey [ ] , klpqlctel [ ] [ ] [ ] , iseyrhycy [ ] , eyrhycysl [ ] , klpdlctel [ , [ ] [ ] [ ] , fafkdlfvv [ , ] and klpdlctel [ , [ ] [ ] [ ] from the e -derived epitopes, rahynivtf [ ] , ledllmgtl [ ] , tlheymldl [ , [ ] [ ] [ ] , llmgtlgiv [ , , , ] , qaepdrahy [ ] , gtlgivcpi [ , ] , fqqlflntl [ ] and tlqdivlhl [ ] from the e -drived epitopes were reported as t-cell epitopes experimentally. besides, ivyrdgnpy, cyslygttl, klpqlctel and iseyrhycy from the e -derived epitopes, and rahynivtf and gtlgivcpi from the e -derived epitopes were also reported as hla ligands [ ] . others are novel epitopes that they also require experimental studies for validation. as far as we know, this is the first time that in a laborious in silico study for epitope prediction, e , e and e oncoproteins of hrhpv , , and have been investigated altogether. moreover, in previous studies, usually only one predictor tool was used for making epitope prediction, or if several tools were used, no integrated approach was employed to make the conclusion. we believed that our predicted epitopes are valuable candidates for further in vitro and in vivo therapeutic vaccine studies. additionally, the introduction of the ten epitopes for each hpv genotype oncoprotein in the first step of the study shows which region of each oncoprotein is rich of the epitope, and thus, is more suitable for use in the design of slps. notably, the previous in vivo studies have been conducted using slps of hrhpv-e and/or-e oncoproteins, in particular hpv oncoproteins [ , [ ] [ ] [ ] [ ] [ ] . furthermore, the two-step plan of this in silico study, which each step includes several analyses to find proper epitopes by an integrated approach, would provide a basis for rational epitope prediction. however, it could be more efficient by adding other useful analyses. further studies are recommended on the peptide binding assays, the design of polyepitope constructions including e , e and e epitopes, the expansion of the minimal ctl epitopes to longer peptides (slps), the use of various adjuvants, involvement of delivery routes, mouse immunization with the designed constructs, evaluation of immune responses such as cytokines, antibodies, ctls and tumor growth for finding the best construct for clinical trials. it is important that 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approaches a systematic bioinformatics approach for selection of epitope-based vaccine targets a novel multi-epitope peptide vaccine against cancer: an in silico approach epitope-based vaccine target screening against highly pathogenic mers-cov: an in silico approach applied to emerging infectious diseases advances in peptide-based human papillomavirus therapeutic vaccines therapeutic vaccination with papillomavirus e and e long peptides results in the control of both established virus-induced lesions and latently infected sites in a pre-clinical cottontail rabbit papillomavirus model vaccination with cytotoxic t lymphocyte epitope-containing peptide protects against a tumor induced by human papillomavirus type -transformed cells cytotoxic t lymphocytes raised against a subdominant epitope offered as a synthetic peptide eradicate human papillomavirus type -induced tumors scheme for ranking potential hla-a binding peptides based on independent binding of individual peptide side-chains 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hla-a + epithelial cancers immunization with a poly (lactide co-glycolide) encapsulated plasmid dna expressing antigenic regions of hpv and results in an increase in the precursor frequency of t cells that respond to epitopes from hpv , , and identification in humans of hpv- e and e protein epitopes recognized by cytolytic t lymphocytes in association with hla-b and determination of the hla-b -specific binding motif up-regulation of hla class-i antigen expression and antigen-specific ctl response in cervical cancer cells by the demethylating agent hydralazine and the histone deacetylase inhibitor valproic acid human t-cell responses to hla-a-restricted high binding affinity peptides of human papillomavirus type proteins e and e synthetic peptides of human papillomavirus type e harboring hla-a . motif can induce peptide-specific cytotoxic t-cells from peripheral blood mononuclear cells of healthy donors establishment of an hla-a* human papillomavirus type tumor model to determine the efficacy of vaccination strategies in hla-a* transgenic mice different methods of identifying new antigenic epitopes of human papillomavirus type e and e proteins naturally processed and hla-b -presented hpv e epitope recognized by t cells from patients with cervical cancer human ctl epitopes encoded by human papillomavirus type e and e identified through in vivo and in vitro immunogenicity studies of hla-a* -binding peptides dendritic cell-based tumor vaccine for cervical cancer ii: results of a clinical pilot study in individual patients human ctl epitopes encoded by human papillomavirus type e and e identified through in vivo and in vitro immunogenicity studies of hla-a* -binding peptides identification of a naturally processed hla-a* hpv e t cell epitope by tumor cell mediated in vitro vaccination role of hla-a motifs in identification of potential ctl epitopes in human papillomavirus type e and e proteins prospects of combinatorial synthetic peptide vaccine-based immunotherapy against cancer experience with synthetic vaccines for cancer and persistent virus infections in nonhuman primates and patients mechanisms of peptide vaccination in mouse models: tolerance, immunity, and hyperreactivity therapeutic vaccination against human papilloma virus induced malignancies immunologic treatments for precancerous lesions and uterine cervical cancer therapeutic cancer vaccines the authors sincerely thank dr. ali namvar and miss elnaz agi for their valuable guidance and comments during preparation of the paper. key: cord- -s oqphvn authors: baral, prabin; pavadai, elumalai; gerstman, bernard s.; chapagain, prem p. title: in-silico identification of the vaccine candidate epitopes against the lassa virus hemorrhagic fever date: - - journal: sci rep doi: . /s - - - sha: doc_id: cord_uid: s oqphvn lassa virus (lasv), a member of the arenaviridae, is an ambisense rna virus that causes severe hemorrhagic fever with a high fatality rate in humans in west and central africa. currently, no fda approved drugs or vaccines are available for the treatment of lasv fever. the lasv glycoprotein complex (gp) is a promising target for vaccine or drug development. it is situated on the virion envelope and plays key roles in lasv growth, cell tropism, host range, and pathogenicity. in an effort to discover new lasv vaccines, we employ several sequence-based computational prediction tools to identify lasv gp major histocompatibility complex (mhc) class i and ii t-cell epitopes. in addition, many sequence- and structure-based computational prediction tools were used to identify lasv gp b-cell epitopes. the predicted t- and b-cell epitopes were further filtered based on the consensus approach that resulted in the identification of thirty new epitopes that have not been previously tested experimentally. epitope-allele complexes were obtained for selected strongly binding alleles to the mhc-i t-cell epitopes using molecular docking and the complexes were relaxed with molecular dynamics simulations to investigate the interaction and dynamics of the epitope-allele complexes. these predictions provide guidance to the experimental investigations and validation of the epitopes with the potential for stimulating t-cell responses and b-cell antibodies against lasv and allow the design and development of lasv vaccines. to in the t-loop (residues - ) and hr (residues - ) regions of gp . site b contains residues to of the fusion peptide and residues to of hr (residues - ) of gp , . although the antibody predominantly binds to gp , gp is required to maintain the proper prefusion conformation of gp for antibody binding . identification of epitopes is an essential step for understanding disease etiology, immunotherapy, immunodiagnostics, and the discovery and development of epitope based-vaccines. an epitope-based vaccine has fewer side effects compared to conventional vaccines. experimental identification of a promiscuous epitope involves many expensive and time-consuming steps, including the production of antibodies to map antigenic regions on a target protein, animal models, and determination of the crystal structure of antigen-antibody complexes using x-ray crystallography. computational identification of epitopes is often employed as a powerful and fast approach to facilitate the identification of potential epitope candidates that can decrease the number of validation experiments and time , . multi-epitope based vaccine development has already proven effective against several viral infections and cancer , . in this study, we have identified and characterized t and b-cell epitopes for the lasv gp using different sequence and structure-based computational epitope prediction methods. we then selected potential b and t-cell epitopes for the lasv gp based on a consensus approach, and the novelty of the epitopes was examined with the immune epitope database (iedb) tools. subsequently, we identified strongly binding alleles to the mhc-i t-cell epitopes and modeled the allele structures and performed docking to understand the interaction between alleles and epitopes. we further investigated the stability and dynamics of the epitope-allele complexes using molecular dynamics simulations. analyses of root-mean square deviations, hydrogen bond, interaction energy, and solvent accessibility showed that epitope-allele complexes are stable, indicating that the epitopes strongly bind to the alleles. the identified b and t-cell epitopes of lasv gp in the study can be useful for the development of effective vaccines against lassa hemorrhagic fever. selection of lasv gp sequence and d structure. the sequence of gp for different lasv strains was obtained from the niaid virus pathogen database and analysis resource (vipr) . subsequently, multiple sequence alignments were performed between the sequences using clustal omega to select a conserved lasv gp for sequence-based epitope prediction. the corresponding x-ray crystal structure of the mouse/sierra leone/josiah/ lasv gp was obtained from the protein data bank (pdb id: vk ) , for structure-based b-cell epitope prediction. the missing residues were modeled using the charmm-gui - . prediction of b-cell epitopes. sequence-based b-cell epitope prediction was performed with the use of bepipred . , bcpreds and bcepred servers separately. these servers predict epitopes based on physico-chemical properties of amino acids, and these servers accept the primary sequence of lasv gp as an input. . each gp has a gp subunit and a gp subunit (zoomed view). each monomer is colored differently in the gp trimer. in the zoomed view, the gp subunit is lightly shaded to differentiate from the gp subunit, and some of the antibody binding sites (site a, site b) are highlighted (figure generated from the crystal structure of the lasv gp in the protein data bank , pdb id: vk ). structure-based b-cell epitope prediction for the lasv gp (pdb id: vk ) was carried out using three different programs separately: ellipro , epitopia and discotope . these servers predict epitopes regions based on the geometrical and solvent surface-accessibility of a protein structure, and these servers accept the d structure of a protein as input. the consensus epitopes from both sequence and structure-based predictions were selected as potential epitopes for further analysis. sequence-based mhc-i t-cell epitope predictions for lasv gp were carried out by using three different servers, propred-i , ctlpred and netctl . . to predict their alleles, the consensus epitopes among these three prediction methods were analyzed using iedb . the epitopes that strongly bind to the alleles (lowest ic ) were selected for further analysis. sequence-based mhc-ii t-cell epitope predictions for lasv gp were performed with the use of three different servers: propred , netmhcii . ] were obtained from the pdb. the experimental structure for the hla-a* : (a ) allele is not available, and thus, the sequence of this allele was obtained from the uniprot database (uniprotkb id: p ), and subsequently its structure was modeled using swiss-model [ ] [ ] [ ] . the selected consensus mhc- epitopes were extracted from the crystal structure of lasv gp (pdb: vk ). the epitopes and the alleles were prepared for docking using autodock tool version . . . autodock vina . . was used for peptide docking with a grid space that covered the entire allele. the best peptide-allele complexes were selected for further investigation based upon visual inspection of peptide-allele interactions and the autodock vina criteria. the stability and dynamics of the selected peptide-allele complexes were further studied using molecular dynamics simulations. all-atom, explicit-solvent molecular dynamics (md) simulations were performed to investigate the stability and dynamics of the mhc- t-cell epitope-allele complexes using the charmm m force field with the namd . software package . the systems were minimized for , steps followed by ps of equilibration. this was followed by md production runs for ns at a temperature of k using a fs time-step. the long-range ionic interactions were calculated using the particle mesh ewald (pme) method while the covalent hydrogen atoms were constrained by using a shake algorithm . the temperature was controlled by using the langevin temperature coupling with a friction coefficient of ps − and pressure was controlled using the nose-hoover langevin-piston method . visualization, and rendering of trajectories and pictures were performed using vmd . the multiple sequence alignment of the lasv gp sequences resulted in the lasv gp mouse/sierra leone/ josiah/ ) [uniprotkb id: p ] as a highly conserved strain, and we thus selected this strain for the sequence-based mhc-i and mhc-ii t-cell epitope predictions and for both structure and sequence-based b-cell epitope predictions. in addition, a search of this strain with the experimentally determined structure available in the pdb displayed the . Å resolution crystal structure of the prefusion gp trimer of lasv in complex with the human neutralizing antibody . h. [pdb id: vk ] as shown in fig. . this structure was used for the structure-based b-cell epitope prediction. a schematic representation of the epitope prediction cascade is shown in fig. . we have adopted multiple methods to predict and rank the epitopes as they use different criteria for their predictions. some approaches may incorporate some properties that are similar such as solvent accessible surface area, but the predicted epitopes are different. previous studies , have suggested that the consensus approach would improve the specificity and accuracy of the epitope prediction as it can reduce the false positives. therefore, we employ a consensus approach; for example, an epitope can be considered if it overlaps with even a single residue by at least two prediction methods. our consensus approach selected several nanomer epitopes for mhc-i (table s ). although the predicted epitopes for mhc-ii t-cell vary in length, the consensus core region between predicted mhc-ii epitopes is a nanomer (table s ) which is considered an optimal length for the hla. prediction of t-cell epitopes. mhc-i t-cell epitope prediction with the lasv gp sequence was performed using three different methods separately: propred- , ctlpred, and netctl . , and the results are shown in supplementary table s . the epitopes listed by at least two of the methods are listed in table along with their binding affinity (ic ), antigenicity, and allele. among these four consensus epitopes, the nanomer e epitope fatcglvgl shows the lowest average ic value of nm against the a allele as predicted by the iedb, and it has also a reasonable antigenicity score of . . this was followed by the e epitope fsrpspigy, which has an average ic value of nm against the a allele, and also has a better antigenicity score of . compared to the fatcglvgl epitope. interestingly, the e epitope rrgtftwtl is predicted by all three methods though its ic and antigenicity scores are not as good as the other epitopes (table ). all four of these consensus epitopes were docked to the alleles and we performed the md simulations to investigate the stability and dynamics of the allele-epitope complex as discussed later. mhc-ii t-cell epitope prediction with the lasv gp sequence was performed using three different methods separately: propred, netmhcii . , and epitop . , and the results are shown in supplementary table s . propred uses a quantitative matrix approach and netmhcii . uses ann , while epitop . uses quantitative structure-activity relationship models (qsar) to predict the mhc-ii t-cell epitopes. the epitopes that were predicted by at least two methods are listed in table . among these consensus mhc-ii t-cell epitope predictions, the e and e epitopes were predicted by all three methods and have a reasonable antigenicity score of . , indicating that these two epitopes can be potential candidates for the design of mhc-ii t-cell based vaccines. propred and epitop . predict most epitopes as nanomers whereas netmhcii . predicts varying lengths of epitopes (table ) . interestingly, the -mer epitopes predicted by netmhcii have the consensus core nanomer epitopes, suggesting that the core region is responsible for strong binding of the epitope into the mhc-ii binding site - . prediction of b-cell epitopes. in addition to the t-cell epitope predictions, we also predicted the linear b-cell epitopes for the lasv gp using sequence-based methods bepipred . , bcpreds , and bcepred . the bepipred predicts the epitopes based on a random forest algorithm trained on epitopes annotated from antibody-antigen structures. bcpreds predicts epitopes by using svm combined with a different kernel method, including string kernels, radial basis kernels, and subsequence kernels. the bcepred locates b-cell epitopes using four physicochemical properties like hydrophilicity, polarity, exposed surface and beta-turns . the epitope e containing residues was predicted by all three of these sequence methods (table ) but with a negative antigenicity score. we also performed structure-based b-cell epitope prediction using three representative structural and geometrical properties-based methods: ellipro, epitopia and discotope. for this, the experimental d structure lasv gp (pdb id: vk ) with the modeled missing residues was used. ellipro predicts linear and conformational epitopes by incorporating the antigenicity, solvent accessibility, and flexibility of protein structures . epitopia uses a machine learning algorithm to analyze the antigenic features on protein structure and predicts the probable conformational epitope regions . discotope uses amino acid statistics, spatial information, and surface accessibility on the protein d structure to predict residue-by-residue conformational epitopes . the e , e , e and e structure-based epitopes in table are especially interesting as potential candidates as they were predicted by all three methods. in table , we also ranked each epitope based upon how many of the sequence and structure-based methods predicted each epitope, which do not always correlate with the highest antigenicity scores of e , e , e , e and e . robinson et al. have recently reported the cloning of many human monoclonal antibodies derived from memory b cells of lassa fever survivors in west africa. these antibodies specifically bind to both gp and gp epitopes of lasv. the comparison of our predicted b-cell epitopes with those epitopes shows that there are five consensus epitopes ( table ) that share similarity with robinson et al. (table s ) , and another five epitopes that do not share similarity, indicating that our consensus epitope prediction strategy has identified new epitopes. epitope surface mapping. for efficacy of vaccines, the epitopes should be located on an accessible region of the protein so that the epitope will be able to bind with antibodies . this is especially important for the six epitopes that we list in the tables above that do not share any part of their sequence with known epitopes: e , www.nature.com/scientificreports www.nature.com/scientificreports/ e , e , e , e , e . in fig. , we highlight the positions of these epitopes on lasv gp. we also highlight the positions of e and e because the four mhc-i t-cell epitopes have ic information readily available. figure shows that the e , e , e , e , e , e and e epitopes are well located on the exposed regions and thus can interact well with the alleles. www.nature.com/scientificreports www.nature.com/scientificreports/ crystal structure of the hla class i antigen (pdb id: ei ) as the best template for constructing models. the sequence identity between a and the template was %. the best model was then selected based on multiple validation methods, including gmqe (global model quality estimation) and qmean. the gmqe and qmean values , of the model are . , and . , respectively. in addition to these analyses, ramachandran plots and errat were also used for the model validation. analysis of ramachandran plot of the model shows . % of residues are either in favored or in allowed regions ( supplementary fig. s ), indicating that backbone torsion angles of these models are acceptable. the errat overall quality factor score was computed as , which is greater than the normally accepted score range for a high quality model of . these analyses show that the model is within a high quality range and can be used for further analysis. docking of the four consensus mhc-i epitopes (table ) was performed using autodock vina, which enabled the docking of epitopes obtained from the sequence-based mhc- t-cell prediction into the promising allele structures. the autodock vina docking protocol has been previously demonstrated to successfully dock epitopes into allele structures . however, we validated the capability of the docking protocol before docking the epitopes by redocking the epitopes into the allele crystal structure (pdb id: ox ) to see whether the crystal bound conformation of the peptide could be reproduced or not. the docked allele-epitope complex showed the same residue-epitope interactions observed in the epitope bound crystal structure, indicating that the autodock vina docking protocol was capable of reproducing the experimentally observed binding mode of the epitope. we applied autodock vina to each of the four mhc-i allele-epitope complexes. autodock vina found that the highest ranked docking structure had the following binding affinities: − . kcal/mol for a ::e − . kcal/mol table . prediction of the b-cell epitopes. the epitopes predicted by either all three sequence-or structurebased methods are highlighted by boldface. conformational epitopes chosen by all three structure-based methods are indicated in italics. www.nature.com/scientificreports www.nature.com/scientificreports/ for a ::e , − . kcal/mol for a ::e , and − . for a ::e . these epitopes-alleles docking complexes are shown in fig. . in order to investigate the dynamics and stability of the four mhc-i allele-epitope complexes, we performed ns all-atom, explicit solvent md simulations. to quantitatively understand the stability of the allele-epitope complex, we calculated the root mean square deviations (rmsd) of the backbone atoms of the allele-epitope complexes as a function of simulation time as shown in www.nature.com/scientificreports www.nature.com/scientificreports/ figure also includes curves of the rmsd of the backbone atoms of just the allele, and separately, just the backbone atoms of the epitope. all alleles have an rmsd compared to their initial structures of approximately Å, whereas the allele-epitope complexes have a bit higher rmsd of approximately . Å, indicating that the epitopes make the complexes more flexible. interestingly, in the case of a ::e , the allele and the complex show almost the same rmsd, suggesting that the complex is especially stable. to pinpoint why the complexes show a higher rmsd, we further computed the rmsd of only the backbone atoms of the epitope in each the complex. figure shows that the initial configuration of epitopes e and e is compact, and that both of these epitopes rearrange their configuration in the binding site and elongate during the ns md simulation. this elongated configuration is consistent with the investigations of antunes et al. on mhc-i epitopes. since the interactions between protein and epitope peptide are mostly influenced by non-covalent interactions, we computed the number of hydrogen bonds and the interaction energy between the allele and epitope as a function of the md simulation time. the hydrogen bond was calculated between the protein interface atoms with a distance cut-off of . Å and angle cut-off of o between the donor and acceptor heavy atoms. as shown in fig. , the number of h-bonds fluctuates during the md simulations for all the complexes. the a complex has the largest number of h-bonds. table shows that during the last ns of the md simulation trajectory, the a complex averages . h-bonds. additional analysis of the hydrogen bonding between allele and epitope are listed in supplementary table s . figure b shows the interaction energy (electrostatic interaction + van der waals contacts) throughout the entire md simulation and table lists the average over the last ns. the a ::e and a ::e display relatively stronger interaction energies than the a :e and a ::e complexes. the comparison of rmsd, hydrogen bond, and interaction energy information indicates that the e epitope is an especially promising epitope candidate. novelty analysis. the novelty of the four mhc i t-cell epitopes in table , the nineteen mhc ii t-cell epitopes in table , and the ten b-cell epitopes in table identified in this study were analyzed using iedb . the iedb database contains the epitopes that are annotated based on scientific literature. the iedb showed that the e , e , e , e , e , e epitopes, which bind to solvent exposed regions on the protein (fig. ) , have not been previously reported as lasv epitopes or vaccine candidates. in addition, this analysis further indicates that other epitopes (e , e , e , e , e , e , e , e , e , e , e , e , e , e , e , e , e , e , e , e , e , e , e , e ) have partial segments of their sequence reported as subsets of other epitopes, whereas e , e , e are exact match to previously reported sequences. for these epitopes, a comparison showing the overlap between the predicted epitopes in this study and previously known epitopes documented in iedb is given in table s . in addition to the epitopes in the iedb, we compared our consensus predicted epitopes with the previously reported predictions [ ] [ ] [ ] [ ] [ ] [ ] in table s . this comparison shows a varying degree of overlap in the predicted sequences. the www.nature.com/scientificreports www.nature.com/scientificreports/ novelty results confirm that thirty epitopes have not been previously tested experimentally as lasv epitopes, suggesting that their therapeutic potentials in designing vaccines against lasv can be further explored. lasv hemorrhagic fever is endemic in west africa, and no approved effective therapeutics are currently available. therefore, there is an urgent need for the discovery and development of potential antiviral therapeutics. the lasv gp spike has emerged as a promising selective target for the development of novel vaccines as it plays an essential role in the virus-host interaction. several in-silico studies [ ] [ ] [ ] [ ] [ ] [ ] were performed to predict lasv gp epitopes with the use of a single prediction tool for each type of epitope. we have identified new t and b-cell epitopes using a variety of computational approaches, including twelve epitope prediction methods, protein-peptide docking, and md simulations. the mhc i and ii t-cell epitopes were separately predicted with the lasv gp sequence using well-known prediction methods. the predicted mhc i t-cell epitopes then were prioritized based on the consensus score, binding affinity, and antigenicity, while mhc ii t and b-cell epitopes were prioritized based on the consensus score. novelty analysis of the consensus-selected epitopes showed that thirty of these predicted epitopes have either no overlap or only a partial overlap to previously reported sequences. within this list of new epitopes, six sequences have no overlap with any known experimentally tested epitopes in the iedb. in addition, docking and md simulations were performed to further validate the mhc i t-cell epitopes. the simulation results show that the allele-mhc-i epitopes are stable, with favorable hydrogen-bond and interaction energy. of these, epitope e ( fsrpspigy ) segment was found to be especially stable. this study demonstrates that the adopted consensus epitope prediction strategy is valuable for in-silico investigations of known epitopes and the identification of new epitopes. experimental validation of these epitopes may lead to the design and development of effective lasv vaccines. table . allele-epitope interaction parameters calculated by averaging over the last ns of the md simulation trajectory. ictv virus taxonomy profile: arenaviridae lassa fever, a new virus disease of man from west africa. i. clinical description and pathological findings understanding the cryptic nature of lassa fever in west africa structural basis for antibody-mediated neutralization of lassa virus lassa fever, a new virus disease of man from west africa. . isolation and characterization of the virus a case-control study of the clinical diagnosis and course of lassa fever protein structure of lymphocytic choriomeningitis virus: evidence for a cell-associated precursor of the 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lassa fever virus peptides predicted by computational analysis induce epitope-specific cytotoxic-t-lymphocyte responses in hla-a . transgenic mice p.c. conceived the idea. p.b. and e.p. performed and analyzed the computations and simulations, and prepared figures. p.b., e.p., b.g., and p.c. interpreted the results and wrote the manuscript. the authors declare no competing interests. supplementary information is available for this paper at https://doi.org/ . /s - - - .correspondence and requests for materials should be addressed to p.p.c. publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.open access this article is licensed under a creative commons attribution . international license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons license, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons license, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this license, visit http://creativecommons.org/licenses/by/ . /. key: cord- -odfxkfu authors: lim, dong-gyun; hafler, david a. title: molecular mimicry in multiple sclerosis: role of mhc-altered peptide ligands (mapl) date: - - journal: infection and autoimmunity doi: . /b - - . - sha: doc_id: cord_uid: odfxkfu nan multiple sclerosis (ms) is a chronic inflammatory illness affecting the cns white matter that can lead to progressive neurologic dysfunction. together with other organ-specific autoimmune diseases such as type diabetes mellitus and rheumatoid arthritis, ms is thought to be mediated by autoreactive t cells that recognize cns self-antigens. supporting evidence for the autoimmune basis of ms includes the inflammatory nature of the cns lesions, the genetic linkage to the mhc region, similarities to the animal model experimental autoimmune encephalomyelitis (eae), and the therapeutic effects of immunomodulatory drugs [ ] . data from animal studies in the eae model established that cd + thl cells specific to myelin antigens can play a central role in the induction and progression of autoimmune demyelinating disease [ , ] . this finding has fueled efforts to dissect the antigenic specificity and functional characteristics of myelin-reactive cd + t cells in the peripheral blood and cerebrospinal fluid of patients with ms. several peptide epitopes derived from myelin proteins have been found to activate cd + t cells in the circulation of patients with ms. among them, mbp (myelin basic protein) - and mbp - have been classified as immunodominant epitopes in the context of hla-dr haplotype [ ] [ ] [ ] . however, cd + t cells reactive to these self-peptides have also been detected in healthy persons, suggesting that the presence of myelinreactive t cells is not sufficient for the development of ms. while resting autoreactive t cells are a part of normal t cell repertoire, the activation status of these cells is different in patients than in healthy individuals. consistent with the properties of preactivated t cells, myelin-reactive t cells recovered from patients are less dependent on costimulation for activation and express activation markers, such as il- r, on their cell surfaces [ ] [ ] [ ] . these findings lead to the question of what induces the activation of myelin-reactive t lymphocytes in patients with ms. one attractive hypothesis is based on the role of microbial infections in the activation of selfreactive t cells. if invading microorganisms contain protein antigens with sufficient structural homology to human proteins, infection itself may be sufficient to activate pre-existing self-reactive t cells. this is the basic concept of molecular mimicry. several clinical and epidemiological studies support the hypothesis of mimicry as a mechanism of autoimmunity. according to the original observation by fujinami and oldstone [ ] , rabbits immunized with a hepatitis b polymerase peptide that shared six amino acids with the mbp sequence developed cns lesions reminiscent of eae. in humans, upper respiratory infections often precede ms exacerbations. the original concept of molecular mimicry evolved from the recent understanding of the degenerative recognition of antigens by the t cell receptor (tcr). in examining the immune response to mbp, we found that complementary mutations in an antigenic peptide allow for cross-reactivity of autoreacfive t cell clones that may be related to shifts of the tcr structure itself [ ] . using combinatorial libraries, hemmer and co-workers [ ] demonstrated that totally unrelated peptides also activate autoreactive t cells. furthermore, recent data suggest that even different mhc molecules complexed with diverse peptides can activate mbp-reactive t lymphocytes in an agonistic or antagonistic fashion. in the following sections, we will describe examples of this broader concept of molecular mimicry and its clinical relevance in ms. reveal signs of superantigenic activation of myelinreactive t cells in ms. thus, molecular mimicry is still a very attractive theory to explain the frequent association of microbial infection with the development or exacerbation of ms. [ ] . so far, none of these infectious agents have been found to be specific for ms, although an ms-specific agent may yet be discovered. the apparent absence of an ms-specific infectious agent and the autoimmune nature of ms suggest another role of infection in the development of this disease. three mechanisms have been proposed to explain the association of microbial infection and ms. one is the molecular mimicry theory, which has received a great deal of attention and will be discussed in detail in the following sections. another is bystander activation, including epitope spreading [ ] . infections can activate autoreactive t cells through the release of sequestered myelin proteins as a result of infection-related tissue damage, activation of antigen-presenting cells (apcs), and induction of secretion of inflammatory cytokines and chemokines, irrespective of the particular microbial determinants. the third is superantigenic t cell activation. several bacterial and viral products are able to cross-link tcr and mhc molecules independent of specific antigen recognition through the tcr. cross-linking leads to activation of t cells with particular v[ families of tcr. myelin-reactive t cells with a particular tcr vi chain can be activated after infection with microbes whose superantigen recognizes this specific vi chain. again, many studies have failed to . molecular mimicry associated with sequence homology molecular mimicry describes a situation whereby a foreign antigen can initiate an immune response in which a t or b cell component cross-recognizes self. the previous concept for the antigen specificity of t cells predicted that the presence of strict sequence homology between the microbial antigens and self-peptide was necessary to induce autoimmunity ( fig. , top a). there are two approaches to determining the foreign microbial antigens that are cross-reactive to self-antigens. one is the initial identification of causative microbes and their major antigenic epitopes and subsequent demonstration of their cross-reactivity to self-antigens. this approach was applied successfully to reveal that two autoimmune diseases are caused by a molecular mimicry mechanism. autoimmune lyme arthritis is preceded by infection with b. burgdorferi and is associated with hlfa- -reactive t cells that were initially primed and expanded by the ospa( - ) peptide of this spirochete [ ] . herpetic stromal keratitis (hsk) follows infection of the eye with herpes simplex virus and is caused by the activation of autoreactive cells by viral ul ( - ) peptide [ ] . however, it is difficult to apply this strategy for the study of molecular mimicry in ms because, as mentioned previously, no microbial agent has been directly associated with the disease. the other approach is to first identify t cell determinants capable of inducing autoimmunity and then search for the microbial antigens with sequences homologous to those determinants. an initial study adapting this latter strategy successfully demonstrated that a peptide from hepatitis b virus polymerase (hbvp) with six consecutive amino acids in common with an encephalitogenic determinant of mbp (icgygslpqe in hbvp vs tthygslpqk in mbp) could induce subclinical eae in rabbits [ ] . however, proteins sharing a sequence of more than six amino acids are not common in nature, and this type of homologous foreign peptides has not yet been identified in human ms. extensive biochemical and structural characterization of the recognition of mhc/peptide ligand by tcr in humans has greatly influenced the identification of cross-reactive epitopes for the given tcr. while a few amino acid residues of a peptide are important for binding to the mhc molecule, the other one or two amino acid residues serve as critical residues for the recognition by the tcr (fig. , top b) . specifically, in the immunodo-minant peptide ligand (mbp - ) presented by drb* , two hydrophobic residues (val- and phe- ) serve as the primary anchors to the hla-dr molecule, while phe- and lys- have been defined as the primary tcr contact residues for a mbp - -specific t cell clone. mutations in other amino acid residues are tolerated with respect to recognition by the tcr [ ] . on the basis of this information, wucherpfennig and strominger [ ] developed an efficient strategy to search for peptides that share these critical contact motives rather than sequence homology. this search yielded seven viral and one bacterial peptides derived from herpes simplex virus, adenovirus, human papillomavirus, epstein-barr virus, influenza type a virus, reovirus type , and pseudomonas that efficiently activated t cell clones. interestingly, only one of them has been identified as a molecular mimic by sequence alignment. these data clearly indicated that more foreign peptides may act as molecular mimics than was previously thought. conservation of critical mhc and tcr contact residues appears to be a general rule for activation by the peptide of a specific t cell. however, several peptide sequences with no sequence homology have been shown to be able to activate identical t cells (fig. , top c) [ , ] . in addition, the use of synthetic peptide combinatorial libraries has clearly illustrated the extreme degeneracy of tcr recognition of antigen. hemmer et al [ ] examined the response of cd + t cell clones specific for mbp - to a set of -mer peptide sublibraries, each containing degenerated amino acids and one defined amino acid in the positional scanning format. the new knowledge obtained from this study was ) that of highly degenerative recognition of peptides by autoreactive cd § t cells, including identification of stimulatory ligands not sharing a single amino acid in corresponding positions with the antigen used to establish the t cell clone and ) the identification of more potent agonistic peptides than cognate self-peptide. from the database search for natural proteins with deduced peptide sequences, they found four self and three microbial proteins as stimulatory ligands to this t cell clone. most interestingly, one self-peptide (protein-glutamine gamma-glutamyltransferase, - ) and two microbial peptides (human cmv ul , - ; udp-n-acetylenolpyruvoyl-glucosamine reductase of salmonella typhimurium, - ) were defined as even more potent agonists than mbp - . this strategy opened the way for the identification of molecular mimics if relevant autoreactive t cells are defined in ms. the studies discussed above focused on the molecular mimics with respect to the peptide portion of tcr ligand. since tcrs recognize mhc/peptide as a single unit and exhibit greatly degenerative recognition of their ligands, they might recognize a different mhc combined with a cognate or even a different peptide as an agonist (fig. , bottom) . since most people are heterozygous for the hla-dr locus, this type of molecular mimicry is likely to occur in physiological situations. it has been shown that the human mbp peptide - binds to several different dr molecules and that t cells recognize this peptide presented by these different hla-dr molecules [ ] . we systematically exarpdned this type of cross-reaction using a panel of cd § t cell clones specific to taxi - peptide of htlv- in the context of hla-a* [ ] . when cd § t cells were stimulated with cognate taxi - peptide presented by different hla-a subtype alleles, which have one to four amino acid differences at specific positions (table ) , they showed a diverse pattern of t cell function, encompassing agonistic, weak agonistic, or partial agonistic, depending on the individual t cell clones (fig. ) . this is similar to the effects induced by antigenic altered peptide ligands (apl). in addition, atypical partial agonistic t cell function was observed; i.e., a number of cd + t cell clones proliferated in response to taxi - ~ presented by the hla-a* subtype even though they did not exhibit any cytotoxic activity. the analysis of the structural interaction between the tcr and mhc/peptide complex indicated that polymorphic amino acids in the hla-a peptide-binding groove, especially the d-pocket, rather than the dif- a reproduced from ref. [ ] by copyright permission of the rockefeller university press. ferences on the mhc residues in direct contact with the tcr, were responsible for this partial agonism (fig. a ). this was supported by the finding that reciprocal mutations of the tax peptide side chain that engaged the d-pocket restored the agonist functions of the mhc/peptide complex (fig. b) . thus, our study clearly demonstrated that mhc molecules play an important role in t cell activation not only by restricting antigen element but also by altering the antigenic nature of peptide. we termed this type of tcr ligand mhc-altered peptide ligand (mapl). cd § t cell can also exhibit mapl effects on the recognition of variant mhc class ii molecules. germain and colleagues [ ] previously suggested that a peptide presented on a mutant mhc class ii molecule induces a response different from the response induced with the native mhc molecule. we also recently examined the modulation of t cell responses induced by stimulation with different mhc class ii/peptide complexes, demonstrating the existence of significant cross-reactivity of autoreactive cd + t cells in the context of the distinct mhc class ii molecules. the ob a t cell clone is reactive to the immunodominant mbp peptide, mbp - , presented by hla-dra/drb* and was generated from peripheral blood mononuclear cells of a patient with ms [ ] . characterization of this t cell clone showed that it recognizes several single amino acid substitutes of mbp - presented by drb " in a degenerative fashion. moreover, when the responses of this t cell to apls in the context of the other self-dr allele (drm drbi* ) were examined, the obla t cell clone responded to the mbp - v--->k apl, even though it did not recognize the mbp - presented by this allele (fig. , [ ] ). these data indicate that apl-induced cross-reactivity provides a further degree of t cell receptor degeneracy among different dr molecules. it should be noted that the functional outcome of this type of crossreaction could be agonistic, partial agonistic, or even antagonistic depending on the specific combination of mhc molecules and peptides. the cross-reaction induced by mapls containing cognate, altered, or even irrelevant peptides has not been well appreciated as a potential mechanism of molecular mimicry. however, our data generated from the systemic analyses in vitro, as mentioned above, and the increasing awareness of the highly degenerative nature of tcr recognition strongly predict that this type of molecular mimicry might effect the activation of autoreactive t cells. in fact, a recent report provides a good example for this type of molecular mimicry by showing that t cells derived from a patients with ms recognized an epstein-barr virus dna polymerase peptide in the context of drb * as well as the immunodominant mbp - epitope in the context of drb " [ ] . the structural similarity in tcr contact surfaces between these two mhc/peptide complexes explains this cross-reactivity. if this type of molecular mimicry is frequent, the chances for autoreactive t cell activation would be higher than previously thought, while the identification of mimicry peptides would be more complicated. this means that we need to consider both the peptide epitopes and mhc class ii alleles to carry out an exhaustive study of the molecular mimics. it is important to define whether molecular mimicry induces the initiation of autoimmune responses or contributes only to the exacerbation of existing autoimmune responses. unfortunately, no convincing evidence is currently available for a role of molecular mimicry in the initiation of any autoim- mune disease. it should be emphasized that all the cross-reactive foreign peptides have been defined by their stimulatory capacity and selected from protein databases or screening based on the role of individual amino acid residues. therefore, it is not clear whether these peptides can be processed by and presented on apcs after microbial infection. if they can be, it still remains to be defined whether or not cross-reactive t cells can be successfully activated and expanded sufficiently to attack self. even though epiderniological studies suggested that sev-eral viral and bacterial infections often precede the exacerbation of ms [ ] , it is difficult to discriminate between two potential mechanisms for this effect: the molecular mimicry described here and the bystander activation of autoreactive t cells. the latter includes the effect of inflammation, epitope spreading, or a superantigenic effect of invading microbes. in addition, a chronologic relationship is difficult to establish because the starting point of the disease is usually unknown and most suspicious microbes induce persistent infection. the lack of an animal model of spontaneous development of ms is another obstacle to finding answers to these questions. thus, although a great deal of fragmentary evidence is available, a definitive role for molecular mimicry in the pathogenesis of ms has not been proven. potential evidence for the role of molecular mimicry in human ms has recently emerged from a clinical trial of altered peptide ligand therapy [ , ] . in vitro and animal studies found that apls can inhibit the response of t cells to agonistic autoantigen and can block eae [ , ] . furthermore, apls could induce a novel, apl-reactive t cell population that had a th phenotype and cross-reacted with native mbp antigens [ ] . these data supported the use of apls as a therapeutic agent for ms. unfortunately, unexpected detrimental side effects, including a hypersensitivity reaction to apls and exacerbation of disease, necessitated the premature termination of all the trials before completion. an immunological study suggested that the latter side effect came from the expansion of apl-specific t cells with pro-inflammatory phenotype, which cross-reacted with mbp [ ] . this finding is perhaps the strongest argument that, in some situations, molecular mimicry indeed can play a role in the pathogenesis of ms. considering the highly degenerative nature of t cell recognition, it can be proposed that apls may be presented by any of the hla class ii molecules and activate the pro-inflammatory t cells, which cross-react with myelin antigens presented either by apl-restricting or another class ii molecule. however, the previous studies employed a single hla-dr molecule for the selection of the apls. since the majority of patients are heterozygous for hla-dr locus, the effect of apls should be considered in conjunction with the individual's entire hla class ii haplotype. actually, we recently observed that one apl ( v---)k) of mbp - could stimulate the obla tcr in the context of both drb* and drbi* , whereas the original mbp - peptide could not be recognized in the context of drb* [ ] . therefore, stricter criteria, considering all the self-mhc class ii molecules as potential restriction elements, should be applied for the selection of apls. molecular mimicry still remains an attractive hypothesis to explain the initiation and maintenance of ms lesions. epidemiological and immunological studies support this theory. indeed, several microbial peptides that cross-react with self myelin proteins have been identified. in addition, more microbial peptides will be identified in the near future if the mapl concept is considered during screening for cross-reacting antigens. however, as mentioned previously, not all the available data can be taken as direct evidence that this molecular mechanism is at work in the pathogenesis of ms. considering the highly degenerative nature of tcr recognition and the presence of self-reactive t cells in a normal repertoire, why do only a small percentage of people suffer from ms after microbial infections? one possible explanation is the multifactorial origin of ms. a recent twin study convincingly showed the importance of genetic traits as a risk factor [ ] . if genetically predisposed individuals suffer from infections, they may develop ms due to molecular mimicry. recently, there has been a conceptual movement in the ms field that ms is not a single disease entity but rather a syndrome composed of different disorders with different causes [ ] . in addition, major pathogenic mechanisms might differ depending on the disease stage. therefore, the molecular mimicry hypothesis should be re-evaluated according to the new concept of the complexity of t cell cross-reactivity as well as the disease entity. immunological aspects of demyelinating diseases adoptive transfer of myelin-basic protein-sensitized t cells produces chronic relapsing demyelinating disease in mice t-cell clones specific for myelin basic protein induce chronic relapsing paralysis and demyelination t-cell recognition of an immunodominant myelin basic protein epitope in multiple sclerosis myelin autoreactivity in multiple sclerosis: recognition of myelin basic protein in the context of hla-dr products by t lymphocytes of multiple sclerosis patients and healthy donors fine specificity and hla restriction of myelin basic protein-specific cytotoxic t cell lines from multiple sclerosis patients and healthy individuals decreased dependence of myelin basic protein-reactive t cells on cd -mediated costimulation in multiple sclerosis patients. a marker of activated/memory t cells expansion of autoreactive t cells in multiple sclerosis is independent of exogenous b costimulation increased frequency of intedeukin -responsive t cell specific for myelin basic protein and proteolipid protein in peripheral blood and cerebrospinal fluid of patients with multiple sclerosis amino acid homology between the encephalitogenic site of myelin basic protein and virus: mechanism for autoimmunity complementary mutations in an antigenic peptide allow for crossreactivity of autoreactive t-cell clones identification of high potency microbial and self ligands for a human autoreactive class h-restricted t cell clone multiple sclerosis. philadelphia: fa davis virus-induced autoimmune demyelinating disease of the central nervous system identification of lfa- as a candidate autoantigen in treatment-resistant lyme arthritis molecular mimicry by herpes simplex virus-type i: autoimmune disease after viral infection structural requirements for binding of an immunodominant myelin basic protein peptide to dr isotypes and for its recognition by human t cell clones molecular mimicry in t cell-mediated autoimmunity: viral peptides activate human t cell clones specific for myelin basic protein degenerate recognition of a dissimilar antigenic peptide by myelin basic protein-reactive t cells. implications for thymic education and autoimmunity intramolecular mimicry. identification and analysis of two cross-reactive t cell epitopes within a single protein mcfarland he a myelin basic protein peptide is recognized by cytotoxic t cells in the context of four hla-dr types associated with multiple sclerosis allelic variation of mhc structure alters peptide ligands to induce atypical partial agonistic cd § t cell function peptide-major histocompatibility complex class ii complexes with mixed agonist/antagonist properties provide evidence for ligand-related differences in t cell receptor-dependent intracellular signaling t-cell recognition of an immunodominant myelin basic protein epitope in multiple sclerosis cross-reactive human autoreactive t-ceu receptor responses to altered peptide ligands presented by different mhc class ii molecules a functional and structural basis for tcr cross-reactivity in multiple sclerosis clinical viral infections and multiple sclerosis encephalitogenic potential of the myelin basic protein peptide (amino acids - ) in multiple sclerosis: results of a phase ii clinical trial with an altered peptide ligand induction of a non-encephalitogenic type t helper-cell autoimmune response in multiple sclerosis after administration of an altered peptide ligand in a placebo-controlled, randomized phase ii trial. the altered peptide ligand in relapsing ms study group antigen analog-major histocompatibility complexes act as antagonists of the t cell receptor reversal of experimental autoimmune encephalomyelitis by a soluble peptide variant of a myelin basic protein epitope: t cell receptor antagonism and reduction of interferon ~/and tumor necrosis factor ct production an altered peptide ligand mediates immune deviation and prevents autoimmune encephalomyelitis a genetic basis for familial aggregation in multiple sclerosis. canadian collaborative study group heterogeneity of multiple sclerosis lesions: implications for the pathogenesis of demyelination key: cord- -lchdm xr authors: liu, yang; king, nicholas; kesson, alison; blanden, robert v.; müllbacher, arno title: flavivirus infection up-regulates the expression of class i and class ii major histocompatibility antigens on and enhances t cell recognition of astrocytes in vitro date: - - journal: j neuroimmunol doi: . / - ( ) - sha: doc_id: cord_uid: lchdm xr west nile virus (wnv) infection of astrocytes can up-regulate their expression of both class i and class ii major histocompatibility complex (mhc) antigens as determined by flow cytometry with monoclonal antibodies specific for class i and class ii mhc antigens. the up-regulation of class i mhc antigen expression could be partly caused by interferon secreted after wnv infection because the synthetic interferon inducer polyinosinic-polycytidylic acid (poly i : c) has similar effects. in contrast the up-regulation of class ii mhc antigen expression was not induced by poly i : c. the increased mhc antigen expression by wnv infection had significant effects on t cell recognition. thus, wnv and influenza virus a/wsn double-infected astrocytes but not astrocytes infected by a/wsn alone were lysed by influenza virus-immune cytotoxic t cells. similarly, wnv-infected astrocytes were better stimulators than normal astrocytes for a class ii mhc-reactive t cell line, both in terms of t cell proliferation and interleukin release. it is well established that the major histocompatibility complex (mhc) antigens form an essential part of antigenic structures recognised by t iymphocytes (reviewed by zinkernagel and in the case of experimental allergic encephalitis of rats, where transfer of syngeneic t cells specific for myelin basic protein (mbp) results in full expression of the disease (lipton et al., ; tourtellotte et al., ; kreth et al., ; booss et al., ; wekerle et al., ) . the paradox has been partly resolved by findings which indicate that at least some cells from the cns, such as astrocytes, ofigodendrocytes, brain endothelial cells and neurons can be induced to express class i and/or class ii mhc antigens by lymphokines such as "t-interferon (t-ifn) and by certain viral infections such as murine hepatitis virus and theiler's murine encephalomyelitis virus (hirsch et al., ; massa et al., ; suzumura et al., ; rodriguez et al., ) . additional data suggested that such elevation of the mhc antigens on astrocytes and/or brain endothelial cells can potentiate the antigen-presenting capacity of these cells male et al., ) . thus, the induction of expression of mhc antigens on the cells of the cns could be an important regulatory mechanism in the initiation, amplification and targeting of the local immune response. the etiology of a number of neurological disease, most notably, multiple sclerosis, still remains a matter of debate. the preferred hypotheses involve an autoimmune phenomenon in which cellular and/or humoral immune responses are directed against constituents of myelin. however, epidemiological data implicate some kind of acquired factor, most likely an infection, in the etiology of multiple sclerosis (waksman et al., ) . in recent years, evidence has accumulated that viral infections can result in the breakdown of immunological tolerance. thus both autoreactive b cells (haspel et al., a, b) and autoreactive t cells (pfizenmaier et al., ; komatsu et al., ) have been observed during viral infection. in this context it would be of interest to study the possible effect of neurotropic viral infection on the local immune response in the cns. flaviviruses are one group of viruses which are responsible for a number of neurological diseases (monath, ) . in this study, we address the effect of west nile virus (wnv) infection on astrocytes in vitro. west nile virus sarafend strain (wnv) was obtained from dr. i.d. marshall and grown either in vero cells (wnv-v) or in new-born mouse brain (wnv-b). the stocks were prepared and titrated as described (reed and muench, ; taylor and marshall, ) . influenza virus a/wsn was prepared by a standard method (yap and ada, ) . mice were bred under pathogen-free conditions at the animal breeding establishment of the john curtin school of medical research. the strains used were: cba/h (h- k), balb/c (h- d), b .t( r) (kqiqdd), b .aqr (kqlkdd). newborn mice were used at or days of age and adult female mice at - weeks. astrocytes were prepared in essentially the same way as has been described (mccarthy and de vellis, ) . briefly, -to -day-old cba/h mice were anesthetized at - °c for min. after careful removal of the meninges, the brains were broken down by pipetting and were then subjected to treatment with trypsin/dnase i solution ( . ~ trypsin and . ~ dnase i, w/v) for min, shaken occasionally. the action of trypsin was stopped by adding equal volumes of neural medium (dulbecco's minimum essential medium (dmem) supplemented with l-arginine mg/l, l-asparagine mg/l, folic acid mg/l, v-glucose g/l, and non-essential amino acids ~ v/v (flow laboratories, . the suspe-sion was centrifuged and the pellet was dispersed. these cells were cultures in cm nunclon plastic flasks at a density of /ml. at day , the cells were confluent and the medium was changed. one day later, the flasks were shaken for - h ( °c, rpm). the medium was then sucked off and the cultures were washed with puck's saline, trypsinized into single-cell suspensions and cultured again. the secondary cultures were tested by indirect immunofluorescence microscopy with rabbit anti-human glial fibrillary acidic protein (gfap) serum (dako, code a ) and shown to he more than % gfap positive. the secondary cultures were used in this study. after astrocytes were grown to confluency (approximately l s cell/well in linbro -well plastic iissue culture plates), the medium was sucked off and west nile viruses ( plaque-forming units (pfu)/cell of wnv-b and pfu/cell wnv-v, respectively) were added in /li and incubated at o c for i h. at different times after inoculation, cells were harvested and frozen at - ° c. free virus was obtained by freeze-thawing plus ultrasonication as described (taylor and marshall, ) . the protocol is essentially the same as described by de clercz ( ) . briefly, astrocytes were grown in -well tissue culture trays until confluency; the cultures were then exposed to poly i:c (sigma, /lg/ml) for h and washed with dmem medium times. fresh neural medium was added and the cultures were further incubated for h. the supernatants were harvested and ifn activity determined. ifn was assayed by inhibition of viral rna synthesis in l cells as described by morris et al. ( ) . the titer of ifn was defined as reciprocal of the final dilution that gave % inhibition of semliki forest virus rna synthesis. / of : dilution of supernatants from h wnv-b-infected astrocytes were incubated with an equal volume of medium or units of rabbit antiserum to mouse ifn (a +/]) (lee biomedical research, cat. no. ) at °c for h. the ifn activity of these supernatants was titered as described above. spleen cells from b ~t( r) mice (kqlqd d) were stimulated with those from b .aqr (kqlkd d) as described (king et al., ) for three passages. at the fourth passage, an enriched medium for t cell lines (dmem supplemented with x - m -mercaptoethanol, ~ fetal calf serum (fcs) and % concanavalin a (cona)-activated spleen cell supernatant (cs) prepared as described by sinickas et al ( a) ) was added together with allogeneic stimulators. generation of secondary cytotoxic t cells against influenza virus a/wsn and primary allo-reactive cytotoxic t cells has been described in detail (yap and ada, ; king et al., ) . the method for the l target system has been described (yap and ada, ) . wnv-v-infected or uninfected astrocytes were cultured in flat-bottomed tissue culture plates at °c for h. these cultures were then reinfected with influenza virus a/wsn ( eid~o/cell) and labeled with cr by adding /~ medium containing x eids /well influenza virus a/wsn and : dilution of na slcr and incuba~,a at °c for h. the rest of the assay was carried out in the same way as that for l targets. the proliferation of the h- ik-reactive t cell line was determined by [ h]methyl-thymidine (amersham) incorporation as has been described (sinickas et al., b) . both spleen cells and astrocytes were used as stimulators. after growth to confluency in -well tissue culture plates (approximately × cells/well), the astrocytes were irradiated with r from a e°co v-ray source. /~g/ml of indomethacin was added to the enriched medium of t cell lines and /tl of this medium per well was added together with x cells of an h- k-reactive t cell line. h later, . ;tci/well of the [ h]methyl-thymidine was added. after another h, the cultures were harvested onto glass fiber filter strips and the incorporation of label into dna was determined by scintillation counting. wnv-infected or mock-infected astrocytes grown to confluency in -well plates ( x / well) were irradiated with r and were used as stimulators of × t cells in . ml dmem medium per well supplemented with - m mercaptoethanol, #g/ml indomethacin and % fcs. h later the supernatants were collected and their interleukin activity was tested, as described by lafferty et al. ( ) . it is now known that this assay can measure interleukin- (il- ) and/or imerleukin- (il- ) activity (severinson et al., ) . moneclonal antibodies (mcabs) specific for d k, i-a k and i-a a antigens were obtained from the supernatants of cultures of hybridoma clones - - s, - . . . , and mk-d (oi et al., ; ozato et al., ; kappler et al., ) (atcc nos. hb- , t b- and hb- ), respectively, obtained from the american type culture collection. the supernatants were concentrated -fold on an american p membrane. in adoition, an mcab specific for k k (clone - . ) was purchased from becton-dickinson. rabbit anti-mouse immunoglobulin (ramig) was raised in a new zealand white rabbit, separated from whole serum using a protein a-sepharose column (pharmacia) and conjugated to fluorescein-isothiocyanate (fitc) by standard methods (( oding, ) . astrocvtes ~ ere infected with wnv or treated with recombinant ~-ifn ( u/ml) or poly i : c for h. s:,ngle-cell suspensions of astrocytes were obtained from the culture vessel by gentle trypsinization. the cells were counted and viability, as measured by trypan blue dye exclusion, was invariably > ~. all subsequent steps of the labeling procedure were carried out at ° c. samples of × l s astrocytes were incubated in /l of anti-mhc antibody for rain. after the first antibody incubation the cells were centrifuged through a bed of pl fcs, the supernatant removed, and the astrocytes resuspended in /~ of ramig-fitc for a further min at a dilution giving saturation labeling (determined by prior titration). following this incubation, the cells were centrifuged through an fcs bed and resuspended in # dmem for flow cytometry. fluorescence was measured using a facs iv (beckton-dicldnson) equipped with an argon ion laser set at the standard excitation wavelength of nm for fitc. emitted fluorescence between and nm was measured. × cells were analyzed from each labeled sample. in no experiment did any of the astrocyte sample populations show changes detectable by lowangle (cell size) or right-angle (membrane configuration) scatter analysis on the facs. thus we presume that differences in the mhc fluorescence distributions between astrocyte groups reflect a ~:hange in the cell surface mhc antigen concentration. astrocytes grown to confluency in -well tissue culture plates were infected with either pfu/cell of wnv-b or pfu/cell of wnv-v, and viral titers at different times after infection were determined. titers dropped about -fold within h (table ) . however, a clear increase in viral titers was observed by h. the titers reached a plateau at h after infection and remained at a similar level for at least week. further experiments revealed that viral antigens became detectable after h by immunofluorescence microscopy and by h - ~ of the astrocytes were expressing detectable viral antigen. the proportion of viral antigen-positive cells remained at a similar level for at least weeks. these data show that wnv can productively infect some cells in astrocyte populations. astrocyte cultures, either infected with wnv or treated with poly i:c, were tested for their ability to produce ifn. high titers of ifn were detected in the supernatant of astrocytes infected by wnv-b or wnv-v, or treated by poly i : c, all for h ( table ). the ifn titers were -fold . . . . . . . . . . . . • west nile virus prepared from mouse brain. b west nile virus prepared from verb cell line. lower in the supernatant of poly l:c-treated astrocytes than that of wnv-infected astrocytes. furthermore, the ifn produced in wnv-b-infected astrocytes can be neutralized by antibody specific for ifn (a + ~ ), hence the ifn produced is a-and/or p.ifn (fig. ) . the expression of class i mhc antigens on cba/h astrocytes was investigated by flow cytometry. the single-cell suspensions were stained with mcabs specific either for h- d k or h- k k. after the astrocytes were infected by wnv-v for h, the expression of h- k ~ as determined by peak fluorescence increased by more than -fold compared to mock-infected astrocytes (fig. a) . the binding of the mcab to the h- k k was specific as an isotype control mcab specific for h- -a d (hb- ) did not bind significantly to either normal or wnv-infected astrocytes. similar enhancement was observed in h- d k expression (fig. b) . as both wnv-b and wnv-v are potent ifn inducers (table ) , the question arose as to whether the elevation in mhc antigen expression was due *.o the ifn produced. to investigate this question a synthetic ifn inducer, poly i:c was used. as shown in fig. c , poly i : c treatment can significantly enhance the expression of class i mhc antigens, which admits the possibility that the enhanced expression after wnv infection is at least partly the result of virus-induced ifn. as class i mhc antigens are the major restric- in each case astrocytes were treated for h. virus-immune tc (table ). in contrast, astrocytes infected by influenza virus or wnv alone were not lysed by influenza virus-immune tc more than normal astrocytes. the activity of the tc was confirmed as *.hey lysed influenza virus-infected l (h- t) cells, but not mock-infected l cells. this result revealed that wnv infection enhances mhc-restricted virus-specific tc recognition on astrocytes. lysis of wnv-infected astrocytes by balb/c anti-cba/h alloreactive t cells was significantly higher than that of normal astrocytes while comparable to the lysis of y-ifn-treated astrocytes (fig. ) , hence wnv infection enhances ulloreactive tc recognition on astrocytes. the expression of cell surface cl~s ii mhc antigens on cba/h astrocytes was also determined by flow cytometry. astrocytes were labeled with h- i-ak-specifi¢ mcab tib ; control cells were treated with hb- , an mcab specific for h- -a d. normal cba/h (h- k) astrocytes express little or no detectable class ii mhc antigens, as their binding to tib (h- -ak-speeific) is not significantly higher than to hb- (h- i-a dspecific) (fig. a) . however, after the astrocytes were infected by wnv-b for h, a distinct increase in expression of h- -a k antigen was detected fig. b) , although the amount of h- i-a k expressed on wnv-infected astrocytes was not as , and astrocytes treated with "g-ifn (c). c: as for b, but normal astrocytes (a) and astrocytes treated with poly : c (b). d: as above but astroq,'tes infected by wnv-v. ast.~ocytes were subject to pre-treatment with wnv or ifn or poly i : c for h before labeling. much as that on y-ifn-treated astrocytes. the specificity of the labe~ng was ascertained in two ways: (a) hb- (anti-h- i-a °) mcab dad not bind wnv.infected cba/h (h- k) astrocytes (fig. a) ; (b) as hb- is of the igg a subclass, while tib~ is igg b, another igg b mcab specific for murine cd was tested and shown not to bind either wnv-infected or normal astrocytes (data not shown). it seems unlikely that material other than wnv in wnv-b (e.g. murine ifn) is responsible for the up-regulation of class ii mhc antigen, as wnv-v that was passaged times in veto cells (a permanent cell line originating from monkey kidney that does not secrete murine ifn) also consistently up-regulated class ii mhc antigen, though to a lesser extent than wnv-b in this particular experiment (fig. d) . furthermore, poly i :c, which "nduced ifn production and enhanced class i mhc antigen expression in astrocyte cultures (fig. c , table ) dad not induce class ii mhc ant/gen expression (fig. c) . the mechanism of the induction of class ii mhc antigen is at present unknown. an alloreactive b .t( r) anti-b .aqr t cell line specific for h- i k antigens was used to de-table termine if the wnv-induced expression of class ii mhc antigens on astrocytes can be recognised by t cells. the t cell line was induced to proliferate by spleen cells from mice bearing h- i k antigens, e.g. b .aqr and cba/h mice, but not by antologous b .t( r) spleen cells (table , expt. ). wnv-infected cba/h astrocytes also stimulated the proliferation of the h- ik-reactive t cell line (table , expt. ). the stimulation was comparable to cba/h spleen cells; mock-infected astrocytes had no stimulation effect. to test whether the induced stimulation effect of astrocytes was due to induced ¢lass ii mhc antigens on their surface, the h- i-ak-specific mcab tib was added. tib blocked the proliferation of the h- k-specific cell line stimulated by either b .aqr spleen cells or by wnv-v-infected cba/h astrocytes ( wnv-infected and uninfected astrocytes were also compared for their ability to stimulate il- and/or il- release from the h- ik-specific t cell line described above. wnv-infected cba/h astrocytes stimulated significant release of il- and/or l- , whereas uninfected cba/h astrocytes were, much poorer stimulators (fig. ) . the quantity of il- and/or il- released was about -fold higher with wnv-infected astrocytes than with normed astrocytes. specificity of the stimula- wnv-infected cba/h astrocytes stimulated significantly il- and/or il- release from the primary h- ik-reactive t cell population, while co!s the normal astrocytes induced essentially no il- and il. release (fig. ). since it was established that mhc antigens formed an important part of the antigenic struc- ture recognised by t lymphocytes (zinkernagel and doherty, ; schwartz, ) , there "has been great interest in the regulation of mhc antigen expression on potentjal antigen-presenting cells. it is well documented that different virus infections may either inhibit or enhance the expression of mhc antigens, notably, infection by adenovirus (f~i~lbo et al., ) , herpes simplex virus type (jennings et al, ) , ectromeiia virus (gardner et al., ) and measles virus (rager-zisman et al., ) can down-regulate class i mhc antigen expression of their host cells and may make the latter less susceptible to cytotoxic t cells. on the other hand, murine hepatitis virus (massa et al., ; suzumura et al., ), epstein-barr virus (mccune et al., , moloney murine leukemia virus (flyer et ai., ) and theiler's murine encephalomyelitis virus (rodriguez et al., ) can up-regulate class i and/or class ii mhc antigen expression. our results described here revealed that flavivirus infection can up-regulate the e:~pression of cell surface class i and class i i~,~hc antigens. it has been docuraented that interferon can up-regulate class ! and ii mhc antigen. all three classes of ifn l~own (a, t, ~,) have been reported to enhance ,class i mhc antigen expression, while only ~,-ifn can up-regulate the expression of class ii mhc ~mtigens ovong et al., ) . in our experiment, any treat~ent that can induce ifn production, e.,g. wi, v-b and wnv-v infection or poly i : c treatmen~,, can up-regulate class i mhc antigens; it is possible ~hat the up-regulation of class i mhc antigen expression by wnv infection is at least patti , the result of ifn produced after virus infection. in contrast, poly i:c treatment of astrocytes, which induces good ifn production and increases class i mhc antigen expression equal to or more than wnv infection, fails to induce class ii mhc antigen expression. this finding together with the data that ifn produced in wnv-b-infected astrocyte culture can be neutralized by anti-(a + fl)-ifn antibody strongly suggests that the induction of class ii mhc antigen by wnv does not depend on ifn produced. addition of antibodies that neutralize y-ifn to wnv-infected astrocytes culture would help to answer the question if free v-ifn was involved in class ii mhc antigen induction. however, the question as to what extent virus replication per se and induced interferon actually contribute to enhanced class i mhc expression is more problematic. some obviol:s approaches used by others (massa et al., ) , i.e. use of ultraviolet-inactivated virus and neutralizing mcab to the virus cannot provide definile information, because it is not known to what extent uv inactivation will inhibit viral protein and nucleic acid synthesis and the mechanism of viral neutralization is at present not well understood. furtherraore, addition of ifn antibody may not help to exclude the role of ifn due to the autocrine activity without the need of secretion (sanceau et al., ) . it seems unlikely that the binding of h- i-a k-speo_p._c mcab to wnv-infected but not normal astrocytes of cba/h(h- k) was due to the molecular mimicry by a viral product of an epitope on the i k molecule, because an mcab specific for h- -a d can bind to wnv-infected astrocytes from balb/c(h- d) mice but cannot bind to wnv-infected astrocytes from cba/h(h- k) mice; wnv can. repucate productively in astrocytes from both strains of mice (data not shown). a number of studies have shown that treatment which up-regulates the expression of cell surface class i mhc antigens increases the susceptibility of cells to lysis by appropriately sensitised tc cells, probably due to recognition by low-affinity clones that could not exhibit cytotoxic activity without the elevation of mhc antigen expression (shimoukevitz et al, ) . infection by viruses that down-regulate cell surface mhc antigen expression, on the other hand, results in reduced susceptibility of the infected cells to lysis by virus-specific cytotoxic t cells. thus it has l~een shown that herpes simplex virus type infection, which down-regulates cell surface mhc expression, reduces the susceptibility of an sv -and hsv -double-infectezl targets to lysis by sv specific tc cells, as compared to target cells infected with sv alone (jennings et al., ) . adenovirus type infection inhibits the cell surface expression of class i mhc antigen via the e protein. this phenomenon results in less efficient recognition by adenovirus-specific tc cells of target cells infected by this wild-type virus compared to target cells infected by an e deletion mutant which has no effect on host mhc antigen expression (miillbacher and bellett, personal communication) and could be responsible for the tumorigenicity of the wild-type virus (eager et al., ) . a moloney murine leukemia virus that increases cell surface class i mhc antigen expression was also reported to increase the susceptibility of host cells to lysis by alloreactive tc cells (flyer et al., ) . however, no attempt was made to address the effect of up-regulated mhc antigen expression on the recognition of mhc-restricted, antigen-specific t ells. in this paper, we compared wnv.infected astrocytes with uninfected controls for their ability to act as targets of influenza virus-specific tc cells and as stimulators for an ik-reactive t cell line. the results showed that wnv infection has significant effects on t cell recognition of astrocytes. 'ilms, after infection by influenza virus, normal astrocytes are poor targets of influenza virus-specific tc, while doubly infected (with wnv and influenza) astrocytes can be well recognized by influenza-specific tc. wnv-infected astrocytes from cba/h mice can also stimulate ik-reactive t cells significantly better than the normal astrocytes, in terms of both t cell proliferation and interleukin release. the latter function was also enhanced in populations of primary anti-h- i k t cells. these findings are particularly interesting because astrocytes are a large population of cells in the cns, presumably important both for the physiology and immunology of the cns (fontana e~ al., ) . elevated t cell recognition of astroeytes could have an important effect on the organism. these findings also raise interesting questions regarding self tolerance. if t cell tolerance to self antigens is determined quantitatively (blanden et al., ) the up-regulation of mhc antigens in the cns could possibly break t cell self tolerance in the cns. this may explain the autoimmune/virai etiology of some neurological diseases. quantitative considerations of t cell activation and self-tolerance immunohistological analysis of t lymphocyte subsets in the central nervous system in chronic progressive multiple sclerosis interferon induction by polynucleotides, modified polynucleotides, and polycarboxylates expression of histocompatibility antigens h-k. -d, and -l is reduced in adenovirus- -transformed mouse cells and is restored by interferon ~ ) astrocytes as antigen-presenting cells. i. induction of la antigen expression on astrocytes t cells via in-anune interferon and its effect on antigen presentation retrovirus induced changes in major histocompatibility complex antigen expression influence susceptibility to lysis by cytotoxic t lymphocytes the endothelium-astrocyte immune control system cell mediated cytotoxicity against ectromelia virus-infected target cells conjugation of antibodies with fluorochromes: modrficat!ous to the standard methods viral induced autoimmnnity: monoclonai antibb.lies that react with endocrine tissues multiple organ-re.~ctive monoclonal autoantibodies expression of la antigens by cultured estrocytes treated with y-interferon quantitative variation in la antigen expression plays a central role in immune regulation effect of herpes simplex virus type and on surface expression of class ! major histecompatibility complex antigens on infected cells antigen-inducible, h- -restricted interleukin- -producing t cell hybridomas lack of independent antigen and h- re. cognition relationship between surface h- concentration, size of different target cells and lysis by cytotoxic t cells clones of cytotoxic lymphocytes can recognize uninfected cells in a primary response against influenza virus immunohistochemical identification of t lyn~lphocytes in the central nervous system of patients with multiple selerosis and subacute sclerusilag panencephalitis an improved assay for interleukin- (lymphocyte growth factor) produced by mitogen activated lymphocytes theiler's virus-induced demyelination: prevention by immunosuppression preparation of separate astre~ljial and ollgodendroglial cell cultures from rat cerebral tissue enhanced representation of hl-a antigens on human lymphocytes after mitogenesis induced by phytohemagglutinin of epstein-barr virus antigen presentation in brain: mhc induction on brain end~ thdium and astrocytes compared viral particles induce la antigen expression on astrocytes pathobiology of flaviviruses infection of cultured mnrine brain cells by semliki forest virus: effect of interferon-o.~ on viral replication, viral antigen display, major histocompafibility complex antigen display and lysis by cytotoxie t lymphocytes properties of mo~clonal antibodies to mouse ig allotypes, h- and ia antigens hybridoma cell lines secreting monoclonal antibodies to mouse h- and la antigens adenoviruses of subgenera b, c, d, and e modulate cell-surface expression of major histccompatibility complex class i antigens temporary pr~-~,ence of self-reactive cytotoxic t iymphocytes during murine lymphocytic choriomeningitis decreased expression of h- antigens following acute measles virus infection a simple method of estimating fifty percent end points hlunone response geue products (la antigens) on gliai and endothelial cells in virus induced demyelination intracellular v-interferon triggers an antiviral state in transformed l cells immune response (it) gene of the murine major histocompatibility complex interlenkin (iggi induction factor): a multifunctional lymphokin acting also on t cells clonal analysis of cytolytic t lymphocyte-mediated lysis of target cells with inducible antigen expression: correlation between antigen density and requirement for ly- / function l~e cytotoxic response to murine cytomegalovirus. i. parameters in vivo the cytotoxic response to murine cytomegalovirus. ii. in vitro requirement for generation of cytotoxlc t cells coronavirus infection induces h. antigen expression on oligodendrocytes and astrocytes adaption studies with ross river virus: laboratory mice and cell cultures multiple sclerosis: the blood-braln barrier and the measurement of de novo central nervous system of ig synthesis viral etiology of multiple sclerosis: where does the truth fie'? t lyraphocyte auto-inununity in experimental autoimmune encephalomyelitis dist~bution and quantitation of hla-abc and dr (ia) antigens on human kidney and other tissues inducible expression of h- and la antigens on brain cells interferon-v induces the expression of h- and la antigen on brain cells cytotoxic t cells specific for influenza virus infected target cells mhc-restricted cytotoxic t cells: studies on the role of polymorphic major transplantation antigens determining t cell restrictionspecificity, function and responsiveness we thank boehringer ingelheim for the kind gift of recombinant -ifn and barb geary for typing the manuscript. part of the work was carried out when one of us (y.l.) was a recipient of a world health organisation fellowship. key: cord- -jd wxobz authors: běláková, jana; horynová, milada; křupka, michal; weigl, evžen; raška, milan title: dna vaccines: are they still just a powerful tool for the future? date: - - journal: arch immunol ther exp (warsz) doi: . /s - - - sha: doc_id: cord_uid: jd wxobz vaccination is historically one of the most successful strategies for the prevention of infectious diseases. for safety reasons, modern vaccinology tends toward the usage of inactivated or attenuated microorganisms and uses predominantly subunit vaccines. the antigens need to be clearly defined, pure, stable, appropriately composed, and properly presented to the immune system of the host. differing ratios of various proportions between specific cd (+) and cd (+) t cell responses are essential for conferring the required protection in the case of individual vaccines. to stimulate both cd (+) and cd (+) t cells, the antigens must be processed and presented to both antigen-presentation pathways, mhc i and mhc ii. protein antigens delivered by vaccination are processed as extracellular antigens. however, extracellularly delivered antigen can be directed towards intracellular presentation pathways in conjugation with molecules involved in antigen cross-presentation, e.g. heat shock proteins, or by genomic-dna vaccination. in this overview, current knowledge of the host immune response to dna vaccines is summarized in the introduction. the subsequent sections discuss techniques for enhancing dna vaccine efficacy, such as dna delivery to specific tissues, delivery of dna to the cell cytoplasm or nucleus, and enhancement of the immune response using molecular adjuvants. finally, the prospects of dna vaccination and ongoing clinical trials with various dna vaccines are discussed. immunization with plasmid dna (dna-based vaccination) is a relatively novel technique for the efficacious stimulation of a specific cellular and humoral immune response to protein antigens. dna vaccines deliver transgenes, which code for antigens, directly into the cells of immunized host organisms. such antigens are expressed in a similar way as antigens during viral infection [ ] . antigens are processed identically as proteins synthesized in cytoplasm [ , ] and peptide fragments are presented to the immune system on cell-surface mhc i molecules. if the dna vaccine-coded proteins are secreted from the cell, they could be processed by mhc ii and could elicit a specific antibody response [ , ] . dna-based immunization is a new and attractive strategy in the prophylaxis and treatment of infections caused by extracellular and intracellular pathogens. the method of application, dose, boosting schemes, and species used are factors that influence the strength and nature of the elicited immune response. vaccination with plasmid dna may offer several important advantages over traditional vaccines, e.g. the relative stability of dna, the specificity of the antigen produced, and the possibility of guiding the type of elicited specific immune response [ ] . one of the major benefits of dna vaccines is that host cells express a vaccine-coded antigen and thus the antigen presents epitopes which may resemble native viral epitopes more closely than is the case with other vaccination approaches. intracellularly processed epitopes are presented to the host immune system in a way similar to that of a natural viral infection (mhc i presentation followed by cd + t cell responses), but without the risk associated with the administration of infectious agents [ ] . dna vaccines encoding several glycoproteins, i.e. multivalent vaccines, can be delivered to the host in a single dose. a few micrograms to milligrams of plasmid dna are sufficient for inducing a vigorous immune response. dna vaccines can also be manufactured in a relatively cost-effective manner and stored with relative ease [ ] . the temperature-stable storage of dna in a lyophilized form is feasible and more practical for transport and distribution as well [ ] . another important advantage of dna vaccines is their therapeutic potential in ongoing chronic viral infections. dna vaccines are very promising tools for an effective induction of a protective immune response against viral infections (hbv, hcv, hiv) and parasitic infections (malaria, leishmaniasis) [ , , ] . dna vaccines are principally derived from bacterial plasmids [ ] . conventional plasmid dna vaccines consist of two different parts: ) a eukaryotic cistron coding for the target antigen and consisting of a strong promoter/enhancer, cdna coding for the target antigen(s) (full-length or truncated), and a polyadenylation/termination signal, and ) sequences necessary for the manipulation-construction and amplification of plasmid dna in a prokaryotic host (e. coli) and consisting of the origin of replication (usually from e. coli), a multiple cloning site, and an antibiotic-resistant gene used as the selection marker during bacterial amplification [ , , , ] . the majority of eukaryotic promoters used are derived from the human cytomegalovirus (hcmv) or the rous-sarcoma virus (rsv). hcmv and rsv were found to give the highest levels of antigen expression after intramuscular (i.m.) dna injection [ ] . other promoters tested are tissue-specific promoters, e.g. the myocyte-specific desmin promoter [ ] , the actin promoter, major creatine kinase promoter, alphaglobin promoter, chicken beta-actin promoter, and adenovirus promoter. these have been tested primarily for i.m. dna application. efficacious expression of protein from dna vaccines is dependent on the presence of dna vaccine in the nucleus. the effective targeting of plasmid dna to the cell nucleus is dependent on the presence of dna sequences recognized by proteins or peptides called nuclear localization signals (nlss) [ ] , which form a dna-nls complex recognized by cytoplasmic proteins called importins which dock dna-nls complexes to the nuclear membrane receptor/transporter (nuclear pore complex) as a nuclear import substrate [ ] . nuclear dna transport is typical for viral dna. the most effectively recognized dna sequence is localized within the sv early promoter enhancer region of simian virus dna. this dna sequence was shown to be effective in the nuclear transport of plasmid dna [ ] . other dna sequences have been identified and tested, but their recognition through cellular nlss followed by the transport of plasmid to the nucleus is generally poor. an alternative approach is based on the chemical linkage of plasmid dna with an nls. a variety of nlss were tested (large sv t-antigen, m sequence of heterogeneous nuclear ribonucleoprotein, adenovirus fiber protein residues), but they generally obtained unsatisfactory results in in vivo experiments [ ] . this was most likely linked to the size-exclusion limit for active nuclear transport of such artificial dna-nls complexes. studies of the viral dna nuclear transport mechanism are promising because some viral dna, although more than ten times larger than standard vaccination dna plasmids, are effectively transported to the nucleus [ ] . polya-termination signals, which provide stabilization of mrna transcripts, are commonly taken from bovine growth hormone or from sv [ , ] . dna vaccines stimulate both exogenous (mhc class ii-restricted) and endogenous (mhc class i-restricted) antigen-presentation pathways. mhc i-restricted cytotoxic t lymphocytes (ctl) may be induced after dna vaccination by: a) directly transfected somatic cells (myocytes, keratinocytes, or any mhc ii-negative cells) which present expressed antigen on mhc i, b) directly transfected professional antigen-presenting cells (apcs) such as dendritic cells (dcs) which, besides mhc i presentation of the expressed antigen, effectively stimulate naive t cells via a variety of co--stimulatory molecules and cytokines, or c) by cross--priming when protein expressed by dna-transfected somatic cells is taken up by the surrounding professional apcs and presented to t cells [ , ] . the cross--priming phenomenon, discovered by bevan in , is at present used as a general explanation for the re-presentation of exogenously derived cell-associated antigens on both mhc i and mhc ii molecules [ , ] . when the dna vaccine reaches the cell nucleus, transcription is initiated and subsequent translation of the coded protein takes place on cytosolic ribosomes. the proteasome complex processes the expressed protein and released peptides are transported to the endoplasmic reticulum through the membrane transporter complex tap- and tap- . inside the endoplasmic reticulum, the peptides are bound to mhc i molecules [ , ] , which subsequently migrate to the cytoplasmic membrane and present the peptides to the surrounding cd + t cells [ ] . professional apcs, such as macrophages, b cells, and dcs, play a central role in the regulation of the immune response to any vaccine. in contrast to somatic cells, apcs can present the antigen to both mhc i and mhc ii molecules and thus stimulate t-helper cells (cd + ), which control other t cell or b cell responses [ , ] . dcs are much more effective than other apcs because of their unique ability to prime naive t cells. thus dcs are most likely the key cells initiating the immune response to the dna vaccine [ ] . intradermal (i.d.) and gene-gun application would directly target the plasmids to langerhan's cells, which represent immature dcs located in the stratum spinosum of the epidermis. dcs can migrate to lymphatic organs (the spleen and the lymph nodes) where they activate antigen-specific t lymphocytes. apart from high levels of mhc i and mhc ii, dcs express the stimulatory molecules b . and b . (cd and cd , respectively) that are required for immune response initiation [ ] . if non-apcs such as myocytes take up the dna vaccine (following i.m. injection), the expressed antigen is delivered to t cells by cross-presenting dcs [ , , , ] . cross-presentation and cross-priming are responsible for inducing the immune response to apoptotic and/or necrotic bodies [ ] , which may also be associated with dna vaccination. thus dna immunization results in the induction of both humoral (antibody) and cellular (t cell) immune responses. the major advantage of dna vaccines is their ability to generate a strong cellular immunity with preference for mhc i-restricted cd + cytotoxic t cells and mhc ii-restricted t helper type (th ) cell responses [ ] . on the other hand, dna vaccines can induce th responses and results from a number of studies indicate that dna vaccination can be effective in inducing long-term antibody responses [ , ] . this effect may depend on the type of antigen coded by the vaccine [ ] . effective dna vaccination should generate a long--term memory immune responses. there are several possible ways for dna vaccines to induce a long-term response: a) antigen is continuously expressed at a low level sufficient for antigen presentation and b) plasmid dna as well as antigen are completely gone and the response is antigen independent. memory cells generated by dna vaccines probably differ qualitatively from those achieved by other forms of vaccination, such as protein plus adjuvant [ , ] . memory cd + t cell and b cell populations are the most relevant for vaccine development. the most critical factors for current dna vaccines are: ) the efficacy with which the dna vector reaches the target cells' nuclei (transfection efficacy) and ) the amount of actual protein synthesized in dna vaccine--transfected cells. it has been estimated that injection of microgram doses of dna plasmid results in the production of only nanograms of protein [ ] . it is difficult to quantify the number of plasmids that enter the cell nucleus and the number of plasmids that are degraded before they enter the nucleus. it is estimated that more than % of the dna plasmids never reach the cytoplasm and only about . % of cytoplasmatically localized dna plasmids enter the nucleus, where gene expression is initiated [ ] . identifying and overcoming each hurdle along the dna vaccine entry pathway (low uptake across the plasma membrane, inadequate release of dna molecules within the cell's cytoplasm resulting in reduced dna stability, and a lack of nuclear targeting) can improve dna vaccine efficacy [ ] . dna vaccination was described in when wolff et al. [ ] demonstrated induced gene expression after direct i.m. injection of naked plasmid dna into experimental mice. naked dna was then injected into various tissues with the aim of comparing the intensity of proteosynthesis and the host immune response. besides i.m. injection the most frequently tested injection routes were i.d., intravenous, subcutaneous, epidermal, intraepidermal, intraperitoneal, injection into lymphatic follicles, and injection into the thyroid gland [ , , , ] . different routes of administration lead to markedly different levels of protein expression as well as different levels of intensity and quality (th , th , antibody) of the immune response. the skin was found to be one of the best sites for immunization due to the ease of skin injection and the high concentration of dcs (in the skin langerhans cells), macrophages, and lymphocytes, which are necessary for the induction of the immune response [ ] . in contrast, muscle is generally not equipped with dcs and elicitation of the immune response probably relies on cross-presentation, the effectiveness of which is limited by the dose of available antigen [ ] . thus methods which increase antigen expression by increased uptake of the dna by muscle cells could dramatically improve the applicability of i.m. dna vaccination. dna delivery methods can be classified into two general types: ) mechanical and electrical strategies for introducing naked dna into cells, including microinjection, particle bombardment, and the use of electroporation, and ) dna delivery systems which can be classified into biological viral dna delivery systems and chemical non-viral delivery systems (dna-binding polymers and liposomes). physical methods for increasing naked dna delivery dna vaccination into skin and muscle. wang et al. [ ] tested the effect of i.m. injection followed by electroporation on plasmid uptake in mice. the electroporation increased muscle cell plasmid uptake by approximately -to -fold. direct i.d. or i.m. dna injection vaccination very often results in the induction of a th response characterized by interferon (ifn)-γ synthesis and predominately igg a antibodies in mice [ , ] . the doses applied by needle injection are normally in the range of about - µg of naked plasmid dna. in the case of gene-gun dna application, only . - µg of plasmid dna is sufficient to induce antibody or ctl responses. the gene-gun or biolistic system uses compressed heli-um to propel micrometer-sized colloidal gold particles coated with precipitated plasmid dna (dna-coated microparticles) directly into the epidermal cells. in mice, intraepidermal gene-gun dna inoculation generates a prominent th response with interleukin (il)- production and an excess of the igg isotype [ , , ] . the approximately log of the dna dose may explain the dominance of the th response because low plasmid doses result in a low cpg motif moiety, which is important for a th response elicitation [ , ] . needle-free jet injection (biojector) is another dna delivery approach which has been investigated extensively as a method of i.d. immunization of laboratory animals, such as mice, pigs, rabbits, dogs, and monkeys [ , ] . gramzinski et al. [ ] analyzed the effect of various routes for immunizing with a dna vaccine encoding hepatitis b surface antigen (hbsag). experiments confirmed that needle-free injection of a dna vaccine (biojection of dna) induces a greater immune response to hbsag antigen than i.d. or i.m. injection using the classic needle-syringe approach [ ] . similar results were obtained in the earlier experiments of baizer et al. [ ] . the needle-free injection of dna has been tested in several human clinical trials as well [ ] , e.g. a gag-pol candidate hiv dna vaccine [ ] . the delivery of dna vaccines to the liver. a high level of dna vaccine expression in liver cells was achieved by the rapid injection of naked plasmid dna in relatively large volumes via the tail vein, the portal vein, the hepatic vein, and the bile duct in mice and rats [ , ] . this liver-specific approach has been designated "hydrodynamic delivery" and it is increasingly being used as a research tool for elucidating the mechanisms of gene expression and the role of genes and their cognate proteins in the pathogenesis of diseases in laboratory animal models [ , , ] . the procedure has also been shown to be effective in large animals such as dogs and non-human primates [ , ] . the hydrodynamic approach is proving to be a very useful research tool not only for gene expression studies, but also more recently for the delivery of small interfering rna [ , , ] . the application of dna vaccine to the liver is associated with enormous protein expression followed by a strong antibody-and cell-mediated immune response. when the same dose of dna was administered by hydrodynamic application or by i.m. or i.d. injection, the antigen-specific antibody levels induced by the hydrodynamic application were approximately times higher (raska et al., unpublished result) . dna vaccine minimization to minicircle dna. minicircles are small circular dna molecules that are derived from parental dna plasmids by specific recombination. antibiotic resistance genes, selection markers, and bacterial origins of replication are fully removed by the specific recombination [ , ] . minicircles contain only the gene of interest, making them promising tools for dna vaccination and gene therapy. chen et al. [ ] have shown that minicircles can express high ( -and -fold more) and persistent levels of target protein in mouse liver compared with their parent plasmids. this could be attributed to a higher transfection efficacy and the low to minimal content of cpg in minicircle dna. avoidance of bacterial dna also increases the safety of dna application because no antibiotic-resistant genes can be passed to pathogenic bacteria present in host tissues. non-viral carrier systems: dna/polyplexes. dna carriers tested for dna vaccination are various molecules which complex with dna by ) electrostatic forces between negatively charged dna molecules and a positively charged carrier (or cationic ions on a negatively charged carrier), ) analogous to the natural dna-protein interaction, or ) artificial covalent linkage between dna and a carrier [ ] . the dna/carrier complexes protect dna from serum dnases, increase transmission of dna through the cytoplasmic membrane of target cells, allow targeting to specific tissues, and some of them induce the escape of dna entrapped in endosomes by promoting endosomal disruption (weak bases such as chloroquine, the proton-sponge effect of many polymers) [ ] . non-viral dna vaccine delivery systems are based on ) electrostatic complexation of dna with cationic polymers (poly-l-lysine, protamine sulfate, polyethyleneimine, chitosan, polyethylene glycol, poly--(d,l-lactide-co-glycolide)), complexes commonly termed dna/polyplexes, ) electrostatic complexation and condensation of dna with artificial cationic lipids or lipopolyamines (dc-chol, dotma, dotap, dospa, dogs) which are mixed together with zwitterionic helper lipids responsible for the fusion of complexes with the target cell membrane (chol, dope, dppc), complexes commonly termed dna/lipoplexes or dna/lipopolyplexes, ) complexation of dna with artificial anionic lipids (dmpg) through electrostatic interaction mediated by na + and k + ions which are supplemented with zwitterionic helper lipids (dppc), complexes commonly termed fluid dna/liposomes, ) association of dna with proteins or peptides (histones, peptide tyrosine-lysine-alanine-(lysine) -tryptophan-lysine, fab fragments of anti-dna antibodies, cationic viral proteins µ and vp ) which in combination with cationic polymers or lipids facilitate nuclear targeting, and ) complexation of dna with dendrimers with a very low degree of polydispersity (pamam), complexes commonly termed dna/dendrimers (table ) [ , , , , , , ] . cationic liposomes and polymers are accepted as effective vectors for gene delivery with low immunogenicity, unlike viral vectors [ ] . liposomes are often used for systemic (intravenous, i.m., i.d.) or topical (nasal, oral) dna administration [ , , , , , ] . the final dna/lipopolyplex structures, dna concentration, ratio of cationic moiety to dna, and supplements such as condensing agents, endosmolytic agents, or nuclear targeting molecules are the most critical factors in the transfection efficacy of each formulation [ , , ] . the development of modified dna/lipopolyplexes suitable for intravenous application and targeting to tissues and organs such as heart, lung, liver, spleen, or kidney is desirable and necessary. the current focus is on decreasing the toxicity of complexes and increasing transfection efficacy and tissue specificity. to target specific tissue, the carriers are modified by linkage to various ligands allowing dna complexes to be recognized by specific tissue and cell populations. targeting to the liver is possible by linking liposomes or other polymers (polylysine, pei, polyamidoamine dendrimers) with a sugar motive recognized by the liver--specific asialoglycoprotein receptor, which is expressed on hepatocytes. such ligands are composed of trimeric terminal galactose [ , , ] . lipid-based dna/complexes are a promising approach in systemic application because of the minimal toxicity of particular lipids (dppc) in contrast to cationic polymers [ ] . on the other hand, lipid-based dna complexes are larger in size and are thus limited in their penetrability into liver--specific compartments (disse spaces), which are the port for hepatocyte targeting [ , , , ] . therefore an effort has been made to decrease the size of final complexes by protamine sulfate or poly-l-lysine, which increased the transfection efficacy in some in vitro experiments by up to times [ , ] . targeting the dna/complexes to dcs, mainly by interaction with the mannose receptor on the dc surface, is immunologically very promising. a specific ligand, mannose, was linked to a variety of polymers and lipids used for dna/polymer preparation which were tested in vitro or in vivo according to their dc specificity and transfectability. unfortunately, the high transfection efficacy commonly achieved in in vitro experiments is still dramatically diminished under in vivo conditions [ , ] . viral carrier systems. one highly efficacious delivery system for dna vaccines, or, more precisely, genetic vaccines, is based on recombinant viral vectors derived either from attenuated viruses used for preventive vaccination (vaccinia, poliovirus, hepatitis b virus, measles virus) or from viruses such as human adenovirus (hadv), adeno-associated virus (aav), alphavirus, vesicular stomatitis virus (vsv), or poxviruses other than vaccinia [ ] . in contrast to plasmid dna vaccines, virally vectored genetic vaccines induce a specific immune response not only against the expressed transgene, but also against the viral capsid and/or envelope and this response is often effective even after the first immunization. therefore, repeated immunization, often necessary for elicitation of a satisfactory immune response against the transgene product, could be inefficient. virally vectored genetic vaccination is generally performed as the second immunization after dna priming, i.e. a heterologous vaccination approach [ ] . the choice of transgene, viral vector, dose, route of application, prime/boost regimen, and number of immunizations are the most important factors influencing appropriate antigen-specific humoral and cd + and cd + cellular responses. furthermore, delivery efficacy of viral vectors could be dramatically hampered by preexisting immunity to the capsid or envelope proteins in a population. only a few viral vectors (alphavirus, vsv, and some serotypes of hadv or aav) are considered to be unrecognized by preexisting immunity of vaccines. finally, similarly to dna plasmid, a protective immune response elicited in experimental animals by virally vectored genetic immunization may not necessarily be observed in subsequent human clinical trials. this could be attributed to the above-mentioned preexisting immunity, the restricted range of viral hosts, restricted viral tissue tropism, or to many other, not yet well-described factors. poxvirus-derived vectors are some of the most frequently tested. they are safe and genetically stable double-stranded (ds) dna vectors whose entire life cycle occurs in the cytoplasm of somatic cells. they do not enter the nucleus and therefore do not integrate into the host genome. poxviruses are capable of carrying large amounts of foreign genetic material. two vaccinia--derived vectors, the new york vaccinia virus [ , , ] . edible bait vaccine, raboral vr-g, is licensed worldwide for the prevention of rabies in animals. vectors derived from avian poxviruses such as canarypox virus (alvac) and fowlpox virus (fwpv- ) are expected not to be recognized by preexisting immunity in humans. poxvirus-vectored transgenes induce good specific humoral and cd + and cd + cellular responses [ , , ] . recombinant adenoviruses are extensively tested for both vaccination and gene therapy. they contain dsdna in a rigid icosahedral non-enveloped nucleocapsid. the wild-type of adenovirus does not integrate into the genome. nevertheless, hadvs are oncogenic for animals and thus the safety of hadv therapy is still an open question [ ] . replication-incompetent recombinant hadvs are able to carry transgenes of up to kb. adenoviruses are used because of effective cellular uptake and transgene expression. wild and recombinant adenoviruses induce intense inflammatory responses followed by an induction of serotype-specific immunity, which could hamper the effectiveness of a subsequent vaccination with the same serotype [ ] . among the about known hadv serotypes, hadv- is the most explored for recombinant vaccine application. hadv- has one of the weakest pathogenicities associated with mild upper respiratory disease and fever in humans. recombinant hadvs were tested in animal models as a potential vaccine for hiv, malaria, sars, and ebola. heterologous dna-prime/recombinant adeno-boost immunization induced both humoral and cd + and cd + cell responses in experimental animals. a weak response was detected in human volunteers. because the prevalence of hadv- is relatively high in the population, altered surface proteins or chimeric hadv- carrying the surface proteins of another serotype are developed for avoiding the hampering effect of preexisting immunity [ ] . aav are members of the parvoviridae family of single-stranded (ss) dna non-enveloped viruses of the genus dependovirus [ ] . recombinant aavs are nonreplicative but persist within the cells as non-episomal, mainly circular dna. the integration frequency determined in rodent and rabbit muscle tissue is less than approximately - , which is two orders lower than spontaneous mutations in human genes. recombinant aavs could package kb of ssdna, including itr (transgene < . kb) [ ] . within the cell, the ssdna genome is transcriptionally active after conversion to a dsdna template, which takes in vivo a few weeks, a necessary delay before the induction of an immune response. therefore, dsaavs were developed. the maximum capacity for transgene insertion is . kb [ ] . in mice and rhesus macaques, a single i.m. injection of serotype aav expressing hiv antigens induced robust specific antibody and cd + cell responses [ ] . a significantly lower response in humans could be attributed to many factors, including preexisting immunity, because the presence of specific neutralizing antibodies ranges from - % according to age group and geographic location. specific antibodies recognize other serotypes, but their neutralizing activity seems to be less prominent, for example aav- with an affinity for muscle and hepatocytes, aav- for airway epithelium, aav- for muscle, and aav- for hepatocytes. beside vaccination, aav vectors are one of the most frequently tested vectors for gene replacement therapy [ ] . alphavirus vectors were earlier used for in vitro recombinant protein expression. their wild precursors belong to the togavirus family of positive ssrna enveloped viruses which replicate entirely in cytoplasm. a high level of transgene expression from this viral promoter is achieved due to the self-replicating nature of viral rna and the efficient inhibition of translation of host mrna by the viral replicase. because of limited prevalence, the preexisting immunity to the vector seems to be minimal. in experimental animals, immunization with vectors based on sindbis, semliki forest, and venezuelan equine encephalitis viruses induced a cellular and humoral immune response to the transgene. in the case of hiv- antigens the immune response in non-human primates was able to significantly reduce the viral load after challenge [ , ] . the vsv belongs to the rhabdoviridae family of enveloped negative-sense ssrna viruses. vsv-derived vectors are able to generate viral particles that express foreign transmembrane protein on the membrane surface. because vsv proteins do not down-regulate the interferon response of an infected cell, vsv could exhibit promising adjuvant activity. although wild-type vsv could induce neuropathy after intranasal application to mice, recombinant vsvs were proved to be safe in non-human primates after intranasal and i.m. application. immunization of non-human primates with vsv expressing hiv and siv antigens demonstrated efficacy in subsequent challenge with the highly pathogenic shiv . p virus. thus the potential neural toxicity of vsv needs to be finally solved before entering into the clinical trials [ ] . a few additional viral vectors were designed for genetic vaccination. one viral vector of future vaccines will be probably based on the attenuated measles virus, an enveloped negative-sense ssrna virus. at present, genetically stable recombinant vaccine strains are available for cloning up to three transgens (edmonston, schwartz). in animal experiments a recombinant measles vector expressing hiv- envelope antigen induced neutralizing antibodies and envelope-specific cd + and cd + cell responses after a single dose [ ] . although about % of the population experienced a measles infection or vaccination, it is estimated that years after the measles experience the immune response does not preclude successful immunization with recombinant measles vaccines. furthermore, it is expected that within to years measles will be eradicated [ ] . dna/polyplexes for targeting dna vaccine to mucosal surfaces. mucosal immunity establishes the first line of the defense against pathogens which attack the body via mucosal surfaces [ ] . induction of the mucosal immune response, either cellular or humoral, requires local mucosal application of antigen or an antigen-coding dna vaccine. although some mucosal response is detectable after a systemic dna vaccination, i.m. injection and gene-gun delivery of plasmid dna have a limited ability to induce mucosal immune responses [ ] . therefore, various mucosal dna application routes have been tested to achieve sufficient antigen production on mucosal surfaces (intranasal and intratracheal application, inhalation of dna vaccine in aerosol form, application of dna on external genital mucosa, and oral administration) [ , , ] . complexing dna with various polymers enhances dna uptake on mucosal surfaces. dna/polyplexes adhered to the mucosal cells either through specific receptors or through electrostatic interaction between a negatively charged mucosal cell surface and positively charged dna/polyplexes. when dna/lipoplexes were delivered orally or intranasally, they induced a significant mucosal immune response, including secretory iga responses [ , ] . an alternative means of enhancing the efficiency of dna vaccines is the use of genetic adjuvants. genetic adjuvants are most often genes coding for cytokines, chemokines, or co-stimulatory molecules. the cdna can be either administered in separate dna plasmids (monocistronic dna vaccine) or the cdna is cloned into parental dna vaccine plasmid under separate promoters or under promoters shared with antigen-coding dna sequence separated by an internal ribosome entry site element (bicistronic dna plasmid) [ ] . such molecules supply t cells or dcs with the second, antigen-independent stimulatory signal. several co-stimulatory molecules have been tested for enhancement of a) apc activation (cpg, cd l, mip- α), b) ctl response (il- , il- , il- , il- , il- , gm-csf, ifn-γ, cd l, icam- , lfa- ), c) th -type antibody production: igg a in mice (il- , il- , il- , il- , gm-csf, ifn-γ, cd l) or th -type antibody production: igg in mice (il- , il- , il- , tgf-β), and d) cellular response associated with ifn-γ induction (il- , il- , il- , il- , gm-csf, ifn-γ, cd- l, icam- , lfa- ) [ , , , , , , , ] . two additional co-stimulatory molecules of the tumor necrosis factor receptor superfamily expressed on activated t cells are antiapoptotic, i.e. - bb (cd ), expressed on cd + t cells, and its cd + t cell analogue ox (cd ), which protect activated t cells from death and thus enhance the antigen-specific t cell response. stimulating these receptors may be useful in dna vaccine development [ ] . during mucosal dna vaccination, specific parameters are critical for the use of molecular adjuvants. the proinflammatory cytokines il- α, il- , and il- were tested for antibody and mucosal ctl responses. il- has the potential to increase antigen-specific ctl activity and is thus particularly interesting due to its potential role in regulating the homeostasis of memory t cells [ ] . il- and il- were shown to be able to markedly increase iga reactivity to co-expressed heterologous antigen. monocyte chemoattractant protein- was effective in increasing mucosal iga secretion and ctl responses [ ] . rantes, lymphotactin, mip- β, mip- , human neutrophil peptides (hnp- , hnp- , hnp- ), ifn-γ, and ifn-β are important candidates for use as adjuvants [ , ] . another approach used for modification of the immune responses to dna vaccines includes the addition of heterologous gene fragments encoding localization or secretory signals or fusion of the antigen-coding cdna with the sequence coding for ligands which drive the antigen to sites appropriate for immune induction. for example, a number of studies have shown that higher titers of antigen-specific igg (igg in mice) were elicited when the antigen was secreted rather than localized on the cell membrane or within the cell [ , ] . the cellular localization of heterologous antigen may play a role in modulating the immune response, although the role may depend upon the nature of the antigen and/or the model system used. another strategy consists of fusing the antigen-encoding gene with the ubiquitin-encoding gene, thereby accelerating cytoplasmic degradation of the antigen by targeting it to proteasomes and improving class i antigen presentation [ ] . enhancement of the immune response could additionally be reached by fusion of antigen with the hsp -binding viral j-domain during the construction of dna vaccines. this molecule stabilizes fusion antigen by binding to the hsp protein, which in addition serves as an instruction molecule to induce an increase in the cd + t lymphocyte and b lymphocyte response [ ] . further, dna vaccine potency may be improved through fusion of the antigen-coding dna with the endosomal/lysosomal sorting signal sequence (derived from lysosome--associated membrane protein type ; lamp- ), which directs the expressed antigen towards mhc class ii molecules. thus the cd + t cell response could be significantly enhanced [ ] . the above-mentioned use of the bicistronic vector is the most common practice for heterologous production of the binary protein complex, but these methods are primarily in the research stage [ ] . bicistronic vectors could be useful in constructing multivalent vaccines derived from two, and possibly more, different antigens originating from one or more microorganisms. an element common to the majority of plasmids are the cytosine-phosphate-guanosine dinucleotides (flanked by two ´ purines and two ´pyrimidines recognized in mice) [ ] , called cpg motifs, which are unmethylated when the plasmids are amplified in a bacterial host. hypomethylated or unmethylated cpg nucleotides are specific to bacterial dna, but are very rare in eukaryotic dna. cpgs play an important role as an immunomodulatory component of dna vaccines [ ] . they are recognized by the toll-like receptor (tlr) localized in the cytoplasm [ ] . cpgs are potent stimulators of b cell proliferation and antibody secretion. cpg induce apcs (macrophages and dcs) to secrete th -type cytokines il- and ifn-α/β, which activate natural killer cells and t cells (cd + ) [ , ] . rankin et al. showed that cpg motifs or sequences which are effective in mice are ineffective in humans [ ] . this highlights the species diversity within tlr substrate specificity. the amount of cpgs in plasmid backbones could be changed by simple recombinant technology. thus the addition of cpgs to dna plasmids could theoretically decrease the dna vaccine dose used for a single immunization. however, from murine experiments it is clear that too many cpg motifs can actually reduce immunogenicity [ ] . the major safety concerns here are: ) the integration of the plasmid dna into the host genome, thereby increasing the risk of malignancy by activating protooncogenes or inactivating oncosuppressors, ) transfer of antibiotic-resistant genes to surrounding bacteria, ) the induction of an immune response to transfected cells, resulting in the development of an autoimmune disease, ) the stimulation of cytokine responses that alter the host immune homeostasis, and ) the induction of tolerance rather than immunity [ , , , ] . at the present time there is still no evidence for plasmid dna integration into the host genome although traces of plasmid dna are detectable in host cells up to one year after dna vaccination. current methods used for the detection of plasmid integration into genomic dna are not sufficiently sensitive or specific and this question remains open [ ] . the spread of antibiotic resistance is another important question. dna plasmid could be detected far from the original site of injection (in the case of i.m. or i.d. application). the responsible carriers were transfected lymphocytes and macrophages. such cells, of course, could spread antibiotic resistance genes to bacteria. there is, however, no direct experimental evidence to date for this phenomenon [ ] . both plasmid integration and antibiotic resistance transfer could be effectively minimized by minicircle dna technology, as mentioned above [ ] . autoimmune response a) against dna plasmid, mediated by anti-dna antibodies or b) against transfected cells, mediated by a type i immune response was experimentally modeled in mice [ ] . dna vaccination was able to moderately increase the dna-specific serum antibody titers for a limited period of time, but clinical signs of autoimmunity were not confirmed. cpg motifs, on the other hand, may induce a truly harmful autoimmune response when injected together with autoantigens such as the myelin basic protein (inducing encephalomyelitis) or a chlamidia-derived antigen (inducing myocarditis) in experimental models. the above effects were not found in healthy animals treated with therapeutic dna vaccine doses and no sign of toxicity was found in human volunteers exposed to dna vaccines during clinical trials [ , , ] . the use of cytokine genes in modern multicistronic dna vaccines could theoretically disrupt the immune homeostasis and increase susceptibility to other infections. it could also be associated with exacerbation of autoimmune or allergic diseases. experimental observations have confirmed that the cytokines are released locally and that serum cytokine levels are unchanged [ ] . induction of tolerance rather than immunity after dna vaccination is a problem for both extreme age groups. very young animals (mice under days of age) and very old ones (mice older than years) respond weakly to dna vaccination. in newborn mice, dna vaccine encoding circumsporozoite protein of the malaria plasmodium induced tolerance which was long lasting [ ] . thus dna vaccination schedules for both extreme age groups would need to be separately tested, possibly modified by use of cytokines or addition of costimulatory molecules. the majority of ongoing human dna vaccination trials are focused on assessing vaccine safety and immunogenicity [ , ] . as estimated from the us database at www.clinicaltrials.gov as of june , approximately one hundred dna vaccination clinical trials have been registered (irrespective of their trial phase). a large proportion of dna vaccines use viral vectors as delivery systems. the dna vaccine gendicine, produced by sibiono genetech, uses an adenovirus vector for delivery of dna encoding the p suppressor. it is registered by the chinese fda for treatment of head and neck squamous cell carcinoma [ ] . another adenovirus-vectored p -encoding dna vac-cine, advexin, produced by introgene therapeutis, is being tested in phase iii clinical trials for the same indications in the us. if we focus on plasmid (naked) dna vaccines, approximately clinical trials had been registered by www.clinicaltrials.gov as of may . ten have attained phase ii. the majority of dna vaccines are focused on hiv- infection. other infectious diseases include hepatitis b, malaria, ebola hemorrhagic fever, west nile virus infection, and avian flu. the other trials deal with various cancers (hepatocellular carcinoma, renal cell carcinoma, pancreatic cancer, chronic lymphocytic leukemia, breast cancer, ovarian cancer, prostate cancer, bladder cancer, synovial sarcoma, leiomyosarcoma, lung cancer, and melanoma). all ongoing clinical trials have confirmed minimal toxicity and good tolerance to dna vaccines. however, the still poor immune response to the majority of clinically tested dna vaccines is a great challenge for further optimization using novel antigen, delivery systems, and prime-boost-based schedules. dna vaccines remain a promising approach for inducing both humoral and cellular immune responses. one of the more interesting aspects of dna vaccination is the mechanism of inducing antibody-and/or cell--mediated immune responses. this is associated either with antigen presentation on vaccine transfected cells or with cross-presentation of the antigen by dcs, which are the only known cells capable of inducing both cd + and cd + t cell response. dna vaccines are effective inducers of host immune protection against viral, bacterial, fungal, and parasitic infections and may be suitable for cancer therapy. multicistronic dna vaccines that, for example, coexpress cytokines may be able to modulate any autoimmunity or allergic reactions. one basic obstacle to applying dna vaccines in human medicine is their still relatively poor immunogenicity, linked to low transfection efficacy and low antigen production. thus the development of effective delivery systems is the main research challenge in this area of immunology. dna vaccination: transfection and 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learned from the hepatitis b virus surface antigen mechanism of plasmid delivery by hydrodynamic tail vein injection. i. hepatocyte uptake of various molecules dna vaccines: future strategies and relevance to intracellular pathogens regulation and review of dnavaccine products gene vaccines mucosal adjuvants structural impact of hydrodynamic injection on mouse liver recruitment and expansion of dendritic cells in vivo potentiate the immunogenicity of plasmid dna vaccines safety and immunogenicity of a gag-pol candidate hiv- dna vaccine administered by a needle-free device in hiv- -seronegative subjects non-viral gene therapy: polycation-mediated dna delivery molecular adjuvants for mucosal immunity tlr pathway is involved in adjuvant effects of plasmid dna-based vaccines dna vaccine constructs against enterovirus elicit immune response in mice generation of mhc class i-restricted cytotoxic t lymphocytes by expression of a viral protein in muscle cells: antigen presentation by non-muscle cells rapid and highly efficient transduction by double-stranded adeno-associated virus vectors in vitro and in vivo detection of integration of plasmid dna into host genomic dna following intramuscular injection and electroporation tricistronic viral vectors co-expressing interleukin- (il-- ) and cd (b - ) for the immunotherapy of cancer: preclinical studies in myeloma the mechanism of naked dna uptake and expression long-term persistence of plasmid dna and foreign gene expression in mouse muscle direct gene transfer into mouse muscle in vivo oral administration of recombinant adeno-associated virus elicits human immunodeficiency virus-specific immune responses expression of naked plasmid dna injected into the afferent and efferent vessels of rodent and dog livers systemic linear polyethylenimine (l-pei)-mediated gene delivery in the mouse this work was supported by grant msm (ministry of education, youth, and sport, czech republic). key: cord- -m nbs c authors: yong, voon wee; antel, jack p. title: major histocompatibility complex molecules on glial cells date: - - journal: nan doi: . / - ( ) -n sha: doc_id: cord_uid: m nbs c while glial cells of the central nervous system do not constitutively express class i or ii major histocompatibility complex (mhc) molecules, astrocytes and microglial cells can be induced by a variety of factors to express these antigens. oligodendrocytes have inducible class i but not class ii elements. there are considerable differences in regulation of mhc antigen expression between glial cells from rodent and human brains, both in situ and in vitro. the consequence of glial cell mhc expression for immune interactions in the cns is discussed in the context of glial cell antigen presentation capacity and neural cell susceptibility to cell-mediated immune effector mechanisms. major histocompatibility complex (mhc) antigens comprise a highly polymorphic group of cell surface glycoproteins essential for the process of antigen recognition by t cells . such recognition involves a trimolecular complex of antigen, in the form of a processed peptide, associated with (located in a groove or cleft formed by) an mhc molecule, which is presented to the t-cell receptor of t cells expressing the a and ß chains of the receptor . the mhc recognition process is a requirement for initiation of the immune response through presentation of antigen to t helper (cd +) cells by specialized cells termed antigen-presenting cells, and for either cd + or cd + t cells to carry out effector responses dependent on cell-cell contact, particularly cytotoxic responses directed at specific cell targets . the genes encoding mhc molecules are located on the short arm of chromosome in human (hla region) and on chromosome in mice (h- region) . these genes can be divided into major classes, class i and class ii . among the over human class i genes are the hla-a,b,c, genes (h k,d,l in mouse) that encode a glycoprotein heavy chain that becomes associated with , -microglobulin ; the latter polypeptide is encoded outside the mhc region . the class ii gene region in human (hla-d, corresponding to i-region in mouse) is comprised of at least sub-regions, dp, dq and dr, each of which encodes at least one of the a and one of the ß chains that, together, comprise a class ii mhc molecule . genes outside the mhc region may also contribute to the overall process of immune recognition (minor histocompatibility antigens) . class i mhc glycoproteins are `normally' widely expressed on nucleated cells, except for endogenous cells within the central nervous system (cns) (see discussion below) ; class ii antigens are expressed primarily on cells involved in immune function, such as b cells, cells of the macrophage/monocyte lineage, dendritic cells, activated t cells in humans and specialized phagocytic cells within various tissues, e .g. langerhan's cells in the skin . usually, but not invariably, class i molecules are the mhc recognition molecules for cd + t cells (suppressor/cytotoxic) whereas class ii molecules are recognized by cd + t cells (helper/inducer) . the process of t cell recognition of antigen in the context of mhc molecules is a central means whereby an individual distinguishes self from non-self . lack of class ii mhc expression, as occurs in type ii bare lymphocyte syndrome, results in severe immunodeficiency . at the other extreme, aberrant expression on normally class ii-negative cells carries the risk of inappropriate presentation of self-antigens and is thought to be involved in the pathogenesis of many autoimmune diseases . furthermore, individual susceptibility to a variety of autoimmune diseases in humans has been reported to be influenced by the genetically-determined mhc repertoire, particularly those of the class ii mhc region . ' the cns has traditionally been considered a relatively immunologically privileged site, partly because neural cells were thought not to express mhc antigens, based primarily on early immuno-histochemical analyses of normal tissue in situ . ' the selective advantage to the organism for such non-expression remains unexplained . expression in human cns of mhc antigens, both class i and class ii, appears under pathological conditions, including not only those considered primarily inflammatory disorders ' but also in neurodegenerative diseases where the pathogenesis is not thought to be immunemediated . ' central issues raised by these observations include which cns cells can be induced to express mhc molecules ; what conditions regulate this expression ; and what is the functional significance of such expression with regard to regulation of immune reactivity within the cns and to endogenous cns cells becoming susceptible targets of t cell-mediated immune effector mechanisms . here we focus on mhc antigen expression on filial cells within the cns . we review how both technical and biological variables, including analyses in situ versus in vitro, normal versus pathological tissues, age and species factors, may have contributed to apparently discrepant observations . we present our experience with adult human filial cell cultures obtained from non-inflammatory surgical biopsies and consider the relevance of glial cell expression of mhc with regard to antigen-presentation and target-recognition . in particular we discuss how these may be related with susceptibility to cell-mediated autoimmune diseases of the cns . mhc expression in studies of brain sections human brain sections initial immunohistochemical studies of normal human brain sections did not detect the presence of mhcimmunoreactive cells . ' this has been corroborated by more recent reports . ' in contrast, several studies have described class ii mhc immunoreactivity on neural cells in normal human brain sections . the cell types expressing class ii mhc molecules have been variably identified (either by morphology, ultrastructural features and/or immunohistochemistry) as astrocytes or microglial cells ; ' oligodendrocytes and neurons have consistently been found to be negative for class ii mhc antigen expression in situ . ° the majority of examinations continue to report lack of expression of class i mhc molecules in normal neural tissue . , , although the detection of mhc molecules in normal human brain tissue sections remains equivocal, there is agreement that mhc expression is up-regulated (increased intensity of immunoreactivity and in proportion of immunoreactive cells) in many disease states . these include multiple sclerosis, where inflammatory processes are thought to be involved in the pathogenesis, ' and in conditions where no classical inflammation is apparent, such as neurodegenerative diseases like alzheimer's and brain tumors . [ ] [ ] [ ] the cell types with up-regulated class i or ii mhc expression remain controversial, with suggestions that these are either astrocytes or microglial cells ' or both . the expression of mhc on glial cells has been used as an index that these cells have become `activated' and are participating in the pathological reaction . in human disease states, oligodendrocytes and neurons remain mhc-negative . " °t echnical differences may contribute to discrepant observations using tissue sections . mhc molecules are cell-surface antigens, which can be easily destroyed by inappropriate fixation . prolonged exposure to formalin, used routinely to process postmortem human brain tissue, can mask or destroy the epitope . many studies have used frozen sections and often the morphology of cells was suboptimal, leading to possible misidentification of mhc immunoreactive cells . the choice of fixative (paraformaldehyde or bouin's being preferred) and the duration of fixation have recently been highlighted as important determinants when assessing mhc expression in situ . class ii mhc immunoreactivity can best be detected with fixation intervals of less than h ; staining intensity steadily declined with prolonged fixation until after h, little or no immunoreactivity was observed . under the presumed optimal staining conditions, only microglia expressed class ii mhc in the normal human brain . clinical variables may also influence the detection of mhc antigens in brain sections . pre-mortem intercurrent systemic diseases, particularly infections, can affect class ii mhc expression within the cns (u . traugot and p . lebon, personal communication) . in the presumed postmortem normal brain sections used for mhc detection, it is difficult to exclude the possibility that the tissue donors did not have any underlying pathology, such as subclinical viral infections, that could have modulated mhc antigen expression . a confounding factor in the reports of mhc expression in the cns is species differences . unlike v. w. yong and j. p. antel human brain sections, normal rodent brains in situ do not show immunoreactivity for mhc except on scattered endothelial cells of large blood vessels . whereas class ii mhc-positive astrocytes and microglia are observed in active lesions in multiple sclerosis, astrocytes in the lesions of experimental allergic encephalomyelitis (eae), an animal model of multiple sclerosis, do not appear to express class ii mhc (ia) while microglia do ; - astrocytes and microglia are both class i mhc immunoreactive in eae. in wallerian degeneration following eye enucleation or facial nerve resection, microglia but not astrocytes express la antigens ; , is following facial nerve resection microglia are also immunoreactive for class i mhc . the majority of rat cns cells newly induced to express mhc class ii following days of continuous intravenous administration of gamma-interferon, which is a potent inducer of class ii antigens on astrocytes in vitro (see below), were found to be microglia and not astrocytes . normal mouse oligodendrocytes in situ have been reported to express class ia, although this was not confirmed by others . one immunoelectron microscopic study has described cytoplasmic reactivity for class la in a small proportion in view of the many potential difficulties of experiments in vivo, cultured cells have been used to address the issue of expression of mhc on neural cells . here, live cells can be subjected to immunohistochemical protocols without prior fixation, and the cell type of interest can be reliably identified by using cell typespecific antibodies in double immunofluorescence analyses. bulk isolated cultures also allow for more detailed analyses using molecular biology tools . by immunohistochemical analyses largely performed using cells cultured from neonatal animals, the consensus for rodent cells is that under basal culture conditions class i mhc antigens are expressed on a substantial number of astrocytes , and microglial cells but not oligodendrocytes . , class i can be induced on rodent oligodendrocytes by viral infections and gamma-interferon . , immunoreactive expression of class ii mhc (ia) on rodent astrocytes, microglia or oligodendrocytes in basal culture conditons appears to be absent or negligible . , - analyses at the messenger rna level similarly reveal that cultured astrocytes do not constitutively express mrna for class ii mhc . induction of la on rodent astrocytes occurs after treatment with gamma-interferon, , , , tumor necrosis factor and certain strains of viruses . the administration of gamma interferon in vivo results in the detectable expression of la antigens on rodent astrocytes when isolated and cultured, even when none are detected in vivo . similarly, gammainterferon treatment of mouse or rat microglia in vitro results in la expression ; , neonatal or adult brainderived microglia respond in a similar manner . the gamma-interferon-induced la expression on glial cells seems to be mediated by the protein kinase c signal transduction pathway . in contrast to astrocytes and microglia, rodent oligodendrocytes appear refractory to la expression even when stimulated with gamma-interferon, or supernatants derived from activated mononuclear cells or virus-infected cells . , it is of interest to note that the o- a progenitor cell (which gives rise to the type- astrocytes and oligodendrocytes at least in vitro) and the type- astrocytes can be induced by v. w. yong and j. p. antel gamma-interferon to express la ; oligodendrocytes are refractory . these authors suggest that differentiation of the o- a progenitor cells along the oligodendrocyte pathway is associated with a loss of inducibility of ia antigens . in conjunction with studies of mhc induction, down-regulation of mhc expression has also been examined . for rodent astrocytes, the induction of la by gamma-interferon or tissue necrosis factor is inhibited by adrenaline through ß -adrenergic mechanisms, fi vasoactive intestinal peptides or by transforming growth factor-ß and - . most studies of mhc antigen expression to date, including analyses in situ and in vitro, have used antibodies to recognize mhc determinants . previously, we have observed that cytotoxic t cells could recognize class i mhc on murine astrocytes (i.e . functional recognition), although expression of such molecules was not convincingly demonstrated by antibody immunolabelling . these findings could reflect not only the lack of sensitivity of the immunohistochemical techniques used to detect mhc but also that the determinants recognized by antibody and those recognized by t cells need not be identical . the latter possibility could account at least in part for the failure of some antibodies to mhc molecules to block cytotoxic t cell responses directed against specific cell targets . a much more limited number of studies have explored whether human glial cells in vitro are immunoreactive for mhc antigens . in two studies using autopsy-derived human adult cells, the large proportion (close to % in some cases) of astrocytes and oligodendrocytes expressed class i mhc molecules (hla-a,b,c) whereas a smaller proportion of cultured astrocytes ( - %) from all donors expressed class ii mhc (hla-dr) . of interest, - % of oligodendrocytes from of the donor preparations were immunoreactive for hla-dr . , o in contrast, a study using cells from two autopsy adult samples and another reporting on one autopsy case, did not find hla-dr expression on astrocytes . to circumvent possible post-mortem effects and the variability introduced by pre-mortem intercurrent systemic infections that may affect mhc antigen expression (see above) we have been using glial cells cultured from brain samples surgically resected during treatment for intractable epilepsy . except for the seizures, these subjects were normal . their young age group ( ± years) corresponds to one that is at risk for developing suspected autoimmune diseases such as multiple sclerosis . for class i mhc molecules, close to % of oligodendrocytes, astrocytes and microglia were immunoreactive . _ for hla-dr, virtually all microglia showed positivity ; no oligodendrocytes were found to express hla-dr (figure ) . the analyses of hla-dr expression on biopsy-derived adult human astrocytes showed that this class ii mhc molecule was detected on all human biopsy-derived cultures ; however, different series of human astrocytes expressed hla-dr bearing astrocytes remained relatively constant when analysed at different time points, up to days in vitro . the differential level of expression of hla-dr between human series was not correlated with the age of the human subjects, the extent of gliosis in the resected material or the duration for which the cells have been maintained in culture . when adult human astrocytes were subclassified by morphology (these did not fall neatly into the type- and type- astrocytes characterized in rodent), hla-dr expression was prevalent on process-bearing rather than the flat astrocytes (figure ) , the expression of hla-dr on cultured adult human glial cells in our studies probably represents an induction process in vitro, because freshly isolated cells did not have detectable hla-dr and because the type of serum (example, human ab) and its concentration in the culture medium affected the proportion of astrocytes that expressed hla-dr . it would be of interest to determine whether the individual variability of hla-dr expression on human astrocytes reflects a genetically determined property, as seems to be the case with rodents where inducibility of ia molecules on cultured astrocytes correlates with susceptibility to eae (see below) . in contrast to their human counterparts, rat astrocytes in our hands did not express class ii mhc when isolated and maintained in identical culture conditions to the adult human cells . this result is in agreement with the multitude of reports cited above that rat astrocytes have undetectable levels of la under baseline culture conditions . thus, whereas the expression of mhc on adult human cells probably represents an induction process in vitro, rodent cells are not similarly induced ; this suggests differences in inductive capability, probably at the molecular level, of mhc between species . such a result would reflect the observations in situ (see above) that mhc antigens are more readily detected on human neural cells than those of rodents . in correspondence with rodent cells, class ii mhc expression on human adult astrocytes can be upregulated by treatment with gamma-interferon ; , this effect can be blocked by treatment with betainterferon . more recently, it has been shown that interleukin- can decrease constitutive expression of class ii mhc in a glioblastoma multiforme line . human fetal cells have also been examined for expression of mhc molecules . this literature is small but it seems that both class i and ii mhc can be detected on fetal astrocytes ; ß a small proportion of microglia were immunoreactive for hla-dr (class i was not examined) . oligodendrocytes and neurons in human fetal cultures were negative for mhc expression . summary of tissue culture studies expression of mhc antigens by glial cells is more readily detected in tissue culture studies than in the brain in situ (table ) . although one explanation may be the feasibility of determinations in vitro, it is more likely that the induction of mhc molecules is a result of cell culture . another possibility is that in culture as yet uncharacterized in situ inhibitors of mhc antigen expression are lost . class ii mhc expression on astrocytes and microglia in the human system is observed under baseline culture conditions, unlike in rodent cells (table ) ; we speculate that the molecular factors that control inducible elements of mhc gene expression in the human system are less tightly regulated than those in rodent cells . however, in response to treatment with gamma-interferon, a potent cytokine, induction of mhc seems to be similar across species (table ) . there does not appear to be changes in mhc expression due to age factors, from the limited number of studies currently available, , , significance of mhc expression by glial cells antigen presentation neonatal murine astrocytes and microglia, especially after gamma-interferon treatment, can present antigen, e .g. myelin basic protein, to previously sensitized cd + t cell lines in an mhc class iirestricted manner . , , , astrocytes, even after treatment with gamma-interferon, are, however, unable to induce activation of primary cd + t cells in a mixed lymphocyte reaction ; such responses were achieved by `professional' antigen presenting cells, such as dendritic cells and activated b cells . microglial cells prepared from human brain biopsies are also able to induce a response by previously nonactivated human peripheral blood-derived t cells in a mixed lymphocyte reaction . the capacity of endogenous glial cells to present antigen probably contributes to the observed persistence of activated myelin-sensitized t cells within the cns, as compared to non-neural antigen-reactive t cells . immune-mediated injury to oligodendrocytes or its myelin membrane is postulated to account for the demyelination which characterizes the acquired human disease multiple sclerosis, the induced human disease acute disseminated encephalomyelitis (postvaccination, post-infectious encephalomyelitis) and the induced animal disease eae . in the last, the disease can be passively transferred using myelin antigen-reactive cd + t cells . the mechanisms through which oligodendrocyte/myelin injury occurs are not precisely defined ; mechanisms implicated include direct cd + t cell-mediated injury, cellmediated injury by other t and non-t (macrophage/ microglia) cells recruited to the lesion sites, and injury mediated by soluble factors released by the accumulated inflammatory cells . rat t cells, reactive to myelin basic protein and comprised predominantly of cd + t cells (or cd + t cell hybridomas), have been shown to be cytotoxic to cultured rat oligodendrocytes in a strain-, presumably mhc-, restricted manner . the cytotoxic effect of the cd + t cell lines require the presence of antigen presenting cells, which possibly enhance effector-target interactions ; the effect could not be reproduced by soluble factors . myelin basic protein-reactive cd + -t cells can acquire cytotoxic capability, at least in vitro, and have been shown to lyse cells expressing histocompatible class ii mhc antigens (e.g. b cells transformed with epstein-barr virus) in the presence of myelin basic protein . the target oligodendrocytes did not express class ii mhc although they were induced to express class i antigens when cocultured with t cells . the presumably small number of cd + t cells in the mbp-reactive cd + t cell lines could also have contributed to oligodendrocyte lysis, consistent with our previous observations that allo-reactive cd + -t cells can lyse human oligodendrocytes in vitro in an mhc class i restricted manner . a further potential mechanism whereby cd +-t cells may be cytotoxic to non-mhc class ii expessing cells, such as oligodendrocytes, involves the possibility of their acquiring cytotoxic capability following prolonged activation while losing their mhc restriction ; such cells have been generated in vitro and termed promiscuous killers . we have found that t-a/ß chain-expressing cd + and cd + t cells can induce promiscuous killing of human oligodendrocytes (t.c .g. ruijs, e .a . brown and j .p. antel, unpublished) . the conditions for generating these cells involves maintaining persistent activation with interleukin- ; such conditions in vitro may well parallel those found in vivo in the chronic inflammatory lesions characteristic of multiple sclerosis . the promiscuous cytotoxic effect canot be reproduced with supernatants from such cultures . the classic antigen-restricted t cell expresses the a and ß chain of the t cell receptor, as indicated previously . a t cell population identified by expression of the -y/s chains of the t cell receptor has been observed to accumulate in excess numbers in regions of demyelination in cns brains . these cells can mediate potent cytotoxicity of human oligodendrocytes in vitro . these t-y/s cells are not restricted by either classical class i or ii mhc molecules. the recognition molecule of these cells remains unknown, with much interest focused on heat shock proteins . however, further candidates may be class i-like molecules such as cd or possible equivalents of the mouse qa and tl gene products . a potential link between mhc class ii up-regulation on glia and genetic susceptibility to autoimmune disease was suggested by findings that astrocytes from rodent strains susceptible to eae expressed higher levels of mhc class ii after exposure to gammainterferon in vitro than do astrocytes from resistant strains, although this has recently been disputed . glia from eae susceptible mouse strains are more potent presenters of mpb to sensitized t cells than are glia from eae resistance mice ; no differences were found in this function using spleen cells . susceptibility to immune-mediated encephalomyelitis initiated by jhm-virus infection in rats is a function of inducibility of la (but not class i) antigen on astrocytes . such observations have raised speculations that exposure to cytokines (especially gamma-interferon) or viruses could contribute to cns cells becoming capable of presenting auto-antigen to infiltrating t cells or becoming susceptible targets of such t cells . for human adult astrocyte cultures, it remains to be determined whether the differential expression of hla-dr by various specimens could be correlated with particular hla haplotypes associated with genetic susceptibility to the development of autoimmune diseases of the cns, such as multiple sclerosis (dr locus) . whether the observed deleterious effect of systemically administered gamma-interferon on the course of relapsing multiple sclerosis reflects effects on the cns as well as on the systemic immune system, remains speculative . a novel means of inducing overexpression of mhc molecules in the cns in vivo involves the creation of transgenic animals . a transgenic mouse line created by insertion of a class i mhc gene under control of the myelin basic protein promoter, with resultant restricted expression of the transgene to oligodendrocytes, displayed marked dysmyelination but without inflammation . this model may be more akin to genetic models of impaired myelin production, rather than to immune-mediated myelin destruction as claimed . summary offunctional significance of mhc antigen expression on plia mhc antigen expression on glia has been linked with susceptibility to development of autoimmune cns diseases . the potent inducers of mhc expression on glia include viruses and immune system-derived soluble factors (cytokines), which themselves are the suspected initiators or mediators of such diseases . mhc expression on filial cells is of functional significance with regard to these cells acquiring the capacity to present antigen to t cells and thus promoting (or conversely, potentially inhibiting) immune reactivity, and to these cells themselves becoming susceptible targets of specific immune effector mechanisms . the dynamic expression of mhc molecules within the cns contributes both to the capacity of endogenous glial cells to regulate immune reactivity within this compartment and to such cells becoming susceptible targets of immune effector responses . the functional importance of mhc molecules in antigenspecific t cell responses is well illustrated by the potent effects of systemically administered anti-class ii mhc antibodies, which inhibit the development and disease course of eae . the increasing availability of molecular genetic techniques to manipulate expression of mhc per se within the cns provides new opportunities to assess the role of these molecules in influencing neural-immune interactions . with regard to human immune-mediated cns disease, optimal immunotherapies would be those which are organ-specific, i .e . spare overall systemic immune capability . the apparent effect of biological response modifiers on mhc expression on glial cells implicate v. w. yong and j. p. antel them as potential selective therapies . it still has to be defined whether down-regulation of mhc can be more readily achieved on cns cells with their inducible rather than constitutive expression of mhc, than on systemic antigen-presenting cells that constitutively express them . conversely, in some circumstances, up-regulation may be of advantage, for example to augment filial tumor-directed immune responses . as the molecular mechanisms regulating mhc expression in the cns are further defined, additional means will hopefully become available for therapeutic manipulation of this dynamic system . a molecular basis for mhc class ii-associated autoimmunity the tissue distribution and cellular location of transplantation antigens tissue distribution of la antigens and their expression on lymphocyte subpopulations on the presence of la-positive endothelial cells and astrocytes in multiple sclerosis lesions and its relevance to antigen presentation multiple sclerosis : relevance of class i and class ii mhc-expressing cells to lesion development reactive microglia are positive for hla-dr in the substantia nigra of parkinson's and alzheimer's disease brains detection of hla-dr on microglia in the human brain is a function of both clinical and technical factors expression of class ii major histocompatibility antigens on reactive astrocytes and endothelial cells within the gliosis surrounding metastases and abscesses microglia are the major cell type expressing mhc class ii in human white matter multiple sclerosis : oligodendrocytes in active lesions do not express class ii major histocompatibility complex molecules monoclonal antibody analysis of mhc expression in human brain biopsies : tissue ranging from 'histologically normal' to that showing different levels of glial tumor involvement expression of la molecules by astrocytes during acute experimental allergic encephalomyelitis in the lewis rat the distribution of ia antigen in the lesions of rat acute experimental allergic encephalomyelitis in situ detection of class i and ii major histocompatibility complex antigens in the rat central nervous system during experimental allergic encephalomyelitis . an immunohistochemical study expression of la antigen on perivascular and microglial cells after sublethal and lethal motor neuron injury macrophage function during wallerian degeneration of rat optic nerve : clearance of degenerating myelin and la expression effects of dexamethasone on microglial activation in vivo : selective downregulation of major histocompatibility complex class ii expression in regenerating facial nucleus rat ependyma and microglia cells express class ii mhc antigens after intravenous infusion of recombinant gamma interferon expression and synthesis of murine immune response-associated (ia) antigens by brain cells immune response gene product (ia) on glial and endothelial cells in virus-induced demyelination tumor necrosis factor induces expression of mhc class i antigens on mouse astrocytes astrocytes as antigen-presenting cells . part ii . unlike h- k-dependent cytotoxic t cells . h- ia-restricted t cells are only stimulated in the presence of interferon-y fc receptor density, mhc antigen expression and superoxide production are increased in interferon-gamma-treated microglia isolated from adult rat brain coronavirus infection induces h- antigen expression on oligodendrocytes and astrocytes the expression of mhc antigens on oligodendrocytes : induction of polymorphic h- expression by lymphokines expression of la antigens by cultured astrocytes treated with gamma-interferon inducible expression of h- and ia antigens on brain cells cultured human and rat oligodendrocytes and schwann cells do not have immune response gene associated antigen (ia) on their surface viral particles induce la antigen expression on astrocytes antigen presentation and tumor cytotoxicity by interferon-gamma treated microglial cells induction and regulation of class ii major histocompatibility complex mrna expression in astrocytes by interferongamma and tumor necrosis factor-alpha enhanced interferon-gamma-induced iaantigen expression by filial cells after previous exposure to this cytokine cytokines up-regulate la expression in organotypic cultures of central nervous system tissue regulation of hla class ii molecule expressions by -y-ifn the differentiation of o- a progenitor cells into oligodendrocytes is associated with a loss of inducibility of ia antigens norepinephrine inhibits -y-interferon-induced major histocompatibility class ii (ia) antigen expression on cultured astrocytes via / -adrenergic signal transduction mechanisms transforming growth factors type ßl and suppress rat astrocyte autoantigen presentation and antagonize hyperinduction of class ii major histocompatibility complex antigen expression by interferongamma and tumor necrosis factor-alpha susceptibility of astrocytes to class i mhc antigen-specific cytotoxicity antigen expression by glial cells grown in culture expression of la antigens on the surface of human oligodendrocytes and astrocytes in culture induction of human leukocyte antigen-a,b,c and -dr on cultured human oligodendrocytes and astrocytes by human y-interferon immunohistochemical studies of adult human glial cells human oligodendrocytes are susceptible to cytolysis by major histocompatibility complex class irestricted lymphocytes morphologic heterogeneity of human adult astrocytes in culture : correlation with hla-dr expression expression and modulation of hla-dr on cultured human adult astrocytes interferon-$ impairs induction of hla-dr antigen expression in cultured adult human astrocytes interleukin- ß decreases hla class ii expression on a glioblastoma multiforme cell line differential expression and regulation of major histocompatability complex (mhc) products in neural and glial cells of the human fetal brain human microglial cells : characterization in cerebral tissue and in primary culture, and study of their susceptibility to hiv- infection astrocytes present myelin basic protein to encephalitogenic t cell lines la restricted encephalitogenic t lymphocytes mediating eae lyse autoantigen-presenting astrocytes major histocompatibility complex-expressing nonhematopoietic astroglial cells prime only cd * t lymphocytes : astroglial cells as perpetuators but not initiators of cd ' t cell responses in the central nervous system cytotoxic effect of myelin basic protein-reactive t cells on cultured oligodendrocytes colocalization of lymphocytes bearing ys t-cell receptor and heat shock v. w. yong and j. p. antel protein hsp ' oligodendrocytes in multiple sclerosis the ys t cell receptor and class lb mhc-related proteins : enigmatic molecules of immune recognition hyperinducibility of la antigen on astrocytes correlates with strainspecific susceptibility to experimental autoimmune encephalomyelitis induction of class ii major histocompatibility complex antigens on a population of astrocytes from a mouse strain (balb/c) resistant to experimental allergic encephalomyelitis immunologic differences in murine glial cells and their association with susceptibility to experimental allergic encephalomyelitis inducibility of la antigen on astrocytes by murine coronavirus jhm is rat strain dependent dysmyelination in transgenic mice resulting from expression of class i histocompatability molecules in oligodendrocytes we wish to acknowledge the collaborations of the following individuals : s .u . kim key: cord- -kfd wqdx authors: wen, p.; loeffler, j.s.; morris, j.h.; lampson, l.a. title: the effects of irradiation on major histocampatibility complex expression and lymphocytic infiltration in the normal rat brain and the l gliosarcoma brain tumor model date: - - journal: j neuroimmunol doi: . / - ( ) -w sha: doc_id: cord_uid: kfd wqdx the effects of irradiation on major histocompatibility complex (mhc) expression and lymphocytic infiltration in the normal rat brain and the l gliosarcoma brain tumor model were examined. doses of irradiation administered were biologically equivalent to that used in the treatment of patients with malignant gliomas. no significant change in immune parameters was observed following irradiation in the normal rat brain. in the l gliosarcoma model irradiation did not suppress mhc expression or lymphocytic infiltration. these findings suggest that prior exposure to therapeutic irradiation need not adversely affect subsequent immunotherapies, and provide a foundation for future studies of immunomodulation in the irradiated brain. the limitation of current therapy for patients with malignant glioma has led to renewed interest in immunotherapy as an adjunctive mode of treatment. immunotherapy is likely to be most effective in the early weeks following maximal cytore-duction with surgery and radiation therapy. the immunosuppressive effects of ionizing radiation are well known (markoe and saluk, ) . irradiation has been shown to decrease major histocompatibility complex (mhc) expression and lymphocytic infiltration in melanoma (jager et al., ) and reduce the effectiveness of some forms of immunotherapy (ettinghausen et al., ) . however, under certain circumstances, irradiation can enhance the host immune response, and increase the efficacy of immunotherapeutic regimens (hellstrom et al., ; tilkin et al., ; north, ) . recent work has defined a growing number of pathological and experimental situations in which the brain's immune environment is altered (lampson, ). yet few studies have addressed the possible effects of irradiation on immune parameters in the normal or tumor-bearing brain. in this study, we examined the effects of irradiation, biologically equivalent to that used in the treatment of patients with malignant gliomas, during the time period when immunotherapy is most likely to be attempted. mhc expression and lymphocyte infiltration were examined in irradiated tumor-free brains and in the l gliosarcoma brain tumor model. rats. female cdf rats ( - g) were purchased from charles river lab. (wilmington, ma, u.s.a.). antibodies. immunocytochemistry was carried out with rabbit serum against glial fibrillary acidic protein (gfap) (dako) and the monoclonal antibodies ox (class ii), ox (class i mhc), ox (lca), w / (t cells), w / (t helper), ox (t cytotoxic/suppressor), ox (b cells) and ox (microglia) using a modification of the abc vectastain procedure (whelan et al., ) . briefly, sections were fixed in absolute methanol (- °c for min) and incubated in succession with % normal goat serum ( rain), diluted primary antibody ( °c, overnight), biotinylated horse antimouse igg (pre-incubated for rain with % normal rat serum to reduce background staining) ( min) and abc complex ( h). the primary antibodies were diluted / except for ox which was diluted / . specific binding was visualized with a solution containing . % diaminobenzidine tetrahydrochloride, . % hydrogen peroxide, and . m imidazole for min. p /x culture supernatant was used as a negative control. for gfap staining, the sections were serially incubated with % normal goat serum, rabbit anti-cow gfap antibody ( : , ) and biotinylated goat anti-rabbit igg. the remainder of the procedure was unchanged from the one outlined above. normal rabbit serum was used as a negative control. irradiation of tumor-free brain. the posterior half of the brains of anesthetized rats was irradiated with a single dose of cgy using a ge maximar xray generator. this dose was biologically equivalent to the high doses of fractionated radiation used clinically in the treatment of patients with malignant gliomas (ellis, ; kramer et al., ; henderson et al., ) . lead foil blocks shielded the anterior half of the brain, which acted as an internal control. two unirradiated rats served as additional controls. the animals were sacrificed on days (immediately after irradiation), , and and weeks , , , , , , and . /~m coronal frozen sections of the brains were obtained through the irradiated parietal lobes and the shielded frontal lobes and examined for histologic changes (hematoxylin and eosin; h& e) and demyelination (luxol fast blue), and immunocytochemically. sections from the corresponding areas of the brains of the unirradiated rats served as additional controls. normal lymph nodes and brains from two rats that had , units of recombinant t-interferon (ifn-t) (biogen biologicals) injected into the parietal lobes days prior to being sacrificed served as positive controls. irradiation of tumor-bearing animals. the l gliosarcoma cell line was originally derived from tumors induced in cdf rats by weekly intravenous injections of n-nitrosomethylurea (schmidek et al., ) . x l cells were stereotactically implanted into the right caudate nucleus of cdf rats using a modification of the method of kobayashi (kobayashi et al., ) . on day after implantation half the animals were anesthetized and the whole brain irradiated with a single dose of cgy. - irradiated and - non-irradiated rats were then sacrificed on days , , , , , , , and . although an attempt was made to match irradiated with non-irradiated controls as closely as possible, none of the nonirradiated tumor-bearing animals survived beyond day ( days after implantation). coronal frozen sections were obtained through the center of the tumors and assayed immunocytochemically. with each antibody, the percentages of positively staining cells/total number of all cells from six random high-powered fields within the tumor (minimum of cells) were obtained and the average determined (whelan et al., ) . in the normal rat brain, irradiation did not produce significant histologic changes, demyelination or gliosis within the time period studied. the monoclonal antibodies to class i and i! mhc antigens produced dense staining of the lymph node controls, and of dendritic cells in control brains that had been treated with ifn- . no differences were detected in the staining patterns in irradiated brain areas compared to unirradiated areas or normal brains: class i was restricted to blood vessel walls, and class ii to occasional cells associated with the leptomeninges, choroid plexus and blood vessels, and rare dendritic cells. the antibody to lca (ox ) produced strong staining in lymphoid controls, but stained only occasional inflammatory cells in the leptomeninges in both irradiated and unirradiated areas and control brains (fig. ) . in unirradiated tumor-bearing animals, no strong class i activity was seen in the tumor cells or the surrounding neural cells. in contrast, many class ii + cells were seen, especially at the periphery of the tumors. the number of class ii + cells increased with time after tumor implantation, reaching % of the ceils within selected areas of the tumor mass. most of the class ii + cells had a dendritic morphology suggestive of microglia. rare cells ( %) had the morphologic characteristics of macrophages, lymphocytes, and occasionally astrocytes. neither the numbers nor the proportions of cells were significantly changed following irradiation. these identifications were confirmed by serial sections stained with ox and antibody to gfap and with ox and ox . two kinds of evidence suggest that the class ii-positive cells were not tumor cells. in vitro, no class ii was detected on l cells, even after incubation with ifn-t, suggesting that the l cells may not have the potential to express class ii mhc molecules. in work to be reported separately, l cells were transfected with the escherichia coli fl-galactosidase gene so that they could be identified histochemically (lampson et al., ) . double-labelling studies using the modified cells did not reveal class ii-positive tumor cells within the brain. many infiltrating leucocytes were also seen in the tumor-containing areas. as many as % of the cells within the tumors were lca ÷. analysis of lymphocyte subsets showed many t cells, including helper cells (up to % of all cells within the tumor mass), fewer cytotoxic/suppressor cells (up to %) and rare b cells (less than %). in control studies, l cells themselves did not crossreact with the antibodies to lymphocyte subsets. irradiation produced only slight necrosis. there was no significant effect on class i mhc expression in tumor-bearing rats. no significant change was noted in class ii mhc expression or lymphocytic infiltration on the first days after irradiation. from day onwards there was a slight increase in the numbers of class ii-positive cells and lymphocytes in the irradiated tumors. however, these differences did not reach statistical significance in the wilcoxon sign rank test (figs. and ). under normal circumstances there is little detectable mhc expression on neural cells or lymphocytic infiltration in the brain. however, both enhanced mhc expression and lymphocytic infiltration can occur in a variety of conditions, enabling neural cells to become subjected to cellmediated immune reactions (sobel et al., ; wong et al., ; lampson and hickey, ; suzumura et al., ; lampson, ; traugott, ; mcgeer et al., ; streit et al., ) . in this study, we have found that a dose of irradiation biologically equivalent to that used in the treatment of malignant glioma did not produce major histologic changes, leucocytic infiltration, or induction of mhc antigens in the brain in the time covered. surprisingly, our study also suggested that irradiation did not decrease class ii mhc expression or lymphocytic infiltration in the l gliosarcoma brain tumor model. these findings lay a foundation for future studies of immunomodulation in the brain when, as in the case of brain tumor patients, high doses of radiotherapy have been given previously. nominal standard dose and the rat systemic administration of recombinant interleukin stimulates in vivo lymphoid proliferation in tissues regression and inhibition of sarcoma growth by interference with a radiosensitive t-cell population radiation therapy of l rat brain tumors decreased expression of hla class ii antigens on human uveal melanoma cells after in vivo x-ray irradiation an improved rat brain tumor model therapeutic trials in the management of metastatic brain tumors by different time/dose fraction schemes of radiation therapy molecular basis of the immune response to neural antigens monoclonal antibody analysis of mhc expression in human brain biopsies: tissue ranging from 'histologically normal' to that showing different levels of glial tumor involvement interactions between leucocytes and individual brain tumor cells in the rat brain radiation immunobiology and immunomodulation principles and practice of radiation oncology reactive microglia are positive for hla-dr in the substantia nigra of parkinson's and alzheimer's disease brains y-irradiation facilitates the expression of adoptive immunity against established tumors by eliminating suppressor t cells morphological studies of rat brain tumors induced by n-nitrosomethyhirea the immunopathology of experimental allergic encephalomyelitis. ii. endothelial cell ia increases prior to inflammatory infiltration peripheral nerve lesion produces increased levels of mhc antigens in the cns corona virus infection induces h- antigen expression in oligodendrocytes and astrocytes reduced tumor growth after low dose irradiation or immunization against blastic suppressor cells multiple sclerosis: relevance of class i and class ii mhc expressing cells to lesion development hla class and fl -microglobulin expression in frozen and formaldehyde-fixed paraffin sections of neuroblastoma tumors distribution of fl -microglobulin in olfactory epithelium: a proliferating neuroepithelium not protected by a blood-tissue barrier interferon-), induces the expression of h- and la antigens on brain cells key: cord- - x juiw authors: christe, philippe; morand, serge; michaux, johan title: biological conservation and parasitism date: journal: micromammals and macroparasites doi: . / - - - - _ sha: doc_id: cord_uid: x juiw nan pandas, tigers, right whales and gorillas are emblematic and charismatic species worldwide and a consensus exists for the need of their conservation. the same is true for economically important species such as salmonids and sturgeons. but do we really need the conservation of parasitic species? not really! during our childhood, we all have favourite stuffed animals (representing one of these emblematic species) we take to sleep. however, do you ever see a child squeezing a stuffed worm, flea or tick? the lack of affect for cryptic species and the perception of parasites as disgusting creatures among the public certainly leads to a disinterest among governmental and conservation agencies to preserve them. fortunately, in , donald a. windsor expressed his concern for this matter in the famous slogan: "equal rights for parasites!" (windsor ). five years later he pleaded once more for parasite conservation in an editorial that appeared in "conservation biology" (windsor ) . during the same time, the ominous term "co-extinction" was proposed to characterize the dual extinction of a host and its specialized parasite (stork and lyal ) . despite the passing of years since these passionate declarations and the exponential increase of an interest in conservation biology, we can point out that parasitic species are far from being in a leading position among current conservation priorities. very few parasites are listed on the iucn red list of threatened species (iucn, ; whiteman and parker ) . some parasite extinctions have been even intentionally provoked as revealed by the will to remove parasites from hosts in captive breeding programs (stork and lyal ) . to convince resource managers that parasites are an important component of all ecosystems, the following arguments, which mainly rely on their potential utilitarian effects, are advocated by the parasites' defenders. first, parasites are living organisms and are de facto part of biodiversity. they shape community structure by reducing competitive abilities and vulnerability to predation of their hosts and have strong impact on ecosystem functioning (hudson ) . moreover, parasites could maintain biodiversity by mediating competitive interactions between different members of an ecosystem. because the rate of molecular evolution is usually faster in parasite dna than that within the homologous loci of their hosts (moran et al. ; nieberding et al. ) , the study of the evolution of parasite dna sequences could provide valuable information on past population dynamics, evolutionary history and current demographic processes of endangered hosts (whiteman and parker ) . parasites could thus be used as a biological "magnifying glass" (nieberding et al. ) . another utilitarian effect of parasites is their potential use as indicators of environ-mental quality and ecosystem health (marcogliese ) . indeed, parasites may be used as accumulation indicators of heavy metal contamination, particularly in aquatic ecosystems (sures et al. ). in addition, parasite species and composition revealed perturbations in ecosystem structure and function (marcogliese ) . furthermore, the use of parasite in human medicine is a new promising field of investigation, as illustrated by the use of helminths as therapeutic agents for inflammatory disease (hunter and mckay ). wild animals in their natural habitat have to cope not only with predictable environmental changes such as the cycles of seasons and their associated modifications in resource availability and temperature but also with unpredictable events such as catastrophes, spread of new diseases and human disturbances. whereas animals react adaptively by behavioural and physiological modifications to predictable changes, unpredictable disturbances may have negative effects on population dynamics of living organisms. increasingly rapid disappearing and fragmentation of habitats, which may be considered as unpredictable environmental changes, translates in a cascade of negative effects and can result in physiological stress on animals (suorsa et al. (suorsa et al. , . the first physiological responses of an animal to stressful stimuli include cardiovascular effects and a hormonal response involving synthesis and secretion of glucocorticosteroids (romero ) . consequently, a corticosteroid response might be a good indicator of a stress response (hofer and east ) . it is, therefore, not surprising that corticosteroid level is measured in many studies in ecology and conservation biology that have evaluated the effect of different environmental and human perturbations on the stress level of wild animals (creel et al. ; creel et al. ; mostl and palme ; romero ; palme et al. ) . the consequences of a stress response on parasite resistance are complex and alter host immunocompetence in different ways (apanius ). the immune system appears to be down regulated under stressful environmental conditions (von holst ), particularly under severe chronic stress with prolonged periods of high cortisol concentrations (mostl and palme ) . stress stimuli may arise due to different factors in a perturbed environment. habitat fragmentation may be related to chronic food shortage (zanette et al. ) . thus degradation of environmental conditions may decrease resource availability that in turn affects body condition and immune defences (chandra and newberne ; klasing ; christe et al. ) . as body condition is usually positively correlated with immune defences , individuals with poor body condition will be especially vulnerable to attacks of parasites christe et al. ) . edge effect due to fragmentation may also be a source of stress because predators may have easy access to dense forest patches which were previously inaccessible. it has been experimentally demonstrated that exposure to predators reduced the ability of hosts to cope with parasitism mediated through effects on immune function (navarro et al. ) . consequently parasitism may be favoured in fragmented habitat through the effect of predators. thus, parasites, which can also be considered as an environmental stressor, may reinforce the effect of habitat degradation and participate in the reduction of a population. in addition to habitat degradation and fragmentation, anthropogenic factors such as environmental pollution, hunting, tourism and leisure activities exert a negative pressure on wildlife and are thought to cause stress (fowler ; mullner et al. ) . clearly, more studies are needed to investigate the relationship between anthropogenic factors, level of stress and parasitism in endangered populations. conservation biology deals with two major paradigms: population invasion and population decline. both are related to each other (i.e. decline may be a result of an invasion) and both emphasize the potential roles of parasites and/or pathogens (prenter et al. ) . theoretical, experimental and empirical studies have established clearly that parasites play important roles in regulating population dynamics (scott ; scott and dobson ; albon et al. ; rosa et al. in this volume) and structuring free-living communities (minchella and scott ; morand and arias gonzalez ; hudson and greenman ; tompkins et al. ) . parasites then have a large impact on biological conservation (dobson and may ; mccallum and dobson ; sasal et al. ) , as parasites and pathogens may compromise reintroduction or translocation programs (viggers et al. ) . they may have a higher impact on threatened species generally characterized by a lower level of genetic variability, particularly on genes associated to immune system (hedrick, ) . small-sized host populations may be prone to extinction due to stochastic events. several processes, including the allee effect ; see below), may increase the probability of extinction of small populations. these processes operate when host population size decreases to under a critical or threshold level, below which populations are almost doomed. population viability analysis is one approach that has been developed for management purposes of small-sized endangered populations. threshold size has been also extensively studied in the case of hostparasite dynamics (dobson ) . the basic reproductive number, r , is the major concept in host-parasite population dynamics. this quantity is defined as the number of new infections occurring after introduction of one parasite, or one infected host, into a naïve and susceptible host population. r is positively linked to host density in the case of direct-transmitted parasites (see rosa in this book). parasites, or infection, can spread in the population when r > and as r depends on host density, the condition of parasite invasion corresponds to a case when the density of host population exceeds a threshold density. obviously, host-parasite dynamics are viewed in terms of parasite invasion or parasite invisibility. the task of disease management is then to decrease r below one, i.e., below the threshold density. the interplay between host and parasite thresholds has not been considered adequately. deredec and courchamp ( ) emphasized the importance of the relative position of the host and parasite thresholds: when the parasite threshold is higher than that of the host, the parasite is driven to extinction and the host population is relieved of its parasite; when the host population threshold is higher than that of the parasite, the host is driven to extinction while the parasite continues to exert strong pressure on the host until it reaches its own threshold. hence, mathematical epidemiology and population dynamics are important tools for investigating thresholds and persistence of both hosts and parasites. they may help in determining the conditions to maintain a high level of parasite threshold in comparison to the host threshold. microparasites are generally considered as an important threat in conservation biology (daszak et al. ; cleaveland et al. ). all conservation textbooks refer to the canine distemper virus, rinderpest and the avian malaria as examples of pathogen-driven extinction. introduced diseases have been implicated in the local extinction of a number of species (mccallum and dobson ; vitousek et al. ) and the global species extinction of hawaiian birds (vanriper et al. ) and the thylacine (guiler ) among others. daszak et al ( ) , in their review, mentioned microparasites and no macroparasites as important threats for conservation and as zoonotic threats for human health through spill-over. the lack of reference to macroparasites may suggest that they are indeed less important, and that their survey is not of major interest, with the notable exception of ectoparasites (ticks, fleas) because of their roles as vectors of numerous virus, bacteria and protozoans. moreover, results based on comparative analyses in carnivores show that host species that live in low density within a restricted geographic area experience low parasitic pressure in terms of parasite species diversity, suggesting that parasites may not represent a particularly important risk for these host species (torres et al. ) . in contrast, widespread host species that live in high density are exposed to a wide range of parasite species that may affect drastically the population dynamics of these carnivores, suggesting that macroparasites may regulate them at least locally. these results lead to the paradox that parasite infection, and particularly that of macroparasites, is less crucial for small and isolated populations than for large populations. this paradox is apparent and resolved by considering the investment in immune defences, which is directly related to the prevalence and/or diversity of parasites as a mean to control infection (martin et al. ) . evidence comes again from comparative studies, which suggest that hosts allocate their investment in immune function as a function of their probability of exposure to parasites (møller and legendre ; møller et al. ) . large populations are composed of highly immunocompetent individuals and small populations of low immunocompetent ones. hence, parasites and pathogens are threats to small and isolated populations because of poor performance of their immune system against pathogen introduction, but parasites (and parasite diversity) are probably necessary to maintain high levels of immune defence, which helps against new pathogens. the allee effect may be defined as "a positive relationship between any component of individual fitness and either numbers or density of conspecifics" . the beneficial effects of conspecifics not only include antipredator vigilance, predator dilution, social thermoregulation, reduction of inbreeding but also social facilitation of reproduction through helpers courchamp et al. ) . when population size reaches a low density, animal species that are subject to an allee effect will suffer from a reduction in some aspects of their fitness that in turn will affect negatively growth rate of populations. because of their potential role in extinctions of declining species, the allee effects have thus become much studied in conservation biology lafferty and gerber ) . interestingly, allee effects and parasitism have several features in common that are of interest when studying population dynamics in conservation biology (deredec ) . for example, theoretical models demonstrated the importance of host density in the probability for a parasite to become established in a host population (see above) and empirical studies have shown a positive relationship between host sociality or density and parasite prevalence and intensities (anderson and may ; brown and brown ; møller et al. ; stanko et al. ; altizer et al. ) . thus, animal species that aggregated as a behavioural response to the strong allee effects, would be more prone to suffer the negative effects of parasites. parasite species may also be subject to the allee effects that influence the occurrence and the severity of epidemics as illustrated by patchy distributions of worms in hosts as a result of the necessity for female worms to find a mate in order to reproduce (cornell et al. ). it has been suggested that sexual selection, in particular female mate preferences, could lead to an allee effect (møller and legendre ) . if only males of poor quality are available for mating in a small population, females may refrain from reproduction or reproduce at a low rate. as a consequence of mating with a male of a non-preferred phenotype, females could decrease their parental investment resulting in poor reproductive success (møller and legendre ) . parasite-mediated sexual selection has been the focus of numerous studies since the influential hypothesis of hamilton and zuk ( ) . a meta-analysis of the available studies related to this topic has revealed a negative relationship between parasite load, immunocompetence and the expression of male secondary sexual characters (møller et al. ) . thus, parasites, by decreasing the expression of male secondary sexual characters, may contribute and reinforce the potential allee effects created by sexual selection. mediation of competition by parasites is one mechanism of parasite interference (anderson ; hudson and greenman ; poulin ) . parasite-mediated competition is inferred when two different host species have different susceptibilities to the same non-specific parasite species. the presence of a given host species may decrease the fitness of the other host species simply by transmitting a pathogen to the more vulnerable host species (hudson and greenman ) . the abundance of the more vulnerable host to the parasite is then depleted, potentially under the host threshold. moreover, as the parasite infects two host species, the parasite threshold is obviously low. this "apparent" competition, mediated via a shared pathogen, differs from the classical competition for limited resources. strong evidence of this competition was obtained not only from experiments but also from the field (tompkins et al. ) , e.g. red and grey squirrels in england (tompkins et al. b ) and pheasant and grey partridge in england (tompkins et al. a) . parasite-mediated competition may operate for introduced host species, as they can be best competitors simply by introducing and transmitting a new parasite to the naïve native species. this can lead to a non-fit combination that can be more pathogenic (hudson and greenman ; prenter et al. ) . in this case, the invader uses parasites as biological weapons. immune-naïve residents will be weakened or even killed by the new pathogens. the most famous example of such a process comes from the history of the expansion of european humans through america where million of native people were killed by the influenza and other pathogens that accompanied conquistadors. parasite mediated competition is not the only way by which parasites may interfere in competition processes. recently, it was shown that many introduced species lost most of their parasites from their native habitats when introduced to new ones (torchin et al. ; torchin et al. ). this could be responsible for the demographic explosion of some introduced species, formulated as the "parasite release hypothesis". the parasite release hypothesis was proposed as an ecological mechanism to explain the success of introduced species. as the introduced species lose their parasites when invading new habitats, they have a competitive advantage over local species. mitchell and power ( ) and torchin et al. ( ) found that parasitism is significantly reduced in organisms in their introduced range, supporting the "parasite release hypothesis". one cause to explain that invaders may leave behind their parasites is that many parasites have complex life cycle stages with more than one host. if one of those hosts is absent in the new colonized area, the life-cycle of the parasite would be disrupted. in the invasion process, invasive host species harbouring a high diversity of parasites in their native habitat have advantages as they lose a large number of parasites and pathogens (see above). invasive host species have another advantage if they have invested in strong immune defences in their natural range, which may then subsequently confer a better capacity to control parasites that they may acquire in the introduced habitat. hosts having evolved strong immune defences are prime candidates for successful invasion (and also more resistant towards invaders). this hypothesis was proposed in the case of introduced plants and recently for the case of introduced animals (lee and klasing ; møller and cassey ) . in contrast, hosts that are exposed to a low diversity of parasites may invest less in immune defences. maintaining a strong immune system for threatened host species, or for individual hosts maintained in captivity in the view of reintroduction, is a new task for conservation biologists. habitat fragmentation and its degradation is probably one of the main factors leading to the disappearance of many species. indeed, it often leads to a decrease in population sizes as well as to the appearance of barriers to gene flow between isolated populations. the small populations that result from this fragmentation often suffer from reduction of genetic diversity associated with genetic drift and inbreeding effects. this loss of genetic variation can result in a rapid reduction of fitness (lower possibility to adapt to long term changes in environment, poor reproductive ability associated with a lower sperm quality, higher juvenile mortality, lower general survival, etc) (o'brien ) . several recent studies (cassinello et al. ; keller and waller ; spielman et al. ) also showed that populations with a low genetic variability are generally more susceptible to infectious viruses, bacteria and other pathogens. the case of the cheetah (acinonyx jubatus) is probably one of the best known concerning this phenomenon., the two major subspecies of cheetah (a. jubatus jubatus from southern africa and a. jubatus raineyi from eastern africa) display markedly reduced levels of genetic variability compared to other mammal species (o'brien ) . this would result in intensive inbreeding. when a breeding colony of this species was contaminated by feline infectious peritonitis (fip) in oregon state (usa), % of the captive animals showed morbidity symptoms and % of them died (o'brien ) . in contrast, in domestic cats, the mortality incidence of this virus is very rare (around %). according to o'brien ( ) , the high sensitivity of this cheetah colony to the fip would be directly linked to the very low (almost monomorphic) level of variation of the major histocompatibility (mhc) genes characterising the cheetah. a wide variety of gene classes (where the mhc is the most notable but see also the eosinophil-associated rnase (ears) genes, the tumor necrosis factor gene promoter, the interleukine receptor or the -interferon receptors ; hill ; zhang et al. ) are normally variable in natural populations and could contribute to disease resistance. mhc genes encode cellsurface glycoproteins, binding antigens derived from pathogens and parasites and constitute the most polymorphic genes in vertebrates (parham ; charbonnel et al. in this volume) . they present antigens to tlymphocytes which develop the appropriate immune responses. two major groups of mhc genes are recognised: the mhc class i genes are specific to the immune defence against intracellular pathogens by binding peptides mainly derived from viral proteins or cancer infected cells. the mhc class ii genes present with t-lymphocytes, peptides essentially derived from extra-cellular parasites (bacteria, nematodes, cestodes, etc.). the variability of mhc genes is correlated with the diversity of the t-lymphocyte receptors, which, in turn, determine the resistance of an organism to pathogens and parasites (parham ) . therefore, the cheetah, with its very low variability of mhc genes, is not well protected against the fip and probably against many other pathogens and therefore is at a high risk of extinction. however, according to several recent studies, several processes would help to maintain high levels of mhc genes diversity. indeed, these studies demonstrated that the anti-gen binding sites (abs) display more non-synonymous than synonymous substitutions compared to what would be observed under neutral theory (in this condition, the rate of synonymous substitution is predicted to be larger than the rate of non-synonymous substitution as the latter change the amino acid composition and would be likely deleterious) (sommer ) . this phenomenon cannot be explained by higher mutation rates in this region (hughes and yeager ) and the hypothesis accepted at present is that this particular nucleotide diversity in mhc genes would be the result of balancing selection. this would allow the maintenance of large numbers of alleles in populations and also the persistence of allelic diversity over long periods of time. following this strategy, the binding of a large set of antigens would be possible. two main types of balancing selection have been proposed to explain high levels of genetic diversity in mhc genes of vertebrates: -"overdominance" strategy (hedrick ; richman ) , where the heterozygotes are expected to have higher fitness than parental homozygotes as the latter will carry less divergent allelic sequences and, therefore, will have less chance to resist a large panel of antigens and/or multiple types of pathogens and parasites. -"frequency dependent selection" strategy (hedrick ) . this occurs when an allele or genotype is favoured at one frequency, but disadvantaged at another frequency. this hypothesis is based on the fact that hostparasite dynamics is considered as a co-evolutionary race. pathogens adapt to infect the most common genotype, leaving rare genotypes least infected. if alleles are favoured when they are rare, but selected against when they are common, this will result in a balanced polymorphism (sommer ) different studies confirmed the effect of balancing selection on the high mhc diversity. one of the best examples concerns the nicolas island fox (urocyon littoralis dickeyi) (aguilar et al. ). on the basis of different neutral markers (microsatellites, minisatellites and allozymes), this species is considered as one of the most monomorphic among sexually reproducing species. regarding the low variability of these markers, this species would have many problems of fitness as well as low resistance to pathogens. however, it is characterised by a surprising high level of mhc diversity which makes it much more resistant to what could be expected. this observation is interpreted as being the result of intense periodic balancing selection at the mhc which may have allowed the persistence of variation within this species despite strong genetic drift. under some circumstances (for example, particular historical events such as bottlenecks or founder effects), strength of selection acting on mhc genes can be insufficient to maintain variation in small or fragmented populations over a long period of time (sommer ) . in these cases, the power of genetic drifts can be stronger than the power of selection. this can lead to a loss of genetic diversity not only on the neutral markers but also on the mhc genes. this would explain the very low genetic variability in highly threatened species such as the cheetah (acinonyx jubatus) (see above), the asian lion (pantera leo persica) (o'brien, ) , the common hamster (cricetus cricetus) in the netherlands (smulders et al. ) , the scandinavian beaver (castor fiber) (ellegren et al. ) , the northern elephant seal (mirounga angustirostris) (hoelzel et al. ) and the scandinavian moose (alces alces) (ellegren et al. ) . under these circumstances, threatened species present a high risk of extinction as they can be very sensitive to new diseases and changes in environment. however, other studies demonstrated that endangered species such as the przewalski's horse (equus przewalski) (hedrick et al. ) , the arabian oryx (oryx leucoryx) (hedrick et al. ) and the malagasy giant jumping rat (hypogeomys antimena) (sommer ) are characterised by a low number of mhc alleles but which are separated by a high level of nucleotide and amino acid divergence. analysis at the abs showed that non-synonymous substitutions were higher than synonymous ones, suggesting selection leading to an increase of amino acids changes in the abs region and thus to higher divergences between mhc alleles (sommer ) . these studies indicated that other selection processes are able to maintain some mhc polymorphism (not on the number of alleles but rather on the genetic difference between the existing alleles) even in species surviving bottlenecks. this would be sufficient to prevent immediate pathogen-induced declines. however, such kind of adaptive processes to changing conditions is probably limited and does not predict the outcome effects of introduced pathogens, which differ from commonly encountered diseases. probably, the maintenance or even renewal of variation in functional important regions of the mhc, either from mutation, recombination or immigration from other populations, would be an important genetic component to allow an appropriate immune response (sommer ) . however, too strong genetic bottlenecks, leading to important inbreeding depressions, do not permit such kind of processes to operate and this explains why some species like the cheetah or the asian lion are so sensitive nowadays to diseases. as mentioned by mccallum and dobson ( ) , "diseases and parasites pose particularly severe problems in captive populations, in which animals are held at high density, may be stressed and may be exposed to crossspecies transmission". during the last years, a great amount of zoos worldwide have participated in the management of endangered species., many threatened species have captive populations that act as insurance against extinction in the wild and, indeed, captive breeding programs have saved some endangered species from extinction (e.g. père david's deer, european bison, etc) (frankham et al. ) . because parasites may have negative effects on their host, veterinarians in zoos take great care to reduce or even to remove entirely parasite loads on captive animals. as the ultimate goal of breeding programs in zoos is to increase threatened populations or to reintroduce individuals into the wild, parasites play an important role. what could be the consequences of maintaining hosts during many generations in a parasite-free environment? the potential risk is to release into the wild individuals that have lost their defences against pathogens and diseases. once in the wild, they will be in contact with a vast array of parasite species and may be unable to resist to their detrimental effects. maintaining some parasites on individual hosts kept in captivity could be a way to solve part of this problem. macroparasites, because of chronic infections, have evolved several kinds of immune evasion strategies (charbonnel et al. in this volume) . some strategies of immunomodulation displayed by many macroparasites may have some beneficial effects on their hosts by regulating th /th cytokine responses (weinstock et al. ). th responses induce inflammatory cell activity to control intracellular infections while th responses drive humoral immune responses to control extra-cellular parasites (see weil et al. in this volume) . mice with helminths have blunted th responses while helminths promote th responses associated with production of interleukin (il- ), which helps impede th cell differentiation. thus, induction of il- could underlie the alterations seen in host immunity (i.e. high inflammatory activities). helminths also appear to protect the host from aberrant th diseases such as asthma and food allergy (weinstock et al. ) , and there is now an immunological basis for protection by helminths. human epidemiological data and several animal studies support the notion that helminths protect the host from immunological disease (elliott et al. ) , particularly those caused by the activation of the th response by microparasites. for example, helminths protect mice and rats from experimental autoimmune encephalomyelitis, as welll as other diseases of immunity. thus, natural exposure to helminths may guard animals from developing severe immunological diseases, suggesting that helminths should be useful in conserving both endangered and captive species. gompper and williams ( ) proposed a series of measures to maintain endangered parasite species originating from threatened hosts in captive breeding program. however, they pointed out that because most of the public disapprove of protecting parasite species, attempts to conserve unique species of parasites could result in a hostile public response against efforts to preserve hosts. therefore they proposed a series of measures aimed to save parasite species without damaging attempts to conserve hosts. one of those measures was to find alternative hosts to maintain parasite populations for potential reintroduction once the host population was restored. however, the problem of parasite conservation concerns mainly highly host-specific parasites. to find alternative hosts on which specialist parasite populations would be viable may be a difficult task because experiments on cross-species infection have shown a strong decrease on both parasite survival and reproductive success on the foreign host, even if this new host species belongs to the same host genus (giorgi et al. ) . the consequence of human population growth is closer contact between human and reservoir hosts of numerous diseases. the spread of disease to endangered wildlife species due to contact with humans and domestic animals, and vice versa, increases as humans and their domestic animals get in more contact with these species due to habitat fragmentation. emergence of new diseases and particularly those from small mammals such as rodents or bats are of great public health concern (leroy et al. ) . conservation medicine, a new theme within the field of conservation biology, has been viewed as the application of medicine to improve the conserva-tion of wildlife and ecosystems . conservation medicine, according to otsfeld et al. ( ) is "devoted to understanding the interactions among human-induced and natural changes in ( ) climate, habitat and land use; ( ) pathogens, parasites, and pollutants; ( ) biodiversity and health within animal communities; ( ) health of humans" . the "anus horribilis" for bats worldwide illustrates the importance of this new field of investigations. while it was discovered in china that bats are the reservoir of sars virus (lau et al. ; li et al. ) , it was found in africa that they are probably the reservoir for ebola virus (leroy et al. ) . we strongly hope that this chapter will convince ecologists and conservation biologists that pathogens and parasites, mostly investigated by veterinarians and physicians, should not be ignored or eradicated because of their crucial importance to wild and domestic animals and humans. high mhc diversity maintained by balancing selection in an otherwise genetically monomorphic mammal the roles of parasites in the dynamics of a reindeer population 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function, and sexual selection coevolutionary arms races: increased host immune defense promotes specialization by avian fleas faster evolutionary rates in endosymbiotic bacteria than in cospeciating insect hosts is parasitism a missing ingredient in model ecosystems? hormones as indicators of stress exposure to ecotourism reduces survival and affects stress response in hoatzin chicks (opisthocomus hoazin) predation risk, host immune response, and parasitism a parasite reveals cryptic phylogeographic history of its host a role for molecular genetics in biological conservation conservation medicine. the birth of another crisis discipline stress hormones in mammals and birds. comparative aspects regarding metabolism, excretion, and noninvasive measurement in fecal samples virtual reality in the mhc the functional importance of parasites in animal communities: many roles at many levels? roles of parasites in animal invasions evolution of balanced genetic polymorphism physiological stress 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with physiological stress in an oldgrowth forest passerine effects of forest patch size on physiological stress and immunocompetence in an area-sensitive passerine, the eurasian treecreeper (certhia familiaris): an experiment parasites as accumulation indicators of heavy metal pollution field evidence for apparent competition mediated via the shared parasites of two gamebird species differential impact of a shared nematode parasite on two gamebird hosts: implications for apparent competition parasite-mediated competition among red-legged partridges and other lowland gamebirds parapoxvirus causes a deleterious disease in red squirrels associated with uk population declines kuris am ( ) parasites and marine invasions introduced species and their missing parasites parasite species richness of iberian carnivores: influences of host density and range distribution the epizootiology and ecological significance of malaria in hawaiian land birds the importance of diseases in reintroduction program introduced species: a significant component of human-caused global change the concept of stress and its relevance for animal behavior using parasites to infer host population history: a new rationale for parasite conservation heavenly hosts equal rights for parasites food shortage in small fragments: evidence from an area-sensitive passerine evolution of the rodent eosinophilassociated rnase gene family by rapid gene sorting and positive selection key: cord- -lj us dq authors: flower, darren r.; davies, matthew n.; doytchinova, irini a. title: identification of candidate vaccine antigens in silico date: - - journal: immunomic discovery of adjuvants and candidate subunit vaccines doi: . / - - - - _ sha: doc_id: cord_uid: lj us dq the identification of immunogenic whole-protein antigens is fundamental to the successful discovery of candidate subunit vaccines and their rapid, effective, and efficient transformation into clinically useful, commercially successful vaccine formulations. in the wider context of the experimental discovery of vaccine antigens, with particular reference to reverse vaccinology, this chapter adumbrates the principal computational approaches currently deployed in the hunt for novel antigens: genome-level prediction of antigens, antigen identification through the use of protein sequence alignment-based approaches, antigen detection through the use of subcellular location prediction, and the use of alignment-independent approaches to antigen discovery. reference is also made to the recent emergence of various expert systems for protein antigen identification. of smallpox were reported annually from across the globe, leading to about million deaths a year. yet, today, the disease has been completely eradicated. in the last years, there have been no known cases. poliomyelitis or polio is the other largescale disease which has come closest to eradication. its success too has been formidable: in , the pan american health organization effectively eradicated polio from the western hemisphere, since when the global polio eradication programme has significantly decreased the overall incidence of poliomyelitis through the rest of the world. in , there were approximately , cases spread through countries; in the past years, global figures amounted to less than , annually. yet, in spite of such remarkable success, death from vaccine-preventable diseases remains unacceptably high [ ] . there are over common infectious diseases responsible for one in four deaths globally. rotavirus and pneumococcus are pathogens causing diarrhoea and pneumonia, the leading causes of infant deaths in underdeveloped countries. in the next decade, effective, widespread vaccination programs against such pathogenic microbes could save the lives of . million children under years of age. hepatitis b causes , deaths in adults and children aged over . seasonal, non-pandemic influenza kills upwards of half a million globally each year. for those aged under in particular, a series of diseases causes an extraordinary and largely preventable death toll. for example, tetanus accounts every year for , deaths, pertussis is responsible for over , deaths, hib gives rise to in excess of , deaths, diphtheria accounts for , deaths, and yellow fever over , deaths. arguably, the most regrettable, the most lamentable situation is that of measles. measles accounts for the unneeded deaths of , under-fives and over , adults and older children. despite this, the situation is by no means bleak. by the close of , approximately million had been vaccinated against hib and million children against hepatitis b. during its first decade, vaccinations against polio, hep b, hib, measles, pertussis, and yellow fever funded by gavi had prevented the unnecessary loss of over million lives. there are approximately vaccines licensed for use in humans, around half of these are widely prescribed. yet, most of these vaccines target the prevention of common childhood infections, with the remainder addressing tropical diseases encountered by travellers to the tropics; only a relatively minor proportion combat endemic disease in under-developed countries. balancing the persisting need against the proven success and anticipated potential, vaccines remain an area of remarkable opportunity for medical advance, leading directly to unprecedented levels of saved and improved lives. from a commercial perspective, the vaccine arena has long been neglected, in part because of the quite astonishing success limned above; today, and in comparative terms at least, activity within vaccine discovery is feverish [ , ] . during the last years, tens of vaccines and vaccine candidates have moved successfully through clinical trials, and vaccines in late development number in the hundreds. in stark contrast to antibiotics, vaccine resistance is negligible and nugatory. despite the egregious and outrageous success enjoyed by vaccines, many major issues persist. the world health organisation long ago identified tuberculosis (tb), hiv, and malaria as the three most significant life-threatening infectious diseases globally. no vaccine has been licensed for malaria or hiv, and there seems little realistic hope for such vaccines appearing in the immediate future. bacille calmette guérin (bcg), the key anti-tb vaccine, is of limited efficacy [ ] . levels of morbidity and mortality generated by diseases already targeted by vaccines remain high. influenza is the key example, with a global annual estimated death toll in the region of half a million. in the twenty-first century, the world continues to be threatened by infectious and contagious diseases of many kinds: visceral leishmaniasis, marburg's disease, west nile, dengue, as well as sars potentially pandemic h n influenza, and over human and emerging zoonotic infections, as well as the persisting threat from hiv, tb, and malaria mentioned above. all this is further compounded by the additional risk arising from antibiotic-resistant bacteria and bioterrorism, not to mention major quasi-incidental issues, such climate change, an accelerating growth in the world's population, increased travel, and the overcrowding seen within the burgeoning populations concentrated into major cities [ ] . for reasons we shall touch on below, the discovery of vaccines is both more urgent and more difficult than it has ever been. in an era where conventional drug discovery has been seen to fail-or at least as seen by cupiditous investors, for whom the current model of pharmaceutical drug discovery is broken-vaccines are one of a number of biologically derived therapies upon which the future economic health of the pharmaceutical industry is thought to rest. the medical need, as stated above, is clear. set against this is the unfortunate realisation that vaccines exist for most easily targeted diseases, those mediated by neutralising antibodies, and so outstanding vaccine-targets are those of more intractable diseases mediated primarily by cellular immunity. to address those properly requires what all discoveries required: hard work and investment; but they also need new ideas, new thinking, and new vaccine discovery technology. amongst, these are computational techniques, the most promising of which are those targeting the discovery of novel vaccine antigens: the candidate subunit vaccines of tomorrow see fig. . . vaccines are agents-either molecular (epitope-or antigen-based vaccines) or supramolecular (attenuated or inactivated whole pathogen vaccines)-which are able to create protective immunity against specific pathogenic infectious microorganisms and any diseases to which they might give rise. protective immunity can be characterised as an enhanced but highly specific response to consequent re-infection-or infection by an evolutionarily closely related micro-organismsmade by the adaptive immune system. such increased or enhanced immunity is facilitated by the quantitative and qualitative augmentation of immune memory, which is able to militate against the pernicious effects of infectious disease. vaccines synergise with the herd immunity they help engender, leading to reduced transmission rates as well as prophylaxis against infection. the term "vaccine" derives from vacca (latin for cow). the words vaccine and vaccination were coined specifically for anti-smallpox immunization by the discoverer of the technique, edward jenner ( - ). these terms were later extended by louis pasteur ( - ) to include a far more extensive orbit or remit, including the entire notion of immunisation against any disease [ , , ] . several fundamentally distinct varieties of vaccine exist. these include inter alia inactivated or attenuated whole pathogen-based vaccines; subunit vaccines are based on one or more protein antigens, vaccines based upon one or more individual epitopes, carbohydrate-based vaccines, and combinations thereof. hitherto, the best-used and, thus, the most successful types of vaccine were built from attenuated-"weakened" or non-infective or otherwise inactivated-pathogenic whole organisms, be they bacterial or viral in nature. well-known examples include the following: the bcg vaccine which acts prophylactically against tuberculosis and albert sabin's anti-poliomyelitis vaccine based on attenuated poliovirus. the vast majority of subunit vaccines are immunogenic protein molecules, and are typically discovered using a somewhat haphazard search process. concerns over the safety of whole-organism vaccines long ago prompted the development of other kinds of vaccine strategy, including those based upon antigens as the innate or immanent active biological constituent of either single or composite vaccines. the vaccine which targets hepatitis b is a good exemplar of a so-called subunit vaccine as it is based on a protein antigen: the viral envelope hepatitis b surface antigen. other types of as-yet-unproven vaccines include those based on epitopes and others based on antigen-presenting cells; many have entered clinical trials, but none have fulfilled their medical or commercial potential. whole antigen discovery. when looking at a reverse vaccinology process, the discovery of candidate subunit vaccines begins with a microbial genome, perhaps newly sequence, progresses through an extensive computational stage, ultimately to deliver a shortlist of antigens which can be validated through subsequent laboratory examination. the computational stage can be empirical in nature; this is typified by the statistical approach embodied in vaxijen [ ] . or this stage can be bioinformatic; this involves predicting subcellular location and expression levels and the like. or, this stage can take the form of a complex mathematical model which uses immunoinformatic models combined with mathematical methods, such as metabolic control theory [ ] , to predict cell-surface epitope populations it is often difficult to capture the proper scientific meaning and use of recondite terms, often borrowed from common usage or archaic language. so, let us be more specific. an immunogen-a molecular moiety exhibiting the property of immunogenicity-is any material or substance capable of eliciting a specific immune response. an antigen, on the other hand, is a molecular moiety exhibiting the property of antigenicity. it is a substance or material recognised by a primed immune system. such a persisting state of immune readiness may be mediated by humoral immunity (principally via the action of soluble antibodies) or by cellular immunity (as mediated by t-cells, antigen presenting cells (apcs), or other phagocytic cells), or a combination of both, in what is often referred to as a "recall" response. immunogenicity is vital: it is the signature characteristic or property that prompts a certain molecular moiety to evoke a significant immune response. here, we shall strictly limit use of "immunogen" and "antigen" to a sole meaning. here, an "antigen" or an "immunogen" will mean a protein that is capable of educing some kind of discernible response from the host immune system. specifically, and for practical reasons, we will almost exclusively be referring to proteins derived from a pathogenic micro-organism. at present, the prophylaxis engendered by all current effective vaccines-all except bcg-is primarily mediated by the humoral immune system, via soluble antibodies. however, the disease mechanisms of most serious diseases for which vaccines are not available are usually mediated by cellular immunity. thus, for untreated disease, we seek to identify immunogenicity generated principally by cellular responses or by a combination of cellular and humoral responses, rather than by humoral immunity alone. to some extent, subunit vaccines can be thought to represent something of a compromise between vaccines based on attenuated or otherwise inactivated wholeorganisms and the many more recent and more innovative vaccine strategies typified by epitope or poly-epitope vaccines. vaccines based around whole pathogens have long engendered safety concerns [ ] [ ] [ ] . from the lubeck disaster and the cutter incident [ ] [ ] [ ] to the recent mmr debacle, issues over safety, real or imagined, have always dogged the development of vaccines [ , ] . indeed, during the eighteenth century the pre-vaccination practice of variolation against smallpox prefigured much of the current debate over the perceived danger of vaccines [ ] . while the case for vaccines is unanswerable, we should not be complacent. any live vaccine, however extensively attenuated, can revert to a pathogenic, diseaseinducing form. this is currently an on-going issue for polio vaccination [ ] . other issues, particularly the chemical or biological contamination of vaccines during manufacture, remain enduring and persistent problems. undesired immunogenicity, the type leading to severe and pathological immune responses, rather than enduring immune memory, is a concern for both whole-organism and subunitbased vaccines, as well as putative biologics [ ] . immunologists and vaccinologists have thus long sought alternatives to the use of whole organisms as vaccines. subunit vaccines and conjugate vaccines are one such. vaccines based on epitopes, singly or in combination, are another. the diversity of innovations in vaccine design holds much potential for success, but, thus far at least, has proved spectacularly unsuccessful in a clinical context. logically, a vaccine that relies solely on, at most, a few well-chosen epitopes, should be effective, efficacious, and, above-all, safe. epitopes, as peptides, may be cytotoxic and might possibly prompt some kind of inopportune immune response but cannot be infective or revert to infectivity. in many ways, epitopes are closer in size and share many properties with synthetic small molecules; possibly dealing with their pharmacokinetics as such may be better than thinking of them as biologic drugs. in practice, of course, epitope-based vaccines, like subunit vaccines, suffer from poor immunogenicity, necessitating the use of a complex combination of adjuvants and complicated delivery systems. for diverse reasons, including immunogenicity, stimulating protective immune responses against intracellular pathogens remains problematic when using nonreplicating vaccines. why should this be? first, the immune response is very complex, involving both the innate and adaptive immunity, and significant interaction between them. in all probability, and particularly when viewed in the context of the whole population, many epitopes and danger signals are involved; likewise, the many different immune actors, be they acting at the cellular or molecular levels, interact with each other and are subject to complex mechanisms of genetic, epigenetic, and system-level control and regulation. it may be that only the large and complex organism-sized vaccines can induce the range of immune responses necessary across the population to induce protection, since they comprise a potential host of immunogenic molecular moieties, not just a single immunodominant epitope see fig. in that which follows, we shall seek to explore the availability and accessibility of informatic techniques and informatic tools used to identify candidate subunit vaccines of microbial origin. yet, we shall start by adding context with an examination of experimental approaches to antigen discovery: so-called reverse vaccinology. reverse vaccinology already relies on informatics, but, in a sense at least, what we would like to do using informatics is to reproduce as much as is possible the steps inherent in successful reverse vaccinology in silico rather than in vitro. reverse vaccinology, and the necessary computational support, is a much more prevalent means of identifying subunit vaccines [ ] . see fig. . . even today, many experimentalists retain a deep and atavistic distrust of all computation. experimentalists seldom trust the reliability and dependability of computational methodology, choosing to trust instead in what they believe to be infallible, if actually rather elusive, empirical reliability of observations, experiments, and the whole paraphernalia of laboratory experimentation. yet, things are in the process of changing, and this change is likely to accelerate as we move forward into a future that looks more parsimonious and uncertain by the day. vaccines have come a long way from the days when they were prepared directly from the fluids of smallpox pustules or extracts of infected spinal cords. yet vaccine discovery and development remains firmly empirical. many modern vaccines still comprise entire inactivated pathogens. while vaccines targeting papillomavirus, tetanus, hepatitis b, and diphtheria are subunit vaccines, few are recombinant proteins devoid of contaminants. some would argue that the only molecular vaccines are glycoconjugates: oligosaccharides conjugated to immunogenic carrier proteins. conventional empirical, experimental, laboratory-based microbiological ways to identify putative candidate antigens require cultivation of target pathogenic micro-organisms, followed by teasing out their component proteins, analysis in a series of in-vitro and in-vivo assays, animal models and with the ultimate objective of isolating one or two proteins displaying protective immunity. unfortunately, in reality, the process is more complex, and more confusing, and much more confounding as this brief synopsis might suggest. cultivating pathogens outside the environment offered by their host organism can be difficult, even impossible. not every protein is readily expressed in adequate quantities in vitro, and many proteins are only expressed in an intermittent basis during the time course of infection. thus, a considerable number of potential, putative, and possible vaccine candidate antigens could be missed by conventional experimental approaches. reverse vaccinology [ ] [ ] [ ] [ ] has the potential to analyse genomes for potential antigens, initially scanning "open reading frames" (orfs), then selecting proteins because they are open to surveillance by the host immune system. this usually involves some complex combination of informatic-based prediction methodologies. recombinant expression of the resulting set of identified molecules can overcome their reduced natural abundance, which has often prevented us recognising their true potential. by enlarging the repertoire of native antigens, this technology can help to foster the development of a new cohort of vaccines. reverse vaccinology was originally established and has been established by studying neisseria meningitidis, which is responsible for meningococcal meningitis and sepsis. vaccines are currently available for all serotypes, except that serogroup b. n. meningitidis orfs were found initially [ , ] ; proteins were then identified, expressed in vitro and found to be surface exposed. seven proteins elicited immunity over many strains. the culmination of this work was a "universal" vaccine for serogroup b based on five antigens [ ] . this protovaccine, when used with alum as adjuvant, induced murine bactericidal antibodies versus % of meningococcal strains drawn from the world population of n. meningitidis. strain coverage increases to over % when used with cpg or mf as adjuvant. another key illustration is porphyromonas gingivalis, an anaerobic gramnegative bacterium found in the chronic adult inflammatory gum disease periodontitis. initially, orfs were identified [ ] ; of these, protein sequences were open to immune surveillance and were positive for several sera. two antigens were found to be protective in mice. yet another fascinating instance is provided by streptococcus pneumoniae, a prime cause of meningitis, pneumonia, and sepsis [ , ] . in this study, potential orfs were initially identified, with of these proteins being readily expressed. finally, six proteins were seen to induce protection against the pathogen. more recently, other and more advanced experimental techniques, such as microarrays, are beginning to come on-stream, opening up a gallimaufry of possible technologies to the new but maturing field of reverse vaccinology. the following gives but a taste of what is to come. using ribosome display to undertake in-vitro protein selection, weichart et al. [ ] identified within the methicillin-resistant col strain of the virulent human pathogen staphylococcus aureus genes, the majority of which were secreted or surface-localized proteins; of these, % had cell envelope function, % were transporter proteins, and % were virulence factors or toxins. using an ingenious combination of advanced proteomics techniques and in-vitro assays, giefing et al. [ ] identified novel vaccine candidates which prevented infections in children and in the elderly caused by a variety of pneumococcus serotypes; four demonstrating major protection versus sepsis in animals. two leads-stkp (a serine/threonine protein kinase) and pcsb (a structural protein with a role in cell wall separation of group b streptococcus)-showed clear cross-protection as potential candidate vaccines against four separate pneumococcal serotypes. using a whole proteome microarray, and in order to identify protein antigens, eyles et al. [ ] probed serum from balb/c mice previously immunized with a vaccine comprising: killed francisella tularensis and two immunomodulatory adjuvants. eleven out of the top twelve immunogenic antigens were known already as immunoreactive, although further proteins were discovered using this experimental approach. in further work from this consortium, titball and co-workers [ ] constructed a protein microarray of , burkholderia pseudomallei proteins, treated it with patient samples, identifying antigens. this smaller set was treated with a further distinct sera from groups of patients, identifying putative candidate antigens. this survey, brief though it is, helps to highlight the potential power of reverse vaccinology for vaccine discovery. however, since the number of antigens is high, given all the potential difficulties in characterising and expressing them, it is important to note that both computational and experimental techniques and methodologies will doubtlessly omit important and interesting proteins from further analysis, though not necessarily for the same or similar reasons. thus, with the burgeoning discipline of reverse vaccinology, both computational and experimental techniques are in need of constant development and improvement. compared to its role to drug discovery, genomics, and a host of other bioscience sub-disciplines, bioinformatics support for the preclinical discovery and development of vaccine is in its infancy; yet, as interest in vaccine discovery increases, the situation changes. there are two key types of bioinformatics support for vaccine design, discovery, and development. at the technical level, the first of these cannot be properly or meaningfully distinguished from general support for target discovery. it includes the annotation of pathogen genomes, more conventional host genome annotation, and the statistical analysis of immunological microarray experiments. the second form of support concentrates on immunoinformatics, that is, the informatics analysis of immunological problems, principally epitope prediction. b-cell epitope prediction remains defiantly basic or is largely dependent on a sometimes unavailable knowledge of three-dimensional protein structure. both structure- [ ] and data-driven [ ] prediction of antibody-mediated epitopes evince poor results. however, methods developed to predict t-cell epitopes now possess considerable algorithmic sophistication. moreover, they continue to develop and evolve, as well as extend their scope and remit to address new and ever larger and more challenging epitope prediction problems. presently, accurate and reliable t-cell epitope prediction is restricted to predicting the binding of peptides to the major histocompatibility complex (mhc). class i peptide-mhc prediction can be reasonably accurate, or is for properly characterised, wellunderstood alleles [ ] . yet a number of key studies have demonstrated that class ii mhc binding prediction is almost universally inaccurate, and is thus erratic and unreliable [ ] [ ] [ ] . a similar situation persists for structure-driven prediction of mhc epitopes [ , ] . irrespective of poor predictive performance, several other problems exist for epitope prediction. for t cell prediction in particular, a prime concern is with the availability or rather lack of availability of relevant data. it is now known that immunogenic t cell epitopes, thought previously to be peptides no more than amino acids in length, can be or more residues long. longmer epitopes now greatly expand the number of possible peptides open to inspection by t cells [ ] [ ] [ ] [ ] . the inadequate results generated by b cell epitope prediction algorithms may indicate that a fundamental reinterpretation of extant b cell epitope data is necessary before improved methods become feasible. these factors, when taken together, are consistent with the notion that methods relying only on the possession of certain epitopes will not be fully effective when tasked with antigen or immunogen identification. this is supported by information indicating a lack of correspondence between selected antigens and experimentally verified protective proteins. there are many means of identifying antigenic proteins. most focus on the properties of protein sequence and structure, but arguably one of the most insightful is instead to examine properties, both local and global, of the underlying nucleic acid. one notable way is to look for evidence of the horizontal or lateral transfer of so-called pathogenicity islands or pais. horizontal transfer, such as transformation, conjugation, or transduction, is distinct from the vertical transfer of genetic material from an ancestor within its lineage. it typically involves an organism incorporating genetic material from an evolutionarily distant organism without being its offspring. pais are a specific type of genomic island; that is, part of a genome acquired through direct transfer between microbes. a genomic island can occur in distantly related species and may be mono-or multi-functional; there are many sub-classes classified by function. other examples include antibiotic resistance islands, metal resistance, and secretion system islands. the gene products of pais are crucial to the propagation of disease pathogenesis, much as the pais themselves are key to the evolution of pathogenesis. pathogen-associated type iii and type iv secretion systems are, for example, often found together in the same pai. detecting such large (> kb) and discrete clusters of genes clusters, habitually possessing a characteristically atypical g/c content, at least when compared with the remainder of the genome, leads, in turn, to the individual identification within clusters of virulence-associated protein antigens. prokaryotic pais are frequently associated with trna-encoding genes, many are flanked by repeat structures, and many contain fragments of mobile genetic elements such as plasmids and phages. pais can be identified by combining analysis of nucleotide composition and phylogeny, amongst others. composition-based approaches rely on the natural variation between genome sequences from different species. regions of the genome with abnormal composition, as demonstrated by nucleotide or codon bias, may be potentially transferred horizontally. such methods are prone to inaccuracies; these result from inherent genomic sequence variation, such as is seen in highly expressed genes, and the observation that over time the sequences of genomic islands alter to mirror the composition of host genomes. evolution-based approaches seek regions that may have been transferred horizontally by comparing related species. put at its simplest: a putative genomic island present in one species, but absent from several related species, is consistent with horizontal transfer. of course, the island may have been present in the last common ancestor shared by the species compared and subsequently been lost from the other species. a less likely explanation would be that the island arose by mutation and selection in this species and no other. to decide, a body of extra evidence would need to be explored, such as the size of the pai, the mechanistic ease of deletion, the consistent presence of the island in more distantly related species, the relative pathogenicity of island-less species, and the divergence of the genome relative to that of other related species. many methods, which seek to quantify and leverage these somewhat vague notions, are now available [ ] [ ] [ ] . such analysis at the nucleic acid level shares many features in common with approaches used to identify cpg islands in eukaryotic genomes [ ] [ ] [ ] [ ] . recently, langille et al. tested six sequence-composition genomic island prediction methods and found that islandpath-dimob and sigi-hmm had the greatest overall accuracy [ ] . island path was designed to help identify prokaryotic pais, through the visualisation of common pai characteristics such as mobile element-associated genes or atypical sequence composition [ ] . sigi-hmm is a very accurate sequence composition-based genomic island predictor, which combines a hidden markov model (hmm) and codon usage measurement to identify genomic islands [ ] . in another work, yoon et al. coupled heuristic sequence searching methods, which aimed simultaneously to identify pais and individual virulence genes, with composition and codon-usage bias [ ] . exploiting a machine learning approach, vernikos and parkhill sampled the structural features of genomic islands using a hypothesis-free, bottom-up search, with the objective of explicitly quantifying the contribution made by each feature to the overall structure of different genomic islands [ ] . arvey et al. sought to identify large chromosomal regions with atypical features using a general divergence measureable to quantify the compositional difference between genomic segments [ ] . islandpick is a comparative genomic island predictor, rather than a composition-based approach, that can identify very probable genomic islands and very probable non-genomic islands within investigated genomes but does require that several phylogentically related genomes are available [ ] . observing pais as having a g + c composition closer to their host genome, wang et al. used so-called genomic barcodes to identify pais. these barcodes are based on the fact that the frequencies of -mers to -mers, and their reverse complement, are very stable across a whole genome when using a window size of over , bps and that this constituted a characteristic signature for genomes [ ] . the ready detection of pais, as a tool in computational reverse vaccinology, has been greatly aided by the deployment of several web-based resources. a key example of a server that successfully integrates several accurate genomic island predictors is islandviewer [ ] , which combines the methods: islandpick [ ] , islandpath [ ] , and sigi-hmm [ ] and is available at the url: http://www. pathogenomics.sfu.ca/islandviewer/query.php. the gui facilitates the visualisation of genomic islands and downloading of data at the gene and chromosome levels in a variety of formats. another important, web-accessible resource is paidb or the pai database. this is a wide-ranging database of pais, containing distinct pais and genbank accessions present in strains of pathogenic bacteria [ ] . paidb may be accessed via the url: http://www.gem.re.kr/paidb. thus, alternative techniques and methodologies are required in order to select and to rank proteins likely to be protective antigens and thus candidate vaccines. below, we shall explore three key approaches: subcellular location prediction, alignment-dependent sequence similarity searching, and alignment-independent empirical statistical approaches. in this section, we consider, perhaps, the clearest and cleanest way to identify potential new antigens in any microbial genome to alignment-dependent sequence similarity searching. there are two complimentary but distinct ways of identifying the immunogenicity of a protein from its sequence. one is to look for significant similarity to proteins of known immunogenicity. this idea seems so straightforward as to be almost facile. the other approach is somewhat less obvious conceptually but almost as straightforward logistically and involves seeking to identify antigens as proteins without discernible sequence similarity to any host protein. let us turn to the first of these two alternatives. let us begin by stating or rather reiterating the obvious. if we know the sequence of an existing antigen or antigens, we can use sequence searching to find similar sequences in the target genome [ , ] . any candidate antigens selected by this process can then be selected for further verification and validation. the same old, familiar caveats apply here: are chosen thresholds appropriate? are high-scoring matches an artefact or are they real and meaningful? the litany of such conditions is all too familiar to anyone well versed in sequence similarity searching. clearly, when a sequence search is run, using blast or fasta , for example, an enormously long list of nearly identical proteins might ensue, or one that does not get any hits at all, or almost any intervening result might be obtained. as reflective practitioners, we must judge which result can be classified as useful and which cannot, and in so doing, identify sets of suitable thresholds, above which we expect usefulness and below which we might anticipate little or no utility. thresholds are contingent upon the sequence family studied, as well as being dependent solely on the problem investigated. thus heuristically identified cut-offs are desirable, but much thinking and empirical investigation are required to select appropriate values. of course, the process adumbrated above presupposes that sufficient antigenic protein sequences are known. compilation of this data is the role of the database. recently, extensive literature mining, coupled with factory-scale experimentation, has created many functional immunology databases, although databases, such as syfpeithi [ , ] , focussing on cellular immunology-primarily mhc processing, presentation, and t cell recognition-have existed for - years. arguably, the best extant database is the hiv molecular immunology database [ ] , although clearly the depth of the database is at the expense of generality and breadth. other recent databases include mhcbn [ , ] and epimhc [ ] , amongst many others. two databases, warrant particular attention: antijen [ ] , formerly known as jenpep [ , ] ; and iedb [ ] . implemented as a relational postgresql database, antijen integrates a wideranging set of data items, much of which is not stored by other databases. in addition to the kind of cellular immunological information familiar from syfpeithi, such as mhc binding and t cell data, antijen additionally archives b cell epitopes and also includes a significant stockpile of quantitative data: kinetic, thermodynamic, as well as functional, including measurements of immunological peptide-protein and protein-protein interactions. the iedb database is considerably more extensive than other equivalent database systems, benefiting from the input of dedicated epitope sequencing projects. iedb has come to eclipse other work in this area. although both antijen and iedb are full of epitope-focussed information of many flavours, they remain incomplete concerning immunogenic antigens. fortuitously, specific antigen-orientated-rather than epitope-focusseddatabases are starting to be available. arguably, the most obvious and most unambiguous example of an antigen is virulence factor (vf): proteins, such as toxins, able to induce disease directly by attacking a host. analysis of known pathogens has allowed recurring vf systems of + distinct proteins. often, sets of vfs exist as discrete, distinct genome-encoded pais, as well as being more widely spread through the genome. clearly, antigens do not need to be vfs in order to be immunogenic and thus candidates for subunit vaccines. instead, they need only be accessible to the immune system. they do not need to directly or indirectly mediate infection. thus, other databases are needed which capture, collate, and archive the burgeoning plethora of antigen-orientated data. recently, we have helped developed a very different database: antigendb [ ] . it contains over antigens collated from the primary scientific literature, as well as other sources. another related database system has been christened violin (vaccine investigation and online information network) [ ] , which allows straightforward curation and the analysis and comparison of research data across diverse pathogens in the context of human medicine, animal models, laboratory model systems, and natural hosts. as we outline above, in addition to identifying sequence similarity to known antigens, another idea gaining ground is that the immunogenicity of an antigen is solely determined by the absence of similarity to host proteins. some think this is the prime determinant of potential protein immunogenicity [ , ] . such ideas are supported by the belief that immune systems are actively educated to lack reactivity to self-proteins [ ] , a process-often termed "immune tolerance"-which is generated via epitope-specific mechanisms [ , ] . what we really want is a meaningful measure of the "foreignness" of a protein correlating with its immunogenicity. usually, "evolutionary distance" substitutes for "foreignness." clearly, such an evolutionary distance must be specified in terms of biomacromolecular structures or sequences. but, is this practically useful for selecting candidate vaccines? another way to formulate this idea is to say that the probability that a protein is immunogenic is exclusively a product of its dissimilarity, at the whole-sequence or sequence-fragment level, to each and every protein contained within the host proteome. most search software is well matched to this problem. in terms of fragment length, the typical length of an epitope might seem logical, since the epitope is the molecular moiety typically recognised during the initial phase of an immune response. yet, even at the epitope level-say a peptide of - amino acid residues-even a single conservative mutation or mismatch in an otherwise identical match might prove significant. single sequence alterations may totally abrogate or significantly enhance neutralising antibodies binding or recognition by the machinery of cellular immunology. we have attempted to benchmark sequence similarity and correlate it with immunogenicity in order to explore the potential of this idea in a quantitative fashion. to that end, we examined the differences between sets of antigens and non-antigen using sequence similarity scores. we looked specifically at sets of known non-antigenic and antigenic protein sequences from six sources: bacteria, viruses, fungi, and parasites, as well as allergens and tumours [ ] [ ] [ ] , comparing pathogen sequence to those from humans and mice using blast [ ] . most non-antigenic and antigenic sequences were non-redundant; implying a lack of homologues between pathogens and host proteomes, although certain parasite antigens, such as catalases and heat shock proteins, had a much greater level of similarity. we were not able to determine a suitable and appropriate threshold based on the hypothesis of non-redundancy to the host's proteome, suggesting that this is not a viable solution to vaccine antigen identification. however, rather than looking at nucleic acid sequences, or at protein sequences using an alignment-based approach, a new set of techniques, based upon alignmentfree techniques, has been and is being developed; as this approach begins to show significant potential, we shall examine it next. proteins accessible to immune system surveillance are assumed to lie external to the microbial organism or be attached to its surface rather than being sequestered and sequestrated within the cell. for bacteria, this means being located on-or in-the outer membrane surface or being secreted. thus, being able to accurately predict the physical location of a putative antigen can provide considerable insight into the likelihood that a particular protein will prove to be an immunogenic and possibly protective. there are two basic kinds of prediction method for identifying subcellular location: manual rule construction and the application of data-driven machine learning methods. data used to discriminate between compartments include sequence-derived features of the protein, such as hydrophobic regions; the amino acid composition of the whole protein; the presence of certain specific motifs; or a combination thereof. accuracy differs significantly between different methods and different compartments, mostly resulting from the deficiency and inconsistency of data used to derive models. gross overall sequence similarity is unable to predict protein sub-cellular location reliably or accurately. even nearly identical protein sequences may be found in distinct locations, while there are many proteins which exist simultaneously at several distinct locations within the cell, often having equally distinct functions at these different sites [ ] . eukaryotes and prokaryotes have quite distinct subcellular compartments. the number of such compartments used in prediction studies varies. a common schema reduces prokaryotic to three compartments (cytoplasmic, periplasmic, and extracellular) and eukaryotic cells to four compartments (nuclear, cytoplasmic, mitochondrial, and extracellular). other structural classifications evince in excess ten eukaryotic compartments. ten compartments maybe a conservative estimate, such is the complex richness of sub-cellular structure. any prediction method must account for permanent, transient, and multiple locations, and, in addition, multi-protein complexes and membrane-bound organelles as possible sites. numerous signal sequences exist. several methods predict lipoproteins. the prediction of proteins translocated via the tat-dependent pathway is important but has yet to be addressed properly. however, amongst binary, single-outcome approaches, signalp is probably the most accurate and reliable method available. it uses neural networks to predict the presence and probable cleavage sites of type ii or n-terminal spase-i-cleaved secretion signal peptides [ ] [ ] [ ] . this signal is common to both prokaryotic and eukaryotic organisms. signalp has recently been enhanced with a hmm intended to discriminate cleaved from uncleaved signal anchors. a limitation of signalp is its proclivity to over-predict: it cannot properly discriminate reliably between a number of very similar yet functionally different signal sequences, regularly predicting lipoproteins and integral membrane proteins as type ii signals. many methods have been devised capable of dividing a genome or virtualproteome between the various subcellular locations of a eukaryotic or prokaryotic cell. psort is a good example; it is a multicategory prediction procedure, comprising many different programmes [ ] [ ] [ ] [ ] . psort i predicts subcellular compartments, while psort ii predicts ten different locations. ipsort deals with several compartments: chloroplast, mitochondrial, and proteins secreted from the cell, while psort-b focuses solely on predicting bacterial sub-cellular locations. another effective programme is hensbc [ ] . hensbc can assign gene products to one of four different types (nuclear, mitochondrial, cytoplasmic, or extracellular) with an accuracy of about eight out of ten for gram-negative bacteria. another programme, subloc [ ] , predicts prokaryotic subcellular location divided between three compartments. another programme is gpos-ploc [ ] , which integrates several basic classifiers. other methods include phobius [ ] , lipop . [ ] , and tatp . [ ] . a comparison of several such programmes, using mycobacterial proteins as a gold standard [ ] , showed subcellular localisation prediction and possessed high predictive specificity. we have developed a set of methods which predict bacterial subcellular location. using a set of methods for lipoprotein, tat secretion, and membrane protein prediction [ ] [ ] [ ] [ ] [ ] [ ] [ ] , three different bayesian network architectures were implemented as software pipelines able to predict specific subcellular locations, and two serial implementations using a hierarchical decision structure, and a parallel implementation with a confidence-level-based decision engine [ ] . the soluble-rooted serial pipeline performed better than the membrane-rooted predictor. the parallel pipeline outperformed the serial pipeline but was significantly less efficient. genomic test sets proved more ambiguous: the serial implementation identified more of the proteins of known location yet more accurate predictions are made overall by the parallel implementation. the implications of this work are clear. the complexity of subcellular structures must be integrated fully into sub-cellular location prediction. in extant studies, many important cellular organelles are not considered; different routes by which proteins can reach the same compartment are ignored; and proteins existing simultaneously at several locations are likewise discounted. clearly, combining high specificity predictors for each compartment appropriately must be the way forward [ ] . many difficulties, problems, and quandaries persist; the most keenly felt is the lack of high-quality, verified, and validated datasets which unambiguously established the location of well-characterised proteins. this dearth is particularly serious for certain types of secreted protein, such as type iii secretion. in a similar manner, considerably more work is required to accurately predict the locations for proteins of viral origin; while certain studies are encouraging [ , ] , the complexity of viral interaction with host organisms continues to confound attempts at analysis. predicting antigens in silico typically utilise bioinformatics tools. such tools can identify signal peptides or membrane proteins or lipoproteins successfully, yet the majority of algorithms tend to depend on motifs characteristic of antigens or, more generally, sequence alignment as the principal arbiter of definitive and meaningful sequence relationships. this is potentially a problem of some magnitude, particularly given the wide range of evolutionary rates and mechanisms amongst microbial proteins. certain protein families do not, however, show obvious or significant sequence similarity, despite having common biological properties, functions, and three-dimensional structures [ , ] . thus alignment-based approaches may not always produce useful and unequivocal results, since they assume a direct sequence relationship that can be identified by simple sequence search techniques. immunogenicity, as a signature characteristic, may be encrypted within the structure and/or sequence instead. this may be encoded so cryptically or so subtlety as to completely confound or at least mislead conventional sequence alignment protocols. discovery of utterly novel and previously unknown antigens will be totally stymied by the absence of similarity to known antigenic proteins. alignment-dependent methods tend to dominate bioinformatics and, by extension, immunoinformatics. several authors have chosen to look at alternative strategies, implementing so-called alignment-independent or alignment-free techniques. the first authors to do so were mayer et al., who reported that protective antigens had a different amino acid composition compared to control groups of nonantigens [ ] . such a result is unsurprising since it has long been known that the structure and sequence composition of proteins adapted to the different redox environments of different sub-cellular compartments [ ] . mayer's analysis was formulated primarily in terms of univariate comparisons of antigens versus controls for different properties. subsequently, we explored bivariate comparison in terms of easily comprehensible scatter-plots. see fig. . for representative examples. what their results ably demonstrate is the potential for the discrimination of antigens and non-antigens by the appropriate selection of orthogonal descriptors. the challenge, of course, is to identify a robust choice of descriptors which are capable of extrapolating as well interpolating when used predictively. progressing beyond this type of analysis, and synergising with our other work on alignment-independent representation [ ] [ ] [ ] [ ] [ ] , we have initiated the development of new methods to differentiate antigens-and thus potential vaccine candidates-and non-antigens, using more sophisticated alignment-free approach to sequence representation [ , ] . rather than focus on epitope versus nonepitope, our approach utilises data on protective antigens derived from diverse pathogens to create statistical models capable of predicting whole-protein antigenicity. our alignment-independent method for antigen identification uses the auto cross covariance (acc) transformation originally devised by wold et al. [ , ] to transform protein sequences into uniform vectors. the acc transform has found much application in peptide prediction and protein classification [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . in our method, amino acid residues are represented by the well-known and well-used z descriptors [ ] [ ] [ ] , which characterise the hydrophobicity, molecular size, and polarity of residues. our method also accounts for the absence of complete independence between distinct sequence positions. we initially applied our approach to groups of known viral, bacterial, and tumour antigens, developing models capable of identifying antigen. extra models were subsequently added for fungal and parasite antigens. for bacterial, viral, and tumour antigens, models had prediction accuracies in the - % range [ , , ] . for the parasite and fungal antigens, models had good predictive ability with - % accuracy. these models were incorporated into a server for protective antigen prediction called vaxijen [ ] (url: http://www.darrenflower.info/ vaxijen). vaxijen is an imperfect but encouraging start; future research will yield significantly more insight as well-characterised protective antigens increase significantly in number [ ] . as we have said, a number of bioinformatics problems are unique to the discipline of immunology: the greatest of these is the accurate quantitative prediction of immunogenicity. this chapter has in its totality been suffused and pervaded by the idea of immunogenicity and the challenge of predicting this property in silico. such an endeavour is confounding, yet exciting, and, as a key instrument in developing better, safer, more effective vaccines, is also of undisputed practical utility. successful immunogenicity prediction is at its simplest made manifest through the identification of b cell or t cell epitopes. epitope recognition, when seen as a chemical event, may be understood in terms of the relationships between apparent biological function or activity and basic physicochemical properties. delineating structure-activity or property-activity relationships of this kind is a key concern of immunoinformatics. at the other end of the spectrum, immunogenicity can be viewed is a cohesive, integrated, system property: a property of the entire and complete immune system and not a series of individual and isolated molecular recognition events. thus, the task of predicting systems-level immunogenicity is in all likelihood manifold more demanding than predicting peptide-binding say. the clinical manifestation of vaccine immunogenicity arises from the complex amalgam of many contributing extrinsic and intrinsic factors, which includes pathogen-side and host-side properties, as well as those just coming directly from proteins themselves. see fig. . . protein-side properties include the aggregation state of candidate vaccines and the possession of pamps. pathogen-side properties are clearly properties intrinsic to the pathogen, including expression levels of the antigen, the time-course of this expression, as well as its subcellular location. socalled host-side properties are innate recognition properties of host immunity, and most obviously include t cell epitopes or b cell epitopes. a bona fide candidate antigen should be available for immune surveillance and thus highly expressed, constitutively or transiently, as well as having several epitopes. a protein without immunogenicity would logically lack all or some of these characteristics. as a prediction problem, this is, to say the least, not uncomplicated; clearly consisting of a great variety of difficult-to-compute stages. in terms of mechanism, many of these stages are poorly understood. yet, each can be addressed using standard computational and statistical tools. they can all be predicted, however, presupposing, of course, the presence of relevant data in sufficient quantity. one of the strongest messages to emerge from this review is that immunogenicity is a strongly multi-factorial property: some protein antigens are immunogenic for one reason, or set of reasons, and other immunogenic proteins will be so for another possibly tangential reason or set of reasons. each such causal manifold is itself complex and potentially confusing. thus, the prediction of immunogenicity is a problem in multi-factorial prediction, and the search for new antigens is a search through a multi-factorial landscape of contingent causes and discombobulating decoys. some of the evidence will be highly precise and quantitative. the kind provided by predictive immunoinformatics, for example. this typically yields exact values for, say, the binding affinity of a peptide to a protein component of the immune system, or an unequivocal yes or no answer to the question: is this peptide sequence an epitope? however, for each such exact prediction, we have some notional associated probability concerning how reliable we regard this result. different methods evince a range of accuracy, which, in practice, equate to probabilities of reliability: we naturally have more confidence and assume a greater reliability for a highly accurate prediction versus one of average predictability, though it can still give wrong predictions and generally inaccurate predictors may work well for a specific subset of the data. other types of forms of evidence will have a distinctly more anecdotal flavour. take, for example, the case of bacterial exotoxins. together with endotoxins, such as lps, and so-called superantigens, exotoxins form the principal varieties of toxin secreted by pathogenic bacteria. exotoxins have evolved to be the most toxic substances known to science: in terms of the median lethal dose, botulinum toxin-the active ingredient of botox and causative agent of botulism, amongst others-is about ten times as lethal as radioactive isotope polonium- and a million times more deadly than mainline poisons, such as arsenic or potassium cyanide. virtually, all such potent bacterial exotoxins comprise two functionally distinct subunits, either separate proteins or distinct domains, usually denoted a and b. the a subunit is habitually an enzyme, such as a protease, which modifies specific protein targets, thus disrupting key cellular processes with host cells. the b subunit is a protein which binds to host cell surface lipids or proteins, enabling the toxin to be internalised efficiently. the high specificity of this dual action lends exotoxins much of their remarkable lethality. exotoxins are also extremely immunogenic, inducing the immune systems to produce high-affinity neutralising antibodies against them, and thus make excellent targets for vaccinology. a toxoid-a toxin which has been treated or inactivated, often by formaldehyde-is in essence a form of subunit vaccine and, as such, requires adjuvant to induce adequate immune responses. vaccines targeting tetanus and diphtheria, which usually need boosting every decade, are based on toxoids, albeit typically combined with pertussis toxin acting as an adjuvant. poisoning by exotoxins, on the other hand, requires treatment with antitoxin comprising preformed antibodies. however, and say that we were offered a newly sequenced pathogen genome, is such a classification for ab toxins helpful when trying to identify a potential exotoxins? the answer is neither yes nor is it no, but lies somewhere between these extremes. assuming we had extant knowledge or a reliable method predicting the presence of structural and functionally distinct domains, this very simple ruleof-thumb would become a useful tool for eliminating large numbers of possible toxin molecules. it would not directly identify an antigen but would enormously reduce the workload inherent in their discovery. as well as needing more and more reliable predictors, we also need a way of combining the information we gather from any set of reliable predictors to which we have access. thus, when analysing a pathogen genome, what we seem to need, at least in order to identify immunogenic proteins, is both a set of reliable and robust tools and a cohesive expert system within which to embed them. such systems, albeit still at a relatively crude and faltering level, do exist. because there is an implicit hierarchy of one prediction being based on others, there is a need to balance and judge different pieces of probabilistic evidence. an effective expert system should be capable of such a feat. to a first approximation, an expert system is a computer programme that undertakes tasks that might otherwise be prosecuted by a human expert ostensively by simulating the apparent judgement and behaviour of an individual or organization with expertise and experience within a particular discipline. an expert system might make financial forecasts, or play chess; it might diagnose human illnesses or schedule the routes of delivery vehicles. to create an expert system, one first needs to analyse human experts and how they make decisions, before translating this into rules that a computer can follow. such a system leverages both a knowledge base of accumulated expertise and a set of rules for applying such distilled knowledge to particular situations in order to solve problems. sophisticated expert systems can be updated with new knowledge and rules and can also learn from the success of its prediction, again mirroring the behaviour of properly performing experts. at the heart then of an expert system is the need to combine evidence in order to reach decisions. combining evidence, and reaching a decision based on that combined evidence, is no easier in the laboratory, be that virtual or actual, than it is in the court room. the problem of combining evidence is encountered across the disciplines, and various solutions have arisen in these different areas. within bioinformatic prediction, a particular variety of evidence combination, so-called meta-prediction, is a now a well-established strategy [ , ] . this approach seeks to amalgamate the output of various predictors, typically internet servers, in an intelligent way so that the combined result is more accurate than any of those coming from a single predictor. indeed, combining results from multiple prediction tools does often increase overall accuracy. a consensus strategy was first proposed by mallios [ ] , who combined syfpeithi [ , , ] , propred [ , ] , and the iterative stepwise discriminant analysis meta-algorithm [ ] [ ] [ ] . multipred [ ] integrates hmms and artificial neural networks (ann). six mhc class ii predictors were combined by dai and co-workers [ ] [ ] [ ] basing its overall prediction on the probability distributions of the different scores. trost et al. have used a heuristic method to address class i peptide-mhc binding [ ] . wang et al. [ ] applied a consensus method to calculate the median rank of the top three predictive methods for each mhc class ii protein initially evaluated so as to rank all possible -, -, and -mers from one protein. this rank was used to identify the top % of peptides from each protein. in probabilistic reasoning, or reasoning with uncertainty, there are many ways to represent espoused beliefs-or, in our domain, predictions-that effectively encode the uncertainty of propositions. these include fuzzy logic and the evidential method, among many others. for quantitative data, information fusion, in its various guises [ ] , is one robust route to effective combination. another requires us to enter the world of bayesian statistics, or, at least, a special thread within it. bayes theory, and the ever-expanding strand of statistics devolving from it, is concerned primarily with updating or revising belief in the light of new evidence, while so-called dempster-shafer theory [ ] is concerned not with the conditional probabilities of bayesian statistics but with the direct combination of evidence. it extends the bayesian theory of subjective probability, by replacing bayesian probabilities with belief functions that describe degrees of belief for one question in terms of probabilities for another and then combines these using dempster's rule for merging degrees of belief when based on independent lines of evidence. such belief functions may or may not have the mathematical properties of probabilities but are seemingly able to combine the rigor of probability theory with the flexibility of rule-based approaches. several expert systems of different flavours and hues have now become available within the vaccinology arena. sundaresh et al. developed a specialist software package for the analysis of microarray experiments that could easily be classified as an expert system and used it in the area of reverse vaccinology. this package, which was written in the open-source statistical package r, was used to help analyse a variety of complex microarray experiments on the bacteria f. tularensis, a category a bio-defense pathogen [ ] . this programme implements a two-stage process for diagnostic analysis: selection of antigens based on significant immune responses coupled with differential expression analysis, followed by classification of measured antigen responses using a combination of k-means clustering, support vector machines, and k-nearest neighbours. we have already discussed vaxijen [ , , ] , and the related server epijen [ ] , which combines various methods for identifying epitopes within extant proteins. these two servers can also be classified as vaccine-related expert systems. nerve is another expert system, which has been developed to help automate aspects of reverse vaccinology [ ] . using nerve, the prioritisation of potential candidate antigens consists of several stages: prediction of subcellular localisation; is the antigen an adhesion?; identification of membrane-crossing domains; and comparison to pathogen and human proteomes. candidates are filtered then ranked and putative antigens graded by provenance and its predicted immunogenicity. the web-based expert system, dynavacs [ ] , was developed to facilitate the efficient design of dna vaccines and is available in the url: http://miracle. igib.res.in/dynavac. it takes a structured approach for vaccine design, leveraging various key design parameters, including the choice of appropriate expression vectors, safeguarding efficient expression through codon optimization, ensuring high levels of translation by adding specific sequence signals, and engineering of cpg motifs as adjuvant mechanisms exacerbating immune responses. it also allows restriction enzyme mapping, the design of primers, and lists vectors in use for known dna vaccines. vaxign is another expert system developed to help facilitate vaccine design [ ] . vaxign undertakes dynamic vaccine target prediction from sequence. methodologically, it combines protein subcellular location prediction with prediction of transmembrane helices and adhesins, analysis of the conservation to human and/or mouse proteins with sequence exclusion from the genomes of nonpathogenic strains, and prediction of peptide binding to class i and class ii mhc. as a test, vaxign has been used to predict vaccine candidates against uropathogenic escherichia coli. however, nerve and its various and varied siblings are tasked with such a confounding and difficult undertaking that they are obliged to fall somewhat short of what is required. an obvious first step in tackling the greater problem is to address first subcellular location prediction. then, we can look at antigen presentation, modelling for each component step, before building these into a fully functional model. we can also develop empirical approaches-such as vaxijen [ , , ] . we must also factor in antibody-mediated issues, properly address pamps, post translational danger signals, expression levels, the role of aggregation, and the capacity of molecular adjuvants to enhance the innate immunogenicity to usable levels. see fig. . . the value of vaccines is not yet unchallenged. however, most reasonable people would, in all probability, agree that they are a good thing, albeit with a few minor provisos. the idea underlying all vaccines is a strong and robust one: it is in the reification-that is, the realisation, manifestation, and instantiation-of this abstract concept that the trouble lies, if indeed trouble there is. existing vaccines are by no means perfect; again, most sensible and well-informed people would no doubt acknowledge this also. one might argue that their intrinsic complexity, and the highly empirical nature of their discovery over decades, and the fraught nature of their manufacture, has much to answer in this regard. why should this be? in part, it is due to the extreme complexity of immune response to an administered vaccine, which is largely specific to each individual or at least is different in different sub-groups within the totality of the vaccinated population. the immune responses is comprised, at least for whole-pathogen vaccines, of the adaptive immune response to multiple b cell and t cell epitopes as well as the responses made by the innate immune responses to diverse molecular structures, principally pamps. when one considers also the degree to which such a repertoire of responses is augmented and modified by the action of additives, be they designed to increase the durability and stability of vaccines or be they adjuvants, which are intended to raise the level of immune reactions. add in stochastic and coincidental phenomena, such as reversion to pathogenicity, and we can see immediately that navigating our way through the vaccine minefield is no easy task. all such problems engendered by this intrinsic complexity are themselves compounded by our comparatively weak understanding of immunological mechanisms, since, if we understood the mechanism of responses well enough, we could and would have designed our vaccines to circumvent these issues. part of the answer to this cacophony of conflicting and confounding quandaries is the newly emergent discipline of vaccinomics. a proper understanding of the relationships between gene variants and vaccine-specific immune responses may help us to design the next generation of personalised vaccines. vaccinomics addresses this issue directly. it seeks to identify genetic factors mediating or moderating vaccine-induced immune responses, which are known to be extremely variable within population. much data indicate that host genetic polymorphisms are key determinants of innate and adaptive response to vaccination. hla genes, non-hla genes, and genes of the innate immunity all contribute, and do so in many ways, to the variation observed between individuals for immune responses to microbial vaccines. vaccinomics offers many techniques that can help illuminate these diverse phenomena. principal amongst these are population-based gene/snp association studies between allele or snp variation and specific responses, supplemented by the application of next-generation sequencing technology and microarray approaches. yet, and for all this nay-saying and gainsaying, vaccines and vaccination have demonstrated their worth time after time; yet, to justify the continuing faith we invest in them, new and better ways of making safer and more focussed vaccines must be found. most current vaccines work via antibody-mediated mechanisms; and most target viruses and the diseases they cause. unfortunately, the stock of such disease targets is dwindling. low-hanging fruit has long since been cut down. only fruit that is well out of reach remains. vaccines based on apcs and peptides are new but unproven strategies; most modern vaccine development relies instead on effective searches for vaccine antigens. one of the clearest points to emerge from such work is that there are many competing concepts, thoughts, and ideas that may confound or help efficient identification of immune reactive proteins. certain such ideas we have outlined. some are indisputably persuasive, even compelling, yet many strategies-and the technical approaches upon which they are based-have singly failed to deliver on their promise. long ago, and based on his lifetime's experience of all things immunological, professor peter cl beverley sketched out a paradigm for protein-focussed vaccine development, which we have formalised further, and which schema is summarised in fig. . . some of his factors overlap with the factors from fig. . . he identified many of the factors that potentially contribute to the immunogenicity of proteins, be they of pathogen origin or another source entirely, and also other features which might make proteins particularly suitable for becoming candidate vaccines. of these, some are as-yet beyond prediction, such as the attractiveness for apcs or the inability to down-regulate immune responses. the status of proteins as evasins is currently only possibly addressable through sequence similarity-based approaches and likewise for the attractiveness for uptake by apcs is again, though possible there exist motifs, structural or sequence, which could be identified. currently, the dearth of relevant data precludes prediction of such properties; and, while it is possible to predict some of these properties with some assurance of success, and others are predictable but only incidentally, overall, we are still some way from realising the dream embodied in fig. failure occurs for simple reasons: we deal with simplified abstractions and cannot hope to capture all that which is required for prediction by looking superficially at a single factor. protein immunogenicity comes instead from the dynamic combination of innumerable contributing factors. this is by no means a facile or easily solved informatics conundrum. a vaccine candidate should have epitopes that the host recognises, be available for immune surveillance, and be highly expressed. factors mediating protein immunogenicity are many; possession of b or t cell epitopes, post-translational danger signals, sub-cellular location, protein expression levels, and aggregation state amongst them. predicting such diverse, complex, confounding properties is-and remains-a challenge. vaccine antigens, once discovered, should, ultimately, and with appropriate manipulation, together with an apt, apposite, and appropriate delivery system and the right choice of adjuvant, become first a candidate for clinical trials, before, hopefully, progressing to regulatory approval. we require an integrative, systemsbiology approach to solve this problem. no single approach can be applied universally and with success; what we crave is the full integration of numerous equally wakefield's article linking mmr vaccine and autism was fraudulent computer-aided 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server/client suite for protein subcellular location based on soap gpos-ploc: an ensemble classifier for predicting subcellular localization of gram-positive bacterial proteins advantages of combined transmembrane topology and signal peptide prediction-the phobius web server prediction of lipoprotein signal peptides in gram-negative bacteria prediction of twin-arginine signal peptides validating subcellular localization prediction tools with mycobacterial proteins toward bacterial protein sub-cellular location prediction: single-class discrimminant models for all gram-and gram+ compartments multi-class subcellular location prediction for bacterial proteins alpha helical trans-membrane proteins: enhanced prediction using a bayesian approach beta barrel trans-membrane proteins: enhanced prediction using a bayesian approach a predictor of membrane class: discriminating alpha-helical and beta-barrel membrane proteins from non-membranous proteins tatpred: a bayesian method for the identification of twin arginine translocation pathway signal sequences lippred: a web server for accurate prediction of lipoprotein signal sequences and cleavage sites combining algorithms to predict bacterial protein sub-cellular location: parallel versus concurrent implementations predicting the subcellular localization of viral proteins within a mammalian host cell virus-ploc: a fusion classifier for predicting the subcellular localization of viral proteins within host and virus-infected cells structure and sequence relationships in the lipocalins and related proteins structural relationship of streptavidin to the calycin protein superfamily analysis of known bacterial protein vaccine antigens reveals biased physical properties and amino acid composition adaptation of protein surfaces to subcellular location hierarchical classification of g-protein-coupled receptors with data-driven selection of attributes and classifiers gpcrtree: online hierarchical classification of gpcr function optimizing amino acid groupings for gpcr classification on the hierarchical classification of g protein-coupled receptors proteomic applications of automated gpcr classification vaxijen: a server for prediction of protective antigens, tumour antigens and subunit vaccines identifying candidate subunit vaccines using an alignment-independent method based on principal amino acid properties dna and peptide sequences and chemical processes multivariately modeled by principal component analysis and partial least-squares projections to latent structures principal property-values for nonnatural amino-acids and their application to a structure activity relationship for oxytocin peptide analogs peptide binding to the hla-drb supertype: a proteochemometrics analysis proteochemometrics mapping of the interaction space for retroviral proteases and their substrates proteochemometrics analysis of substrate interactions with dengue virus ns proteases generalized modeling of enzyme-ligand interactions 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prediction of hla-a -restricted ctl epitope specific to hcc by syfpeithi combined with polynomial method propred analysis and experimental evaluation of promiscuous t-cell epitopes of three major secreted antigens of mycobacterium tuberculosis propred: prediction of hla-dr binding sites predicting class ii mhc/peptide multi-level binding with an iterative stepwise discriminant analysis meta-algorithm class ii mhc quantitative binding motifs derived from a large molecular database with a versatile iterative stepwise discriminant analysis meta-algorithm iterative stepwise discriminant analysis: a meta-algorithm for detecting quantitative sequence motifs neural models for predicting viral vaccine targets building a meta-predictor for mhc class ii-binding peptides a probabilistic meta-predictor for the mhc class ii binding peptides a meta-predictor for mhc class ii binding peptides based on naive bayesian approach strength in numbers: achieving greater accuracy in mhc-i binding prediction by combining the results from multiple prediction tools a systematic assessment of mhc class ii peptide binding predictions and evaluation of a consensus approach combination of fingerprint-based similarity coefficients using data fusion connectionist-based dempster-shafer evidential reasoning for data fusion from protein microarrays to diagnostic antigen discovery: a study of the pathogen francisella tularensis epijen: a server for multistep t cell epitope prediction nerve: new enhanced reverse vaccinology environment dynavacs: an integrative tool for optimized dna vaccine design vaxign: the first web-based vaccine design program for reverse vaccinology and applications for vaccine development enzymes, metabolites and fluxes key: cord- -fuv f authors: de groot, anne s.; moise, leonard; mcmurry, julie a.; martin, william title: epitope-based immunome-derived vaccines: a strategy for improved design and safety date: - - journal: clinical applications of immunomics doi: . / - - - - _ sha: doc_id: cord_uid: fuv f vaccine science has extended beyond genomics to proteomics and has come to also encompass ‘immunomics,’ the study of the universe of pathogen-derived or neoplasm-derived peptides that interface with b and t cells of the host immune system. it has been theorized that effective vaccines can be developed using the minimum essential subset of t cell and b cell epitopes that comprise the ‘immunome.’ researchers are therefore using bioinformatics sequence analysis tools, epitope-mapping tools, microarrays, and high-throughput immunology assays to discover the minimal essential components of the immunome. when these minimal components, or epitopes, are packaged with adjuvants in an appropriate delivery vehicle, the complete package comprises an epitope-based immunome-derived vaccine. such vaccines may have a significant advantage over conventional vaccines, as the careful selection of the components may diminish undesired side effects such as have been observed with whole pathogen and protein subunit vaccines. this chapter will review the pre-clinical and anticipated clinical development of computer-driven vaccine design and the validation of epitope-based immunome-derived vaccines in animal models; it will also include an overview of heterologous immunity and other emerging issues that will need to be addressed by vaccines of all types in the future. the availability of immunome-mining tools has fueled the design and development of vaccines by a process that has come to be termed 'reverse vaccinology,' 'vaccinomics,' 'immunome-derived vaccine' (idv) design, or 'genome-derived vaccine' design (rappuoli and covacci ; petrovsky and brusic ; pederson ; de groot and martin ; doytchinova, taylor and flower ) . this vaccine concept is based on the identification of a minimal set of antigens that induce a competent immune response to a pathogen or neoplasm. recognition of antigens occurs through the presentation of b cell and t cell epitopes derived from the antigen, in the correct immunological milieu. in its minimal form, an idv would contain only adjuvanated b cell and t cell epitopes in delivery vehicles such as liposomes. when these minimal components are packaged in an appropriate delivery vehicle, the complete package comprises an idv. compared to traditional vaccines, idvs have the potential to be safer and more effective since the vaccine focuses the protective immune response on the most essential antigenic elements of the pathogen/neoplasm. a number of idvs have been tested in clinical trials (elliott ; gahery et al. ; asj¨o et al. ; kran et al. ) . because epitope-based idvs are generally considered to be safe, when compared to other vectored or attenuated live vaccines, many have progressed rapidly from pre-clinical concept into clinical trials. in the cancer vaccine field, where epitope-based vaccines are well-established, many such vaccines are currently in phase i/ii clinical trials (pietersz, pouniotis and apostolopoulos ) . this chapter will review the development and validation of idvs; it will also provide a step-by-step guide to develop idv using validated immunoinformatics tools. these tools have the potential for dramatically accelerating the development of new and improved vaccines for the emerging and existing infectious diseases. whole-antigen-based idvs will be covered briefly; however, the main focus will be on epitope-based idvs and a description of the immunoinformatics tools that have been developed to accelerate the pre-clinical phase of vaccine discovery. so as to illustrate the process of pre-clinical vaccine development using these tools, two epitope-based idvs case studies will be presented: (i) a genome-derived vaccine for tularemia and (ii) an epitope-based hpv vaccine for adjunctive treatment of cancer. application of molecular biology techniques led to the sequencing of genomes and improved definition of the proteome (expressed proteins). even though the fundamental concept of the 'immunome' (the subset of fragments of expressed proteins that interface with the host immune system) is relatively well accepted, many important questions remain. due to genetic variation and poorly understood determinants of antigen processing, it has become clear that immunomes can vary substantially from one host to the next, even when major histocompatibility complex (mhc) and antibody germline genes are shared. the extent of the overlap between immunomes (in different hosts) and the general size of the immunome-representing epitopes derived from a particular pathogen or cancer remain to be determined. recently published studies are beginning to address these questions, partly due to the availability of algorithms that facilitate the identification of epitopes from whole genomes. the size of the immunome has been puzzling vaccinologists for decades. certainly, there are examples in the literature that 'a single epitope protects.' for example, crowe et al. ( ) recently found that immunization with a single th epitope provided a one-log reduction in influenza viral titers early in infection. a single epitope has also been shown to protect against viral disease in woodchucks (menne et al. ) , and multiple single epitopes have protected mice against an array of pathogens (an and whitton and olsen et al. ) . a more diverse set of t cell epitopes appear to be critical to immune response to vaccinia (in mice): class i mhc epitopes were shown to contribute to the large majority of the cd + t cell immune response (moutaftsi et al. ) . remarkably, it was found that of these predicted epitopes (derived from more than , candidates) represented over % of the vaccinia-specific cd t cell repertoire. how this number might be extrapolated to humans is unknown, but the result is relevant because epitopes is few enough to be easily packaged and delivered in a vaccine. an epitope-based idv for genetically diverse populations of humans will almost certainly require more than that number of epitopes, particularly if the vaccine is intended to protect against complex bacteria or viruses, or against solid tumors presenting variable antigenic profiles. harnessing the power of immunoinformatics accelerates the tasks of defining the immunome and of identifying and developing new vaccines for human diseases. the task of developing an epitope-based idv can be deconstructed into a series of achievable steps. after selecting a target organism, the next step in the immunome-to-vaccine process is to identify, from within the target genome, a set of potentially antigenic genes/proteins. these sequences can then be screened using a variety of in silico, in vitro, and in vivo mechanisms or a combination of methods. in the case of small viral genomes, it may be possible to include the entire genome in the search universe. in the case of larger bacterial and viral pathogens, the traditional vaccine targets include surface proteins and secreted proteins (which can easily be found using in silico screening programs), toxins (which can be identified through homology matching), and virulence factors (which can be identified through the use of comparative genomics). however, many other types of proteins may also be worthy of consideration: in particular, proteins expressed in high amounts (such as viral capsid proteins), proteins overexpressed during growth or replication, or proteins overexpressed in response to stress conditions. for cancer vaccines, comparisons between cancerous and normal tissue can uncover cancer-specific genes. one approach involves the use of mrna derived from cancerous and normal tissue to probe dna microarrays, followed by selection of genes that are upregulated in cancerous and not in normal tissue (mathiassen et al. ; sepkowitz ) . using 'reverse vaccinology', a term recently coined by rino rappuoli, the immune memory of subjects who have successfully encountered and defeated a pathogen can be interrogated to identify the primary targets of a natural immune response. rappuoli and colleagues identified novel vaccine targets using in silico techniques to screen the target genome for 'surface protein-like' sequences. candidate proteins were then expressed in escherichia coli and screened against human sera isolated from pathogen-exposed subjects. reactive proteins were deemed relevant to immune response (pizza et al. ) . a related approach for discovering candidate vaccine antigens involves analyzing the target pathogen's proteome in silico, using t cell epitope mapping tools. putative t cell epitopes identified can then be screened against peripheral blood mononuclear cells (pbmc) isolated from human subjects who have been infected with the target pathogen (or who have the target cancer). t cell reaction to a particular peptide epitope, typically measured by elisa or elispot assay, implies that the protein from which the peptide was derived, expressed, processed, and presented to the immune system in the course of a 'natural' immune response. using this method, measuring immune response to an epitope reveals a protein antigen. our group describes this approach as 'fishing for antigens using epitopes as bait.' other common in vitro techniques used to identify expressed or overexpressed genes/proteins include: d sds-page, (kaufmann et al. ; sonnenberg and belisle ; hernychova et al. ) , mass spectrometry (tomlinson, jameson and naylor ) , and/or tandem mass spectrometry (barnea et al. ) . in the context of vaccines against infectious diseases, it may be prudent to exclude proteins that are highly conserved across species; such proteins (including housekeeping genes) may cross-react with unrelated avirulent organisms or with self-proteins to which there may be pre-existing tolerance. however, conservation across species should not be confused with conservation within species variants. sequence conservation within species variants is a highly desirable trait for vaccine components and one that the idv approach is particularly well suited to harness. rna viruses in particular (hcv, hiv, coronaviruses) are highly variable pathogens. in these cases, selecting epitopes that are conserved across variants or subtypes may allow for the development of a more broadly applicable vaccine. alternatively, single proteins that are relatively well conserved, when compared to the balance of the target pathogen, can be selected as vaccine candidates. recently, for example, one team has developed a hexon-epitope vaccine that may be effective against a range of adenoviruses, across serotypes (leen et al. ) . once critical antigens have been identified, the next steps in epitope-based idv development are to select epitopes and confirm their immunogenicity. once the adaptive immune system has been engaged, a humoral, or antibodybased response forms the first line of defense against most viral and bacterial pathogens. antibodies recognize b cell epitopes composed of either linear peptide sequences or conformational determinants, which are present only in the three-dimensional form of the antigen. several b cell epitope prediction tools, such as dex and cep, have been proposed and are in the process of being refined (enshell-seijffers et al. ; schreiber et al. ; kulkarni et al. ) . unfortunately, the computational resources and modeling complexity required to predict b cell epitopes are enormous. this complexity is due in part to the inherent flexibility in the complementarity determining regions (cdr) of the antibody and in part due to glycosylation, and other post-translational modifications can result in modification of b cell epitopes. although accurate b cell epitope mapping tools remain elusive, the selection of potent b cell antigens can be accelerated using t cell epitope mapping tools. when considering b cell antigens as potential subunit vaccines, it may be important to also consider their t cell epitope content since the quality and kinetics of the antibody response is dependent upon the presence of t help. b cell antigens, which contain a significant t help, may outperform b cell antigens lacking cognate help. and in some cases, an identified t cell epitope may contain a b cell epitope. although different epitopes activate t and b cells, it has been widely reported that b cell epitopes have been shown to co-localize near, or overlap, class ii (th, cd +) epitopes (graham et al. ; rajnavolgyi et al. ). the adaptive immune system's second line of defense is the t lymphocyte. class i-restricted cytotoxic t cells (cd + ctl) directly engage and attack infected host cells. class ii-restricted t helper cells mediate the growth and differentiation of both t effector cells and antibody-producing b lymphocytes. both class i and class ii t cells carry out their roles in response to t cell epitopes, small linear fragments derived from protein antigens, displayed on the surface of antigen-presenting cells (apc) by various alleles of mhc. while b cells and antibodies generally recognize epitopes on surface proteins only, t cells recognize epitopes derived from a variety of proteins. once taken up by apc, antigenic proteins are broken down by digestive enzymes. during this process very large numbers of peptide fragments are released. any one of these fragments could be a t cell epitope, but only about % of all the fragments generated can implant themselves in the binding groove of the mhc molecule and be presented on the surface of the apc. one of the critical determinants of t cell epitope immunogenicity is the strength of epitope binding to mhc molecules (lazarski et al. ) . peptides binding with higher affinity are more likely to be selected by mhc molecules and to be displayed on the cell surface where they can be recognized by t lymphocytes. using a variety of methods including frequency analysis, support vector machines, hidden markov models, and neural networks, researchers have developed highly accurate tools for modeling the mhc-peptide interface and for accurately predicting t cell epitopes. for a review of t cell epitope mapping tools, see de groot and berzofsky ( ) and the accompanying issue of methods. what all these tools have in common is an ability to quickly screen large volumes of genomic sequences for putative epitopes; this preliminary screen reduces the search space dramatically, typically by at least -fold. the ability to accurately predict t cell epitopes from raw genomic data is fundamental to the development of an idv. however, even a highly accurate prediction is still only a prediction. before including predicted epitopes in a candidate vaccine, it is important to validate their immunogenicity in vitro and in vivo. once identified, peptides representing the selected epitopes are then synthesized. hla binding assays can be used to assess whether peptides derived from immunoinformatics analysis can bind to either mhc class i or class ii by measuring the affinities of predicted epitope sequences for the hla alleles in vitro. in vitro evaluation of mhc binding can be performed by quantifying the ability of exogenously added peptides to compete with a fluorescently labeled known mhc ligand (steere et al. ) and can be adapted for high throughput (mcmurry et al. a) . epivax routinely uses these high-throughput hla binding assays to confirm epitope predictions in vitro. a concordance between hla binding and immunogenicity is often observed (mcmurry et al. ). peptides are used to measure t cell responses in vitro; they can be of variable lengths ( - ). peptides presented in the context of class i mhc are generally limited to or amino acids in length, although some processing is believed to occur during the t cell assay and so -mers are also used for class i assays. in contrast with class i epitopes, which are short and fit tightly in the bounded mhc molecule, class ii (t helper) epitopes lie within an open-ended groove in the mhc ii. as such, a class ii epitope can shift within the groove, thereby accommodating mhc of various haplotypes. the only limit on the size of the peptide is its ability to remain in a linear conformation in the open-ended groove. both mhc class i-and mhc class ii-restricted epitopes (targeting cd + and cd + t cells, respectively) are believed to be important for the development of effective vaccines. cd + t helper cells enhance and amplify cytotoxic t cell (ctl) immune responses and have been shown to be important in the development of cd + t cell memory to a range of pathogens (ahlers et al. ) . ctls generally play a role in the containment of viral and bacterial infection (plotnicky et al. ) , and the prevalence of ctls usually correlates with the rate of pathogen clearance. like peptides, whole antigens too can be used to measure t cell responses in vitro. the recognition of these antigens requires the presence of an apc that is capable of processing and presenting peptides derived from the antigen. if blood from exposed individuals is available, the peptides validated as mhc ligands in binding assays can be tested for their reactivity with t cells, serum, or both. a positive immune response (as measured by elisa, elispot, or intracellular cytokine staining) should be interpreted as a sign that the parent protein interfaces with host immune response in the course of natural infection or disease. following confirmation, the peptides that stimulate a response can be considered vaccine candidates themselves or can be used to select the entire protein for use in a subunit vaccine. these candidates can then be incorporated into a vaccine delivery vehicle with an appropriate adjuvant. elisa and elispot are related methods for detecting t cell responses by the measurement of cytokines secreted by the t cells (gamma interferon, il- , and il- are examples). the expansion (proliferation) of t cells in response to stimulation by peptide:mhc can be measured by ( ) the dilution of a fluorescent dye in subsequent generations of cells (cfse) and ( ) the incorporation of a radioactive label in the proliferating cell's dna (tritiated thymidine incorporation assay). fluorescence activated cell sorting (facs) and intracellular cytokine staining (ics) are the most precise methodologies available for measuring and defining t cell response. for example, t cells that respond to a particular epitope can be directly labeled using tetramers (comprising mhc class ii: peptide complexes). labeled cells can then be sorted and counted, and the phenotype of t cells that respond to the antigen can be determined using cell surface markers and ics (tobery et al. ). factors extrinsic to processing, such as the cytokine milieu induced in response to a particular component of a vaccine (krieg et al. ) or pathogen (ghosh et al. ) , also play a role in the conditioning of the immune response. thus, t cell epitopes may be necessary to drive immune response, but are not sufficient. co-stimulatory molecules that provide a second signal, the right cytokine milieu and other factors directing the nature (th vs. th ) of the immune response, are also crucial (shahinian et al. ; kuchroo et al. ) . adjuvants provide this added 'boost' in the context of vaccines. the same range of delivery vehicles that exist for conventional vaccines can be used for the development of idvs and epitope-based idvs. for example, idvs and epitope-based idvs can be formulated and delivered as pseudo-proteins or peptides in a carrier vehicle such as a liposome or viral-like protein (vlp); alternatively, the sequence of the idv antigens or epitope string can be inserted into a viral or bacterial vector such as adenovirus or salmonella; alternatively, a dna vaccine construct encoding the antigen(s) or epitopes can be developed. the choice of adjuvants for use in humans is relatively extensive and each adjuvant has advantages and disadvantages. the advantages and disadvantages of each type of vaccine delivery vehicle and adjuvant here is beyond the scope of this chapter; readers are referred to a review by fraser et al. ( ) . the next step in the development of epitope-driven idv is to determine whether immunization provides competent immune response. the idv or epitope-based idv is administered and immune responses to the components are evaluated following immunization. even though a range of animal models are used for the evaluation of vaccines, results from immunogenicity studies in these models should be interpreted with caution. although their functions may be similar, the mhc of mice, rodents, and non-human primates differ from human mhc (known as hla in the context of human immune response) at the amino acid level and these differences effect which epitopes can be presented. this helps to explain why different strains of mice (balb/c, c bl/ ) have different immune responses to pathogens as well as vaccines for those pathogens (klitgaard et al. ) . in particular, epitope-based vaccines that are developed using predicted human t cell epitope mapping tools can be tested only in murine models that are hla transgenic. fortunately, a number of transgenic mouse strains that express the most common hla a, hla b and hla dr, molecules have been developed. t cell responses in these mice correlate directly with t cell responses observed in infected/vaccinated humans (man et al. ; shirai et al. ) . hla transgenic mice are now routinely used to assay and optimize (human) epitopedriven vaccines in pre-clinical studies (ishioka et al. ; charo et al. ; livingston et al. ) . despite the limited number of hla class ii alleles for which tg mice have been developed, comparisons of immunogenicity can be done to a high degree of accuracy in the mouse model for selected hla class ii alleles (hla dr , , , ). unfortunately, it appears these mice may have difficulty breeding due to poorly understood consequences of their transgenic heritage, limiting the use of this important model system. the final step in the development of any vaccine is experimental validation of the immunogenicity and protective efficacy of computationally selected antigens. currently, a series of experimental vaccines have shown efficacy in animal models and several idvs are being tested in clinical studies. in the context of infectious disease, genome-derived vaccines that have progressed furthest along the vaccine development pipeline are generally based on whole proteins rather than epitopes (rappuoli and covacci ; pizza et al. ) . however, epitope-based idvs are currently being developed for a range of infectious diseases by the authors' laboratory and by many others (depla et al. ). while it is common knowledge that subunit-based vaccines can protect against infection, similar success with epitope-based approaches is not as widely known. in addition to the studies previously cited, immunization of balb/c mice with three doses of a peptide construct containing an h- (d)-restricted cytotoxic t lymphocyte (ctl) epitope from a murine malaria parasite induced both t cell proliferation and a peptide-specific ctl response mediating nitricoxide-dependent elimination of malaria-infected hepatocytes in vitro, as well as partial protection of balb/c mice against sporozoite challenge (franke et al. ) . in a separate study, immunization of balb/c and cba mice with measles virus ctl epitopes resulted in the induction of epitope-specific ctl responses and conferred some protection against encephalitis following intracerebral challenge with a lethal dose of virus (schadeck et al. ) . these are just a few successful examples of many studies carried out in animal models; however, translation to prevention of disease in humans has been difficult to achieve. in contrast with whole-protein subunit vaccines, idvs and epitope-based idvs have taken longer to make the transition from animal model to the clinic, mainly due to the novelty of the concept and perhaps unfounded concerns that epitopes are not sufficient for the generation of effective immune response. cancer therapy is an exception to this rule. as previously described, eptiopebased idvs have been evaluated in the context of therapy against chronic infection or cancer (ueda et al. ; valmori et al. ) . in the cancer vaccine field, where the concept of epitope-driven vaccines is well established, many more peptide vaccines have successfully passed pre-clinical tests and are currently in phase i/ii clinical trials (pietersz, pouniotis and apostolopoulos ) . new approaches are emerging, which may improve the success rate and, indeed, the results from recent clinical trials prove the principle. one approach is to identify epitopes that are unique to the tumor (prostate, lung, colon) and to pre-screen the patient for response to the peptide. this approach, called personalized vaccination, takes into account the diversity of ctl epitope recognition among patients. whereas the response rates to classical (non-personalized) peptide vaccines have been disappointing, responses to personalized vaccines (in a phase i trial, conducted in japan) have been as high as . % in the advanced cancers and equal to or more than % in malignant glioma and cervical cancers, respectively (itoh and yamada ) . it is noteworthy that just a few epitope-driven vaccines against viral and microbial pathogens have reached the stage of phase i or ii efficacy trials in humans. for example, bionor immuno's hiv p gag peptide vaccine (vacc- x) was demonstrated to be safe and well tolerated in phase i trials (asj¨o et al. ) and dose-dependent and immunogenic in phase ii trials in norway (kran et al. ) . similarly, nardin's epitope-based vaccine for malaria is moving along the clinical trial pathway (nardin et al. ) . in this section, we describe the immunomics tools developed and used by the epivax vaccine development group in recent collaborations with dr. steve gregory of lifespan, dr. ousmane koita of the university of bamako, mali and dr. david weiner of university of pennsylvania. t cell epitopes are linear peptides that bind to mhc molecules. binding is mediated by the interactions between the r-groups of the amino acids in the peptide ligand and the pockets on the floor of the mhc binding groove. because the mhc:peptide interaction is well characterized, pattern-matching algorithms can be used to screen protein sequences for peptides that will bind mhc. the authors of this report currently use the epimatrix system, a suite of epitope-mapping tools that has been validated by more than a decade of use in selecting putative epitopes for in vitro and in vivo studies (see references (de groot et al. ; bond et al. ; dong et al. ; mcmurry et al. ; koita et al. ) . the epimatrix algorithm is based on a set of class i and class ii hla matrices wherein individual frequencies of all amino acids (aa) in each hla pocket position are applied to the prediction of overlapping -and -mer peptides. in a typical analysis, protein antigens are parsed into overlapping -mer frames where each -mer overlaps the last by eight amino acids. each -mer is then scored for predicted binding affinity to one or more class i or class ii hla alleles. in order to compare potential epitopes across multiple hla alleles, epimatrix raw scores are converted to a normalized 'z' scale. peptides scoring above . on the epimatrix 'z' scale (typically the top % of any given sample) are likely to be mhc ligands. since class ii epitopes can be promiscuous, our approach to the prediction of class ii epitopes is to estimate the binding potential of each frame with respect to each of a panel of eight common class ii alleles (drb * , * , * , * , * , * , * , and * ). taken together, these alleles 'cover' the genetic backgrounds of most humans worldwide (southwood et al. ) and they also represent the predominant types of 'pockets' for the most common mhc. recently, the epimatrix system has been utilized to measure the potential immunogenicity of whole proteins. in this context, epimatrix assesses the aggregate epitope density of a given protein with respect to the aggregate epitope density of a set of randomly generated pseudo-protein sequences of similar size (de groot ) . by correcting for the size and expected epitope density, the potential immunogenicity candidate vaccine antigens can be directly compared. further immunogenicity of low scoring proteins may be enhanced by modifying the immunogenic region amino acid sequence so that it contains more t cell epitopes (see illustration of this approach in the hpv vaccine section, below). peptides predicted to bind to multiple hla alleles are known as promiscuous t cell epitopes. the clustimer algorithm is used to scan the output produced by the epimatrix and identifies the polypeptides predicted to bind to an unusually large number of hla alleles. briefly, the scores of each analyzed -mer are aggregated. high-scoring -mers are then extended at the n-and c-terminal flanks until the predicted epitope density of the promiscuous epitope falls below a given threshold value. this particular approach to mapping epitopes has also been useful for discovering 'epibars,' which may be a signature feature of highly immunogenic, promiscuous class ii epitopes. an example of a promiscuous t cell cluster containing an epibar (tetanus toxin - ) is shown in fig. . a single t cell epitope 'cluster' usually ranges from to about amino acids in length and can contain anywhere from to binding motifs. using epimatrix as described above and clustimer, scores above and, in particular, scores above indicate significant immunogenic potential (de groot ) . note the horizontal bar of high z scores at position in fig. . having observed this 'epibar' pattern to be characteristic of promiscuous epitopes, the authors have integrated the pattern into the prospective selection of clusters. promiscuous epitopes also exist, to a certain degree, for class i alleles. some laboratories have demonstrated cross-presentation of peptides within hla 'superfamilies' (such as the a superfamily: a , a , a , a , and a ) described by . the authors have confirmed cross-mhc binding and presentation to t cells in our hiv vaccine studies ). one limitation of conventional vaccination, and to a lesser extent natural infection, is that the immune system focuses strongly on the most mutable immunogen of the virus -typically, the viral envelope. in the case of hiv and other viruses, vaccination with more conserved, subdominant epitopes has been shown to circumvent this hierarchy and potentiate cross-strain protection (ostrowski et al. ; nara and lin ) . in like manner, a conserved t helper-directed vaccine may provide a more 'democratic' way of stimulating immune response, increasing the number targets for t cell recognition, thereby providing t help to antibody response despite potential viral variability (santra et al. ; subbramanian et al. ; scherle and gerhard ; scherle and gerhard ; russell and liew ; johansson et al ) . the genetic variability of some pathogens constitutes a significant challenge to the efforts to design a vaccine driven by cellular immune response de groot et al. ) . the authors have been involved in developing an hiv- vaccine that includes highly conserved (cross-clade) t cell epitopes. the conservatrix algorithm, developed for this application, parses input sequences into component strings (the lengths of the strings may be determined ) indicates the potential of a -mer frame to bind to a given hla allele. all z scores in the top % (> . ) are considered 'hits'. though not hits, scores in the top % are considered elevated; scores below % are masked for simplicity. frames containing four or more alleles scoring above . are colloquially referred to as 'epibars' (see frames :yikanskfi and : ikanskfig). this band-like pattern is characteristic of promiscuous epitopes. the tetanus toxin peptide scores are extremely high for all eight alleles in epimatrix; the deviation compared to expectation is + . by the operator) and then searches the input dataset for matching segments. conservatrix may be used to compare strings derived from different strains of the same organism (hepatitis c, for example, or hiv) or to search a given sequence for a user-supplied target sequence. target sequences may be input as specific sequences or as coded patterns. thus, the operator can use 'wild cards', allowing for one or more of the amino acid residues in any given peptide sequence to be any amino acid [x], or a limited set of amino acids such as [l, v] . results of each analysis are stored in a database and may be browsed or exported to another program for analysis. by selecting highly conserved epitopes, regardless of their distance from the ancestral hiv- genome, we have identified sequences conserved for structural and functional reasons and are therefore less likely to be modified in the course of further evolution of hiv- (peyerl et al. ; koibuchi et al. ). the problem of virus variability also significantly complicates the selection of epitopes that have a population-coverage advantage; such epitopes are termed 'clustered,' 'superfamily,' or 'promiscuous.' to address this problem, the authors developed epiassembler ) to identify sets of overlapping, conserved, and promiscuously immunogenic epitopes and assemble them into extended immunogenic consensus sequences (ics) (see fig. ). in theory, proper processing and presentation of these sequences would allow for the presentation of highly conserved peptides in the context of more than one mhc. the resulting peptide is not a 'pseudosequence' as such, since each constituent epitope occurs in its corresponding position in the native protein. thus, while the full-length 'immunogenic consensus sequence' is not necessarily found in any one variant sequence, the peptide is more representative of the sequence universe. in the case of hiv, for example, we used the ics approach to design a peptide-based vaccine. while the full-composite ics peptides happen to be exactly conserved in a few individual strains of hiv, each peptide represents a significant percentage of circulating strains, because every constituent overlapping epitope is conserved in a large number (range to , ) of individual hiv- strains. as compared with immunogenic consensus sequences, randomly selected counterparts, on average, contain half as many binding motifs and cover a third fewer isolates. to develop vaccines of equivalent antigenic 'payload,' using conventional methods would be prohibitively expensive as it would require including multiple different variants of each antigen. this approach has been useful for identifying highly immunogenic epitopes for hiv vaccine design . by focusing on conserved, mhc-promiscuous t helper epitopes, the ics approach has the potential to efficiently overcome the genetic variability of both virus and host. one of the advantages of idv is that it is possible to omit deleteriously crossreactive epitopes. perhaps, the most famous example of an adverse effect due to cross-reactivity with self was observed following vaccination for lyme disease with the osp a protein. the vaccine has been recently re-engineered with the cross-reactive epitope removed (willett et al. ). in the context of our own work, peptides selected for in vitro evaluation are evaluated for homology with human proteins by blasting the sequences against the human sequence database at genbank (http://www.ncbi.nlm.nih.gov/). blastimer automates the process of submitting sequences to the websites featuring search engines such as the blast engine at ncbi (www.ncbi.nlm.nih. gov/blast). by default, blastimer blasts sequences against all non-redundant x x x x x x fig. the ics assembly operation was performed using epiassembler (bill martin, epivax, ) . left panel: each variant strain is first analyzed and a highly conserved, putatively promiscuous -mer is chosen as the core peptide. mismatches with the selected epitope sequences are represented with the letter x. right panel: additional epitopes are then identified, which overlap with the natural n-and c-terminal flanking regions of the core -mer epitope. the overlap length requirement is decreased by one amino acid iteratively until reaching a minimum of three overlapping amino acids. if more than one suitable overlap is identified, the overlapping peptide with the higher overall epimatrix rank is selected. this process is repeated using the extended peptide as the new core sequence. the cycle can be repeated for the length of an entire protein or can be truncated when the peptide reaches a length that can be easily produced synthetically genbank cds translations, pdb, swissprot, pir, and prf. blastimer assesses the homology between the submitted sequence and the sequence of proteins of other organisms. patent blast, on the other hand, targets a database of sequences gleaned from patents. users of either program may control all of the submission options available to interactive users at ncbi. in both cases, results are recorded in a database and can be browsed, exported, or summarized and rendered in a report format. according to the authors' standard practice, any peptide that shares greater than % identity with peptides contained in the human proteome is eliminated from consideration in a vaccine. a number of methods for enhancing epitope-based vaccines have been described and implemented (thomson et al. ; rodriguez and whitton ) . one approach is to align the epitopes in a protein or dna vaccine construct as a 'string of beads' without any intervening sequences or 'spacers' in a dna plasmid encoding the individual epitopes . however, the lack of 'natural flanking sequences' -has raised concern that their proteolytic processing may be compromised, and that junctional epitopes, peptides other that the specific peptides of interest, may be generated as a result of processing (godkin et al. ) . to address this concern, the authors developed vaccine-cad (see fig. fig. vaccine-cad, illustrated with three sample epitopes represented by the words 'create,' 'new,' and 'epitopes.' the default arrangement of the words results in unintended sequences, represented by the words 'eaten' and 'ewe,' at the junctions between the intended epitopes. reiterative modifications in the arrangement of the epitopes results in the development of a sequence that has no 'pseudoepitopes' (new epitopes that were not intended) at the junctions of the juxtaposed epitopes junctional epitopes, the insertion of spacers and breakers, the requirements for secretion or processing tags, and the evaluation of epitope strings for potential homologies to human protein fragments. an important component in the epitope-driven vaccine process is the selection of epitopes from the regions of pathogens that are presented by mhc molecules for t cell recognition. 'mhc binding motifs,' first identified by r¨otzschke and falk, are the patterns of amino acids in peptides that are known to promote the binding of peptides containing these patterns to the mhc molecules on the surface of apcs rotzschke et al. ) . different mhc molecules have different binding motifs, limiting the set of mhc ligands that can be presented in the context of any given mhc. while consideration of hla alleles may lead to concern about the selection of epitopes for broad coverage of populations, gulukota and delisi ( ) and sette and sidney ( ) have demonstrated that epitope-based vaccines that contain epitopes restricted by selected 'supertype' hla can provide the broadest possible coverage of the human population. furthermore, recent studies by brander and walker indicate that there may be even greater flexibility in the binding of epitopes to mhc than previously recognized; this is also consistent with the data recently presented by frahm et al. ( ) . the inclusion of 'promiscuous epitopes' -epitopes that are recognized in the context of more than one mhc (paina-bordignon et al. ; de groot et al. ; sette and sidney ) in epitope-driven vaccines may therefore overcome the challenge of genetic restriction of immune response. in addition, the repertoire of possible mhc-restricted epitopes recognized by an individual's t cells has been shown to be quite variable, even between hla-matched individuals (jameson, cruz and ennis ; gianfrani et al. ; betts et al. ). aggregatrix, a new algorithm that was recently developed at epivax, iteratively searches for the combination of epitopes that achieves maximal cross-clade representation. the authors performed this analysis for our hiv epitopes, as shown in fig. , evaluating each hla-a -restricted hiv peptide individually and the set in aggregate for coverage of hiv- strains by year ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) , by country of origin, and by clade. as can be seen in this figure, the set of highly conserved a -restricted peptides we have tested and confirmed in elispot assays covered between % and % of strains in a given year, between and % of strains in a given country, and between % and % of strains in a given clade. the hla a peptides cover , , , and % of clades a, b, c, and d, respectively. this is a remarkable breadth of coverage for a limited set of coverage of gaia hiv b peptides--individually and in aggregate-across time, countries, and clades fig. gaia hiv a peptides -individually and in aggregate -percentage coverage of strains by year, country, and clade. each row of the matrix denotes a specific peptide; the peptide's protein of origin is included within the peptide id. each column of the matrix denotes a specific year, country, or clade, grouped as indicated. the percentage coverage of strains is represented on a color gradient, with warm tones indicating values above % and cool tones indicating values below %. the bottom row of the matrix shows the percentage of each protein set that contains at least one peptide from our pool. black boxes indicate that no isolates of the protein are available for that year, clade, or country. the bottom row represents the aggregate percent coverage for the epitope set. each cell of the matrix represents the percentage coverage per peptide, except for the bottom row cells, which represent the aggregate percentage coverage for the peptide set. column headers are listed here for space considerations: left to right, the year columns are , , , , , , , , , , and ; aggregate coverage of strains by year ranges from % ( ) hla a epitopes, given the well-known ability of hiv to mutate away from hla (nguyen et al. ; iversen et al. ) . in studies of immune response to therapeutic proteins, the authors have observed that subject-to-subject variation in t cell response closely relates to ( ) subject hla type and ( ) the number of protein-derived peptides that match the subjects hla. to describe this relationship, the authors developed a metric that may be useful in the development of epitope-based vaccines or in the clinical assessment of immune response to vaccines, called the 'individualized t cell epitope measure' or item. the item can be calculated for each subject who is responding to a given epitope by summing the epimatrix z-scores for each positive peptide for each hla allele in a given subject's haplotype, thus: item score ¼ ½epimatrix score of peptide for hla type þ ½epimatrix score of peptide for hla type þ . . . the same calculation can be performed for larger peptides and proteins by summing all of the scores for the subject's hla type. this calculated score allows for the individualized potential immunogenicity to be predicted, based on the number of putative epitopes contained in a protein that might be presented to their t cells, based on their hla haplotype. using this score, it is possible analyze the contribution of haplotype to the corresponding t cell response. in our prospective evaluations, significant correlations were found between the ifn-gamma response to a given antigen and the item scores for individual subjects (for data published in koren et al. ( ) r= . , p= . ). in addition, correlations between the item score and patient hla were also observed for their antibody titers. further evaluations of this method for predicting individual responses to vaccines and therapeutic proteins are in progress. defining the immunome by identifying t cell epitopes and confirming their immunogenicity is but the first step of vaccine development. a number of conditions extrinsic to the mhc-ligand interaction may influence the final composition of the epitope ensemble. for example, whether or not a predicted epitope is confirmed is related to: ( ) the quantitative expression of the source protein; ( ) the number of different epitopes derived from this protein, which are presented on the surface of the apc (wherry et al. ) ; ( ) the amino acids that flank the epitope (bergmann et al. ; shastri, serwold and gonzalez ; livingston et al. ) ; and ( ) proper cleavage and trimming by proteolytic enzymes in the proteasome and processing pathways (van kaer et al. ; york et al. ; chen et al. ; toes et al. ) . for example, it is very likely that end-to-end epitope presentation, such as was used in the design of the oxavi hiv gag/epitope vaccine, impaired the presentation of the epitopes in immunogenicity studies. vaccine-cad also takes into account the role of flanking residues: studies conducted in murine models have demonstrated that residues flanking an mhc class i epitope strongly influence the delivery of the intact epitope to tap following proteasome degradation (thomson et al. ; hozhutter, frommel and kloetzel ; mo et al. ). in addition, livingston et al. ( ) have tested a standard spacer sequence (-gpgpg-) for vaccine constructs consisting of mhc-ii-restricted, th-cell epitopes; the use of this spacer disrupts junctional epitopes that might compete for degradation or for mhc binding (both g and p are unusual carboxy-terminal anchors for a peptide that binds to class ii mhc). this approach has been used for constructs with up to epitopes, in assays where responses were detected to the majority of epitopes (livingston et al. ). methods of confirming idv . two case studies francisella tularensis is a zoonotic bacterium. it is endemic to certain communities such as martha's vineyard, massachusetts, usa, where it is known as rabbit fever. tularemia represents a potentially dangerous biological weapon owing to its high degree of infectivity, ease of dissemination, and capacity to cause severe illness. despite several decades of research, no vaccine for tularemia is licensed for public use. for a review of tularemia vaccines, see mcmurry et al., b. we have been actively developing an epitope-based tularemia vaccine combining computational immunology with in vitro and in vivo validation (mcmurry et al a) . the starting point of our vaccine was the fully annotated f. tularensis subsp. tularensis (schus ) genome published in by larsson et al. ( ) . a prototype vaccine containing only class ii epitopes has been tested in challenge studies in hla drb * transgenic mice. for this vaccine, the epimatrix algorithm was utilized to identify highly promiscuous t cell epitopes within the tularemia genome. twenty-five class ii-restricted epitopes were selected, synthesized, and screened in vitro using a recombinant soluble hla class ii competition-binding assay (described above). peptides that bound with high affinity were then tested ex vivo in elispot assays with blood obtained from f. tularensis subsp. tularensis-exposed individuals. search-light analysis was also performed on supernatants derived from human cell culture stimulated with peptide using a panel of nine cytokines. forty-two percent of peptides bound to drb* and are likely to bind to several other alleles (in that they were predicted using the clustimer algorithm). elispot assays showed positive ifn-gamma responses to / individual peptides and to peptide pools in nearly all of the human study. the number of epitopes recognized per subject ranged from to and averaged per subject; not every peptide was tested for every subject. peptides that elicit a robust memory response, as evaluated by these various assays, were incorporated into a vaccine construct and tested in challenge studies with hla transgenic mice as a possible vaccine against tularemia. immunogenicity studies in hla transgenic drb * mice were performed using the multi-class ii-epitope dna constructs and/or peptides representing the epitopes, and t cell responses were evaluated in ifn-gamma elispots. hla dr transgenic mice were challenged with five times the ld of f. tularensis lvs; % of vaccinated mice survived, all non-vaccinated mice died (manuscript in preparation). this result demonstrates the potential for a genome-derived epitope-based vaccine to protection from a class a bioterror pathogen. importantly, the protection we observed is accounted for by only of the schus epitopes in the vaccine, as they were conserved in the lvs challenge strain. the schus -specific epitopes that were found to be significantly immunogenic would further contribute to protection in a schus challenge. this result is consistent with other findings that a limited set of epitopes may be sufficient to induce a protective immune response (moutaftsi et al. ) . studies using schus (the wild type tularemia) are planned. the results obtained to date appear to indicate that vaccine design that originates with the whole genome may lead to the development of a protective epitope-based vaccine. cervical cancer is the second leading cause of death afflicting women worldwide; % of cervical patients develop persistent, recurrent, or widely metastatic disease. while a preventive vaccine now exists for hpv, there is a need for a therapeutic vaccine to treat existing cases of hpv, especially in resource-poor areas where access to the preventive vaccine is limited. cellular immune responses are believed to be critical for effective immune response to cancer; accordingly, epivax is pursuing the development of an 'immunotherapeutic vaccine' which would focus on the proteins primarily expressed either before or during carcinogenesis (e and e , e , and e ). in order to maximize immunogenicity across hpv subtypes, our strategy involves analyzing variant strains of hpv protein sequences, using ( ) epimatrix to identify class i and class ii hla motif matches, ( ) conservatrix to identify those motif matches that are conserved, and ( ) epi-assembler to weave together the conserved immunogenic sequences into a full immunogenic consensus sequence (ics) antigen. a full ics vaccine antigen would retain the fundamental structure of its naturally occurring counterparts; however, it will contain more and better epitopes than would occur in any such one counterpart. the authors have identified five conserved epitopes in e and e , which have stimulated significant responses in elispot ifn-g assays. in the proposed vaccine, these epitopes and others will be incorporated in their natural context within the proteins, which could be delivered as dna, proteins, or a primeboost combination. by preserving the natural flanking regions surrounding our epitopes, we hope to retain, in large part, the natural processes surrounding hpv protein degradation, transport, and presentation as they occur during natural infections. the ics approach described here is the same as that illustrated in fig. , but extending over the full natural length of the protein. for a more in-depth review of a similar approach being pursued for influenza, see mcmurry et al. ( ) . the epivax hpv vaccine illustrates yet another aspect of vaccine design: 'megatope' proteins, re-engineered to increase the epitope content. this approach already had some success (okazaki et al. ) . in the case of variable viruses such as hcv, influenza, and hiv, one limitation of conventional vaccination, and of natural infection, is that the immune system often focuses strongly on the most mutable immunogens. idvs can be constructed from alternative antigens, which are more conserved or more protective, circumventing this problem (russell and liew ; scherle and gerhard ; scherle and gerhard ; santra et al. ; subbramanian et al. ) . amino-acid sequences of ns proteins from available hpv isolates identification of immunogenic and conserved t-cell epitopes using conservatrix, epimatrix. (epitopes represented by open symbols) ns protein is optimized to incorporate the most conserved immunogenic epitopes in their natural context within the protein. illustration of our novel strategy to generate an hpv vaccine candidate. the putative epitopes identified during this analysis will be combined to form several consensus sequences, which retain the fundamental structure of these hpv proteins but which also contain an unnaturally large number of conserved t cell epitopes in addition, broadening the t cell repertoire might make it possible to impair viral escape and decrease viral loads sufficiently to disrupt transmission. epitope-driven vaccines also offer distinct advantages over vaccines encoding whole protein antigens, since epitopes are safe and can be packaged into relatively small delivery vehicles. the epitope-driven approach offers platform independence: a delivery vehicle (peptide, dna, multi-epitope construct) can be modified or selected midway into the development process. multiple conserved epitopes, in addition to augmenting the efficacy of a preventive vaccine, could provide a broad and universal cellular immunity, known to be crucial for containment of infection, although perhaps ineffective for protection against infection. despite these advantages, there are a number of reasons that a given pathogen-directed, epitope-based vaccine might fail to reach clinical trials or protect humans: ( ) the limited number of epitopes expressed by the vaccine (i.e., poor payload quantity); ( ) limited conservation of epitopes (leading to limited coverage of variant clinical isolates) ( ) the limited hla population coverage (i.e., poor payload quality); ( ) suboptimal vaccine delivery; and/or ( ) the dearth of suitable animal models. in addition, the concept of epitope-driven vaccines is relatively novel. complete genome sequences have been available for only a little more than a decade now and the tools to process the data for vaccine design are only newer. experimental validation needed to push forward these vaccines into clinical trials is now emerging and promises to enable epitope-based vaccines to claim a prominent place in the vaccine world. the technologies needed to identify immunostimulatory antigens and epitopes from pathogen genomes are already well developed. the principle focus of future research in this area will likely be in fine-tuning these technologies and expanding them to tailor immune responses in individuals. for example, development of epitope mapping algorithms for dq and dp class ii hla alleles will make it possible to completely characterize immunomes. this information will make it possible to generate comprehensive individual t cell epitope measures (item) based on an individual's hla genetic make-up and allow researchers to identify a priori clinically important epitopes and screen clinical cohorts for subjects that are more likely to develop targeted immune responses. furthermore, genome-mapping tools that are currently available are not yet useful for discovering b cell epitopes, whether from proteins or from nonprotein components such as carbohydrates or lipid antigens. immunoinformatics tools that are currently available cannot be used to accurately predict conformational (b cell) epitopes that interact with antibody, although such tools are being refined (enshell-seijffers et al. ) . thus, the immunogens identified using in silico approaches must be evaluated in vitro and also in appropriate challenge models, prior to progressing to vaccine trials. protective immune response probably also involves some engagement of the innate immune system; it has been impossible to differentiate between effective and non-protective epitopes. cytokine milieu may affect the outcome of immunization; thus, a limited number of toll-receptor agonists (imler and hoffmann ) have been identified and these are under study in conjunction with idvs. in the future, toll-receptor signaling 'pathogen-associated molecular patterns' (pamps) might also be modeled and selected using immunoinformatics tools. besides antigen identification, the success of idvs relies heavily on delivery technologies. these areas continue to independently mature and provide important lessons to epitope-based vaccine design. the major areas of research to watch include biological macromolecule (including cytokines), lipopeptide, and polysaccharide adjuvants and particulate (liposomes, exosomes, virosomes, nanoparticles) and cell-based delivery systems. the development of safe and effective vaccines against emerging infectious diseases such as influenza, both seasonal and pandemic, hiv, and tb, in addition to cancers associated with infectious pathogens such as hbv and hcv, is an urgent and achievable public health priority. in addition, vaccines for the prevention and treatment of cancer hold enormous promise for human health. the threat of bioterrorism following the events of september , , provided vaccinologists with a persuasive argument for more rapid development of vaccines against viral and bacterial pathogens that are now included on the nih category a-c biopathogen list (http://www .niaid.nih.gov/topics/ biodefenserelated/biodefense/research/cata.htm). 'emerging infectious diseases' were added to the vaccine wish list following the outbreak of severe acute respiratory syndrome (sars) in guangdong china in . indeed, only a few months following the publication of the sars-coronavirus (sars-cov) genome (marra et al. ; rota et al. ) , researchers began to map vaccine components using new bioinformatics and immunoinformatics tools, coupled with improved immunology techniques and specialized animal models. new vaccines based on this approach are currently being evaluated in animal models, less than a year from the start of the epidemic. future vaccine approaches may need to move away from 'whole' protein vaccines for a wide range of reasons. multiple antigen or epitope vaccinations such as the approach illustrated here could be one way to elicit the sort of strong th response necessary to pathogens following infection, in the context of a therapeutic vaccine. this approach could also be useful for a wide range of pathogens for which genomes have been partially or completely mapped. as described in this chapter, our group is actively pursuing the development of epitope-driven vaccines for hiv koita et al. ) , franciscella tularensis, helicobacter pylori, and smallpox. we have progressed from genome-derived epitope mapping to challenge studies in less than one year for some of these vaccine development programs. epitope-based and whole antigen idvs are now just beginning to enter clinical trials, but this relative disadvantage may be cured with the tincture of time. one reason for the relative paucity of idvs in clinical development is that the immunoinformatics tools for developing these vaccines have really only evolved in the last to years. the average length of time to develop a vaccine may be years or more. while immunoinformatics tools are useful for accelerating the discovery and pre-clinical stage of vaccine development, testing vaccines in animal models and developing clinical trials is a lengthy process. it is likely that idv and epitope-based idv will begin to enter clinical trials and emerge on the market in greater numbers in to years. high-affinity t helper epitope induces complementary helper and apc polarization, increased ctl, and protection against viral infection quantitative and qualitative analyses of the immune responses induced by a multivalent minigene dna vaccine a multivalent minigene vaccine, containing b-cell, cytotoxic t-lymphocyte, and th epitopes from several microbes, induces appropriate responses in vivo and confers protection against more than one pathogen phase i trial of a therapeutic hiv type vaccine, vacc- x, in hiv type -infected individuals with or without antiretroviral therapy analysis of endogenous peptides bound by soluble mhc class i molecules: a novel approach for identifying tumor-specific antigens differential effects of flanking residues on presentation of epitopes from chimeric peptides analysis of total human immunodeficiency virus (hiv)-specific cd (+) and cd (+) t cell responses: relationship to viral load in untreated hiv infection an hla-directed molecular and bioinformatics approach identifies new hla-a hiv- subtype e cytotoxic t lymphocyte epitopes in hiv- -infected thais dna immunization of hla transgenic mice with a plasmid expressing mycobacterial heat shock protein results in hla class i-and ii-restricted t cell responses that can be augmented by cytokines immunoproteasomes shape immunodominance hierarchies of antiviral cd (+) t cells at the levels of t cell repertoire and presentation of viral antigens uneven distribution of mhc class ii epitopes within the influenza virus immunomics: discovering new targets for vaccine and therapeutics from genome to vaccine -new immunoinformatics tools for vaccine design from immunome to vaccine: epitope mapping and vaccine design tools engineering immunogenic consensus t helper epitopes for a cross-clade hiv vaccine from genome to vaccine: in silico predictions, ex vivo verification human immunodeficiency virus reverse transcriptase t helper epitopes identified in mice and humans: correlation with a cytotoxic t cell epitope an interactive web site providing major histocompatibility ligand predictions: application to hiv research hiv vaccine development by computer assisted design: the gaia vaccine immunoinformatics: mining genomes for vaccine components rational design of a multiepitope vaccine encoding t-lymphocyte epitopes for treatment of chronic hepatitis b virus infections hla-a -restricted cd +-cytotoxic-t cell responses to novel epitopes in mycobacterium tuberculosis superoxide dismutase, alanine dehydrogenase, and glutamine synthetase proteomics in vaccinology and immunobiology: an informatics perspective of the immunone phase i trial of a cd + t cell peptide epitope-based vaccine for infectious mononucleosis the mapping and reconstitution of a conformational discontinuous b-cell epitope of hiv- allele-specific motifs revealed by sequencing of self-peptides eluted from mhc molecules consistent cytotoxic-t-lymphocyte targeting of immunodominant regions in human immunodeficiency virus across multiple ethnicities a subdominant cd (+) cytotoxic t lymphocyte (ctl) epitope from the plasmodium yoelii circumsporozoite protein induces ctls that eliminate infected hepatocytes from culture improving vaccines by incorporating immunological coadjuvants new cd + and cd + t cell responses induced in chronically hiv type- -infected patients after immunizations with an hiv type lipopeptide vaccine lipoarabinomannan induced cytotoxic effects in human mononuclear cells human memory ctl response specific for influenza a virus is broad and multispecific naturally processed hla class ii peptides reveal highly conserved immunogenic flanking region sequence preferences that reflect antigen processing rather than peptide-mhc interactions the structural requirements for class ii (i-ad)-restricted t cell recognition of influenza 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of recombinant polyclonal antibodies to inhibitory anti-variable domain antibody responses limited sequence evolution within persistently targeted cd epitopes in chronic human immunodeficiency virus type infection confirmation of immunogenic consensus sequence hiv- t cell epitopes in bamako clinical validation of the ''in silico'' prediction of immunogenicity of a human recombinant therapeutic protein hla-and dose-dependent immunogenicity of a peptide-based hiv- immunotherapy candidate (vacc- x) the role of cpg dinucleotides in dna vaccines b - and b - costimulatory molecule activate differentially the th /th developmental pathways: application to autoimmune disease therapy cep: a conformational epitope prediction server the complete genome sequence of francisella tularensis, the causative agent of tularemia the kinetic stability of mhc class ii peptide complexes is a key parameter that dictates immunodominance identification of hexon-specific cd and cd t-cell epitopes for vaccine and monotherapie optimization of epitope processing enhances immunogenicity of multiepitope dna vaccines definition of a human t-cell epitope from influenza a non-structural protein using hla-a . transgenic mice tumorassociated antigens identified by mrna expression profiling induce protective anti-tumor immunity diversity of francisella tularensis schu antigens recognized by t lymphocytes after natural infections in humans: identification of candidate epitopes for inclusion in a rationally designed tularemia vaccine a call to cellular and humoral arms: enlisting cognate t cell help to develop broad-spectrum vaccines against influenza a tularemia vaccinesan overview analyzing mycobacterium tuberculosis proteomes for candidate vaccine epitopes characterization of t-cell response to woodchuck hepatitis virus core protein and protection of woodchucks from infection by immunization with peptides containing a t-cell epitope distinct proteolytic processes generate the c and n termini of mhc class i-binding peptides a consensus epitope prediction approach identifies the breadth of murine t(cd +)-cell responses to vaccinia virus hiv- : the confounding variables of virus neutralization synthetic malaria peptide vaccine elicits high levels of antibodies in vaccines of defined hla genotypes frequent human leukocyte antigen class i alleles are associated with higher viral load among hiv type seroconverters in thailand epitope enhancement of a cd hiv epitope toward the development of the next generation hiv vaccine efficient protection against mycobacterium tuberculosis by vaccination with a single subdominant epitope from the esat- antigen identification of neutralizing and nonneutralizing epitopes in the porcine reproductive and respiratory syndrome virus gp ectodomain universally immunogenic t cell epitopes: promiscuous recognition by t cells the immunome computational immunology: the coming of age fitness costs limit viral escape from cytotoxic t lymphocytes at a structurally constrained epitope design of peptide-based vaccines for cancer identification of vaccine candidates against serogroup b meningococcus by whole-genome sequencing the immunodominant influenza matrix t cell epitope recognized in human induces influenza protection in hla-a /k(b) transgenic mice a repetitive sequence of ebstein-barr virus nuclear antigen comprises overlapping t cell epitopes which induce hla-dr restricted cd + t lymphocytes reverse vaccinology and genomics enhancing dna immunization exact prediction of natural t cell epitope t cells primed by influenza virion internal components can cooperate in the antibody response to haemagglutinin prior vaccination increases the epitopic breadth of the cytotoxic t-lymphocyte response that evolves in rhesus monkeys following a simian-human immunodeficiency virus infection ctl epitopes identified with a defective recombinant adenovirus expressing measles virus nucleoprotein and evaluation of their protective capacity in mice functional analysis of influenza-specific helper t cell clones in vivo. t cells specific for internal viral proteins provide cognate help for b cell responses to hemagglutinin differential ability of b cells specific for external vs. internal influenza virus proteins to respond to help from influenza virus-specific t-cell clones in vivo d-epitope-explorer ( dex): localization of conformational epitopes within three-dimensional structures of proteins tuberculosis control in the st century hla supertypes and supermotifs: a functional perspective on hla polymorphism differential t cell costimulatory requirements in cd -deficient mice presentation of endogenous peptide/mhc class i complexes is profoundly influenced by specific c-terminal flanking residues ctl responses of hla-a . -transgenic mice specific for hepatitis c viral peptides predict epitopes for ctl of humans carrying hla-a . definition of mycobacterium tuberculosis culture filtrate proteins by two-dimensional polyacrylamide gel electrophoresis, n-terminal amino acid sequencing, and electrospray mass spectrometry several common hla-dr types share largely overlapping peptide binding repertoires antibiotic-refractory lyme arthritis is associated with hla-dr molecules that bind a borrelia burgdorferi peptide magnitude and diversity of cytotoxic-t-lymphocyte responses elicited by multiepitope dna vaccination in rhesus monkeys targeting a polyepitope protein incorporating multiple class ii-restricted viral epitopes to the secretory/endocytic pathway facilitates immune recognition by cd + cytotoxic t lymphocytes: a novel approach to vaccine design minimal epitoeps expressedin arecombinant polyepitope protein are processed and presented to cd + t cells : implications for vaccine design a comparison of standard immunogenicity assays for monitoring hiv type gagspecific t cell responses in ad hiv type gag vaccinated human subjects discrete cleavage motifs of constitutive and immunoproteasomes revealed by quantitative analysis of cleavage products strategy for isolating and sequencing biologically derived mhc class i peptides dendritic cell-based immunotherapy of cancer with carcinoembryonic antigen-derived, hla-a -restricted ctl epitope: clinical outcomes of patients with metastatic gastrointestinal or lung adenocarcinomas simultaneous cd + t cell responses to multiple tumor antigen epitopes in a multipeptide melanoma vaccine altered peptidase and viral-specific t cell response in lmp mutant mice the induction of virusspecific ctl as a function of increasing epitope expression: responses rise steadily until excessively high levels of epitope are attained an effective secondgeneration outer surface protein a-derived lyme vaccine that eliminates a potentially autoreactive t cell epitope proteolysis and class i major histocompatibility complex antigen presentation key: cord- - lz oqb authors: porter, kristen a.; kelley, lauren n.; george, annette; harton, jonathan a.; duus, karen m. title: class ii transactivator (ciita) enhances cytoplasmic processing of hiv- pr gag date: - - journal: plos one doi: . /journal.pone. sha: doc_id: cord_uid: lz oqb background: the pr (gag) (gag) polyprotein of hiv serves as a scaffold for virion assembly and is thus essential for progeny virion budding and maturation. gag localizes to the plasma membrane (pm) and membranes of late endosomes, allowing for release of infectious virus directly from the cell membrane and/or upon exocytosis. the host factors involved in gag trafficking to these sites are largely unknown. upon activation, cd + t cells, the primary target of hiv infection, express the class ii transcriptional activator (ciita) and therefore the mhc class ii isotype, hla-dr. similar to gag, hla-dr localizes to the pm and at the membranes of endosomes and specialized vesicular mhc class ii compartments (miics). in hiv producer cells, transient hla-dr expression induces intracellular gag accumulation and impairs virus release. methodology/principal findings: here we demonstrate that both stable and transient expression of ciita in hiv producer cells does not induce hla-dr-associated intracellular retention of gag, but does increase the infectivity of virions. however, neither of these phenomena is due to recapitulation of the class ii antigen presentation pathway or ciita-mediated transcriptional activation of virus genes. interestingly, we demonstrate that ciita, apart from its transcriptional effects, acts cytoplasmically to enhance pr (gag-pol) (gag-pol) levels and thereby the viral protease and gag processing, accounting for the increased infectivity of virions from ciita-expressing cells. conclusions/significance: this study demonstrates that ciita enhances hiv gag processing, and provides the first evidence of a novel, post-transcriptional, cytoplasmic function for a well-known transactivator. hiv polyprotein gag serves as a scaffold to promote assembly of progeny virions at cellular membranes [ ] and recruits components of the vesicular protein sorting pathway to facilitate virus budding [ , , ] . concomitantly, the virally encoded protease begins to cleave gag, which is required for complete virion maturation and infectivity [ , , ] . gag proteins can be detected at both the pm and the membranes of endosomes among different cell types, suggesting that budding is not limited to one cell-type specific locale [ , , , , , , , , ] . further, host factors which participate in targeting gag trafficking to particular membranes are largely unknown. as gag and infectious virus can originate from two cellular locations, two models for gag trafficking have emerged. the first model proposes that following synthesis, gag traffics to endosomal membranes, and upon exocytosis is deposited on the pm, where it serves as the site for productive virus assembly [ , ] . the second model proposes that gag is first trafficked to the pm, where virus assembly occurs, and then excess gag is internalized to intracellular compartments [ , , , ] , that serve as sites of productive virus assembly [ , ] . mhc class ii heterodimers follow a similar trafficking route, appearing at both the pm and specialized multivesicular bodies (mvbs) called mhc class ii containing compartments (miics) [ ] . mhc class ii is utilized by antigen presenting cells (apcs) to present exogenous processed antigen to cd + t cells [ , , ] . mhc class ii genes, including: hla-dr, -dp and -dq and the accessory molecules, invariant chain (ii) and hla-dm, are transcriptionally activated by the class ii transactivator (ciita), the global regulator of coordinate class ii mhc gene expression [ , ] . as ciita is induced in cd +t cells upon activation, these cells express mhc class ii [ , ] . upon synthesis, hla-dr heterodimers are assembled in the er and the immature complex (hla-dr+ ii) travels through the secretory pathway to miics, where the specialized hla-dm chaperone loads the hla-dr heterodimer with peptide [ , , ] . interestingly, both immature and mature forms of hla-dr can be found at the pm and can be subsequently internalized to miics due to a di-leucine motif in the cytoplasmic tail of ii (immature hla-dr) and a dileucine motif and/or ubiquitination of conserved lysine residues within the hla-dr b chain (mature hla-dr), respectively [ , , , , , , , ] . therefore, a connection between hla-dr and gag trafficking would not be surprising as both have an alternative route to intracellular compartments by way of the pm. indeed, expression of hiv- nef, vpu and gag have been shown to alter hla-dr trafficking [ , , , ] . in addition, hla-dr is preferentially acquired on the viral envelope of budding virions, which enhances virion infectivity and may play a role in bystander apoptosis of t lymphocytes [ , , ] . therefore, hla-dr localization at virus assembly sites is not unexpected. finzi et al. ( ) addressed the contribution of mhc class iiinduced miic formation to pr gag trafficking by monitoring virus budding in the presence of transiently expressed hla-dr heterodimers [ ] . in hek- t cells expressing hla-dr, there was a marked redistribution of gag to intracellular compartments, and reduced virus release at the pm; mature virus budded into hla-dr containing multivesicular bodies and was retained [ ] . we hypothesized that recapitulating endogenous expression of the entire class ii antigen presentation pathway in producer cells via expression of ciita would restore infectious virus release and provide a more physiologically relevant model for hiv- assembly studies. as expected, stable or transient expression of ciita did not induce intracellular retention of gag. however, hla-dr induced gag retention could not be alleviated by transient co-expression with hla-dm and/or ii, or ciita-mediated upregulation of the recycling endosome gtpase, rab b [ ] . further, mutating ubiquitinatible lysine residues or complete truncation of the cytoplasmic tails of the hla-dr a and b chains did not restore virus release. rather, limiting the amount of hla-dr dna transfected into cells restored virus release and alleviated gag retention. curiously, ciita expressing cells produced virus that was significantly more infectious than ciita deficient cells, and this was independent of the class ii antigen presentation pathway. ciita enhances infectivity of virions from producer cells through a novel function, by improving maturation through an increase in viral protease-dependent gag processing. using a panel of ciita mutants, we demonstrate that cytoplasmic ciita increases gag-pol polyprotein levels. overall, our work reveals a novel cytoplasmic, post-transcriptional function of ciita, which is expressed upon t cell activation and is constitutively expressed in macrophages and dendritic cells, all known targets of hiv infection. transient expression of the class ii heterodimer, hla-dr, induces intracellular accumulation of gag and a marked reduction in extracellular virus release from producer cells [ ] . this previous study focused on hla-dr in the absence of ii and hla-dm, critical components of the antigen processing and presentation pathway which influence mhc class ii trafficking. to determine if expression of the complete mhc class ii pathway could overcome gag and virus retention we used ciita to coordinately express these genes. cells were either transiently (pcciita) or stably (a t-ciita) transfected with ciita ( figure s ) and hiv- nl - dna, and gag localization was monitored by immunofluorescence and compared to cells transiently expressing the hla-dr a and b heterodimers (pcdrab b ), or vector-only (pcdna . ). as expected, a weak but uniform gag signal was present in vector-only transfected cells (figure aa ) and transient hla-dr expression induced a marked redistribution of gag as indicated by a dense, punctuate gag staining pattern (figure ad ). conversely, gag accumulation was not observed when ciita was either transiently or stably expressed (figure ab or c, respectively). therefore, transient expression of hla-dr in producer cells does indeed lead to gag retention, which is not observed in the presence of ciita. however, recapitulation of the mhc class ii pathway in trans (pcdrab b +ii+hla-dmab), also resulted in dense accumulation of gag signal (figure ae ), suggesting that ciita-mediated coordinate activation of hla-dr, -dm and ii expression is insufficient to overcome gag retention. flow cytometric analysis confirmed these findings, as cells transfected with hla-dr heterodimers and/or co-transfected with some or all of the components of the class ii antigen presentation pathway stained as gag hi , indicating gag accumulation ( figure b and c). however, this population was absent in cells transiently or stably expressing ciita ( figure b and c) . therefore, absence of gag accumulation in ciita expressing cells is likely not due to its transactivation of the mhc class ii antigen presentation pathway. to assess whether ciita-mediated alleviation of gag retention in class ii expressing cells correlated with restored virus production, virus particle and infectious virus release were measured by p elisa and ghost cell infectivity assays, respectively. virus release, both infectious and particle titers) were reduced when cells were transfected with either hla-dr or other components of the mhc class ii antigen presentation pathway ( figure s ), confirming a correlation between gag retention and reduced virus titers in the presence of hla-dr, as previously demonstrated [ ] . these assays also demonstrate a dramatic increase in infectious virus release from ciita-expressing cells as compared to cells expressing vector only (figure a & s ), despite significantly fewer virus particles being released from a ciitaexpressing cell (as measured by pg/ml of p in the culture supernatant, figure b & s ). therefore, while fewer virus particles are released from a ciita-expressing cell, those particles are significantly more infectious ( figure c ). previously, hla-dr incorporation into the envelope of budding virions was demonstrated to enhance virion infectivity [ , ] . to determine if ciita-enhancement of virion infectivity was due to hla-dr or other components of the class ii antigen presentation pathway, we measured virion infectivity from cells expressing these proteins. however, virions from these cells were just as infectious as virions released from vector only controls, yet not as infectious as virions from cells either stably or transientlyexpressing ciita ( figure d ). therefore, ciita enhancement of virion infectivity is independent of the mhc class ii antigen presentation pathway. together, these data suggest ciita has two effects on the hiv replicative cycle in producer cells, both of which are independent of the mhc ii antigen processing pathway; i) it does not induce hla-dr, mediated intracellular retention of gag and ii) it increases the infectivity of hiv virions. ciita has been shown to upregulate expression of rab b, a small gtpase involved in endocytic recycling [ , ] . therefore, we hypothesized that ciita via the action of rab b, could increase gag recycling back to the pm, thereby alleviating hla-dr-mediated gag retention. commercially available rab antibodies cannot distinguish between rab a and b. however, krawczyk et al. ( ) demonstrated by chromatin immunoprecipitation that ciita specifically associates with the rab b and not the rab a promoter [ ] . immunoblotting demonstrated that there is an increase in rab in ciita-expressing cells ( figure a ), therefore it is likely that this is in the form of rab b. however, when rab b and hla-dr were co-expressed in producer cells with the hiv- nl - plasmid, it did not rescue intracellular retention of gag ( figure b ). in fact, infectious virus release from cells transiently co-expressing rab b and hla-dr was further reduced, suggesting that rab b does not alleviate hla-drmediated retention of gag ( figure c ). up until this point, we had not co-expressed ciita with hla-dr in producer cells to determine if ciita could alleviate retention of gag and/or restore infectious virus release as would be expected. interestingly, when ciita was transiently expressed with the hla-dr heterodimers in producer cells, gag still accumulated intracellularly and infectious virus production remained reduced ( figure b and c, respectively), suggesting that expression of ciita is not sufficient to compensate for hla-drmediated gag retention. however, when hla-dr is endogenously expressed under the control of ciita, retention is alleviated. [ ] . ubiquitination of a conserved lysine residue on the hla-dr b chain induces intracellular accumulation of class ii heterodimers in mhc class ii compartments (miics) [ , ] . thus, the ubiquitination state of hla-dr, might influence gag retention. to address this idea, lys arg (pcdrabk r) and lys / arg (pcdrabk / r) point mutations were made in the hla-dr beta chain. despite these mutations, hla-dr expression on the surface of a t cells was not significantly altered (flow cytometric data not shown), nor was intracellular retention of gag alleviated or infectious virus release restored ( figure a and b, respectively). therefore, we attempted to alleviate gag retention and restore infectious virus release, as demonstrated by finzi et al. ( ) [ ] , by complete truncation of the cytoplasmic tails of both hla-dr a and b (pcdradcyto and pcdrbdcyto, respectively). however, loss of either cytoplasmic tail (pcdradcytob and pcdrabdcyto), or both cytoplasmic tails (pcdradcy-tobdcyto), from the heterodimer did not alleviate gag retention and even further reduced infectious virus release from these cells ( figure a and b, respectively). our results may differ from finzi's because of the differences in cell type, or hla-dr gene alleles, nevertheless they strongly suggest that transient hla-dr expression in producer cells induces gag retention. further, flow cytometry to monitor surface hla-dr expression demonstrates that transient transfection of hla-dr induces an approximate half-log to log-fold increase in hla-dr as compared to cells stably or transiently expressing ciita, respectively ( figure s ). therefore, it is possible that hla-dr induced gag retention is a consequence of hla-dr overexpression in this transient system to test this possibility, virus producer cells were transfected with increasing amounts of hla-dr in the presence of hiv- nl - dna, and infectious virus release was monitored. as hla-dr expression increased, the level of infectious virus release decreased ( figure c ). further, while not statistically significant, there was a positive correlation (r = . ) between gag accumulation (gag hi ) and increasing hla-dr expression ( figure d ), suggesting gag retention is likely related to hla-dr overexpression. similarly, when hla-dm, which is structurally similar to hla-dr, was overexpressed in producer cells, gag retention was also induced ( figure d ) and infectious virus release reduced (data not shown). as endogenous expression of the class ii antigen presentation pathway by ciita does not induce gag retention and limiting expression of hla-dr or -dm likewise has a limited effect on gag retention in producer cells, these data collectively suggest that overexpression of the class ii pathway alters gag trafficking in producer cells leading to reduced infectious virus release. however, these data do not provide an explanation for the increased infectivity of virions released from ciita-expressing cells. viral protease cleavage of the gag and gag-pol polyproteins is required for virion maturation and infectivity [ , ] . figure a ) [ , , , , ] . as there is an increase in virion infectivity from ciita-expressing cells, we hypothesized that the processing of gag polyproteins may be enhanced in these cells. as suspected, analysis of cell lysates from equal numbers of hiv- transfected a t-ciita and a t cells demonstrated a higher ratio of mature cap to pr gag in ciita-expressing cells ( figure b&c), demonstrating that gag processing in the presence of ciita is enhanced. additionally, increased levels of processing intermediates are present in a t cell lysates. specifically, the p (ma-ca-sp ) and p (ca+p ) products were increased (figure b&c) in these cells, indicating that gag processing in the presence of ciita is more efficient. gag processing is also enhanced in cell-free virions from cells either transiently or stably expressing ciita, as demonstrated by the increased levels of cap , and the loss of the p and p intermediate products relative to pr gag ( figure d&e ). collectively, these results suggest that ciita increases gag polyprotein processing, leading to enhanced production of infectious virions. hla-dr is incorporated into the hiv virion envelope [ ] and is transcriptionally activated by ciita, therefore its contribution to gag processing was assessed. virions from hla-dr-expressing cells exhibited less gag processing than virions from ciitaexpressing cells ( figure f , bottom panel). when virions from both cell lines were affinity-purified on hla-dr binding columns, gag processing was reduced in those produced in the hla-drexpressing cells as compared to virions from cells which express ciita ( figure f , upper panel and g), suggesting that hla-dr alone does not contribute to increased gag processing. similar to our previous results, there are fewer processing intermediates present in virions from ciita-expressing cells, suggesting that ciita expression enhances virion infectivity by increasing maturation through more complete gag processing. cleavage of the gag and gag-pol polyprotein is mediated specifically by the virally-encoded protease [ , , ] . we considered that ciita might increase viral protease processing of gag; thus, we examined gag processing and infectious virus release from ciita-expressing hiv producer cells in the presence of a protease inhibitor. if ciita is increasing protease processing, such an enhancement should be overcome by lower doses of the hiv protease inhibitor, lopinavir. as expected, between concentrations of . and . nm of lopinavir infectious virus release from ciita-expressing cells was reduced to that of a t cells treated with vehicle only ( figure a ). to determine if the reduced gag processing correlated with decreased infectious virus release, we monitored gag cleavage by western blotting and as expected, between . and . nm of liponavir, gag processing in virions from ciita-expressing cells was returned to that of vehicle treated, vector-only control cells ( figure s ). these results directly demonstrate that ciita-mediated enhancement of gag processing and infectious virus release is through the hiv protease. the only known function of ciita is as a transcriptional coactivator; therefore, we thought it likely that it drives the expression of a gene which enhances viral protease-mediated gag processing. therefore, we reasoned that expression of ciita mutants which fail to localize to the nucleus should not mediate increased gag processing or enhanced infectious virus release. to test this idea, a panel of ciita mutants (gtp , a magnesium ion coordination site mutant and gtp , a guanine ring-binding domain mutant and l p, a point mutant in the c-terminal leucine rich repeat domain, which are all defective for nuclear localization and fail to activate hla-dra transcription [ , , ] ), were individually co-expressed with hiv- nl - dna, and infectious virus release and gag processing were monitored. interestingly, producer cells expressing these mutants also produced more infectious virus as compared to vector-only control ( figure a ) and had increased gag processing in both cell-bound virions and cell-free virions ( figure b and figure s , respectively). loss of ciita nuclear localization (and therefore coactivation potential), did not hinder increased gag processing and infectious virus release, strongly suggesting that ciita acts cytoplasmically to increase hiv virus maturation independent of its transactivation function. we further evaluated ciita transactivation potential as a mechanism of enhanced virus release utilizing a different model system. nih t balb/c cells (expressing human p to allow for virus like particle production [ ] ) expressing ciita and transfected with an ltr-deficient hiv genome construct (pchiv pal),under control of a cmv promoter had a dramatic increase in virus-like particle production as compared to nih t cells in the absence of ciita ( figure s ). this result provides further evidence that ciita enhancement of virus production is independent of its transactivation potential on the hiv ltr. to determine the mechanism by which ciita mediates protease activity in these cells, we analyzed the expression of the gag and gag-pol in ciita-expressing cells. expression of the hiv protease is extremely toxic to cells, and is thus very tightly regulated during virus replication [ , , ] . the viral protease arises from the alternative gag translation product ( figure a ), gag-pol which, accounts for only - % of gag polyproteins and results from ribosomal frameshifting at a conserved heptamer ''slippery sequence'' and a secondary stem-loop structure on gag-pol transcripts [ , ] . therefore, increased viral protease-mediated processing of the gag polyproteins from ciita-expressing cells may be due to an increase in overall viral protease levels, due to increased gag-pol synthesis. to establish the ability of ciita to mediate the enhanced production of hiv protease, cells were transiently co-transfected with either ciita or pcdna and hiv- nl - dna, and were treated with nm of lopinavir to inhibit the majority of gag polyprotein processing. gag-pol levels relative to gag were then determined by western blotting. while - % of all gag polyprotein in cell lysates of vector-only cells was in the form of gag-pol, expression of ciita increased this level to almost % ( figure c and d) , indicating that ciita increases gag-pol levels in virus producer cells. we also measured the potential of the cytoplasmic ciita mutants to increase gag-pol levels relative to gag and, interestingly, gag-pol made up approximately , and % of all gag polyproteins in gtp , gtp and l p -expressing cells, respectively. this result further demonstrates a novel transcription-independent role for cytoplasmic ciita during hiv infection, where ciita enhances gag-pol protease expression, which subsequently enhances virus maturation and infectivity. [ ] . we speculated that complete expression of the antigen presentation pathway, through expression of ciita, would alleviate this phenotype in virus producer cells. interestingly, we noticed two phenomena: i) ciita did not induce intracellular retention of gag or impair virus release in producer cells despite expression of hla-dr and ii) the virus released from cells expressing ciita was significantly more infectious. upon investigation of ciita-mediated alleviation of hla-dr-induced gag retention, we found that this effect was not due to the lack of ii or hla-dm (key components of antigen presentation), as had been expected, nor was it a consequence of rab b expression in these cells. further, when lysine residues in the hla-dr chain were mutated or both of the hla-dr chain cytoplasmic tails were truncated, gag was still retained and virus release inhibited. in addition, when ciita was expressed in conjunction with hla-dr this effect could not be alleviated, suggesting that gag retention is a consequence of hla-dr overexpression. indeed, retention of gag is directly correlated to levels of hla-dr expression. further, we did not observe gag retention when class ii antigen presentation pathway genes were expressed at endogenous levels via ciita expression. overexpression of hla-dm, which is structurally similar to hla-dr also induced gag retention, suggesting that retention of gag is a likely consequence of altered trafficking of overexpressed class ii antigen presentation pathway components rather than a physiologically relevant phenomenon. despite observing similar hla-dr and gag retention/reduced virus release effects as finzi et al.( ) , our results differed in that we did not observe the requirement for intact hla-dr a and b-chain cytoplasmic tails for the induction of gag retention. in our hands, when the ubiquitinatible lysine residues in the hla-dr b-chain were mutated or the cytoplasmic tails of the hla-dr dimer were completely removed, there was no alleviation of gag retention or virus release. beyond the obvious differences between the previous work [ ] and our own (i.e. provirus construct and cell type), our mutant data may differ from theirs because arginine residues were substituted for lysine and tyrosine residues in the hla-dr a and b chain, respectively, in order to ensure stabilization of the truncated hla-dr molecule at cellular membranes. these differences in mutant constructs may potentially explain discrepancies in our results. we also observed that expression of hla-dm was sufficient to induce gag retention and impede virus release from cells. however, finzi et al.( ) did not observe retention in the presence of hla-dm or hla-do [ ] . this difference may be explained by their use of a bicistronic construct for expression of the hla-dm heterodimer; however, this strategy was also used to express hla-dr, which still induced retention [ ] . irrespective of these differences, gag retention and loss of virus release correlates with increasing hla-dr expression. these results do not exclude the possibility that hla-dr and gag trafficking may be linked. indeed, we have demonstrated that gag co-immunoprecipitates with hla-dr from ciita expressing cells and this interaction is independent of the cytoplasmic tails of hla-dr (data not shown). further, other hiv proteins may be linked to class ii trafficking. stumptner-cuvelette et al. ( ) and chaudhry et al.( ) , have independently demonstrated that hiv nef induces internalization of surface class ii in epithelial and monocytic cell lines, respectively [ , ] . however, we did not observe downregulation of hla-dr from the cell surface, following transfection of the hiv genome into ciita-expressing epithelial cells (data not shown) or hiv infection of ciita-expressing cd + t cells (unpublished data). further, gluschankof and suzan demonstrated that expression of gag-pol restored hla-dr presence at the cell surface in the h -c . line, a t cell clone deficient for surface hla-dr expression [ ] . collectively, our work and that of others suggests a link between hla-dr and gag trafficking, where localization may be cell-type dependent. one of the more interesting findings of this study is that while overall viral titers from ciita-expressing cells decrease the infectivity of these particles is significantly enhanced. increased infectivity was due to improved virion maturation in a viral protease-dependent manner. not only was processing to capsid p more complete in ciita expressing cells, but vector-only control cells and those only expressing hla-dr contained increased levels of processing intermediates. the presence of higher levels of processing intermediates in virus produced in these cells may help explain the reduced infectivity, as studies in both mlv [ ] and hiv [ ] demonstrate that partially cleaved gag products act transdominantly to reduce virion infectivity through reduced reverse transcription of viral rna, despite the presence of functional reverse transcription polymerase [ ] . next we demonstrated that ciita increased gag processing through the viral protease and evaluated whether this enhancement was a consequence of ciita-mediated transcriptional activation. the ciita l p mutant, which fails to translocate to the nucleus [ ] , demonstrated increased gag processing and virus release compared to vector-only control, suggesting a cytoplasmic role for ciita in the later stages of the hiv infection cycle. this does not preclude the possibility that an undetectable level of ciita l p might translocate to the nucleus. however, no l p was detected in the nucleus after a hour treatment with the nuclear export inhibitor, leptomycin b [ ] . further, the predominantly cytoplasmic gtp-binding ciita mutants, gtp and gtp , had a similar increase in infectious virus release versus vector-only expressing cells. previously, it was demonstrated that increased gag-pol levels severely impair virion infectivity through disruption of rna genome dimerization [ ] . further, hiv protease is known to be toxic to cells as it leads to the production of the novel procaspase cleavage product, casp p , which induces apoptosis through loss of mitochondrial membrane potential [ ] . therefore, we would expect that virions from ciita-expressing cells would be reduced in titer and infectivity, as there is increased gag-pol and protease. however, while overall viral particle numbers (as measured by p elisa) from ciita expressing cells were decreased, the infectivity of these virions was significantly increased when calculated by infectious units per pg p . interestingly, at . nm lopinavir, the infectivity of virions from ciita-expressing cells increased over vehicle-treated controls, despite reduced gag processing. further, the mutants of ciita, which produced the highest level of gag-pol, did not demonstrate a linear increase in gag processing or virion infectivity, which may be explained by previous work demonstrateing that increased gag-pol levels impairs viral infectivity [ , ] . therefore it is likely that ciita is capable of increasing gag-pol levels to a point which can impede virus maturation, albeit not enough to reduce it to levels observed from vector-only expressing cells. therefore, overall infectivity of virions is increased from cells expressing ciita despite an altered gag to gag-pol ratio. future studies should focus on how ciita can increase this ratio without severely impairing virus release as well the novel mechanism by which ciita increases gag-pol levels. preliminary studies in this laboratory suggest that ciita enhancement gag-pol may be due to increased ribosomal frameshifting (data not shown). finally, it is tempting to speculate that ciita, which is expressed upon cd + t cell activation and increases viral protease levels, may also contribute to casp p -mediated apoptosis, which may link ciita to the decimation of t cells in the galt during primary viremia [ ] . thymic epithelial cells [ ] , cervical epithelial cells [ ] , human colonic epithelial cells [ ] and oral keratinocytes (normal human oral epithelial cells) [ ] have all demonstrated in vitro the capability of being productively infected with hiv. infection of epithelial cells provides potential in vivo significance to this study, especially considering that thymic epithelial cells constitutively express mhc class ii (and thus ciita) [ ] . in addition, most other cells can be stimulated to express ciita in the presence of ifn-c (i.e. a pro-inflammatory environment), which is induced during hiv infection of the galt [ ] . overall, this study demonstrates that the function of ciita may be more broad than previously thought. given this previously undescribed role in enhancement of virion maturation, the precise consequences of ciita expression during the hiv replicative cycle may provide rationale for the development of novel antiviral therapeutics. a t cells [ ] were maintained as previously described [ ] and ciita-stable a t cell lines were generated by cotransfecting pvu i linearized pdna .flag.ciita [ ] and pcmv his, a mammalian selection vector which encodes the histidinol dehydrogenase gene under control of the cmv promoter into the cells. stable clones were selected in dmem medium containing mm l-histidinol (sigma-aldrich., st. louis, mo) and cloned by limiting dilution assay. ghost cell medium was supplemented with . mg/ml g , . mg/ml hygromycin b and mg/ml puromycin (sigma) as previously described [ ] . using cdna reverse transcribed from a t-ciita rna, we amplified hla-dra, hla-drb and hla-drb sequences (genbank accession nos. nm , nm , and nm , respectively), using primers containing forward restriction site xbai and the reverse restriction site hindiii (table s ). the haplotyping of hla-dr isotypes expressed by a t cells was determined by the transplantation immunology lab, albany medical college. primers were designed to include an intact kozak sequence upstream of the translation start site. hla-dra and b plasmids were then used for site-directed mutagenesis using primers indicated (table s ). transient transfections of all plasmids, including: pdna .flag.ciita [ ] , ciita-gtp and -gtp [ ] and ciita -l p [ ] , p - (coding for the p and p isoforms of invariant chain-a kind gift provided by dr. eric o. long, niaid), hla-dma and b (pmcfr-pac and pdmb/mcfrkindly provided by dr. lisa k. denzin, sloan-kettering cancer center), egfp-rab b (kindly provided by dr. marci scidmore, cornell university), and hiv gag-igfp [ ] (kindly provided by dr. benjamin chen, mount siani school of medicine) were performed using a : ratio fugene hd transfection reagent to dna in serum-free media as suggested by the manufacturer (roche, indianapolis, in). virus plasmid dna provided half of total dna used in transfection reactions. nih t balb/c cells are transfected with . mg ciita and . ml fugene hd ( : ratio of fugene to dna for optimal cells growth) and selected for with l-histidionol for two weeks and analyzed for mhc ii expressing using fluorescence-activated cell sorter (facs) with pe-conjugated mouse igg a (cedar lane) against i-a d and clones were selected by limiting dilution. these cells were co-transfected with p cdna and pchiv pal (which contains all hiv genes except the ltr sequences) at a : ratio. the p cdna was donated by the peterlin laboratory [ ] . both mhc ii expressing vlps and mhc ii-negative vlps were produced and concentrated -fold in a k molecular weight cut-off filter tube (millipore) for minutes at rpm prior to titering via hiv- p ca antigen capture assay kit were performed following the manufacturer's instructions (nih aids research and reference reagent program). cytoplasmic rna from hr cultures of each cell type was collected using the rneasy mini kit according to manufacturer's instructions (qiagen, inc., valencia, ca). ug of dna-free rna was run out on a non-denaturing gel to ensure equal concentrations of each sample followed by reverse transcription of mg of each rna sample using the iscript cdna synthesis kit, following manufacturer's instructions (biorad, hercules, ca). ng of cdna was used to amplify hla-dm, ciita, gapdh, and ii from each cell type to confirm expression. forward and reverse primers sequences used in rt-pcr experiments indicated in table s . cells were analyzed for hla-dr expression as previously described [ ] . briefly, cells were incubated with murine clone l [ ] . densitometric analysis was performed as previously described [ ] and the percentage of total hiv- a ca p ( -h - c) [ ] reactive bands was used as a measure of gag processing [ ] . intracellular gag staining cells were permeabilized with intraprep permeabilization reagent (beckman-coulter, fullerton, ca), following manufacturer's instructions. gag was stained with fitc-conjugated fh - - (beckman-coulter) for approximately m prior to analysis and data interpretation as performed above, with the exception of the percentage of gag+ cells was determined after gating on hla-dr+ cells. virions were purified using the mmacs streptavidin kit (miltenyi biotec inc., auburn, ca.), according to the manufacturer's instructions using biotinylated-l (biolegend). ghost assay [ ] to determine infectious virus production and p elisas to determine virus particle release using the hiv- p ca antigen capture assay kit were performed following the manufacturer's instructions (nih aids research and reference reagent program). cells were transfected with either pcciita or empty pcdna vector . hours prior to lopinavir (nih aids research and reference reagent program) or dmso (vehicle-only) treatment at indicated concentrations. virus and cell supernatants were collected approximately hours post-treatment. for determina-tion of p gag-pol to p gag ratios, producer cells were transfected with ciita constructs (pccitia, gtp , gtp , l p) or vector only control (pcdna) and then transfected with pnl - hours later, prior to being treated with nm lopinavir. cell lysates were used to monitor the levels of pr gag to pr pol via western blotting with hiv- a ca p ( -h - c) after hours. table s primers used in this study. hiv- gag proteins: diverse functions in the virus life cycle tsg : hiv- 's ticket to ride tsg and the vacuolar protein sorting pathway are essential for hiv- budding hiv gag mimics the tsg -recruiting activity of the human hrs protein the activity of the protease of human immunodeficiency virus type is initiated at the membrane of infected cells before the release of viral proteins and is required for release to occur with maximum efficiency partial inhibition of the human immunodeficiency virus type protease results in aberrant virus assembly and the formation of noninfectious particles human immunodeficiency virus (hiv- ) gag processing intermediates trans-dominantly interfere with hiv- infectivity major histocompatibility complex class ii molecules promote human immunodeficiency virus type assembly and budding to late endosomal/multivesicular body compartments assembly of infectious hiv- in human epithelial and t-lymphoblastic cell lines infectious hiv- assembles in late endosomes in primary macrophages more than one door -budding of enveloped viruses through cellular membranes retrovirus budding productive human immunodeficiency virus type assembly takes place at the plasma membrane evidence that productive human immunodeficiency virus type assembly can occur in an intracellular compartment the cell biology of hiv- virion genesis endosomes, exosomes and trojan viruses intracellular destinies: degradation, targeting, assembly, and endocytosis of hiv gag vpu and tsg regulate intracellular targeting of the human immunodeficiency virus type core protein precursor pr gag plasma membrane is the site of productive hiv- particle assembly endosomal trafficking of hiv- gag and genomic rnas regulates viral egress mhc class ii transport at a glance regulation of mhc class ii expression, a unique regulatory system identified by the study of a primary immunodeficiency disease genetic control of mhc class ii expression minireview: specificity and expression of ciita, the master regulator of mhc class ii genes genetic control of mhc class ii expression epigenetic control of ciita expression in leukemic t cells function and regulation of mhc class ii molecules in t-lymphocytes: of mice and men trafficking of mhc class ii molecules in the late secretory pathway mhc class ii molecules on the move for successful antigen presentation achieving stability through editing and chaperoning: regulation of mhc class ii peptide binding and expression identification of the hla-dm/hla-dr interface hla-dm induces clip dissociation from mhc class ii [alpha][beta] dimers and facilitates peptide loading the invariant chain is required for intracellular transport and function of major histocompatibility complex class ii molecules surface expression of mhc class ii in dendritic cells is controlled by regulated ubiquitination dendritic cells regulate exposure of mhc class ii at their plasma membrane by oligoubiquitination human immunodeficiency virus- nef expression induces intracellular accumulation of multivesicular bodies and major histocompatibility complex class ii complexes: potential role of phosphatidylinositol -kinase human immunodeficiency virus type vpu protein interacts with cd and modulates major histocompatibility complex class ii presentation down-modulation of mature major histocompatibility complex class ii and up-regulation of invariant chain cell surface expression are well-conserved functions of human and simian immunodeficiency virus nef alleles hiv- gag polyprotein rescues hla-dr intracellular transport in a human cd + cell line differential incorporation of cd , cd (b - ), cd (b - ), and major histocompatibility complex class i and ii molecules into human immunodeficiency virus type virions and microvesicles: implications for viral pathogenesis and immune regulation distinct mechanisms of cd + and cd + t-cell activation and bystander apoptosis induced by human immunodeficiency virus type virions mhc class ii and hiv pathogenesis: lots of data, few conclusions expression of rab b, a protein governing endocytic recycling, is co-regulated with mhc class ii genes the presence of hostderived hla-dr on human immunodeficiency virus type increases viral infectivity the acquisition of host-derived major histocompatibility complex class ii glycoproteins by human immunodeficiency virus type accelerates the process of virus entry and infection in human t-lymphoid cells identification of ciita regulated genetic module dedicated for antigen presentation endosomal trafficking of hiv- gag and genomic rnas regulates viral egress vivoprocessing of pr gag-polfrom human immunodeficiency virus type (hiv) in acutely infected, cultured human t-lymphocytes comparison of human immunodeficiency virus type pr (gag) and pr (gag-pol) processing intermediates that accumulate in primary and transformed cells treated with peptidic and nonpeptidic protease inhibitors programmed ribosomal frameshifting in hiv- and the sars-cov cellular proteins bound to immunodeficiency viruses: implications for pathogenesis and vaccines hiv- protease specificity of peptide cleavage is sufficient for processing of gag and pol polyproteins the regulation of sequential processing of hiv- gag by the viral protease complete mutagenesis of the hiv- protease gtp binding by class ii transactivator: role in nuclear import gtp-dependent recruitment of ciita to the class ii major histocompatibility complex promoter leucine-rich repeats of the class ii transactivator control its rate of nuclear accumulation human p protein relieves a posttranscriptional block to hiv replication in murine cells cell killing by hiv- protease overexpression of the gag-pol precursor from human immunodeficiency virus type proviral genomes results in efficient proteolytic processing in the absence of virion production maintenance of the gag/gag-pol ratio is important for human immunodeficiency virus type rna dimerization and viral infectivity the human immunodeficiency virus type ribosomal frameshifting site is an invariant sequence determinant and an important target for antiviral therapy nef promotes endocytosis of cell surface mhc class ii molecules via a constitutive pathway mutant murine leukemia virus gag proteins lacking proline at the n-terminus of the capsid domain block infectivity in virions containing wild-type gag hiv- protease processes procaspase to cause mitochondrial release of cytochrome c, caspase cleavage and nuclear fragmentation the gastrointestinal tract is critical to the pathogenesis of acute hiv- infection immunopathogenic mechanisms of hiv infection productive infection of a cervical epithelial cell line with human immunodeficiency virus: implications for sexual transmission galactosyl ceramide (or a closely related molecule) is the receptor for human immunodeficiency virus type on human colon epithelial ht cells human immunodeficiency virus type infection and replication in normal human oral keratinocytes hiv infection and gut mucosal immune function: updates on pathogenesis with implications for management and intervention characteristics of a human cell line transformed by dna from human adenovirus type separation of human immunodeficiency virus type replication from nef-mediated pathogenesis in the human thymus importance of acidic, proline/serine/ threonine-rich, and gtp-binding regions in the major histocompatibility complex class ii transactivator: generation of transdominant-negative mutants primary human immunodeficiency virus type (hiv- ) isolates, like hiv- isolates, frequently use ccr but show promiscuity in coreceptor usage predominant mode of human immunodeficiency virus transfer between t cells is mediated by sustained env-dependent neutralization-resistant virological synapses hla-dr alpha chain residues located on the outer loops are involved in nonpolymorphic and polymorphic antibody-binding epitopes macrophage-tropic human immunodeficiency virus isolates from different patients exhibit unusual v envelope sequence homogeneity in comparison with t-cell-tropic isolates: definition of critical amino acids involved in cell tropism functional analysis of glucan binding protein b from streptococcus mutans human immunodeficiency virus (hiv- ) gag processing intermediates trans-dominantly interfere with hiv- infectivity characterization of a thymus-tropic hiv- isolate from a rapid progressor: role of the envelope key: cord- -jtbxefm authors: mohammed, arwa a.; shantier, shaza w.; mustafa, mujahed i.; osman, hind k.; elmansi, hashim e.; osman, isam-aldin a.; mohammed, rawan a.; abdelrhman, fatima a.; elnnewery, mihad e.; yousif, einas m.; mustafa, marwa m.; elfadol, nafisa m.; abdalla, alaa i.; mahmoud, eiman; yagaub, ahmed a.; ahmed, yassir a.; hassan, mohamed a. title: epitope-based peptide vaccine against glycoprotein g of nipah henipavirus using immunoinformatics approaches date: - - journal: j immunol res doi: . / / sha: doc_id: cord_uid: jtbxefm background: nipah belongs to the genus henipavirus and the paramyxoviridae family. it is an endemic most commonly found at south asia and has first emerged in malaysia in . bats are found to be the main reservoir for this virus, causing disease in both humans and animals. the last outbreak has occurred in may in kerala. it is characterized by high pathogenicity and fatality rates which varies from % to % depending on the severity of the disease and on the availability of adequate healthcare facilities. currently, there are no antiviral drugs available for niv disease and the treatment is just supportive. clinical presentations for this virus range from asymptomatic infection to fatal encephalitis. objective: this study is aimed at predicting an effective epitope-based vaccine against glycoprotein g of nipah henipavirus, using immunoinformatics approaches. methods and materials: glycoprotein g of the nipah virus sequence was retrieved from ncbi. different prediction tools were used to analyze the epitopes, namely, bepipred- . : sequential b cell epitope predictor for b cell and t cell mhc classes ii and i. then, the proposed peptides were docked using autodock . software program. results and conclusions. the two peptides tvyhcsavy and flidrinwi have showed a very strong binding affinity to mhc class i and mhc class ii alleles. furthermore, considering the conservancy, the affinity, and the population coverage, the peptide flidrinwit is highly suitable to be utilized to formulate a new vaccine against glycoprotein g of nipah henipavirus. an in vivo study for the proposed peptides is also highly recommended. nipah virus (niv) is an rna virus that belongs to the genus henipavirus within the family paramyxoviridae and has first emerged in malaysia in , gaining its name from a village called sungai nipah where it was isolated from the cerebrospinal fluid (csf) of one of the patients [ ] [ ] [ ] [ ] . niv is transmitted zoonotically (from bats to humans, or from bats to pigs, and then to humans) as well as human-to-human routes. its clinical presentation varies from asymptomatic (subclinical) infection to acute respiratory illnesses and fatal encephalitis in most of the patients who has been in direct contact with infected pigs. it has also been found that the virus causes central nervous system illnesses in pigs and respiratory illnesses in horses resulting in a significant economic loss for farmers [ , [ ] [ ] [ ] [ ] [ ] . large fruit bats of the genus pteropus seem to act as a natural reservoir of niv based on the isolation of hendra virus which has showed the presence of neutralizing antibodies to the hendra virus on the bats [ , ] . although, there are no more cases of niv in malaysia, several outbreaks have been frequently occurring in india, bangladesh, thailand, and cambodia [ ] . the case fatality rate ranges from % to %, making it one of the deadliest viruses known to infect humans [ , , ] . laboratory diagnosis of nipah virus infection is made using reverse transcriptase polymerase chain reaction (rt-pcr) from throat swabs, cerebrospinal fluid, urine, and blood analysis during acute and convalescent stages of the disease. igg and igm antibody detection can be done after recovery to confirm nipah virus infection. immunohistochemistry on tissues collected during an autopsy can also confirm the disease [ , ] . currently, there are no effective treatments for the nipah virus infection. therefore, a few precautions should be followed such as practicing standard infection control, barrier nursing to avoid the spread of the infection from person to person, and the isolation of those suspected to have the infection [ , , ] . recent computational approaches have provided further information about viruses, including the study conducted by badawi m et al. on zika virus, where the envelope glycoprotein was obtained using protein databases. the most immunogenic epitope for the t and b cells involved in cell-mediated immunity was previously analyzed [ ] . the main focus of the analysis was the mhc class i potential peptides using in silico analysis techniques [ , ] . in this study, the same techniques were applied to keep mhc classes i and ii along with the world population coverage as our main focus. furthermore, in this study, we aimed to design an epitope-based peptide vaccine against nipah virus using peptides of its glycoprotein g as an immunogenic part to stimulate a protective immune response [ ] . nipah virus invades host cells by the fusion of the host cell membranes at an optimal physiological ph for cleavage without requiring viral endocytosis. cell-cell fusion is a pathological lineament of nipah virus infections, resulting in a cell-to-cell spread, inflammation, and destruction of endothelial cells and neurons [ ] . both nipah virus entry and cell-cell fusion require concerted efforts of the attachment of glycoprotein g and fusion (f) glycoprotein. upon receptor binding, nipah virus glycoprotein g triggers a conformational cascade in nipah virus glycoprotein f that executes a viral and/or a cell membrane fusion [ ] . due to the potency of glycoprotein g over f, we have considered this incident to be the target of this study. there are a lot of challenges regarding the development of peptide-based vaccines, and therefore, we have decided to study and propose a new vaccine against the nipah virus, since they make a helpful alternative strategy that relies on the usage of short peptide fragments to induce immune responses [ ] [ ] [ ] [ ] . antigenic epitopes from single proteins may not be really necessary, whereas some of these epitopes may even be detrimental to the induction of protective immunity. this logic has created an interest in peptide vaccines and especially those containing only epitopes that are capable of inducing desirable t cell-and b cell-mediated immune responses. less than amino acid sequences make up the peptides used in such vaccines, which are then synthesized to form an immunogenic peptide molecule. these molecules represent a specific epitope of an antigen. these vaccines are also capable of inducing immunity against different strains of a specific pathogen by forming noncontiguous and immunodominant epitopes that are usually conserved in the strains of the pathogen [ ] . the production of peptide vaccines is extremely safe and cost-effective, especially when they are compared to conventional vaccines. traditional vaccines that prevent emerging infectious diseases (eids) are very difficult to produce because they require the need to culture pathogenic viruses in vitro. however, epitope-based peptide vaccines do not require any means of in vitro culturing which makes them biologically safe, allowing a large scale of bioprocessing to be carried out rapidly and economically. finally, their selectivity allows a precise activation of the immunological responses by means of selecting immunodominant and conserved epitopes [ , ] . the complexity of an epitope-based peptide vaccines' design depends largely on the properties of its carrier molecules' reactogenicity as well as its allergenicity [ , ] . when it comes to the selection of epitopes, it is based on the analysis of the b cells, cytotoxic t cells, and the induction of the helper t cells. then, it is important to identify the epitopes capable of activating t cells vital for stimulating a protective immunity. one of the issues concerning peptide vaccines representing t cells in a human population and that are highly mhc-heterogeneous is to identify the highly conserved immunodominant epitopes that are considered to be among a broad spectrum of vaccines due to their ability to work against multiple serovars of pathogens [ ] . in this study, we have used a variety of bioinformatics tools for the prediction of epitopes along with the population coverage and epitope selection algorithms, including the translocation of peptides into mhc class i and mhc class ii. [ ] were then used to analyze the candidate epitopes. conservation regions were determined using multiple sequence alignments with the help of clustal-w in the bioedit software version . . [ ] . epitope conservancy prediction for individual epitopes was then calculated using the iedb analysis resource. conservancy can be defined as the portion of a protein sequence that restrains in which an epitope is measured at or which that is exceeding a specific level of identity [ ] . the physicochemical properties of the retrieved sequence, molecular weight, and amino acid composition were also determined by using bioedit software. candidate epitopes were analyzed using several b cell prediction methods to determine their antigenicity, flexibility, hydrophilicity, and surface accessibility. the predicted linear epitopes were obtained from the immune epitope database (http://tools.iedb.org/ bcell/result/) [ ] using a bepipred test with a threshold value of . and a window size of . . moreover, surface accessible epitopes were predicated with a threshold value of . and a window size of . using the emini surface accessibility prediction tool [ ] . kolaskar and tongaonkar antigenicity methods (http://tools.iedb.org/bcell/result/) were also proposed to determine the sites of antigenic epitopes with a default threshold value of . and a window size . [ ] . [ ] . the artificial neural network prediction method was chosen to identify the binding affinity of mhc ii grooves and mhc ii binding core epitopes. all epitopes that bind to many alleles at a score equal to or less than , halfmaximal inhibitory concentration (ic ), were selected for further analysis. coverage. the population coverage of each epitope was calculated by the iedb population coverage tool at (http://tools.iedb.org/tools/population/iedb_input). this tool was used in order to determine the fraction of individuals predicted to respond to a given set of epitopes, with known mhc restrictions [ ] . for every single population coverage, the tool computed the following information: ( ) predicted population coverage, ( ) hla combinations recognized by the population, and ( ) , which was in a complex with an azobenzene-containing peptide [ ] . all water molecules and heteroatoms in the retrieved target file uq were then removed. the target structure was further optimized and energy minimized using swiss pdb viewer v. . . software [ ] . molecular docking was performed using autodock . software, based on the lamarckian genetic algorithm, which combines energy evaluation through grids of affinity potential to find the suitable binding position for a ligand on a given protein [ , ] . polar hydrogen atoms were added to the protein targets, and kollman united atomic charges were computed. the targets' grid map was calculated and set to × × points with a grid spacing of . Ǻ. the grid box was then allocated properly in the target to include the active residue in the center. the genetic algorithm and its run were set to as the docking algorithms were set on default. finally, results were retrieved as binding energies and poses that showed the lowest binding energies in which they were visualized using ucsf chimera. parameters. the physicochemical properties of the nipah virus glycoprotein g protein was assessed using bioedit journal of immunology research software version . . . . the protein length was found to be amino acids, and the molecular weight was at . daltons. the amino acids that form the nipah virus glycoprotein g protein are shown in table along with their numbers and molar percentages in (mol%). the ref sequence of the nipah virus glycoprotein g was subjected to a bepipred linear epitope prediction. emini surface accessibility and kolaskar and tongaonkar antigenicity methods in iedb were used to determine bindings to the b cell and in testing its surface and immunogenicity. the results are shown in figures - . mhc class i alleles. the nipah virus glycoprotein g sequence was analyzed using the iedb mhc class i binding prediction tool based on ann-align with half-maximal inhibitory concentration ðic Þ ≤ ; the least most promising epitopes that had a binding affinity with the class i alleles along with their positions in the nipah virus glycoprotein g are shown in table . mhc class ii alleles. the nipah virus glycoprotein g sequence was analyzed using the iedb mhc class ii binding prediction tool based on nn-align with half-maximal inhibitory concentration ðic Þ ≤ . the list of the epitopes and their correspondent bindings to mhc class ii alleles, along with their positions in the nipah virus glycoprotein g, while the list of the most promising epitopes that had a strong binding affinity to mhc class ii alleles and depending on the number of their binding alleles is shown in table . a population coverage test was performed to detect all the epitopes that bind to mhc class i alleles and mhc class ii alleles available in the database in relation to the world, south asia, southeast asia, sudan, and north africa. traditional vaccination approaches depend on the total amount of pathogens that are either live-constricted or inactivated. among the significant issues, these vaccines have brought along pivotal security concerns. in light of the fact that they are being utilized for vaccination, this may have caused them to become actuated and may also cause contamination. additionally, due to the varied hereditary pathogen strains found in the world, vaccines are probably going to lose their viability in various areas or even in certain populations. however, novel vaccine approaches such as dna-and epitope-based immunizations may possibly conquer obstructions for this type of immunization approaches, making them increasingly successful, explicit, and longlasting in vulnerable reactions with insignificant structures and without any undesired impacts [ ] . moreover, many peptide-based vaccines have been effectively proposed through utilizing in silico approaches against madurella mycetomatis, mokola rabies virus, lagos rabies virus, and others [ ] [ ] [ ] [ ] [ ] [ ] . such investigations, in regard to those viruses, have built up immunoinformatics in the computational analysis field. in our present work, potential peptides were suggested to design an epitope-based vaccine for nipah virus, using the latest amino acid sequences of glycoprotein g (glycoside hydrolase family) for a total of strains of nipah virus that were retrieved from the ncbi database (https://www.ncbi .nlm.nih.gov/protein) [ ] on july after the last outbreak at the end of may in kerala-india according to the who report [ ] . figure summarizes the method of the present work. various literatures were surveyed to define the antigenic part of the virus. glycoprotein g was found to be on the outer surface of the virus which was chosen as our target. initially, we have evaluated the binding affinity of the virus to mhc alleles. this was done by submitting the protein reference sequence to iedb mhc, a binding prediction tool, based on the ann align method with i c ≤ [ ] for mhc class i molecules. peptides were found to bind to mhc class i with different affinities. it is well known that a better immune response depends on whether or not the recognition of epitopes by hla molecules with significant affinity is successful. therefore, a peptide recognized by its highest number of hla alleles has the best potential to induce a strong immune response, leading us to take into account the only three peptides found with a % conservancy. the conserved peptide flidrinwi was found to interact with alleles ( the reference sequence of nipah virus glycoprotein g was reanalyzed using the iedb mhc ii binding prediction tool based on nn-align with half-maximal inhibitory concentration ðic Þ ≤ [ ] . the analysis resulted in the prediction of peptides from which fswdtmikf, flidrinwi, and ilsafntvi were potentially proposed according to their high number of binding alleles ( , , and alleles, respectively). additionally, the sequence of nipah virus glycoprotein g was subjected to bepipred linear epitope prediction, emini surface accessibility, and kolaskar and tongaonkar antigenicity methods in iedb. unfortunately, the peptides with the strongest binding affinities, utilizing the three mentioned tests, were absent. population coverage results for the total peptides found and the proposed peptides binding to mhc classes i and ii alleles are summarized in tables and . obtained results from the bindings to mhc i alleles revealed a . % projected population coverage in the world, . % in southeast asia, . % in south asia, . % in north africa, and . % in sudan while the population coverage results for the total number of peptides binding to mhc ii alleles showed only a . % projected population coverage in the world, . % in southeast asia, . % in south asia, . % in north africa, and . % in sudan. the selected peptides were further subjected to both mhc i-and mhc ii-based population coverage analysis in the whole world, southeast asia, south asia, north africa, and sudan as shown in table . among the six primarily selected epitopes, the obtained results showed a very strong potential in proposing the epitope flidrinwi as a vaccine candidate compared to the rest, taking into consideration its overall epitope conservancy, population coverage, and its affinity for the highest number of hla molecules. furthermore, in silico docking was carried out to measure the binding efficacy between the proposed peptides and hla-a * : , in which it has been specifically chosen in relation to their contribution to several immunological and pathological diseases [ ] [ ] [ ] , although numerous investigations have shown a relationship between hla alleles and disease susceptibility, which defines defensive hla allelic associations that possibly permit a recognizable proof that pathogen epitopes are limited by particular hla alleles. these epitopes may then be fused into a vaccine design in the expectation that the immunization will be reproduced naturally [ , ] . calculations of the root mean square deviation (rmsd) between coordinates of the atoms and formation of clusters based on rmsd values have computed the resemblance of the docked structures. the most favorable docking is considered to be the conformation of the lowest binding energy. the least energy predictions of the peptide flidrinwi (- . kcal/mol) and the d structure of the allele and its peptide are shown in figure . furthermore, the monoisotopic mass, sum formula, and molecular weight of the three highly proposed peptides are shown in table . as a result of these interesting outcomes, formulating a vaccine using the suggested peptide is highly promising and encouraging to be highly proposed as a universal epitopebased peptide vaccine against nipah virus. the present study proposed a very promising epitope-based peptide vaccine against glycoprotein g of nipah virus. it is expected to be highly antigenic with a minimum allergic effect. the proposed peptide flidrinwi has a strong binding affinity to both mhc class i and mhc class ii alleles. moreover, it shows an exceptional population coverage result for both mhc class i and mhc class ii alleles in the whole world, southeast asia, south asia, north africa, and sudan. despite having to validate the findings of the current study, an in vivo assessment of the most promising peptides, journal of immunology research journal of immunology research namely, flidrinwi, tvyhcsavy, and fayshleri, is highly recommended and will serve as the ground data for such work as shown in figures - . the data used to support the findings of this study are available from the corresponding author upon request. the authors declare that they have no competing interests. nipah virus epidemic in 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epitope-based peptide vaccine against nipah virus a focus on vaccine development current protocols in microbiology a bioinformatics tool for epitope-based vaccine design that accounts for human ethnic diversity: application to emerging infectious diseases peptide vaccine: progress and challenges ncbi protein sequence database the immune epitope database (iedb) . design and evaluation of primer pairs for efficient detection of avian rotavirus development of an epitope conservancy analysis tool to facilitate the design of epitope-based diagnostics and vaccines improved method for predicting linear b-cell epitopes a semi-empirical method for prediction of antigenic determinants on protein antigens immune epitope database analysis resource immune epitope database analysis resource (iedb-ar) predicting population coverage of t-cell epitope-based diagnostics and vaccines raptorx-property: a web server for protein structure property prediction ucsf chimera-a visualization system for exploratory research and analysis bioorthogonal cleavage and exchange of major histocompatibility complex ligands by employing azobenzene-containing peptides swiss-model and the swiss-pdbviewer: an environment for comparative protein modeling autodock and autodocktools : automated docking with selective receptor flexibility automated docking using a lamarckian genetic algorithm and an empirical binding free energy function a novel approach to vaccine design-epitopebased vaccines epitope-based peptide vaccine against fructose-bisphosphate aldolase ofmadurella mycetomatisusing immunoinformatics approaches epitope-based peptide vaccine design against mokola rabies virus glycoprotein g utilizing in silico approaches immunoinformatic approach for epitope-based peptide vaccine against lagos rabies virus glycoprotein g structural analysis and epitope prediction of hcv e protein isolated in pakistan: an insilico approach a computational assay to design an epitope-based peptide vaccine against saint louis encephalitis virus a highly conserved wdypkcdra epitope in the rna directed rna polymerase of human coronaviruses can be used as epitope-based universal vaccine design key concepts in genetic epidemiology basic molecular genetics for epidemiologists reverse immunogenetics: from hla-disease associations to vaccine candidates table : the most potential t cell epitopes (core sequence) and the number of their binding alleles. key: cord- -o wpcxdp authors: schmidt-küntzel, anne; dalton, desiré l.; menotti-raymond, marilyn; fabiano, ezequiel; charruau, pauline; johnson, warren e.; sommer, simone; marker, laurie; kotzé, antoinette; o’brien, stephen j. title: conservation genetics of the cheetah: genetic history and implications for conservation date: - - journal: cheetahs: biology and conservation doi: . /b - - - - . -x sha: doc_id: cord_uid: o wpcxdp from allozymes in to whole genomes in , genetic studies of the cheetah have been extensive. in this chapter we provide an overview of the available literature. overall, patterns of genetic variation provided evidence of low variability and suggest this loss occurred thousands of years ago. differences between published subspecies were supported genetically. at a local scale, populations were generally considered panmictic with minor genetic structure. although cheetahs have persisted despite low genetic variability, important questions arise from these findings: does the cheetah have the ability to adapt to and evolve with future changes in environmental and infectious pressure? how would cheetahs cope with further loss of genetic diversity? connectivity in the wild should be maintained via prevention of habitat loss, while management of small isolated populations may require reestablishing gene flow. genetics could assist captive-breeding decisions and provide forensic evidence as to the geographical origin of illegally traded animals. the cheetah (acinonyx jubatus) is one of the most recognized examples of the important links between evolutionary history, genetic variation, and conservation. its value to the biodiversity of the world is not only warranted by its unique physical characteristics, such as being the fastest land mammal (chapter ), but also its unique evolutionary lineage as the only extant representative of its genus, acinonyx. concerns over levels of genetic variation among cheetahs were first raised as captive programs grappled with difficulties in breeding cheetahs (chapter ). these observations led to research investigating the biological basis of the low rates of captive breeding success ( %- %) and the concurrent high rate of infant mortality ( %) . this research led to the discovery of low levels of genetic diversity in the cheetah, which were attributed to one or several severe population bottlenecks. as a consequence, debates arose regarding the impact of low genetic diversity on the survival of the species, and the cheetah has been featured in genetic textbooks since the s. early research on cheetah represented one of the first studies in the new field of conservation genetics. in the last century, cheetah numbers have declined drastically due to loss of habitat and prey, persecution due to real or perceived livestock depredation, and removal from the wild to supply captive facilities and private individuals (chapters , , , and ) . the reduction in numbers and fragmented distribution add to the urgency to preserve the genetic diversity left today. in this chapter, we review the current status of cheetah genetics and its impact on the species' conservation. while publications on cheetah genetics may appear contradictory at times, the fundamental conclusions have been consistent for over years, with various measures confirming low genetic diversity (section "genetic diversity"), which was shown to have originated thousands of years ago (section "historic demography"), and with a relatively recent divergence of extant published subspecies (section "subspecies definition and divergence"). the differences debated among geneticists only affect the interpretation of genetic results regarding precise timing of events and extent of reduced genetic diversity. this chapter also covers the cheetah's phylogenetic (evolutionary relation based on genetic data) position among other felids (section "species-level taxonomy"), the genetic structure of the subspecies and within geographical regions (section "phylogeography"), an overview of additional genetic studies including kinship (section "additional insights into cheetah genetics"), and implications of genetic findings for cheetah conservation (section "discussion"). the cheetah is a member of the family felidae ( fig. . ), which comprises living species that are distributed throughout the world, with the exception of australasia and the polar regions (kitchener et al., ) . one of the most striking aspects of molecular genetic studies in the felidae was how rapidly felids evolved into eight different lineages (over a -million-year period), each with unique biogeographical histories. earlier groupings of felid lineages were largely based on morphological features and life-history patterns. the cheetah was generally considered to be an early divergence from the felid radiation due to some of its unique adaptations, including its incompletely retractile claws (chapter ). however, the advent of genetic approaches has provided clarity to more confidently reconstruct felid evolutionary history, and today the cheetah is included in the puma lineage, which was the sixth of eight lineages to branch off during felid evolution [ million years ago (mya); johnson et al., ; li et al., ; werdelin et al., ; fig. . ] . the cheetah's closest living relatives are known to be the puma (puma concolor) and the jaguarundi (herpailurus yagouaroundi) . the cheetah (johnson et al., ; li et al., ; werdelin et al., ; fig. . ) , with whom the cheetah likely shared a common ancestor and evolutionary history prior to their divergence. today the cheetah is found in the old world, and the puma and jaguarundi in the new world (chapter ). the cheetah has chromosomal pairs (i.e., chromosomes) like most felid species . the cheetah is the only extant representative of its genus. genetic variation (polymorphism) forms the raw material of evolution. novel variation rises from dna mutations. dna mutation rates that alter the amino acid sequence and may be detectable in allozyme migration rates (section "allozymes") are several orders of magnitude slower than those observed in the mitochondrial dna (section "mitochondrial dna") or in repetitive elements in nuclear dna, such as microsatellites (section "microsatellites"). while significant gain of genetic variation is limited by mutation rates and takes numerous generations, variation can be lost within a single generation if only a subset of the population reproduces due to either high death rates or a high number of nonreproducing individuals. the first study to indicate reduced genetic diversity of the cheetah documented low variation at protein-based markers compared to other felids and mammals (o'brien et al., ; o'brien et al., ; et al., ) . thus, it was surprising that analyses of southern african cheetahs failed to identify genetic polymorphisms across a total of allozyme loci (o'brien et al., ; o'brien et al., ; however, insights from these studies were limited, because protein-based markers only assess amino acid changes that alter the electrophoretic mobility of the protein. dna changes, such as silent/synonymous substitutions are masked in proteins, resulting in an underestimation of the amount of genetic variation in all species. in addition, protein-based markers are more likely to be linked to functional differences and as such allele frequency differences may be susceptible to selective pressure (environmental conditions affecting survival of organisms with a particular characteristic). the subsequent development of dna-based markers provided more detailed information about the degree of genetic diversity in cheetahs. concurrently with allozyme studies, functional studies demonstrated that reciprocal skin allografts between unrelated cheetahs and siblings showed no signs of acute graft rejection, whereas xenografts (from domestic pokorny et al. ( ) ; in gir; sachdev et al. ( ) . et al., ) . these results were attributed to reduced functional allelic variation at the cheetah's major histocompatibility complex (mhc), an important immune gene family, which encodes cell surface proteins responsible for distinguishing foreign from self molecules. the inferred reduced functional variation was ultimately supported by molecular studies (section "major histocompatibility complex"). as sequencing techniques became available, low levels of genetic variation were also observed in mitochondrial dna (mtdna). mtdna is independent from the nuclear genome, and represents the maternal demographic history. the mitochondrial genome evolves faster than the nuclear coding genome, with the control region (cr) being the most rapidly evolving region of the mitochondrial genome. mtdna, in particular the cr, has been informative for investigations of diversity patterns, population structure, and phylogeography (phylogenetic structure in relation to location). the complete cheetah mtd-na genome has , bp (burger et al., ) and has % similarity with the mtdna genome of the domestic cat (lopez et al., ) . in the s, a study based on restriction fragment length polymorphism (rflp) inferred low levels of nucleotide variation ( . % diversity) in cheetah mtdna relative to comparable studies in other species (menotti-raymond and o'brien, ; table . ). nucleotide variation was even lower when a short sequence of the mtdna coding region was included with the mtdna-cr (charruau et al., ) , or when the entire mtdna genome was evaluated (dobrynin et al., ; table . ). dobrynin et al. ( ) found a % reduction in nucleotide variation across cheetahs ( namibian, tanzanian) relative to other mammals (dobrynin et al., ; microsatellites (nuclear dna markers consisting of variable numbers of tandem repetitions of - nucleotides; also called str or short tandem repeats) accumulate new variation quickly as they have high mutation rates (several orders of magnitude higher than dna coding for proteins), and are usually not associated with any function (i.e., they do not code for a protein) and thus are not subjected to selection pressure. these markers have been used widely in conservation genetics, population genetics, and wildlife forensics. most non-domestic felid studies have selected a subset from polymorphic microsatellite loci, which were mapped and characterized in the domestic cat (menotti-raymond et al., ) . initial studies, with microsatellite markers in cheetahs, demonstrated low observed heterozygosity ( . ; menotti-raymond and o'brien, ; table . ). expected heterozygosity was slightly higher ( . - . ) when measured in randomly selected microsatellite markers in cheetahs (driscoll et al., ; table . ). these estimates were comparable to the heterozygosity levels of other felids in small isolated populations (e.g., pumas from idaho, lions from serengeti and ngorongoro crater), but lower than in domestic cats (driscoll et al., ; table . ) . subsequent studies of cheetahs only included a subset of these microsatellite markers, which were not selected randomly, but instead for their known relatively high level of polymorphism, leading to higher heterozygosity estimates. hence, these increased heterozygosity estimates do not reflect the genetic diversity of the cheetah species per se, but are summarized in table . for informational purposes. terrell et al. ( ) suggested a reduction in genetic diversity in the wild population from a dataset spanning years. the study was based . the cheetah on individuals born between and from south africa and namibia, which were genotyped (characterized on a genetic level) with microsatellite markers. while microsatellite markers demonstrated levels of heterozygosity in the cheetah that were not always significantly lower than for other species (table . ), this does not contradict findings of low genetic diversity in cheetahs at other, more slowly evolving markers. it merely indicates that the variation at microsatellite loci is more recently evolved in origin. the length of time needed to accumulate this new microsatellite variation can inform estimates of the timing of events that led to the loss of genetic variation (section "historic demography"). the mhc is one of the most polymorphic loci known in vertebrates and has important immune functions. an increasing number of studies have documented an association between the diversity of mhc genotypes or individual alleles with disease susceptibility in wildlife, thus confirming the importance of the selection pressure from pathogens on the mhc (reviewed in sommer, ) . most comprehensive studies of the mhc in felids (also known as the feline leucocyte antigen), have been conducted in the domestic cat (winkler et al., ) . the cat is a good model as the general architecture of the mhc appears relatively conserved within each class of vertebrates; the number of mhc class i or ii genes, however, can vary substantially among species. the domestic cat mhc region is located on chromosome b and includes mhc i and mhc ii genes (yuhki et al., ) . the cheetah mhc sequence resolved genes with complete homology to the domestic cat for all mhc ii and most mhc i genes. its structural organization was also found to be highly similar to that of the domestic cat (dobrynin et al., ) . an early study of cheetah mhc i based on rflp markers showed reduced genetic diversity in cheetah (observed heterozygosity = . - . ) compared to other species, which was only comparable to that of lions from isolated populations (gir forest and ngorongoro crater; yuhki and o'brien, ; table . ). only mhc i alleles were identified in individual cheetahs through sequencing (yuhki and o'brien, ; table . ) and mhc ii-drb alleles were identified in individuals through reference strand-mediated conformational analysis (drake et al., ; (table . ). while a -fold increase in the number of mhc i alleles identified in may appear like an increase in genetic diversity, the identification of only additional alleles despite a -fold increase in the number of study animals is in fact further confirmation of low levels of allelic diversity in the cheetah. the low level of allelic diversity was further confirmed by comparison to other species, which harbor more alleles in fewer individuals (table . ). more recently, a %- % reduction in single nucleotide variants (snvs) was observed when the complete mhc sequence of cheetahs ( namibian, tanzanian) was compared to that of human (homo sapiens), dog (canis familiaris), and an outbred domestic cat; only variants affecting the amino acid sequence were detected in the mhc i coding region of cheetahs (dobrynin et al., ; dobrynin et al. ( ) described patterns of diversity across the entire genome of the cheetah. five commonly employed metrics confirmed the genic and genomic lack of diversity of the species: snv incidence was % less than that observed in a feral domestic cat; snv density was - × less than in the domestic cat, . the cheetah european wildcat (felis silvestris silvestris), or human (however it was higher than in lions from the gir forest); regions of continuous homozygosity (identical alleles at a given locus/set of loci) were - × longer than in the domestic cat; heterozygosity levels were %- % of the levels observed in the domestic cat, tiger (panthera tigris), and human; snvs in coding genes were % reduced compared to the domestic cat or european wildcat (dobrynin et al., ; table . ; fig. . ). since the initial discovery of reduced genetic diversity in the cheetah in the s, conservation, and scientific interest have turned toward identifying its cause (section "historic demography") and assessing the impact of low genetic diversity on the cheetah's chances of long-term survival (section "importance of low genetic diversity on cheetah survival"). the cumulative results, indicative of reduced genetic diversity in the cheetah, were consistent with a genetic bottleneck or a series of demographic reductions over time and space. menotti-raymond and o'brien ( ) proposed a scenario of distant past, rather than recent, reduction in the global population size. the demographic event causing this drastic loss of diversity was estimated to have occurred during the end of the pleistocene ( , - , years ago; table . ). this proposal was based on the time estimated for the near-reconstitution of genetic variation at rapidly evolving minisatellite (nuclear dna marker consisting of variable numbers of tandem repetitions of - nucleotides) loci. the authors obtained similar estimates with mtd-na rflp data calibrated on estimates of divergence of the species from the panthera genus. this estimated timeframe was later corroborated with microsatellite markers (driscoll et al., ) , with the time needed for nuclear alleles to reach fixation in the mhc (castro-prieto et al., ) , and with whole genome data (dobrynin et al., ) (table . ). in the asiatic cheetah population a more recent, independent, bottleneck was inferred based on a significant heterozygosity excess observed in the microsatellite loci tested (charruau et al., ) . several alternative hypotheses to the single bottleneck scenario have been proposed to explain the severe loss of genetic variation. first, that the uniformity resulted from a persistent low effective population size (ne; theoretical number which roughly reflects the number of animals genetically contributing to the population), possibly resulting from the high reproductive variance linked with the cheetah's presumed polygynous mating system (pimm et al., ) . second, that low effective population sizes were maintained by a continuous cycle of extinction of subpopulations followed by recolonization, that is, metapopulation dynamics (gilpin, ; hedrick, ; pimm et al., ) . the availability of snvs derived from whole genome data of individuals from both eastern and southern africa permitted more robust analyses of historical demographic patterns (dobrynin et al., ) . these analyses support the premise that cheetah populations expanded uniformly following a founder event , years ago. this was followed by a more recent split into an eastern and southern population, which were subjected to a bottleneck around , - , years ago. an alternative scenario of a gradual decline in the effective population size was supported by analyses of diploid whole genome sequence data to estimate past population sizes (table . ). fabiano et al. (in preparation) suggested a gradual decline in population numbers, commencing at least , years ago, based on different coalescent-based approaches applied to published microsatellite profiles in the namibian cheetah. while there was evidence . the cheetah of a continuous decline during this time period, some methods suggest an accelerated decline around , and , years ago (table . ). prior to the availability of genetic data, the species' taxonomy was based on morphological and geographical information. according to these taxonomic criteria, the extant cheetah populations were classified into four african and one asiatic subspecies (smithers, ) a. j. jubatus and a. j. raineyi were the first two subspecies to be assessed with molecular tools. initial allozyme analyses in detected some minor differences (o'brien et al., ; table . ). the separation of the two sub-saharan populations was further supported with mtdna-cr (freeman et al., ) , microsatellites (driscoll et al., ) , and whole genome variation (dobrynin et al., ; . the cheetah single subspecies (kitchener et al., ) . additionally, kitchener et al. ( ) put forward that as additional data become available the four subspecies that the iucn cat specialist group currently recognizes may be further merged in the future. however, this does not affect the amount of diversity found between the populations identified to date, which we will present here. a. j. soemmeringii was first compared to a. j. jubatus in based on mtdna (freeman et al., ) (kelly et al., ) ; years (modified from kelly et al., ) ; years (marker and o'brien, ) . c cal: reference time point used as calibration; µ: mutation rate given per generation; mya, million years ago. d /µ × g (nei, ) ; coal, coalescent methods, msvar . (storz and beaumont, ) , vareff (nikolic and chevalet, ) , diyabc-fda (cornuet et al., ) ; dadi, diffusion approximation to the allele frequency spectrum (gutenkunst et al., ) ; fixation n e , fixation of a neutral nuclear marker is expected after n e generations (nichols, ) ; psmc, pairwise sequential markovian coalescent (li and durbin, ) ; smm, stepwise mutation model (valdes et al., ) . e an accelerated decline may be present , and , years ago. a. j. hecki was not specifically assessed, as none of the studies was able to include confirmed a. j. hecki samples from west africa. historically, the distributions of each subspecies were defined based on morphological differences and presumed connectivity between populations. once the subspecies were confirmed genetically, the distribution of each subspecies and their genetic structure could be verified (fig. . a) . charruau et al. ( ) obtained a sample collection of cheetahs from countries from the extant and historical cheetah range. these samples were expected to represent four of the five cheetah subspecies recognized at that time. a. j. jubatus was confined to individuals from southern african countries, which included botswana, south africa, and namibia. these samples consistently clustered (grouped) together, with both nuclear (microsatellite) and mtdna data. depending on the type of analyses, a single cheetah sample from the democratic republic of congo grouped with a. j. jubatus, or slightly outside. the mtdna haplotype (linked group of variants that is inherited together) group of a. j. jubatus was the most diverse ( haplotypes) of the investigated sample collection and was centrally positioned in the mtdna haplotype networks, with the haplotypes of the other subspecies radiating from it. haplotypes assigned to a. j. raineyi were confined to east african countries, which included kenya and tanzania. however, the maternal lineages (mtdna) fell into separate haplotype groups, one of which clustered with a. j. jubatus, separately from other a. j. raineyi haplotypes. as a consequence, a. j. raineyi has been included in a. j. jubatus by kitchener et al. ( ) a. j. venaticus was confined to extant samples from iran. historical samples of asiatic cheetahs (oman, jordan, india, iraq, medieval iran) clustered with the extant iranian cheetah samples, as observed with both nuclear and mtdna data. additionally, one sample from the extinct population in northeastern egypt also clustered with the asiatic cheetah samples of a. j. venaticus. historical samples from libya appeared distinct based on mtdna data. with microsatellite data the libyan sample clustered more closely to, but separately from, the asiatic cheetah (bootstrap support of % for the divergence between the branch of the asiatic samples and the libyan sample). cheetahs from southern egypt, western sahara, and algeria shared the mtdna haplotype with the libyan samples, and were separate from the a. j. venaticus haplotypes. thus the mtdna data supported the asiatic subspecies . the cheetah (marker and o'brien, ) ; cal, reference time point used as calibration; clo, reference time point used for molecular clock; µ, mutation rate given per generation; mya, million years ago. d migr, isolation with migration rate = , conservation method (wakeley and hey, ) ; (δµ) , microsatellite genetic distance (goldstein and pollock, ; zhivotovsky and feldman, ) ; apd, average percent difference; coal, coalescent method (gaggiotti and excoffier, ) ; d, nei's raw number of nucleotide differences between populations (nei and li, ) ; d a , net number of nucleotide differences between populations (nei and li, ) ; d sw , stepwise weighted genetic distance (shriver et al., ) ; ima, isolation with migration, demographic method (hey and nielsen, ) ; k p, kimura's -parameter model (kimura, ) ; prop unique, proportion of unique alleles. . the cheetah boundaries suggested by nowell and jackson ( ) for a. j. venaticus, with cheetahs from the northern sahara being of a differtent subspecies (belbachir, ; krausman and morales, ) . the cheetahs from the northern sahara may be of the same subspecies as the west african cheetah (a. j. hecki), but this could not be confirmed, as no west african samples were available. a. j. soemmeringii was confined to northeast african countries, which included sudan, djibouti, ethiopia, and somalia. although these samples clustered together, some substructuring was identified. in freeman et al. ( ) , of the a. j. soemmeringii mtdna haplotypes was more similar to the a. j. raineyi haplotype ( substitution apart) than to the other a. j. soemmeringii mtdna haplotypes ( substitutions apart). this likely reflects a more complex migration/colonization history, imperfect lineage sorting, or weakly defined boundaries of subspecies. all the a. j. soemmeringii mtdna haplotypes shared a -amino acid deletion in the mtdna-nd protein, which, if confirmed, could be used as diagnostic site to trace illegally traded specimens. globally, there was no evidence of gene flow between the subspecies in recent generations, as no admixture was detected with bayesian analysis of population structure. genetic differentiation was further supported by the neighbor-joining (nj) phylogenetic tree based on microsatellite markers. patterns of genetic variation identified broad regional populations in southern africa: namibia, the kalahari, and south africa, when analyzing microsatellite loci in cheetahs from botswana, namibia, and the northwestern parts of south africa (fig. . b ; kotze et al., ) . f st values between namibia and south africa ( . ) and namibia and the kalahari ( . ) were higher than those between south africa and the kalahari ( . ). however, these values remain below the f st values recommended for high statistical certainty of population assignment ( . - . ; manel et al., ) . also, while the significance of the differentiation between populations (p < . ) suggests that accurate population assignment may be possible, attempts to assign cheetahs of unknown origin to a specific region (from a bayesian exclusion test method and a frequency-based method) were inconclusive. the efficacy of assignment testing in cheetahs will most likely be improved through more comprehensive sampling (more subpopulations and more individuals per region; manel et al., ) . mtdna data may also help to resolve questions of origin within southern africa, given that haplotypes were identified in a bp sequence of mtdna in botswana, namibia, and south africa (charruau et al., ) . the namibian cheetah population was the first national population to be assessed genetically. marker et al. ( ) analyzed microsatellite markers in unrelated individuals whose distribution covered most (over %) of the cheetah's natural distribution in the country (fig. . b ). no regional structure was detected when analyzing samples individually. when individuals were grouped by region, very modest support was given to a tentative grouping of the north-western regions (outjo, grootfontein; nj tree, bootstrap support of %) and of the southern regions (windhoek and gobabis; bootstrap support of %). multiple component analysis distributed the regions somewhat according to their geographical location, with the most northern region (outjo) appearing to be most distinct, but f st values for okahanja and outjo (f st = . ) were below the recommended threshold for separate population assignment ( . - . ; manel et al., ) . overall it was concluded that the namibian cheetah population is panmictic (without barrier to breeding and without population structure), with gene . the cheetah flow maintained between the studied regions and precluding the emergence of any major structure. connectivity was further supported by identification of two migrants to and from the outjo region. more recently, castro-prieto et al. ( ) reached similar conclusions through analyses of exon of mhc class i, and class ii-drb genes that were genotyped with the single strand conformational polymorphism method in individuals from the north-central region and individuals from the east-central region (fig. . b ). all identified alleles and resulting haplotypes were present in both regions. no regional differentiation was detected for mhc ii-drb, although haplotype frequency for mhc class i varied between north-central and eastcentral namibia with a moderate f st support (f st = . ; p < . ). the difference in allele frequency at mhc class i (which targets intracellular pathogens) was attributed to different viral selective pressures in the two regions. botswana dalton et al. ( ) conducted an analysis of the botswanan population ( fig. . b) in unrelated animals with microsatellite loci. although this study included a small sampling number, it still provided essential insights into the lack of population structure in botswana. all animals were assigned to one unique group. absence of substructure was further supported from the analysis of molecular variance ( % of variation shared among localities) and low genetic distance between the two largest sampled populations (ghanzi vs. jwaneng f st = . ; p < . ). weak subdivision among the geographical populations suggests that gene flow occurs, which can be attributed to natural cheetah movements. the moremi population appeared slightly "distinct" from the neighboring ghanzi population (f st = . ), which was tentatively attributed to the presence of the okavango delta as potential natural barrier between these two populations. genetic analyses of fecal samples in the serengeti identified multiple paternities for subsequent litters ( females), as well as within litters ( / litters); observed adoption events could also be confirmed genetically (gottelli et al., ) . in namibia of females with cubs were confirmed as biological mothers, and of presumed sibling groups without a dam were confirmed to be related (marker et al., ) ; in all exceptions ( mothers and sibling groups) individuals had been sampled from captive facilities with suspected humaninduced animal grouping. coalition males appeared to be related in namibia based on of male coalitions (marker et al., ) ; while in botswana, of wild-caught coalitions appeared to include at least one unrelated individual (dalton et al., ) . all three unrelated namibian groups and one of the unrelated botswanan groups dispersed at the time of release, suggesting that the grouping may have been an artifact of capture. it was also shown that females with higher levels of genetic relatedness had greater homerange overlap (marker et al., ) , suggesting a matriarchal society. the identification of the infectious agent responsible for one of the better documented viral outbreaks in the captive cheetah population (chapter ) was made possible due to samples properly stored since the outbreak in the early s (pearks wilkerson et al., ) . using a phylogenetic approach, the virus was identified as a coronavirus, very similar to domestic cat coronavirus responsible for feline infectious peritonitis. however, in cheetahs, morbidity . the cheetah was % (symptoms included diarrhea, jaundice, and seizures) and overall mortality was % within years ( % in cubs). this was significantly higher compared to the %- % mortality in domestic cat or the corresponding coronavirus (sars) outbreak in humans in , making this the deadliest documented death toll of a coronavirus. the authors attributed the high death toll to the lack of genetic diversity and the naïveté of the cheetah population to this virus, which appeared to have jumped from the domestic cat into the cheetah species. a new species of infectious blood-borne parasite, babesia lengau, was characterized genetically in cheetah (bosman et al., ) . while, as opposed to its effect in domestic cats, babesia is not currently known to cause any pathology in the cheetah, additional research is continuing to investigate the effect of babesia on specific health parameters (schmidt-küntzel et al., in preparation) . a single nucleotide polymorphism (snp), described in the serum amyloid a (saa) gene, was genotyped in captive cheetahs to assess whether there was a correlation between amyloidosis disease status (chapter ) and the snp genotype (franklin et al., ) . it was found that the snp had a semidominant effect on the associated protein level within each study population (n = ), but that the institution at which the animals were housed had an even larger effect. in addition, there was no significant association between genotype and disease status (n = ). thus, the genetic impact of saa on amyloid levels in cheetahs is minimal and outcompeted by other factors. no correlation could be detected between variants in the mtdna genome and myelopathic pathology (burger et al., ) . and to date no correlation could be identified between oxalate nephrosis and the coding regions of published candidate genes (cheetah conservation fund and national zoological gardens of south africa, unpublished data). the cheetah has long been known for its poor sperm quality, with less than % viable sperm observed in reproductive studies (chapter ). in a whole genome study comparing the cheetah sequence to that of other species, several mutations affecting gene function were found in a-kinase anchor protein (akap ), a gene involved in spermatogenesis, and were shown to be likely fixed (only allele present in the population) in the cheetah (dobrynin et al., ) . those mutations may be in part responsible for the documented poor sperm quality. other phenotypes (heritable traits that can be seen/measured) of interest observed in the cheetah are kinked tails, crowded incisors, palatal depression, and coat variations (chapter ). while no molecular work has been performed on the morphological traits to date, insight was gained on several coat related phenotypes. genes from the keratin-associated protein family were found to be expressed at higher levels in the yellow background of the cheetah fur (kaelin et al., ) , which is consistent with observations that fur of the black spots is softer relative to the coarser textured yellow background. conversely, genes responsible for pigmentation were expressed at higher levels in the black spots relative to the less pigmented yellow background of cheetah fur (hong et al., ) . another gene whose expression was increased in the black spots was a paracrine hormone, which was hypothesized to be involved in coordination of the spot pattern (kaelin et al., ) . the genetic basis for the king cheetah coat variant was determined to be a mutation in the transmembrane aminopeptidase q gene (kaelin et al., ) . rare cases of gross morphological deformities have occurred . the cheetah in the past, but no literature is available to substantiate whether they were based on low genetic diversity, inbreeding in a captive setting, or teratogenic influences. in a recent genome-wide analysis of the cheetah (dobrynin et al., ) signatures of positive selection (by comparison with lion, tiger, cat, human, and mouse) were identified in close to a thousand genes. ten of the genes were involved in muscle contraction (both cardiac and striated muscles), specifically the mitogen-activated protein kinase pathway (which is linked to stress), and in the regulation of catabolic processes, indicating that the cheetah underwent some degree of specialization in these pathways. in the same study over million nucleotides of segmental duplications were identified, and affect genes that are believed to be involved in energy balance, nutrition, and sensory adaptation. in addition, gene expansion was observed in the mhc extended class i region, which includes vomeronasal receptors, as well as olfactory and g-coupled receptor genes; these gene expansions were tentatively linked to behavior (pheromones) and physiology (e.g., ldh-a and ldh-b are linked to a carnivorous diet). both segmental duplications and gene expansions lead to temporary redundancy, allowing new gene functions to arise. evidence of historical positive selection on antigen binding sites that interact directly with pathogen-derived proteins was detected for both mhc classes, particularly mhc i (castro-prieto et al., ) . signatures of selection in the mhc were also identified in the whole genome study (dobrynin et al., ) . a study of the cytochrome p gene (cyp d ), involved in drug metabolism, showed considerable genetic diversity and signs of relaxed selection pressure in felids, including cheetahs (schenekar et al., ) . the most notable and still poorly understood feature of cheetah evolutionary history is how the cheetah has persisted in spite of remarkably low levels of genetic variation. genomic variation is generally considered to be crucial for long-term survival of species as it provides potential for adaptive responses (natural selection of advantageous genetic variation) to environmental changes, such as climate change (chapter ), and adaptability of immunity to disease outbreaks (o'brien and evermann, ) . therefore, the initial discovery of genetic uniformity of the cheetah was quickly followed by concerns about the species' chances of long-term survival. however, the discovery that the event or events leading to the loss of diversity could be placed over , years ago and that cheetah numbers had recovered by the th century, indicate the cheetah's ability to survive and thrive, despite reduced levels of genetic diversity, over extended periods of time. however, this does not guarantee the cheetah's survival in the future, as lack of genetic diversity limits the ability to adapt and evolve, in particular in the light of major changes in environmental conditions or pathogenic pressure. reduced genetic variation, particularly at adaptively important mhc loci, has been associated with high susceptibility to infectious diseases in captive cheetahs o'brien et al., ; o'brien and evermann, ) . a prime example is the high death toll caused by a coronavirus outbreak in a north american zoo (section "genetic investigations of infectious diseases affecting cheetahs," chapter ). despite this, free-ranging cheetahs from eastern and southern africa show robust health (caro ; munson et al., ; munson et al., ; thalwitzer et al., ) and do not seem to have compromised immunocompetence (castro-prieto et al., ) . in addition, in the wild the cheetah's . the cheetah large home ranges (chapter ) reduce the risk of infectious disease transmission. however, this may not be sufficient to protect the species in the event of an emerging disease, especially given the low levels of mhc diversity. at an individual level manifestation of deleterious traits caused by excessive levels of homozygosity (inbreeding depression) appear to be limited compared with the puma population in florida (florida panther), which suffered from atrial defects, poor sperm quality, cryptorchidism, and high disease load (roelke et al., ) . the cheetah is only known to suffer from poor sperm quality; none of the other traits observed in the cheetah (e.g., kinked tails, crowded incisors; section "investigations of the molecular basis for heritable traits") are detrimental to individual health, or the capacity to survive and reproduce. this, and evidence of positive signatures of selection on genes involved in muscle contraction and stress metabolism (section "signatures of selection, copy number variation, and changes in gene families"), suggests that the low levels of genetic diversity were caused, or followed by, strong selective pressures, which perhaps purged deleterious alleles from the species (e.g., hedrick and garcia-dorado, ). despite limited sperm quality, cheetah matings produce sufficient viable cubs (up to cubs per litter every years; chapter ) to maintain and even increase the population. however, further loss of genetic diversity could impair reproductive success, which is the ultimate requirement for species survival. indeed, increased infant mortality was observed in captive inbred individuals . with the reduction of its natural range, cheetah numbers are declining (durant et al., ; chapter ) , and most cheetah populations today are fragmented with loss of connectivity between them (chapter ). small populations, such as the critically endangered iranian cheetah (a. j. venaticus; chapter ), which has the lowest amount of genetic diversity of all the currently recognized cheetah subspecies (charruau et al., ) , are particularly at risk of losing further genetic diversity. therefore, it is crucial to maintain or regain sufficiently large population sizes and connectivity, while preserving existing variation through viable long-term storage of sperm and oocytes (chapter ). whenever possible, animals should remain in, or if captured, be returned to the wild (chapter ). captured animals that are not suitable for release back into the wild should be considered for breeding programs (section "genetic diversity and ex situ cheetah conservation"). wild cheetah populations of the same subspecies and geographical region were generally panmictic; minor population structure was only observed in allele frequencies of the rapidly evolving immune response genes. as such, translocations of wild caught individuals performed as part of conservation actions within these populations, only mimic natural connectivity (which may be restricted by anthropogenic barriers to gene flow). an additional level of complexity arises when populations are from different subspecies. while in principle exchange between populations of different subspecies should be avoided as they may be considered evolutionary significant units (moritz, ) , a compromise between preserving the existing structure and the urgency to rescue a small population at risk of disappearing, may have to be reached. relatively recent times of subspecies divergence and the merging of two subspecies in , are additional considerations during such decision making processes (section "subspecies definition and divergence"). this dilemma may have to be addressed for the iranian population at some point if the numbers remain below individuals (chapter ). additional information on the genetic health of the existing population is critically and urgently needed to determine if this population is likely to survive without management actions. captive cheetah populations have been established in part to serve as a reservoir for the wild population (chapter ). breeding decisions are guided by the genealogical data managed through the regional and international cheetah studbooks (chapter ). reputable captive programs (chapter ) aim to retain % of genetic diversity over years (lacy, ) . however, this may not be sufficient in the long term as the small number of founders only represents a subset of the genetic diversity found in the wild (founder effect) and the % genetic diversity lost is irreversible. this loss of diversity can only be compensated for by the recovery of lost breeding lines through "reinjection" of viably preserved reproductive material (e.g., gametes) of founders into the captive population, addition of new founder individuals (or gametes) obtained from the wild, or inclusion of unrelated captive individuals to the breeding pool. as with wild populations, interconnectivity of captive populations should be maintained as much as possible, through regional and interregional exchange of animals or reproductive material. as the cost of genetic research continues to decrease, it will provide the possibility to assess the genetic makeup of all captive cheetahs, and thus the integration of animals of unknown origin into the cheetah breeding pool, if genetic evaluation determines that they represent a new breeding lineage. of note, inbred individuals (high homozygosity levels) can also be used for further breeding if they represent a unique breeding lineage, as homozygosity is not heritable. the field of conservation genetics will see more advanced analyses emerging, including the evaluation of heritable traits, landscape genetics, and increased precision in assessing the extent and timing of the events that cause the loss of genetic diversity. non invasive samples are increasingly employed to provide answers regarding populations that have not yet been intensely studied (chapter ). additional research involving contemporary and museum samples is currently under way to fill existing knowledge gaps regarding the published subspecies (léna godsall bottriell, personal communication). however, it is crucial to remember that data obtained from genetic studies published to date agree sufficiently in confirming the low genetic diversity of the species at nonrepetitive loci (section "genetic diversity"), dating the origin of the low diversity to more than , years ago (section "historic demography"), providing support for genetic differences, although short divergence times, between the populations corresponding to most published subspecies (section "subspecies definition and divergence"), and showing only minimal population structure within geographical regions (section "phylogeography"). this in turn enables a joint message in terms of recommendations for cheetah conservation, as well as in situ and ex situ management (sections "genetic diversity and in situ cheetah conservation" and "genetic diversity and ex situ cheetah conservation"). we hope that by presenting all available data on cheetah genetics, this chapter provides clarity to the results and conclusions arising from the field of conservation genetics, and contribute to the global efforts for the cheetah's long-term survival. les grandes questions relatives à la conservation des grands félins d'algérie: cas du guépard et du léopard babesia lengau sp. nov., a novel babesia species in cheetah (acinonyx jubatus, schreber, ) populations in south africa analysis of the mitochondrial genome of cheetahs (acinonyx jubatus) with neurodegenerative disease cheetahs of the serengeti plains: group living in an asocial species phylogeography, genetic structure and population divergence time of cheetahs in africa and asia: evidence for long-term geographic isolates inference on population history and model checking using dna sequence and microsatellite data with the software di-yabc (v . ). bmc bioinf social and genetic population structure of free-ranging cheetah in botswana: implications for conservation the use of reference strand-mediated conformational analysis for the study of cheetah (acinonyx jubatus) feline leucocyte antigen class ii drb polymorphisms genomic microsatellites as evolutionary chronometers: a test in wild cats the global decline of cheetah and what it means for conservation serum amyloid a protein concentration in blood is influenced by genetic differences in the cheetah (acinonyx jubatus) sequence variation in the mitochondrial dna control region of wild african cheetahs (acinonyx jubatus) a simple method of removing the effect of a bottleneck and unequal population sizes on pairwise genetic distances the genetic effective size of a metapopulation launching microsatellites: a review of mutation processes and methods of phylogenetic inference genetic analysis reveals promiscuity among female cheetahs inferring the joint demographic history of multiple populations from multidimensional snp frequency data understanding inbreeding depression, purging, and genetic rescue integration within the felsenstein equation for improved markov chain monte carlo methods in population genetics digital gene expression for non-model organisms the late miocene radiation of modern felidae: a genetic assessment specifying and sustaining pigmentation patterns in domestic and wild cats a simple method for estimating evolutionary rate of base substitutions through comparative studies of nucleotide sequences . a revised taxonomy of the felidae. the final report of the cat classification task force of the iucn cat specialist group the power of resolution of microsatellite markers and assignment tests to determine the geographic origin of cheetah (acinonyx jubatus) in southern africa acinonyx jubatus achieving true sustainability of zoo populations phylogenomic evidence for ancient hybridization in the genomes of living cats (felidae) inference of human population history from individual whole-genome sequences complete nucleotide sequences of the domestic cat (felis catus) mitochondrial genome and a transposed mtdna tandem repeat (numt) in the nuclear genome demography of the serengeti cheetah (acinonyx jubatus) population: the first years detecting wildlife poaching identifying the origin of individuals with bayesian assignment test and multilocus genotypes captive breeding of the cheetah (acinonyx jubatus) in north american zoos ( - ) molecular genetic insights on cheetah (acinonyx jubatus) ecology and conservation in namibia second-generation integrated genetic linkage/radiation hybrid maps of the domestic cat (felis catus) dating the genetic bottleneck of the african cheetah evolutionary conservation of ten microsatellite loci in four species of felidae defining 'evolutionarily significant units' for conservation serosurvey of viral infections in free-ranging namibian cheetahs (acinonyx jubatus) extrinsic factors significantly affect patterns of disease in free-ranging and captive cheetah (acinonyx jubatus) populations molecular evolutionary genetics mathematical model for studying genetic variation in terms of restriction endonucleases biochemical genetic variation in eight endangered or threatened felid species gene trees and species trees are not the same variation of effective population size. r package version (vareff) wild cats. status survey and conservation action plan interactive influence of infectious disease and genetic diversity in natural populations conservation genetics of the cheetah-lessons learned and new opportunities an atlas of mammalian chromosomes genetic basis for species vulnerability in the cheetah the cheetah in genetic peril east-african cheetahs-evidence for two population bottlenecks the cheetah is depauperate in genetic variation coronavirus outbreak in cheetahs: lessons for sars plausible alternatives to bottlenecks to explain reduced genetic diversity mhc class i and mhc class ii drb gene variability in wild and captive bengal tigers (panthera tigris tigris) the consequences of demographic reduction and genetic depletion in the endangered florida panther. curr major histocompatibility complex class i polymorphism in asiatic lions isolation and characterization of the cyp d gene in felidae with comparison to other mammals a novel measure of genetic distance for highly polymorphic tandem repeat loci the mammals of africa. an identification manual the importance of immune gene variability (mhc) in evolutionary ecology and conservation testing for genetic evidence of population expansion and contraction: an empirical analysis of microsatellite dna variation using a hierarchical bayesian model continued decline in genetic diversity among wild cheetahs (acinonyx jubatus) without further loss of semen quality seroprevalences to viral pathogens in free-ranging and captive cheetahs (acinonyx jubatus) on namibian farmland allele frequencies at microsatellite loci: the stepwise mutation model revisited estimating ancestral population parameters felid phylogenetics and evolution genetic characterization of fla, the cat major histocompatibility complex sequences, annotation and single nucleotide polymorphism of the major histocompatibility complex in the domestic cat dna variation of the mammalian major histocompatibility complex reflects genomic diversity and population history exchanges of short polymorphic dna segments predating speciation in feline major histocompatibility complex class i genes microsatellite variability and genetic distances key: cord- -fod xkd authors: summerfield, artur; mccullough, kenneth c. title: the porcine dendritic cell family date: - - journal: dev comp immunol doi: . /j.dci. . . sha: doc_id: cord_uid: fod xkd considering the pivotal roles played by dendritic cells (dcs) in both innate and adaptive immune responses, advances in the field of porcine immunology dc biology have recently progressed rapidly. as with the more extensively studied murine and human dcs, porcine dc can be generated from bone marrow haematopoietic cells or monocytes, and have been analysed in various immunological and non-immunological tissues. both conventional dc (cdc) and plasmacytoid dc (pdc) have been characterized. the function of porcine monocyte-derived dc has not only been characterized in terms of antigen presentation and lymphocyte activation, but also their response to various ligands of pattern recognition receptors. these have been characterized in terms of the induction of dc maturation and pro-inflammatory, th -like or th -like cytokines secretion. porcine pdc most effectively sense virus infections and are characterized by their capacity to produce large quantities of ifn-α and the pro-inflammatory cytokines tnf-α, il- and il- . as such, the dc family as a whole is a powerful ally in the host battle against pathogen attack. nevertheless, dc in particular tissue environments or under particular stimuli can down-regulate immune response development. this is not only important for preventing over-activation of the immune system and also for ensuring tolerance against self or “friendly” substances including food components, but may also be used as a mechanism of pathogens to evade immune responses. dendritic cells (dcs) are a heterogeneous group of potent antigen-presenting cells (apcs) with the unique capacity to prime naive t-cell responses [ ] . in order to fulfil their role as sentinels of the immune system, they express several families of specialized pattern recognition receptors (prrs) for particular pathogenassociated molecular patterns (pamps). these include toll-like receptors (tlrs), nucleotide-binding oligomerization domain (nod)-like receptors (nlrs), retinoic acid-inducible gene i (rig-i)like receptors (rlrs) and c-type lectin receptors (clrs) all reacting directly with pathogen components [ ] [ ] [ ] . in addition, many other receptors exist such as fc receptors and activated complement component receptors, reacting with complexes of pathogen antigen with antibody or complement. another element essential to dc biology is their migratory behaviour in response to chemokine gradients. dcs are the main cellular element controlling t lymphocyte activation and regulation. furthermore, they are involved in b-cell responses, possibly through the delivery of native antigen to b lymphocytes [ ] [ ] [ ] , but certainly through the production of b-cell stimulatory factors important for b-cell proliferation, differentiation and isotype switching [ , ] . dcs also play a functional role for nk cell activation [ , ] , and are in a two-way communication with neutrophils [ ] . understanding dc biology requires the consideration of the heterogeneity and independence of dc functional plasticity. an important element therein is the functional and phenotypic differentiation of ''conventional dc'' (cdc) from ''plasmacytoid dc'' (pdc). the term cdc summarizes all dc subsets with ''professional antigen presenting'' function, while pdc represent the ''professional interferon-a producers'' [ ] . indeed, pdc are also referred to as ''natural interferon producing cells'' (nipc), a functional entity first described years ago [ ] . however, more recent studies have demonstrated that also pdc have important antigen-presenting functions and that the two dc subsets complement each others by having a distinct regulation of mhc class i-and ii-dependent antigen presentation [ ] [ ] [ ] . furthermore, dcs show a high level of heterogeneity, particularly in the specialized roles of various dc subsets dependent on their tissue localization and local immunological environment, which guides their function (table ) . one general functional consideration of all dc subsets is their critical roles as immunological sentinels. being strategically located at sites of pathogen entry, such as mucosal surfaces and considering the pivotal roles played by dendritic cells (dcs) in both innate and adaptive immune responses, advances in the field of porcine immunology dc biology have recently progressed rapidly. as with the more extensively studied murine and human dcs, porcine dc can be generated from bone marrow haematopoietic cells or monocytes, and have been analysed in various immunological and nonimmunological tissues. both conventional dc (cdc) and plasmacytoid dc (pdc) have been characterized. the function of porcine monocyte-derived dc has not only been characterized in terms of antigen presentation and lymphocyte activation, but also their response to various ligands of pattern recognition receptors. these have been characterized in terms of the induction of dc maturation and proinflammatory, th -like or th -like cytokines secretion. porcine pdc most effectively sense virus infections and are characterized by their capacity to produce large quantities of ifn-a and the proinflammatory cytokines tnf-a, il- and il- . as such, the dc family as a whole is a powerful ally in the host battle against pathogen attack. nevertheless, dc in particular tissue environments or under particular stimuli can down-regulate immune response development. this is not only important for preventing over-activation of the immune system and also for ensuring tolerance against self or ''friendly'' substances including food components, but may also be used as a mechanism of pathogens to evade immune responses. ß elsevier ltd. all rights reserved. dermal layers, dc can rapidly interact with pathogen pamps resulting in cell activation, pathogen uptake and degradative processing of the pathogen (antigen). a critical element therein is the concomitant uptake of antigen with pamp recognition. the latter represents the ''danger'' signalling which the dc requires to be activated. in the absence of the ''danger'' signal, the dcs tend to function more as tolerogenic dc, inducing lymphocyte anergy. such processes are involved in responses to self-antigens and tolerance of food antigens. following interaction with a pathogen (and its pamps), cdc endocytic activity-particularly macropinocytosis-is enhanced during the first h after stimulation, followed by down-regulation as the dc mature. the latter relates to a prolonged capacity for efficient presentation of the endocytosed and processed antigen [ ] . with completion of the dc maturation process, important biological changes occur to the dc. chemokine receptor expression is modified, enabling migration to the inductive sites of the adaptive immune system. for example, the ccr and ccr receptors for inflammatory chemokines such as ccl and ccl are down-regulated, while the ccr receptor for ccl and ccl is up-regulated [ ] . the latter provides dc with the signal for entry into the inductive sites of the adaptive immune system such as the lymph node. dc activation also results in an increase of cell surface mhc and co-stimulatory molecule expression-such as cd and cd -as well as the production of immunoregulatory and/or inflammatory cytokines. mhc class ii antigenic peptide complexes are stabilized on the cell surface to ensure efficient stimulation of t-cell responses. overall, the induced functional changes are usually associated with increased t-lymphocyte stimulatory capacity, although this depends on the stimulus received [ ] . clearly, the interaction of microbial pathogens with dc can provide insight into the pathogenesis of and defence against infectious diseases. moreover, the central role of dc in immune defence development makes them a prime target for vaccines and immunotherapies. recent advances in porcine immunology have allowed the characterization of the porcine dc system in this direction (as witnessed by the identified cell types shown in table ). this has also been possible through the availability of antibodies against cell surface markers classified in three international swine cd workshops [ ] [ ] [ ] and studies using crossreactive antibodies-summarized in table . by such means, rapid advancement in the current knowledge on porcine apc and the diversity of porcine dc function has been forthcoming. the subsequent sections of this review will present these advancements. similar to other species, porcine dc can be generated by stimulating blood monocytes with interleukin- (il- ) and granulocyte macrophage-colony-stimulating factor (gm-csf). after - days of culture, non-adherent or loosely adherent cells with dendritic morphology can be harvested [ , ] . generation of monocyte-derived dc (modc) in vitro can also employ a gm-csf/ ifn-a combination. this can prove more potent than the gm-csf/ il method when seeking dc for restimulating virus-specific cytotoxic t-cells [ ] . addition of ifn-a to the gm-csf/il- cocktail also influences the dc, resulting in an enhanced t-cell a two subsets. b variable, two subsets with some animals (see also fig. ). c can be negative in mucosal tissue [ ] . d not determined. e four subsets based on cd a/cd ri expression and dependent on localization in lp, pp and mln [ ] . f negative on lp dc [ ] . g two subsets: cd a + cd + cd + and cd a À cd À cd À . stimulatory capacity in mixed leukocyte cultures [ ] . in fact, cytokine modulation is important for manipulating the type of dc generated. tgf-b permits the generation of cells with langerhans cell characteristics [ ] , while pamps modulate mrna expression levels for particular tlrs [ ] . bautista et al. [ ] have also demonstrated that il- can be replaced by il- for the generation of modc. phenotypically, porcine modc are characterized as cd + cd + cd + cd / + cd a + and mhc class ii + [ , , [ ] [ ] [ ] (table ) . from a comparative immunological point of view, it was unexpected to find cd , because human cd is considered to be a typical monocyte/macrophage rather than a dc marker [ ] . nevertheless, modc from other species such as cat, cattle and dog have also been shown to express cd [ ] . of the other markers, the cd a was expected due to its classification as the porcine swine workshop cluster (swc ) antigen expressed on cells of the myelomonocytic lineage [ ] . it is expressed on many monocytic and granulocytic cells quite early during their differentiation [ ] . functionally, this marker represents the signal regulatory protein alpha (sirp-a). altogether, the co-expression of cd a and cd along with relatively high levels of both cd / and mhc class ii represent phenotypic characteristics of porcine modc but no marker clearly differentiating them from monocyte-derived macrophages has been identified. porcine modc generated with gm-csf and il- relate to human modc in that they are in an immature state, and represent a convenient cell culture model to study the dc maturation process. akin to their human counterparts, porcine modc up-regulate cd / , mhc classes i and ii, and t-cell stimulatory activity upon maturation, while inflammatory chemokine receptors such as ccr and macropinocytic activity are down-regulated [ , , , [ ] [ ] [ ] . porcine dc, which phenotypically and functionally resemble modc, can also be generated from bone marrow haematopoietic cells (bmhc), stimulated with gm-csf plus tnf-a, or gm-csf alone, for - days-bmdc [ ] . addition of stem cell factor to the gm-csf/tnf-a cocktail increases the yield of dc obtained [ ] . in contrast to gm-csf, flt ligand (flt l) stimulation induces the differentiation of both cdc and pdc, similar to the situation with human and mouse bmhc [ ] (guzylack-piriou and summerfield, unpublished data). furthermore, flt l-induced bmhc-derived cdc phenotypically and functionally differ from gm-csf-derived dc either generated with monocytes or bmhc. flt l induces the differentiation of cd À cdc which are more sensitive to stimulation by tlr /tlr , tr , tlr , tlr and tlr ligands in terms of cytokine responses and maturation [ ] (guzylack-piriou and summerfield, unpublished data). this may relate to the central role of flt l in the generation of dc from a clonogenic bmhc dc precursor [ ] . porcine blood is similar to human blood in representing an important source of apc. several populations have been identified, including monocytes, dc precursors and fibrocytes. all of these express cd a, and in a model of antigen presentation of inactivated foot-and-mouth disease virus (fmdv) to t lymphocytes the activation was dependent on the presence of cd a + cells [ , ] . similar results were also obtained with classical swine fever virus (csfv) and protein antigens derived from this virus [ , ] , as well as with tetanus toxoid [ ] . separating the cd a + pbmc into a major population of cd + monocytes and a minor population of cd À cells showed that the latter contained the cd À blood cdc along with the cd + pdc (table ; [ ] ). it is notable that although the blood cdc and pdc differ phenotypically (cd expression), they are similar in their lack of cd expression, which distinguishes them from in vitro generated modc. however, this simple discrimination is not absolute. the composition of blood apc is more complex, as summarized in fig. . accordingly, it is necessary to appreciate each of the blood apc in turn, to obtain a better understanding of their roles and function in immune defences. porcine blood carries a subpopulation of pbmc, which are cd a + cd À cd À , with characteristics of cdc-high levels of mhc class ii and cd / ( fig. and table ), nonadherence and potent t-cell stimulatory capacity [ ] . after in vitro culture, these cells strongly up-regulate mhc and co-stimulatory molecules, and their dendritic morphology becomes clear. based on these characteristics, we have proposed that this population contains the precursors of blood cdc. these cells have variable expression of cd and cd and can be differentiated from monocytes by lower levels of cd a ( fig. and [ ] ). although the majority of porcine blood monocytes are cd À cd À cd + cd + cd a + (table and fig. ), a detailed analysis of the monocytic population has identified at least four subsets based on cd and cd [ ] . it is currently not clear how the blood cdc population phenotypically defined as cd a+cd À cd À cell relates the cd À cd + monocytic cell. cd is proposed as a macrophage marker, which is up-regulated during the differentiation of monocytes to macrophages [ ] but down-regulated if monocytes are induced to differentiate into dc by gm-csf/il- [ ] . this would argue for two distinct subsets of cells, a point which is not surprising considering the diversity of the dc family, but still requires further clarification (see also dominguez et al., this volume). based on the frequent expression of the cd r marker on dc in the mucosa [ , ] , this marker has also been proposed for identification of cdc in the blood and other organs as a cd a + cd r + population [ , ] . however, this definition may not be sufficient as no functional studies have been described, and it does not consider that a subset of monocytes also express cd r [ ] . although several cell types can produce type i ifn upon viral infection, pdc are particularly adept at secreting very high levels of type i ifns [ ] . representing less of . % of the pbmc [ ] , porcine pdc were identified through their ifn-a responses to transmissible gastroenteritis virus (tgev) [ ] . charley and lavenant [ ] originally described them as being non-adherent, non-t, non-b, cd +, mhc-class-ii positive cells. subsequently, they went on to describe their ontogeny [ , ] and migration into lymphoid tissue after viral challenge in vivo [ , ] . recently, using the ovine and porcine models, it has been demonstrated that pdc also migrate in the afferent lymph of cannulated animals [ ] . phenotypically, porcine pdc can be clearly identified as cd a low cd high cd À cd À within the peripheral blood population (table ). they express no or low levels of the t-cell markers cd , cd and cd , as well as no cd [ ] , but can carry low levels of cd and moderate levels of cd (fig. ). related to their interaction with immune complexes porcine pdc express fc receptors including cd (fig. , [ ] ) and cd [ ] . in contrast to porcine pdc, ovine pdc do not express cd and cd a but are characterized by high expression of cd rb [ ] . porcine pdc appear to be the major dc population producing high quantities of ifn-a and tnf-( in response to cpg motifs [ ] . this clearly relates to the human dc system, placing the porcine dc system in line with that of humans and therefore distinct from the murine model [ ] . certainly related to their human and mouse counterparts is their ability to respond to many viruses by the production of large quantities of ifn-a (see below). with the major site for pathogen entry being through the mucosa of the respiratory and digestive tract, it is not surprising that most lymphocytes are in the mucosalassociated lymphoid tissues (malt). consequently, it is essential to regulate the mucosal immune responses against harmless microorganisms and food antigens in contact with the mucosae. this is a major role played by mucosal dc and is reflected by their anatomical localization in the mucosal tissues. they can mediate tolerance to self-antigens and harmless entities, while acting as sentinels sensing the danger posed by invading pathogens and deleterious entities. the first report on porcine mucosal dc described a putative dc in peyer's patches (pp) as mhcii + cell lacking t-and bcell markers [ ] . in the lamina propria (lp) of the small intestine the presence of an mhcii+cd a + cd r + cd + dc subset was demonstrated [ ] . bimczok and colleagues [ ] further char-acterized dc in other immunological sites of the gut and proposed four phenotypically distinct subsets of dc based on the expression of cd r and cd a. lp dc are mainly cd a + cd r +, pp dc are mainly cd a + cd r À in subepithelial domes and cd a À cd r À in interfollicular regions, and mesenteric lymph node (mln) dc are mostly cd a À cd r + . interestingly, only the cd r + dc subsets were present in lymph, suggesting that dc migration to mln originates largely from the lp. with respect to antigen sampling a rare population of lp dc extending cytoplasmic processes between enterocytes have been described [ ] . in the pp, it appears that antigen transfer from m cells to dc is an important process, as many dc in the subepithelial dome have been demonstrated to be adjacent to m cells [ ] . in contrast to the lack of cd a on many dc in the inductive interfollicular areas of the mln and pp, cd a is expressed on porcine dc is at peripheral sites of pathogen entry and antigen contact such as the skin, lp and pp subepithelial dome [ , , ] . this would indicate a possible down-regulation of this molecule during the maturation and migration process, and would be supported by the observed expression of cd a on modc, bmdc generated using either gm-csf or flt l, as well as circulating blood cd a + blood apc can be differentiated into cd + monocytic cells and cd À dc. two major subpopulations of cd À monocytic cells can be defined based on cd expression (depicted light blue; see also dominguez et al., this issue). furthermore, a small cd + cd + cd + subset with unknown function can be identified (dark blue). porcine blood dc express relatively low levels of cd a and lack cd and cd . while cdc are cd À (green), pdc express high levels of cd (red). (b) expression of cd , mhc class ii, cd , cd , cd and cd on cd a high cd À monocytes (light blue dots), cd a high cd + monocytic cells (dark blue dots), cd a low cd À cdc (green dots) and cd a low cd + pdc (red dots). a representative animal is shown. comparison of five different spf pigs of the similar age ( - month old) revealed high variability in the expression levels of mhc class ii, cd , cd and cd on the dc subpopulations. dc [ , , ] . nevertheless, with in vitro maturation studies using modc, bmdc as well as blood dc [ , , , , ] no loss of cd a was observed indicating that additional factors would be required for this process. an alternative explanation would be that cd a À dc represent lymph node tissue resident dc which are phenotypically distinct from in vitro generated dc. in humans, rat, cattle and sheep, the expression of cd a differentiates functionally distinct dc subsets. while cd a + dc are more stimulatory for t cells, it is possible that the cd a À subsets is specialized in the phagocytosis of apoptotic cells [ ] [ ] [ ] [ ] [ ] . future studies are required to clarify such functional differences in the pig. dcs from the porcine upper respiratory tract have also been described. in the tracheal mucosa many dc are located above the basal membrane and inside the epithelial layer where they form a dense network with many cytoplasmic processes probably related to their important role as immunological sentinels in this organ [ ] . the majority of these cells co-express cd and mhc class ii but not cd r . jamin et al. [ ] recently described putative dc in the tonsils by co-expression of cd r and cd (dc lamp) or coexpression of cd r and cd a. nevertheless, from this study it is unclear whether a cd a À dc would exist in this organ. it is also not yet clear how cd a is expressed in dc of non-mucosal lymphoid tissue. several functional properties have been assigned to mucosal dc originating from the gut including the capacity to imprint the mucosal homing receptors a b integrin and ccr on t and b lymphocytes, the secretion of cytokines of the mucosal microenvironment such as il- and tgf-b, the promotion of t regulatory and th rather than th responses, and the induction of iga secretion [ ] [ ] [ ] [ ] [ ] . considering that the capacity of gut dc to produce retinoic acid (ra) is a requirement for many of these functions and that gut dc are likely to be themselves under the influence of ra derived from gut epithelial cells, we have tested whether porcine modc can acquire the function of gut mucosal dc. after treatment of porcine modc with ra the dc acquired the capacity to promote a b integrin and ccr on t lymphocytes, to secrete tgf-b and to promote iga responses [ ] . we have extended these studies and also demonstrated that ra induces the expression of retinaldehydrogenase, a rate-limiting enzyme in the synthesis of ra. the drug-mediated inhibition of this enzyme in ra-treated dc abrogated their capacity to promote mucosal homing receptors expression on lymphocytes (saurer and summerfield, unpublished data). this underlines the important role of tissue-specific factors in governing dc function. nipc/pdc have been identified as ifn-a-positive cells by immunohistochemistry in the intestinal epithelial layer, the lp, near the pp and in the mln early after infection with transmissible gastroenteritis virus (tgev) [ ] . since this was associated with high levels of serum ifn-a and only few ifn-a producing cells were identified in other organs it appears that during an enteropathic virus infection ifn-a would almost exclusively originate from gut pdc triggered locally. the rapid ifn-a response of pdc as early as h after infection would imply that pdc are present in mucosal tissue fulfilling their role as sentinels. this relates to the recently identified ccr expression and migration of mouse pdc to the small intestine under steady-state conditions [ ] and also to the presence of pdc identified as cd a + cd + cells in tonsils and mln of healthy pigs [ ] . bautista et al. have isolated dc migrating from porcine skin explants. phentotypically these cells resemble modc in terms of cd a co-expression with cd and the high levels of mhc class ii and cd / (table ) . moreover, isolated skin-derived dc show variable expression of cd r , cd and cd [ ] . immunohistochemical analysis of porcine thymic tissue has shown dc to be large cells located in the medullary and the corticomedullary regions, as evidenced by the presence of surrounding hassall's corpuscles. porcine thymic dc have also been partially purified and characterized [ ] . they too relate to modc and skin dc in their cd , cd a and mhc class ii expressions, but additionally express cd (table ) , which can also be found on blood dc (fig. ) . croizet and colleagues [ ] demonstrated the value of the porcine model for characterizing dc from non-lymphoid organs, such as the thyroid dc. besides the more ''classical'' apc, a relatively recent addition has been described. fibrocytes are a blood-derived cell population with fibroblastoid morphology, which is distinct from dc. fibrocytes have been described for mice, humans and pigs [ , , ] , and represent . - % of nucleated cells in peripheral blood. they express cd and cd , which would suggest a haematopoietic origin, possibly myeloid. the presence of cd , cd and cd a on porcine fibrocytes [ ] , and the differentiation of human fibrocytes in vitro from a blood-derived cd + population [ ] support this hypothesis. porcine fibrocytes originate from a cd + pbmc subpopulation [ ] . fibrocytes are important during wound healing, rapidly entering sites of injury together with inflammatory leukocytes [ ] . they are also an important source of cytokines and chemokines important for t lymphocyte and dc development. these include il- , il- , macrophage-colony-stimulating factor, macrophage inflammatory protein- a, mip- b and monocyte chemoattractant protein- [ ] . it has been suggested that fibrocytes may play an early role in the induction of antigen-specific immunity [ , ] , in particular the activation of t helper lymphocytes [ ] . porcine fibrocytes were also shown to be potent apc, relating to their expression of mhc class ii, cd and cd /cd , as well as their endocytic activity [ ] . these cells activate cd + t cells, but also efficiently stimulate virus-specific cd + ctl. in fact, fibrocytes are effective at low ratios with t lymphocytes, ratios at which modc are less efficient [ ] . porcine fibrocytes also respond to tlr ligands by producing large quantities of il- [ ] . these tlr ligands include lps (tlr -ligand), lipopeptide (diacylated form recognized by tlr /tlr heterodimers; triacylated form recognized by tlr / tlr heterodimers), and tlr ligands [ ] . the importance of dc within the innate immune system is due to their recognition of pamps through their prrs. tlr play a central role in this concept of innate immune recognition. this results in a robust cytokine and chemokine response, along with dc activation and maturation, all essential for adaptive immune response development (fig. ) . a simplified concept is that antigen presentation in the absence of dc activation leads to tolerance or shortlived immune responses without immunological memory development [ ] . the current evidence shows that concomitant recognition of the antigen and a ''danger'' signal is essential for the dc to promote adaptive immune defence development [ ] . with self and food antigens, the absence of the ''danger'' signal ensure that the dc involved are tolerogenic. for efficacious immune defence development, the cell subsetspecific recognition of pamps plays a critical role, resulting in the dc-derived cytokines necessary for both the innate response and the strength and quality of the adaptive response. in addition to phenotypic differences, it is also in this area of pamp recognition that species-dependent differences are observed. consequently, it is important to understand how porcine apc recognition of pamp compared with human apc, and to determine the distribution of tlr within the porcine dc system. in fig. , a schematic overview of the functional specialization and cytokine production of porcine cdc and pdc is represented. the current knowledge on the cytokine responses of modc is also included in the overview provided by table . one of the important responses induced by tlr ligation on dc is the induction of maturation. as a simple read-out of this process increased levels of surface molecules involved in antigen presentation such as mhc class ii, cd and cd / are often used. nevertheless, the interpretations of such results alone do not permit definitive conclusions on the maturation status of a dc in terms of t-cell stimulatory activities [ ] . porcine modc as a model of cdc relate to those from other species in their response to tlr ligands by upregulation of mhc and cd / (table ). these include tlr ligands pam cys lipopeptide, pseudomonas opri lipoprotein, lipoteichoic acid (lta) and peptidoglycan [ , ] , tlr ligands such as synthetic doublestranded (ds) rna polyinosinic-polycytidylic acid (polyic) [ , , , ] , tlr ligand lipopolysaccharide (lps) [ , , ] and the tlr ligands r and polyuridylic acid [ ] . similar to human dc, both porcine modc and blood cdc have been reported to be unreceptive to the tlr ligand cpg-odn, when analysed for mhc class ii and cd / expression [ ] . nevertheless, modc express tlr mrna [ , ] , and cpg-odn can induce the mrna of tlr , tlr , ifn-g and il p , as well as increased levels mhc class ii as well as cd / [ ] . such discrepancies between the studies may have been caused by sequence differences in the odn employed or may reflect differences in the responsive status of the cells employed. for example, the relatively modest increase of cd / and mhc class ii expression on modc after tlr stimulation is synergistically enhanced in the presence of ifn-a [ ] . in this study it was also shown that a full phenotypic maturation only occurred during antigen presentation to t lymphocytes. we have also observed that in contrast to modc, flt -ligand derived bmdc are clearly more responsiveness to a range of tlr ligands including tlr , , , and (guzylack-piriou and summerfield, unpublished data). tlr ligation can induce cytokine mrna species for il- , il- p , il- and ifn-g in dc, although this depends on the tlr ligand employed [ ] . while polyic, lps, lta, and cpg induced the th promoting cytokines il and ifn-g, the th -like cytokines il- and il- were induced only by lps and lta (table ) . raymond et al. [ ] also demonstrated that similar to human and mouse dc, the cytokine response of porcine dc can be modulated by cytokines. for example, il- p mrna can be enhanced with tnf-a, il- and ifn-g and reduced with il- treatment of dc. along the same line, the th /th cytokine profile will depend on the type of antigen encountered [ ] . at the protein level modc have been reported to produce tnf-a and il- after stimulation with poly(ic), lps, opri and pam cys (both tlr ligands), while production of il- and il- has been difficult to detect by elisa [ , , , ] . related to the stimulation by these specific tlr ligands is the cytokine response of modc to heat-inactivated actinobacillus pleuropneumoniae in terms of il- , il- and il- secretion [ ] . modc also produce type i ifn after stimulation with poly(ic) and mrna transfection [ , , ] . the latter is dependent on the secondary structure of the mrna molecule forming dsrna structures [ ] . it is important to note that modc maturation and cytokine production is highly variable and subject to immunomodulation. another example for this is the observation that ra has a potent synergistic effect on il- secretion induced by tlr ligands [ ] . this relates to the fact that the dc family has a high diversity to deal with the varying environments and signals it receives in vivo from the local tissue environment and from pathogens. with in vitro analyses, it is only possible to reproduce a small fraction of this diverse scene. therefore, what we are observing in vitro is true for the conditions being created, but can not be taken as a general rule for all dc subsets under all conditions in vivo. as with cdc, the pdc can also upregulate mhc class ii and costimulatory molecules after in vitro culture [ ] and when stimulated with tlr ligands [ ] . however, the notable trait of pdc is their ability to produce inf-a similar to the human and mouse immune system, porcine pdc are the most potent producers of inf-a after stimulation with certain pamps. these pamps are the cpg type a motifs [ ] and tlr / ligands such as r [ ] . such characteristics show a particularly close relationship to the human dc system [ ] ; pdc respond to tlr and ligands, whereas cdc such as modc tend to recognize more tlr - ligands. in contrast, murine dc and macrophages also respond well to tlr ligands, although it is again the pdc which produce large quantities of ifn-( [ ] . dependent on the stimulus, porcine pdc also produce large quantities of tnf-a, il- and as mentioned above il- [ , ] . it is this il- induction, which distinguishes porcine pdc from their human counterparts [ ] . the dc family is particularly effective at sensing viruses through cytosolic and endosomal prr, which detect viral nucleic acid. important cytosolic receptors include the dsrna-dependent protein kinase r (pkr) and the rlr's rig-i and mda- [ ] . generally, such receptors recognize viral replication occurring in situ-being cytosolic, these prr will detect the intermediates, which are also usually cytosolic. while rig-i can sense triphosphorylated singlestranded rna, mda- appears to be more specific for dsrna, but the fine specifity within the helicase system is not entirely clear [ ] . although these receptors are ubiquitously expressed, their important role in the interaction of cdc with viruses has been demonstrated [ ] . this is different with the endosomal prr, all members of the tlr family including tlr , , and , which are expressed only on cells playing a specialized role in innate immune responses such as dc. tlr represents a receptor for dsrna, tlr and for single-stranded rna and tlr for cpgmotif containing dna. tlr will not only sense viruses with dsrna genomes but also endocytosed dsrna replicative intermediates produced during the replicative cycle of singlestranded rna viruses, released from dying cells in the vicinity. as summarized in table , the studies with porcine cdc and pdc demonstrate that similar to human and mouse, pdc produce high levels of ifn type i to most viruses studied including classical swine fever virus, fmdv, influenza virus, lentiviral vectors (lv), pseudorabies virus (prv) and tgev, while such responses are absent or weak with cdc. the only exceptions described to date are the porcine circovirus , which can suppress ifn response in pdc [ ] , and sendai virus which is a potent inducer of ifn type i in cdc [ ] . an important basis for the responsiveness of pdc to viruses is the expression of endosomal tlr and tlr , which can be triggered by dna and rna viruses respectively independently of viral replication [ ] . in addition, the constitutive expression of irf , the master regulator of type i ifn, represents a unique feature of pdc [ , ] . a good example showing the importance of pdc recognition of viruses is the response seen with csfv. this monocytotropic, haemorrhagic rna virus replicates efficiently in cdc without apparently inducing their activation or maturation [ ] . the lack of dc activation is not due to the absence of a trigger, because the virus generates a dsrna intermediate in infected modc theoretically capable of stimulating rlrs. csfv actively prevents the cdc response to dsrna through its non-structural n pro protein targeting the irf- pathway [ ] , on which cdc depend for induction of ifn [ ] . in contrast, in pdc csfv will induce ifn-( production [ ] . as mentioned above, these cells do not rely on irf- due to their high levels of constitutive irf- [ ] . the production of large quantities of ifn-( in the serum of csfv-infected pigs [ , ] presumably originating from pdc [ ] indicates the importance of pdc for systemic ifn-( responses, particularly when cdc activities are impaired by the pathogen. in fact, a number of rna viruses encode proteins interfering with cellular antiviral machinery and therefore prevent activation of cdc [ ] . also with porcine reproductive and respiratory syndrome virus (prrsv), a productive infection of cdc has been observed [ ] [ ] [ ] [ ] . this apparently does not result in the secretion of type i ifn, although ifn mrna is induced, indicating a block of the ifn system at the translational level [ ] . whether prrsv can activate pdc has not yet been described. in addition to the tlr-recognition of viral pamps, pdc possess other receptors to sense viruses. this has been demonstrated for tgev, an rna-genome coronavirus, which can activate pdc using a surface receptor interacting with the viral m glycoprotein [ ] . use of inactivated virus and subunit structures has demonstrated that this activation is independent of viral nucleic acid [ ] , excluding a role for tlr and tlr . moreover, mutation of the glycosylation site in the m protein yields a virus incapable of inducing pdc, but still capable of replication [ ] . also studies in our own laboratory support the conclusion of triggering through a cell surface receptor-inhibitors of endosomal acidification such as chloroquine and bafilomycin prevent cpg-induction, but not tgev-induction of ifna production by pdc [ ] . similar observations have been made with other viral proteins such as hiv-derived gp and human pdc [ ] . although porcine pdc like human pdc are highly efficient producers of ifna and effective at sensing virus infections, not all viruses will activate pdc so efficiently. nonenveloped viruses such as fmdv-an rna virus of the picornaviridae related to poliovirus and rhinoviruses-are less efficient to activate pdc. it should be noted that these viruses either fail to replicate in dc, or produce only an abortive infection [ ] [ ] [ ] . nevertheless, pdc can be efficiently activated by fmdv under particular conditions. in the presence of opsonizing factors such as virus-specific immunoglobulin (ig), which mediate fcgrii-enhanced uptake of virions, pdc activation was observed-an event dependent on the presence of intact and active viral rna [ ] . such an activity would provide important antiviral innate defences at a time early post-vaccination when the adaptive response had begun producing specific antibody, but is inadequate to protect the host from disease or virus replication. this function of pdc would have an additional advantageassisting cdc in promoting the development of an efficacious adaptive immune defence for protecting the host. fmdv can also infect cdc including skin dc and modc [ , ] , which results in low levels if ifn-b secretion. just as in vitro analyses cover only a proportion of likely events in vivo, focusing on phenotypic modulation of dc and cytokine profiles will reveal only part of the story. during immune defence development, these modulations of the dc serve a purpose beyond the direct attack on the pathogen by the dc. that additional purpose is promoting the functional interaction with the adaptive immune system. the interaction between t lymphocytes and dc is a bilateral process. on one side there is the central role of dc in presenting antigen and stimulating t cells. this has been shown in several models including mixed leukocyte reactions [ , ] , superantigen presentation [ ] as well as antigen specific t-cell restimulation [ , , , , ] (see also table ). on the other side, t lymphocytes provide important signals to dc. with porcine modc cultures we have observed that after the co-culture of cytokinematured dc with t cells in the presence of antigens, a further upregulation of mhc class ii expression was obtained reaching levels clearly above those obtained with any stimuli in the absence of tcells [ ] . interestingly, tnf-a pre-treatment of the dc was as efficient as tnf-a/ifn-a cocktails to sensitize the dc for this process. these in vitro observations indicate that modc maturation is a regulated multistep process. from a practical point of view this means that although tnf-a alone is not sufficient to induce modc maturation, the t-cell responses induced by tnf-a-treated dc can compensate to provide stimuli reaching those obtained with more potent maturation cocktails such as tnf-a/ifn-a or tlr ligands [ ] . moreover, the t-lymphocyte activity provides an additional advantage in that cocktails such as tnf-a/ifn-a, as well as those combined with tlr can have the drawback of ''exhausting'' the dc. similar to other species, it has been demonstrated that dc can modulate the type of t-cell response induced. for example, the th /th profile will be influenced by the type of antigen presented as well as by the cytokine environment [ ] . another example of such modulation is the observation that cholera toxin-treated dc have the capacity to suppress t-cell proliferation [ ] . this was associated with decreased mhc class ii expression and increased il- secretion of the dc and was reversible by addition of tnf-a suggesting an immunoregulatory process. prrsv has also been described by several authors to modulate dc and monocytes towards reduced t-cell stimulatory capacity after in vitro infection [ , ] . also here reduced expression of mhc and costimulatory molecules together with increased il- levels have been described [ ] [ ] [ ] ] . such studies are valuable to understand viral pathogenesis but care must be taken to avoid any contamination of the cultures with mycoplasma as modc cultures apparently efficiently support mycoplasma growth, which can result in a potent antiproliferative activity [ ] . dc not only determine the type of t-cell response but also their homing characteristics. as described in section ''mucosal dc'', modc treated with ra promote a b and ccr expression, representing essential gut homing receptors [ ] . classically, b lymphocytes equipped with their surface ig receptor will recognize native unprocessed antigen and would only require t-cell help for clonal expansion and differentiation into antibody producing cells. in this sense, b cells should show only an indirect requirement for apc such as cdc. nevertheless, apc produce a number of cytokines, which have a direct stimulatory effect on b cells. these are classical cytokines such as il- , il- and ifn-(/b, but also more recently identified cytokines such as the b-cell activating factor (baff) and a proliferation-inducing ligand (april)-members of the tumour necrosis factor superfamily [ ] . in addition, several studies also demonstrated the ''delivery'' of native unprocessed antigen by dc to b cells [ ] [ ] [ ] . our own studies with porcine dc in an in vitro model of fmdv-specific ig synthesis demonstrated an important direct role of apc's during antigenspecific restimulation of immune b lymphocytes. purified b cells produced virusspecific ig only in the presence of tcells and apc. monocytes and modc but not pdc supported b-cell differentiation into antibody-secreting cells. while il- could replace t-cells, addition of baff could compensate for a lack of apc. in fact, blocking of baff receptor abrogated the apc-derived help for bcell responses [ ] . in addition, dc also influence isotype switching. as mentioned above, ra-treated modc promote virus-specific iga secretion in vitro [ ] . not surprisingly, similar to the interaction with t lymphocytes, the interplay of dc with b lymphocytes is also bilateral. an important role is certainly played by the ''mé nage à trois'' of fcr expressed on dc, antibody and antigen. this permits an amplification of antigen uptake and presentation, and can also mediate efficient cross-presentation of antigen for stimulation of mhc class i-restricted t-cell responses. furthermore, the fcrsystem sensitizes dc for inflammatory and antiviral cytokine responses. one example is the response of pdc to fmdv mentioned above (table ). these pdc only respond by producing ifn-( to fmdv when the virus is complexed with antibodies [ ] . another example is with csfv, where pdc sensitized with cytophilic antibodies show enhanced ifn-( production in response to lower virus quantities than when no cytophilic antibodies are present [ ] . in both cases fcgrii is involved. recent advances in the characterization of the porcine immune system, particularly in porcine dc biology, have permitted the use of the porcine model for many immunological studies. although the library of reagents for such studies is still restricted compared to that for mouse and human studies, knowledge of porcine immunology is well advanced. with the unveiling of the sequence for the porcine genome, there will be clear advantages for using the pig. in particular, the modc model has a number of advantages and applicability. large numbers of dc can be generated without killing the animal. for example, a typical figure of - million dc can be generated using monocytes isolated from ml of blood. the facility to repeat blood sampling with the pig enables the use of these dc in antigen presentation assays, to monitor autologous tcell responses in immunization experiments with outbred animals [ ] . moreover, with the modc being in an immature state, another advantage is the possibility to study dc maturation in response to cytokines, tlr ligands and infections. considering that dc are a rare cell type within the leukocyte populations, the large size of the pig, its lymphoid organs and the availability of larger volumes of blood together with repeated samplings offer an advantage allowing considerable immunological progress. in addition to this facility of recovering large volumes of blood regularly from the same animal, cannulation procedures for porcine lymph vessels at both peripheral and mucosal sites are now available. this approach allows us the much sought ability of studying dc migrating from peripheral sites over periods of several days [ , ] , a procedure not possible with humans and certainly cumbersome with mice considering their size and the quantities of material obtained. moreover, the pig is more closely related to the human-both genetically and physiologically-when compared to mice. this is reflected by immunological similarities such as the prr and their cellular distributions [ , ] . finally, the advantages which porcine immunology has to offer have gained a further boost with the advent of novel technologies such as rna interference (rnai) [ ] , which can be combined with the generation of transgenic pigs [ ] . localization of distinct peyer's patch dendritic cell subsets and their recruitment by chemokines macrophage inflammatory protein (mip)- alpha, mip- beta, and secondary lymphoid organ chemokine myeloid c-type lectins in innate immunity cooperation of toll-like receptor signals in innate immune defence signaling pathways downstream of patternrecognition receptors and their cross talk dendritic cells interact directly with naive b lymphocytes to transfer antigen and initiate class switching in a primary t-dependent response critical role of itim-bearing fcgammar on dcs in the capture and presentation of native antigen to b cells cell surface recycling of internalized antigen permits dendritic cell priming of b cells immunobiology of dendritic cells dendritic cells, baff, and april: innate players in adaptive antibody responses pathogen-induced private conversations between natural killer and dendritic cells close encounters of different kinds: dendritic cells and nk cells take centre stage two way communication between neutrophils and dendritic cells ipc: professional type interferon-producing cells and plasmacytoid dendritic cell precursors human natural interferon-alpha producing cells direct proteasome-independent cross-presentation of viral antigen by plasmacytoid dendritic cells on major histocompatibility complex class i dendritic cell preactivation impairs mhc class ii presentation of vaccines and endogenous viral antigens intrinsic and cooperative antigenpresenting functions of dendritic-cell subsets in vivo enhanced dendritic cell antigen capture via toll-like receptor-induced actin remodeling dendritic cells in a mature age characterization of swine leukocyte differentiation antigens characterization of swine leukocyte differentiation antigens summary of the first round analyses of the third international workshop on swine leukocyte differentiation antigens porcine dendritic cells generated in vitro: morphological, phenotypic and functional properties functional and phenotypic characterization of distinct porcine dendritic cells derived from peripheral blood monocytes fibrocytes are potent stimulators of anti-virus cytotoxic t cells characterisation of porcine monocyte-derived dendritic cells according to their cytokine profile toll-like receptor, mhc ii, b and cytokine expression by porcine monocytes and monocytederived dendritic cells in response to microbial pathogenassociated molecular patterns il- replaces il- in development of monocyte derived dendritic cells (modc) of swine differentiation of porcine dendritic cells by granulocytemacrophage colony-stimulating factor expressed in pichia pastoris in vitro differentiation of porcine blood cd À and cd + monocytes into functional dendritic cells dendritic cells in different animal species: an overview a porcine cell surface receptor identified by monoclonal antibodies to swc is a member of the signal regulatory protein family and associates with protein-tyrosine phosphatase shp- porcine bone marrow myeloid cells: phenotype and adhesion molecule expression cholera toxin promotes the generation of semi-mature porcine monocyte-derived dendritic cells that are unable to stimulate t cells efficacy and functionality of lipoprotein opri from pseudomonas aeruginosa as adjuvant for a subunit vaccine against classical swine fever double-stranded secondary structures on mrna induce type i interferon (ifn alpha/beta) production and maturation of mrna-transfected monocytederived dendritic cells c-kit positive porcine bone marrow progenitor cells identified and enriched using recombinant stem cell factor summerfield a. toll-like receptor and myd knockdown by lentivirus-mediated rna interference to porcine dendritic cell subsets identification of clonogenic common flt + m-csfr + plasmacytoid and conventional dendritic cell progenitors in mouse bone marrow porcine peripheral blood dendritic cells and natural interferon-producing cells essential role of antigen-presenting cell-derived baff for antibody responses immunological properties of recombinant classical swine fever virus ns protein in vitro and in vivo an in vitro immune response model to determine tetanus toxoid antigen (vaccine) specific immunogenicity: selection of sensitive assay criteria phenotypic and functional heterogeneity of porcine blood monocytes and its relation with maturation the porcine a antigen is homologous to human cd and related to macrophage differentiation professional and non-professional antigen-presenting cells in the porcine small intestine sitespecific expression of cd b and sirpalpha (cd a) on dendritic cells: implications for their migration patterns in the gut immune system lactic acid bacterial colonization and human rotavirus infection influence distribution and frequencies of monocytes/ macrophages and dendritic cells in neonatal gnotobiotic pigs characterization of conventional and plasmacytoid dendritic cells in swine secondary lymphoid organs and blood workshop studies on monoclonal antibodies in the myeloid panel with cd specificity characterization of blood mononuclear cells producing ifn alpha following induction by coronavirus-infected cells (porcine transmissible gastroenteritis virus) ontogeny of interferon alpha secreting cells in the porcine fetal hematopoietic organs in vivo study of interferon-alpha-secreting cells in pig foetal lymphohaematopoietic organs following in utero tgev coronavirus injection in vivo induction of interferon-alpha in pig by noninfectious coronavirus: tissue localization and in situ phenotypic characterization of interferon-alphaproducing cells interferon-alphaproducing cells are localized in gut-associated lymphoid tissues in transmissible gastroenteritis virus (tgev) infected piglets plasmacytoid dendritic cells migrate in afferent skin lymph fcgammarii-dependent sensitisation of natural interferon-producing cells for viral infection and interferon-alpha responses type-a cpg oligonucleotides activate exclusively porcine natural interferon-producing cells to secrete interferonalpha, tumour necrosis factor-alpha and interleukin- of men, mice and pigs: looking at their plasmacytoid dendritic cells isolation and characterisation of pig peyer's patch dendritic cells phenotype and distribution of dendritic cells in the porcine small intestinal and tracheal mucosa and their spatial relationship to epithelial cells characterization and functional analysis of skin-derived dendritic cells from swine without a requirement for in vitro propagation plasmacytoid dendritic cell activation by foot-and-mouth disease virus requires immune complexes role of natural interferon-producing cells and t lymphocytes in porcine monocyte-derived dendritic cell maturation a discrete subpopulation of dendritic cells transports apoptotic intestinal epithelial cells to t cell areas of mesenteric lymph nodes dendritic cell heterogeneity in vivo: two functionally different dendritic cell populations in rat intestinal lymph can be distinguished by cd expression identification of two distinct populations of dendritic cells in afferent lymph that vary in their ability to stimulate tcells enrichment for a cd hi sirp-subset in lymph dendritic cells from the upper aero-digestive tract bidirectional negative regulation of human t and dendritic cells by cd and its cognate receptor signalregulator protein-alpha: down-regulation of il- responsiveness and inhibition of dendritic cell activation mucosal dendritic cells gut lymphocyte migration: we are halfway 'home' generation of gut-homing iga-secreting b cells by intestinal dendritic cells small intestine lamina propria dendritic cells promote de novo generation of foxp treg cells via retinoic acid reciprocal th and regulatory t cell differentiation mediated by retinoic acid in vitro induction of mucosa-type dendritic cells by all-trans retinoic acid ccr is a homing receptor for plasmacytoid dendritic cells to the small intestine dendritic cells enriched from swine thymus coexpress cd , cd and major histocompatibility complex class ii and actively stimulate alloreactive t lymphocytes culture of dendritic cells from a nonlymphoid organ, the thyroid gland: evidence for tnfalphadependent phenotypic changes of thyroid-derived dendritic cells circulating fibrocytes define a new leukocyte subpopulation that mediates tissue repair the peripheral blood fibrocyte is a potent antigen-presenting cell capable of priming naive t cells in situ peripheral blood fibrocytes: differentiation pathway and migration to wound sites responsiveness of fibrocytes to toll-like receptor danger signals regulated production of type i collagen and inflammatory cytokines by peripheral blood fibrocytes interaction of borrelia burgdorferi with peripheral blood fibrocytes, antigen-presenting cells with the potential for connective tissue targeting dendritic cells in immunity and tolerance-do they display opposite functions? the danger model: a renewed sense of self interaction of classical swine fever virus with dendritic cells effects of antigen and recombinant porcine cytokines on pig dendritic cell cytokine expression in vitro silencing of natural interferon producing cell activation by porcine circovirus type dna reading the viral signature by tolllike receptors and other pattern recognition receptors beyond doublestranded rnatype i ifn induction by prna and other viral nucleic acids the role of viral nucleic acid recognition in dendritic cells for innate and adaptive antiviral immunity subset-dependent modulation of dendritic cell activity by circovirus type natural interferon-alpha producing cells: the plasmacytoid dendritic cells irf- is the master regulator of type-i interferondependent immune responses classical swine fever virus npro interacts with interferon regulatory factor and induces its proteasomal degradation role of doublestranded rna and n(pro) of classical swine fever virus in the activation of monocyte-derived dendritic cells high ifnalpha responses associated with depletion of lymphocytes and natural ifnproducing cells during classical swine fever classical swine fever virus induces activation of plasmacytoid and conventional dendritic cells in tonsil, blood, and spleen of infected pigs the virus battles: ifn induction of the antiviral state and mechanisms of viral evasion phenotypic and functional modulation of bone marrowderived dendritic cells by porcine reproductive and respiratory syndrome virus porcine reproductive and respiratory syndrome virus productively infects monocyte-derived dendritic cells and compromises their antigen-presenting ability effects of porcine reproductive and respiratory syndrome virus-infected antigen-presenting cells on tcell activation and antiviral cytokine production differential type i interferon activation and susceptibility of dendritic cell populations to porcine arterivirus coronavirus pseudoparticles formed with recombinant m and e proteins induce alpha interferon synthesis by leukocytes interferon alpha inducing property of coronavirus particles and pseudoparticles single amino acid changes in the viral glycoprotein m affect induction of alpha interferon by the coronavirus transmissible gastroenteritis virus human immunodeficiency virus type gp and other activation stimuli are highly effective in triggering alpha interferon and cc chemokine production in circulating plasmacytoid but not myeloid dendritic cells constitutive expression of alpha interferon by skin dendritic cells confers resistance to infection by foot-andmouth disease virus dendritic cells internalisation of foot-and-mouth disease virus: influence of heparin sulphate binding on virus uptake and immune response induction innate immune responses against foot-and-mouth disease virus: current understanding and future directions interferon-alpha production by swine dendritic cells is inhibited during acute infection with footand-mouth disease virus porcine reproductive and respiratory syndrome (prrs) virus-specific interferongamma(+) t-cell responses after prrs virus infection or vaccination with an inactivated prrs vaccine identification of classical swine fever virus protein e as a target for cytotoxic t cells by using mrnatransfected antigen-presenting cells porcine reproductive and respiratory syndrome virus (prrsv) infects mature porcine dendritic cells and upregulates il- production mycoplasma contamination and viral immunomodulatory activity: dendritic cells open pandora's box efficient transgenesis in farm animals by lentiviral vectors phenotype of porcine monocytic cells: modulation of surface molecule expression upon monocyte differentiation into macrophages molecular cloning and characterization of a novel cd gene from the pig innate immune responses following emergency vaccination against foot-andmouth disease virus in pigs the fusarium toxin deoxynivalenol disrupts phenotype and function of monocyte-derived dendritic cells in vivo and in vitro dendritic cells harbor infectious porcine circovirus type in the absence of apparent cell modulation or replication of the virus key: cord- - m wz i authors: rall, glenn f.; mucke, lennart; nerenberg, michael; oldstone, michael b.a. title: a transgenic mouse model to assess the interaction of cytotoxic t lymphocytes with virally infected, class i mhc-expressing astrocytes date: - - journal: j neuroimmunol doi: . / - ( ) - sha: doc_id: cord_uid: m wz i astrocytes provide crucial support for neurons and their impairment by viruses or their interactions with anti-viral or autoimmune responses could contribute to neurological disease. we have developed a transgenic mouse model to assess lymphocyte-astrocyte interactions. the major histocompatibility complex (mhc) class i molecule, d(b), was expressed in astrocytes under the transcriptional control of regulatory sequences from the glial fibrillary acidic protein (gfap) gene. baseline cerebral mhc class i mrna levels from transgenic mice were elevated over those of non-transgenic controls, and a prominent increase in cerebral mhc class i expression occurred following focal, injury-induced astroglial activation within transgenic brains but not in non-transgenic controls. facs analysis of explant astrocyte cultures from established transgenic lines demonstrated astroglial expression of the gfap-d(b) fusion gene at the protein level. functional antigen-presenting capacity was conferred by the d(b) transgene, as virus-infected primary astrocytes obtained from transgenic balb/c mice (k(d)i(d)d(d)l(d)) expressing the d(b) molecule were lysed by d(b)-restricted anti-viral ctl. the central nervous system (cns) has been considered an immune privileged site compared with other organs by virtue of the blood-brain barrier that restricts access of macromolecules and non-activated lymphoid cells from the periphery into the cns parenchyma and because resident cns cells such as neurons, oligodendrocytes and astrocytes express negligible to undetectable levels of class i major histocompatibility (mhc) molecules (reviewed in lampson, ) . mhc class i molecules function to present viral protein fragments (peptides) on the surface of cells so they can be recognized as foreign and be destroyed by mhc-matched, anti-viral ctl (zinkernagel and doherty, ; townsend et al., ) . however, lack of mhc expression may not be absolute as astrocytes and oligodendrocytes can be focally induced to express mhc class i in response to certain cns infections (massa et al., ; suzumura et al., ; olsson et al., ; liu et al., ) and during inflammatory demyelinating disease (traugott and lebon, ) . further, activated t lymphocytes can traverse the intact blood-brain barrier and enter the brain parenchyma (wekerle et al., (wekerle et al., , hickey et al., ; oldstone and southern, ) . together, these data suggest that activated antiviral or autoimmune t cells can come in close proximity to resident cns cells, and their interactions and/or release of cytokines may result in focal induction of mhc molecules on these cns cells. to better understand the potential role of class i mhc expression and antigen presentation by resident cns ceils, we have expressed a murine class i mhc molecule (d b) in transgenic mice under the control of a variety of cns cell-specific promoters. here we report the establishment of transgenic mouse lines in which a constitutive and inducible class i mhc molecule is expressed in cns astrocytes. astrocytes provide crucial support for neurons and oligodendrocytes through such diverse functions as the production of neurotrophic factors and the elimination of neurotoxins (reviewed in eddleston and mucke, ) . consequently, their impairment or destruction by either antiviral or auto-immune responses would disturb cns homeostasis. notably, a variety of dna and rna viruses can infect astrocytes in vivo (epstein et al., ; stowring et al., ; gyorkey et al., ; mirra and del rio, ; carbone et al., ; itoyama et al., ; rinaman et al., ; epstein et al., ) . the gfap-d b mice should provide a useful tool to study immune interactions with virally infected astrocytes in vivo. from dr. r.b. wallace, city of hope research institute, duarte, ca); / -microglobulin cdna from clone p / -m (daniel et al., ) , obtained from dr. p. kourilsky, institute pasteur, paris, france; a cdna fragment of mouse beta-actin bp - (tokunaga et al., ) amplified from murine genomic dna by pcr; and sv bp - (lebowitz and weissman, which identifies sv sequences at the ' end of the gfap vector and serves as a marker for the transgene. male and female c b / and balb/cbyj mice of various ages (newborns to year of age) were used. animals were maintained in conditions consistent with aaalac regulations throughout the course of the investigation. focal mechanical brain lesions were placed by penetration of one or both hemispheres with a sterile gauge needle as described (mucke et al., ) . for this procedure, mice were anesthetized with methoxyflurane. standard protocols (ausubel et al., ; sambrook et al., ) were used for the construction of the gfap-d b fusion gene. all junctions created by the subcloning were sequenced prior to microinjection. fertilized oocytes were obtained from (c b / × c b / ) or (balb/c x balb/c) females. purification of the transgene, preparation of mice and microinjection and reimplantation of fertilized oocytes were carried out as described (mucke et al., ) . the entire sequence of the gfap gene was included in the construct to avoid deletion of potentially important intragenic regulatory elements (sarkar and cowan, ) . transgenic mice were identified by slot blot or southern blot hybridization of genomic dna extracted from tail tissue. poly(a) + rna was extracted from brains and analyzed by northern blot and sequential hybridization with different probes (mucke et al., ) . the following dnas were p-labeled with the random hexanucleotide primer method (sambrook et al., ) and used as probes: a partial d b cdna from clone ph (reyes et al., ) which cross-hybridizes with both h- b and h- d mhc class i transcripts (obtained tissues were removed from transgenic and nontransgenic littermates and snap frozen in liquid nitrogen. total rna was later extracted using the gtc-acid phenol method (chomczynski and sacchi, ) . ng of total rna was reverse transcribed (m-mlv reverse transcriptase, gibco-brl/life technologies, gaithersburg, md) at °c for min using the random hexamer extension method (perkin-elmer cetus, emoryville, ca). the resulting cdna was then subjected to cycles of pcr ( °c annealing; °c extension; °c denaturation) in the presence of taq polymerase and oligonucleotide primers designed to amplify either a product specific for the gfap-d b transgene (primers a and b) or an internal standard, gapdh (primers c and d). the sequences of the primers are as follows: a, ' aggtt ggagc ggaga cgcat '; b, ' gcgct ctggt tgtag tagcc '; c, ' tggta tcgtg gaagg actca tgac '; d, ' agtcc agtga gcttc ccgtf cagc '. primary astrocytes were isolated by a procedure modified from mccarthy and de vellis ( ) . briefly, neonatal brains were mechanically dissociated in dmem containing % fetal bovine serum. following days of incubation in poly-l-lysine coated tissue culture flasks, cells were shaken at °c at rotations per min for h to remove non-adherent cells. when fixed with % paraformaldehyde in pbs and labeled with antibodies against gfap (dako, carpinteria, ca) as described (mucke et al., ) , at least % of the adherent cells stained positive for this astroglial marker. all astrocytes were used within days of culture and were passaged no more than three times. for facs analysis, uninfected primary astrocytes were trypsinized, washed and incubated with medium alone or with a : dilution of primary antibodies: b . r (anti-d b) or - - s (anti-l ~) (accurate chemical & scientific corporation, westbury, ny). binding of primary antibodies was revealed with a fitc-conjugated secondary antibody (dilution : ). all reactions were incubated on ice for min. the cells were washed extensively and analyzed on a becton dickinson facs . dead cells were excluded by addition of ng/ml propidium iodide to the samples prior to fluorimetry. chromium release assays were carried out as described elsewhere (oldstone, ) . in brief, primary astrocytes were infected with the armstrong ca clone b of lcmv (lcmv arm) (dutko and oldstone, ) at a multiplicity of infection (moi) of . this moi ensured that all cells became infected as determined by infectious center assays (not shown). h later, the astrocytes were labeled with cr and exposed to lcmv primed h- matched or h- mismatched splenocytes at different effector-to-target cell (e:t) ratios. ctl specific for lcmv were raised by injecting - -month-old c b / and balb/c mice intraperitoneally with × plaque-forming units of lcmv arm. splenic lymphocytes obtained from these mice - days later were used in chromium release assays. the specific cr release was calculated according to the following formula: experimental release -spontaneous release × maximum release -spontaneous release each sample was done in triplicate and the variance within triplicates was % or less. different groups of astrocytes compared in ctl assays were established and processed simultaneously. to establish transgenic mice with astroglial expression of an mhc class i molecule, a minigene encoding the mhc class i antigen d b was placed under the regulatory influence of a modified murine glial fibrillary acidic protein (gfap) gene (fig. ) . earlier work documented that the gfap gene effectively targets the expression of different proteins to astrocytes in vivo, including lacz (mucke et al., ) , human alpha-l-antichymotrypsin , the hiv coat protein, gpl (toggas et al., ) , and the cytokine il- (campbell et al., ) . for construction of the gfap-d b transgene, the ' and ' ends of a partial cdna encoding the greater portion of the d b molecule (reyes et al., ) were ligated with fragments of genomic d b sequence derived from the mo/d b clone (allen et al., ) (obtained from dr. r. flavell, yale university, new haven, ct). the resulting d b minigene consists of all exons as well as the first two introns of the d b gene. a polyadenylation signal is provided by ' untranslated genomic d b sequence. the d b minigene was fused with the modified gfap gene (mucke et al., ) after addition of noti( ') and saii( ') linkers and the structure of the resulting construct was characterized by restriction analysis and sequencing of promoterminigene junctions. the gfap-d b fusion gene was then microinjected into fertilized oocytes from c b / (h- b) and balb/c (h- d) mice as described (mucke et al., ) . founders, separate lines of transgenic mice were established and screened for expression of the transgene. four of the five lines were derived from c b / × c bi/ (h- b) zygotes (lines nos. , , , ) while one line (no. ) was derived from a balb/c × balb/c (h- d) zygote. to determine tissue expression of the gfap-d b transgene, reverse transcriptase/polymerase chain reaction (rt-pcr) was done on multiple tissues from transgenic and nontransgenic littermates including brain, spleen, pancreas, kidney, thymus, liver and testes. to determine rna expression in the peripheral cns, sciatic nerve was also analyzed. two sets of primers were used. one set, primers a and b, shown in fig. c , was specific for gfap-d b sequences and distinguished between spliced and unspliced products due to the presence of a -bp intron between the two primers. the other set of primers, primers c and d, served as a pcr control, and amplified gapdh, a protein expressed in all tissues. brain rna from transgenic mice but not from nontransgenic littermates gave rise to a pcr product indicative of spliced rna ( fig. a) . a faint band was also detected in the thymi of these mice. no other organ, including sciatic nerve, gave rise to the transgene-derived pcr fragment. when the brain was dissected into four major regions (the cerebral hemispheres, the olfactory bulb, the cerebellum and the midbrain/brainstem), all portions of the brain resulted in the correctly spliced product (fig. b) . thus, expression of the transgene is primarily restricted to the brain, and is expressed throughout the brain. by northern analysis, offspring from four (lines nos. lesioned: + + the gfap-d b expression at the protein level was too low to be detected in situ by immunostaining. however, expression of the correctly folded d b molecule on the surface of astrocytes could be demonstrated by facs analysis (fig. ) using b . r , a monoclonal antibody that detects the correctly folded d b molecule (allen et al., ). an increase in mean fluorescence (from . to . ) was found in primary transgenic astrocytes relative to non-transgenic astrocytes (fig. a, arrows) . as a control, no significant difference in mean fluorescence was identified between transgenic and non-transgenic astrocytes when an l d specific monoclonal antibody was used (fig. b) . . two mice received focal brain lesions, as described in materials and methods, days prior to sacrifice. two unlesioned mice served to establish baseline levels of the different transcripts. a probe for beta-actin was used to assess amounts of rna per lane. the brains of gfap-d b transgenic mice compared with non-transgenic controls (fig. ) . in normal non-transgenic mice, there was no detectable increase in the cerebral level of mhc class i mrna h following focal mechanical brain injury (fig. ) . in gfap-d b transgenic mice, however, there was a marked increase in mhc class i mrna levels in response to such injuries. this injury-induced upregulation of cerebral mhc class i expression was also found in transgenic lines nos. , and ( - mice analyzed per line) (data not shown). the astroglial expression of a functional, transgenederived d b molecule was demonstrated by infection of explant cultures of astrocytes from transgenic mice with lcmv for h followed by incubation of astrocytes with lcmv-specific, db-restricted ctl. table shows that astrocytes obtained from h- d balb/c mice that expressed the h- b d b minigene were specifically lysed by h- b ctl while astrocytes obtained from h- d mice not expressing the d b molecule were not ( % vs. % s~cr release at e : t ratios of : ). because the exposure to serum-containing media induces astrocytes to express endogenous mhc molecules (keane et al., ) , both transgenic and nontransgenic lcmv-infected astrocytes were effectively lysed by h- -matched ctl bearing the same h- haplotype as the target cell ( % vs. % ]cr release at e : t ratios of : ). similar results were obtained in three other independent assays using three different batches of astrocytes. in this report, mhc class i minigene to astrocytes using sequences derived from the gfap gene. transgenic mice expressing d b mhc molecules on astrocytes displayed similar survival rates as age and sex matched nontransgenic littermates over a -year period of observation (data not shown). further, transgenic mice expressing the mhc molecule in astrocytes showed no signs of cns impairment and histological analysis of their brains was indistinguishable from that of non-transgenic controls, suggesting that class i mhc expression alone is not sufficient to induce spontaneous neuropathology. the gfap-d b transgene is expressed in a functional manner as indicated by ctl assay (table ) . lcmv-infected astrocytes from gfap-d b transgenic mice that are normally kdidd d were specifically lysed by anti-viral, h- b restricted ctl while astrocytes from normal balb/c mice were not. because the transgene-derived o b molecule is the only h- b mhc class i antigen that could be expressed on astrocytes of transgenic balb/c (h- d) mice, lysis of these astrocytes by h- b-restricted ctl demonstrates that lcmv peptide processing and interaction with the o b minigene product is appropriate for recognition by h- b anti-lcmv ctl. transgenic mice which express a class i mhc gene product (k b) driven by the gfap promoter have been established previously (schonrich et al., ) . our model incorporates the entire gfap gene, which may include intragenic regulatory elements required for astrocyte-specific expression. further, our choice of the d b gene was chosen based on the well-characterized anti-lcmv response to target cells expressing the d b molecule. the inability to detect the o b molecule in vivo with antibody likely reflects the insensitivity of the immunohistochemical assay since d b was detected with antibody when facs analysis was used in ex vivo astrocyte preparations (fig. ) . notably, ctl recognition and lysis despite undetectable levels of mhc class i and other immunoregulatory molecules have been noted (skias et al., ; oldstone et al., ) . a strength of this model is the inducibility of the gfap promoter, exemplified here by the increase of transgenic class i mhc levels following mechanical trauma (fig. ) . a variety of neural injuries have also been shown to up-modulate gfap expression (smith et al., ; de la monte et al., ; manuel±dis et al., ; delacourte, ) . therefore, insults which result in an upregulation of the gfap gene should correspond with focal induction of class i mhc expression on astrocytes. experiments using allografts into the rat cns and under the kidney capsule (mason et al., ) have shown that rats mounted a brisk anti-graft response to the kidney graft while the cns graft rejection occurred more slowly. this suggests that the microenvironment of the cns, not the antigen-presenting capacity of the cells 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persistent infections with lymphocytic choriomeningitis virus (lcmv) rapid activation of astrocyte-specific expression of gfap-lacz transgene by focal injury transgenic models to study the pathogenic role of mutated and non-mutated forms of human amyloid proteins in the development of alzheimer's disease (ad) animal virus pathogenesis. a practical approach trafficking of activated cytotoxic t lymphocytes into the central nervous system: use of a transgenic model virus infection triggers insulin-dependent diabetes mellitus in a transgenic model: role of anti-self (virus) immune response induction of class i and class ii transplantation antigens in rat brain during fatal and nonfatal measles virus infection the complete amino acid sequence of the murine transplantation antigen h- d b as deduced by molecular cloning spatiotemporal responses of astrocytes, ramified microglia, and brain macrophages to central neuronal infection with pseudorabies virus overexpression of nef as a marker for restricted hiv-i infection of astrocytes in post mortem pediatric central nervous tissues molecular cloning: a laboratory manual intragenic sequences affect the expression of the gene encoding glial fibrillary acidic protein down-regulation of t cell receptors on self-reactive t cells as a novel mechanism for extrathymic tolerance induction susceptibility of astrocytes to class i mhc antigen-specific cytotoxicity immunocytochemical staining for glial fibrillary acidic protein and the metabolism of cytoskeletal proteins in experimental allergic encephalomyelitis detection of visna virus antigens and rna in glial cells in foci of demyelination coronavirus infection induces h- antigen expression on oligodendrocytes and astrocytes neuropathologic alterations induced in brains of transgenic mice by expression of the hiv-i envelope protein gpl nucleotide sequence of a full-length cdna for mouse cytoskeletal beta-actin mrna cytotoxic t cells recognize fragments of influenza nucleoprotein multiple sclerosis: involvement of interferons in lesion pathogenesis cellular immune reactivity within the cns immune reactivity in the nervous system: modulation of t-lymphocyte activation by glial cells restriction of in vitro t cell mediated cytotoxicity in lymphocytic choriomeningitis virus within a syngeneic or semi-allogenic system we thank jenny price, william johnson and edward rockenstein for excellent technical support, and gay schilling for help with the preparation of the manuscript. key: cord- - z yeb authors: nan title: abstracts des . internistenkongresses date: journal: med klin (munich) doi: . /s - - -y sha: doc_id: cord_uid: z yeb nan so far, platelets were regarded static cells without further movement once they were adherent to fibrinogen or other extracellular matrices. migration of adherent platelets has not been discussed by the scientific audience so far. methods and results: -dimensional gel-electrophoresis of platelets revealed that fibrinogen-adherent platelets synthesize or modify multiple proteins. most of these proteins include cytoskeletal proteins and proteins of the migration apparatus. we concluded that adherent platelets might be able to migrate on the surface of an extracellular matrix or endothelium. to demonstrate platelet migration we exposed fibrinogen-coated coverslips with adherent platelets to continuous flow under high shear rates as in the arterial vascular system. all non-adherent platelets were removed from the experiment. under continuous flow we observed shear stress induced mechanotaxis of human platelets some of which started to actively migrate along the direction of flow. migrating platelets were still tightly adherent to the extracellular matrix and would have otherwise been swept away by the continuous flow. we found, that these migrating platelets underwent cytoskeletal rearrangement and formed pseudopodia. to support that our observation is an active cellular process, we blocked cellular signal transduction on pi -kinase level with ly /wortmannin. after exposure to ly /wortmannin, we could no longer observe platelet migration under flow conditions. we subsequently examined chemokine-directed migration of platelets inlcuding sdf , rantes and ena- . we created a chemokine source constantly releasing chemokines and thereby generating a gradient. as for the sdf (stromal cell derived factor- ; μg/ml) source we observed that about % of platelets adherent around this artificial source started to move towards the sdf- source. platelets were actively migrating for several hours in this in-vitro setting. during migration platelets polarized and formed pseudopodia with modification of several proteins of the migratory aparatus under sdf exposure. when the sdf- source was removed, platelets migrated without a specific direction. also with blockade of the cxcr receptor, the specific receptor for sdf- , directed platelet migration could no longer be observed. we are currently performing inhibitory experiments on different levels of cell signalling to characterize the migratory apparatus of platelets. the role of other cytokines associated with cell migration including rantes and ena- will be investigated. we apply immunofluorescence, western-blot, -dimensional gel electrophoresis and mass spectrometry to differentiate the migratory apparatus of migrating cells. conclusion: platelets that adhere to fibrinogen are not irreversibly fixed on matrix surface. they are able to move into a specific direction guided by shear stress induced mechanotaxis or directed along a chemokine gradient . by these mechanisms, platelets might be able to migrate into a platelet clot to further stabilize the hemostatic plug. moreover, platelet migration may be a physiological mechanism to precisely cover denuded vascular lesions and further play a role in the recruitment of platelets to the site of vascular lesions or inflammation. influence of genetic variation in four obesity candidate genes on body mass index and metabolism j. aberle , p. peitsmeyer , n. a. beck , f. u. beil . medizinische klinik, hamburg objective: it is believed that is in most cases obesity derives from an obesogenic environment on the basis of a genetic predisposition. a large number of genes and genetic variations have been associated with obesity. research methods and procedures: in a cohort of caucasion men and women with a body mass index (bmi) of kg/m² or more we investigated four candidate genes at baseline and following a months low fat caloric restriction diet by polymerase chain restriction and restriction digestion: the g/a variant of the cannabinoid receptor (cb ), the p t polymorphism in fatty acid amide hydrolase (faah), the l p variation in neuropeptide y (npy) and the - c>g polymorphism in resistin. results: comparing groups according to genotype for each gene separately revealed a significantly greater reduction of body weight and bmi in carriers of at least on mutant allele in cb as well as a greater reduction in triglycerides in resistin mutant allele carriers. we furthermore investigated gene-to-gene interaction: adding faah mutant alleles to carriers of at least one mutant allele in cb , or resistin significantly influenced triglycerides, total-or ldl-cholesterol levels respectively. combination of npy n-allele with either faah t-allele or cb a-allele as well resulted in an increase of triglyceride levels. discussion: our results support the hypothesis of obesity as a complex genetic disorder with several genes involved in the development of an obese phenotype and obesity related comorbidities. rt-pcr, die konzentrationen sezernierter proteine mittels immunoassay quantifiziert. ergebnisse: die adiponektinstimulation von rasf resultierte in veränderungen der genexpression sowohl auf mrna-als auch proteinebene. unter diesen regulierten genen waren proinflammatorische zytokine ( schlussfolgerung: adiponektin nimmt einfluss auf rasf, indem u.a. die expression und sekretion von proinflammatorischen und prodestruktiven/matrixabbauenden mediatoren modifiziert werden. diese erkenntnisse könnten dabei helfen, die pathophysiologischen mechanismen in der ra besser zu verstehen und potentielle zielmoleküle zur therapeutischen intervention zu identifizieren. in dieser globalen studie wurde der nutzen einer verlängerten thromboseprophylaxe nach totalem hüftgelenkersatz untersucht. es wurde die kurzzeit-thromboseprophylaxe mit enoxaparin mit einer verlängerten thromboseprophylaxe von bis zu wochen mit einem neuen, oralen, direkten faktor-xa- . bei studienbeginn und nach tagen wurde die wundfläche vermessen und eine mm große biopsie aus der wunde entnommen, aus der mit hilfe der real-time pcr (taqman ® ) die mmp-mrna bestimmt wurde. die wundflüssigkeit wurde mit hilfe der gaze täglich gesammelt und mittels zymographie aufgearbeitet. ergebnisse: die expression von mmp- mrna zeigte keine veränderungen in beiden gruppen. die mmp- und - -konzentrationen in der wundflüssigkeit unterschieden sich nicht signifikant in beiden gruppen zu beginn der studie. allerdings wurde eine signifikante reduktion der aktiven form von mmp- zwischen dem . und . behandlungstag in der pi-gruppe verzeichnet (p= , ). die ergebnisse von mmp- pro zeigten in der therapiegruppe ebenfalls eine sinkende tendenz ( % reduktion zu % anstieg in der kontrollgruppe) erreichten aber nicht das signifikanzniveau. außerdem zeigte sich eine reduktion der wundgröße in der pi-gruppe von % im vergleich zur kontrollgruppe (p= , ). schlussfolgerung: es traten keine veränderung der mmp-mrna-expression unter pi-therapie auf. allerdings fanden wir eine signifikante reduktion der biologisch aktiven form von mmp- in der wundflüssigkeit. die lokale behandlung mit einem pi beeinflusst also primär nicht die expression von proteasen, sondern scheint die wundflüssigkeit so zu modulieren, dass eine geringere aktivität von mmps auf der wundoberfläche zu finden ist und durch eine geringere proteolytische aktivität eine verbesserte wundheilung eintritt. einleitung einer antiretroviralen therapie in zwei ländlichen regionen in rwanda (n= ), fibrose in mehreren regionen (n= ). nur die gruppe, die baseline keine fibrose aufwies zeigte unter dreijähriger ert eine normalisierung der wanddicke (von , ± , auf , ± , mm; p< , ) und eine weitgehende funktionelle normalisierung der regionalen herzfunktion (strain rate radial: von , ± , s- auf , ± , s- , p= , ; vergleichskollektiv , ± , s- ). die anderen beiden gruppen zeigten zwar in hinblick auf die wandstärken einen positiven effekt, hinsichtlich der herzfunktion konnten sie jedoch bei bereits deutlich erniedrigten funktionswerten zum baseline-zeitpunkt lediglich stabilisiert werden (reduktion der wandstärke nach jahren ert: gruppe wenig fibrose= mm; gruppe viel fibrose= mm) schlussfolgerung: die enzymersatztherapie ist eine effektive langzeitbehandlung bei patienten mit fabry kardiomyopathie. hierbei fällt insbesondere auf, dass eine rechtzeitige therapie, bevor sich eine myokardiale fibrose entwickelt hat, indiziert ist. klinikgestützte poststationäre mobile gesundheitsservices; "hospital-to home®" -im poststationären versorgungsmanagement chirurgischer patienten adult-onset still´s disease (aosd) represents a rare systemic inflammatory disorder with an estimated incidence between and cases per million inhabitants in western europe. due to this small incidence only few data on diagnosis and therapy are available. the etiology of aosd remains elusive. currently the hypothesis of an exacerbated immune response based on dysregulation of cytokine mediated signalling cascades is favoured among scientists. according to resent results the proinflammatoy cytokine interleukin (il- ) seems to play a central role in pathogenesis of the more common juvenile as well as adult-onset still´s disease. in view of that, we report about a fast and sustained response of a year old woman with aosd under first-line treatment with the il- receptor antagonist anakinra. the fast mode of action compared to conventional dmards and tnf inhibitors paired with an acceptable safety profile had prompted us to initiate a primary il- receptor blocking therapy. the observed rapid clinical response (cessation of fever within hours) and normalization of highly elevated acute phase laboratory parameters within a few days encourage a first line use of anakinra in aosd-patients. longer observation periods and larger series of patients will be needed to answer open questions like optimal length of anakinra-treatment, long-term outcome and complications. cardiovascular complications in patients with diabetes mellitus are associated with increased oxidative stress in vascular tissue. a key event for no synthase (nos) uncoupling involves downregulation of bh synthesizing enzyme gtpcyclohydrolase i (gch-i). we here investigated the effects of at -receptor block ade with chronic telmisartan therapy on gch-i expression, nos uncoupling and endothelial function in streptozotocin (stz)-induced diabetes mellitus (type i). diabetes was induced by single i.v. injection of stz ( mg/kg, weeks) in male wistar rats ( g). telmisartan ( mg/kg/d) therapy did not improve blood glucose and body weight. aorta from diabetic animals had vascular dysfunction as revealed by isometric tension studies. ros produced by nadph oxidase, mitochondria and xanthine oxidase were increased in the diabetic group. nadph oxidase subunits mrna and protein expression was increased in response to stz. importantly, the expression of the gch-i and enos activating ser phosphorylation was decreased by stz. therapy with telmisartan normalized all these parameters almost completely. the results of the present studies demonstrate for the first time that at receptor treatment of diabetic animals with telmisartan is able to prevent downregulation of the important bh synthesizing enzyme gch-i, which prevents enos uncoupling and an activation of cardiovascular superoxide sources. der einfluss der sauren sphingomyelinase auf die expression von matrix-metalloproteinase- in intestinalen epithelzellen und fibroblasten background: the calcineurin (cn)/nf-at signaling cascade takes a crucial role during t-cell activation and the development of myocardial hypertrophy. in addition to nfat, the phosphatase cn is also translocated into the nucleus. for nuclear import, specific carrier proteins, so-called importins, recognize nuclear localization sequences (nlss) in the sequence of the respective target proteins. we developed a synthetic import blocking peptide (ibp) which is identical to the nls sequence of cn. ibp prevented the interaction between cn and its importin and subsequently inhibited nuclear import of cn. methods and results: according to immunohistochemical studies both the subcellular localization of cn, the inhibitory effect of ibp and also the specificity of the blocking peptide were demonstrated. while the inhibitory effect within the t-cell activation is analyzed by [ h]-thymidine incorporation ( ± , vs. ± [%]), the impact on angiotensin ii stimulated cardiomyocytes was examined on the transcriptional (nfat reporter assay; ± vs. ± [%]) and on the translational level ([ h]-leucine incorporation; ± vs. ± [%]). by using ibp, the expression of hypertrophy markers, e.g. myosin heavy chain β, was suppressed, the cell size was not increased. interestingly, phosphatase activity was not effected (cna assay, biomol). the translocation of diverse transcription factors, like nfat, nfκb or erk , was not effected, which illustrated the specifity of ibp. the results could be confirmed in a pilot animal study for heart failure. discussion: ibp prevents the nuclear translocation of calcineurin both in t-cells and in cardiomyocytes. the inhibitory activity is specific for calcineurin und does not affect the cn phosphatase activity. ibp has also an effect in vivo, where it has been demonstrated to suppress the development of myocardial hypertrophy in an animal model. in summary, ibp suppresses t-cell activation und prevents the development of myocardial hypertrophy. proteasome inhibitors suppress angiogenesis by altering endothelial vegfr- expression the ubiquitin-proteasome system is the major pathway for intracellular protein degradation in eukaryotic cells that controls a wide range of cellular regulatory proteins, including transcription factors and cell cycle regulatory proteins. recent evidence established the importance of the proteasome also in tumor development, showing anti-tumor and anti-angiogenic actions by selective inhibitors in vivo. as signaling via the vegfr pathway is critical for angiogenic responses to occur, we explored whether anti-angiogenic effects via proteasome inhibition were mediated in part through diminished endothelial vegfr expression. our studies show that different proteasome inhibitors (mg , alln and lactacystin) all blocked vegfr expression in a time-and concentration-dependent manner, which was paralleled by respective inhibition of capillary like structure formation and endothelial cell migration. in contrast, tie- or vegfr- expression was not significantly affected by proteasome inhibitor treatment. the suppressive effects on vegfr expression were neither conveyed by increased shedding nor by shortened protein half-life, suggesting that transcriptional mechanisms accounted for the observed effects. in line with this conclusion, proteasome inhibition significantly suppressed vegfr mrna accumulation. in addition, inhibitor treatment considerably decreased transcriptional activity of '-deletional vegfr promoter gene constructs. proteasome inhibition-mediated repression was conveyed by a gc-rich region, harboring one consensus sp binding site. subsequent emsa analyses demonstrated diminished constitutive sp -dependent dna binding in response to proteasome inhibition. hence, vegfr- expression may constitute a critical molecular target of proteasome inhibitors that mediates their anti-angiogenic effects in vivo. loss of ifn-γ inducibility of the mhc class ii antigen processing pathway in head and neck cancer: evidence for posttranscriptional as well as epigenetic regulation abnormalities of the major histocompatibility complex (mhc) antigens by tumor cells impair the cellular immune response and promote tumor evasion from immune surveillance. so far, studies analyzing the mhc class ii expression levels in hnc are limited. therefore, we investigated the constitutive and interferon (ifn)-γ-regulated expression profiles of mhc class ii apm in various hnc cell lines and also analyzed the mhc class ii expression in hnc lesions. hnc cell lines analyzed in vitro lacked constitutive mhc class ii surface expression. despite the ifn-γ-mediated induction at the mrna level, six out of ten cell lines did not show any relevant mhc class ii surface expression. this phenomenon might be attributed to a posttranscriptional dysregulation of specific mhc class ii apm components. one cell line displayed a loss of ifn-γ induced ciita-expression that corresponded to impaired mhc class ii surface expression, which could be linked to hypermethylation of the ifn-γ-responsive ciita-promoter iv. in vivo, immunohistochemistry analyses of patients revealed that about % of hnc tissues exhibit a negative or only marginally positive staining, whereas % displayed a heterogenous or highly positive mhc class ii surface expression. there was no statistical correlation between tumor differentiation and the mhc class ii expression in hnc lesions. taken together these results suggest a high frequency of mhc class ii abnormalities in hnc in vitro and in vivo, which could occur at different steps of the antigen processing pathway. this information may have a significant impact on practical and clinical aspects of tumor immunotherapeutic strategies. ciita versus ifn-γ induced mhc class ii expression in head and neck cancer cells effective and safe reduction of blood pressue by the combination of amlodipine /valsartan mg in patients with hypertension and metabolic risk factors not controlled by amlodipine mg or felodipine mga subanalysis of the express-m trial introduction: atrial fibrillation (af) is the most common cardiac arrhythmia and frequently occurs in patients with coronary heart disease. prophylactic oral anticoagulation (oac) is indicated in patients with af and at intermediate or high risk for thrombembolic events. however, an anticoagulant regimen has not been standardized for patients with af after coronary stent implantation, which may require additional dual antiplatelet therapy. methods: we identified retrospectively patients with af who underwent pci in our department from - . we compared baseline variables and incidence of a combined endpoint (stroke, mi, death, bleeding) in patients receiving clopidogrel and aspirin (n= , group ), versus patients receiving the combination of clopidogrel and aspirin with low molecular weight heparin (lmwh) (n= , group ), versus patients receiving the combination of clopidogrel and aspirin with oac (n= , group ) at discharge. results: the groups were not relevantly different regarding classical risk factors, however, patients discharged with triple therapy including oac seemed to be at ( ) higher risk: patients in group had decreased left ventricular ejection fraction and increased inflammatory state as measured by plasma fibrinogen. in a median follow up of , years (range from months to . years) severe bleeding events, myocardial infarctions, strokes, and cardiovascular deaths occurred. kaplan meyer analyses showed that the occurrence of severe events was not significantly different (p= . ). conclusion: in our analyses no treatment regimen seemed to be superior. however, in the follow-up period incidence of myocardial infarctions and strokes were high in patients with solely clopidogrel and aspirin treatment, and incidence of cardiovascular deaths was high in patients receiving the combination of clopidogrel and aspirin with lmwh. the number of patients receiving the combination of clopidogrel and aspirin with oac (triple therapy) was low, however, only one severe event was found in the follow-up period in this group. the current data underline the importance for prospective trials investigating the optimal anticoagulant-/antiplatelet treatment in patients with af after coronary stent implantation. valvuloplastie der kalzifizierten aortenstenose: neue erfolge durch weiterentwicklung der herkömmlichen methode background: we have shown that assessment of cardiac output (co) by the inert gas rebreathing method provides reliable measurements as compared to magnetic resonance tomography (mrt) in patients with sinus rhythm. during atrial fibrillation, however, co measurement based on the determination of stroke volume may be impaired by heart rate variation, potentially leading to divergent results. the present study therefore aimed to compare co measurements by inert gas rebreathing to mrt in patients with atrial fibrillation. methods: in a prospective case-control study, we included consecutive patients with atrial fibrillation undergoing cardiac mrt, and control patients pair-matched for gender, co by mrt, height and weight from a collective of patients with sinus rhythm undergoing mrt. the co of reclining patients was determined by inert gas rebreathing with dinitric oxide and sulfur hexafluoride (innocor, innovision, odense dk) immediately before or after the mrt. the data determined by mrt for co served as reference value comparing the methods by means of bland-altman analysis. the study collective covered a wide range of patients ( to years, median , men in the atrial fibrillation group vs. to , median in the sinus rhythm group). bland-altman analysis showed a good correspondence of the two methods for co with an average deviation of . ± . l/min in the atrial fibrillation group vs. . ± . l/min in the sinus rhythm group. no statistically significant difference could be found between the two groups (p = . , mann-whitney-test). conclusion: co measurements by mrt and by the rebreathing method with dinitric oxide and sulfur hexafluoride provide closely related results even in the presence of atrial fibrillation. the rebreathing method therefore allows an easy and reliable non-invasive determination of the co in this patient collective. the importance of the method for diagnosing and treating cardiac diseases remains to be assessed by further studies. when analyzing the plasma lipid profile for each polymorphism, we found the following differences in subjects with gallstones: significantly higher triglyceride and cholesterol levels in carriers of the common abcb allele and significantly lower hdl-cholesterol concentrations in carriers of the common rs allele. however, differences were also detected in controls with regard to cholesterol and total hdl-cholesterol concentrations. our results do not support a link between common abcb and abcb polymorphisms, lithogenic dyslipidemia and gallstone risk. evaluation der prävalenz schlafbezogener atemstörungen bei patienten mit vorhofflimmern und unauffälliger linksventrikulärer pumpfunktion mittels kardiorespiratorischer polygraphie hintergrund: in erregbaren herzzellen sind spannungsgesteuerte natriumkanäle für die auslösung und weiterleitung von aktionspotenzialen verantwortlich. natriumkanäle bestehen aus einer die pore bildenden α-untereinheit und zusätzlichen β-untereinheiten. verschiedene isoformen der α-untereinheiten haben ganz bestimmte entwicklungs-und lokalisationsmuster im herzmuskel, und sie besitzen spezifische pharmakologische und funktionelle eigenschaften. das kugelfischgift, tetrodotoxin (ttx), ein spezifischer natriumkanalblocker, inhibiert konzentrationsabhängig verschiedene α-isoformen. die kardiale isoform nav . wird durch mikromolekulare konzentrationen blockiert (ttxresistent), während neuronale ttx-sensitive isoformen bereits durch konzentrationen im nanomolaren bereich inhibiert werden. die α-untereinheiten sind mit zusätzlichen β-untereinheiten assoziiert. methoden und resultate: humanes vorhofmyokard wurde mittels der patchclamp-technik, kontraktilitätsmessungen, immunhistochemie und western blot untersucht. wir konnten zeigen, dass nicht nur kardiale ttx-resistente (ttxr) natriumkanäle am menschlichen herzen vorkommen, sondern ebenfalls neuronale ttx-sensitive (ttxs) isoformen exprimiert werden. ttxs-natriumkanäle tragen zu % zum gesamtnatriumstrom bei, während die restlichen % des stroms von ttxr-kanälen übernommen werden. funktionelle einschränkungen durch die blockade von neuronalen natriumkanälen konnten in kontraktilitätsmessungen ebenfalls nachgewiesen werden. immunhistochemische experimente bestätigten isoformenspezifische lokalisation der α-untereinheiten nav . bis . sowie aller bekannten β-untereinheiten ( - ). schlussfolgerung: im humanen herzen kommen neben kardialen auch neuronale natriumkanäle vor. sie tragen funktionell % zum gesamtnatriumstrom im menschlichen herzen bei und haben ebenfalls einen einfluss auf die kontraktilität. p /p -mapk-aktivierung in hypokontraktilen gefäßen zirrhotischer ratten: geänderte mechanismen der aktivierung jedoch gleichzeitig die g-protein abhängige aktivierung der p /p mitogenactivated protein kinase (mapk). die g-protein abhängige p /p aktivierung durch den angiotensin-ii typ rezeptor (at r) erfolgt dagegen über protein-kinase c. methoden: leberzirrhose bei ratten wurde durch gallengangsligatur (bdl) induziert. isolierte aorten wurden in vitro mit bzw. ohne angiotensin-ii und verschiedenen inhibitoren inkubiert. protein-expressionen und -phosphorylierungen wurden über western-blot analyse untersucht. die interaktion zwischen dem at r und b-arrestin wurde in co-immunopräzipitations-experimenten untersucht. ergebnisse: trotz ausbleibender angiotensin-ii induzierter aktivierung der g-protein abhängigen kontraktilen signalwege in aorten von bdl ratten kommt es in aorten zirrhotischer und nicht-zirrhotischer ratten zu einer angiotensin-ii stimulierten aktivierung der p /p mapk. diese angiotensin-ii stimulierte erk-aktivierung ist in aorten von bdl ratten, nicht jedoch der nicht-zirrhotischen kontrollen, resistent gegenüber hemmung der protein-kinase c und a. gleichzeitig führte die stimulation mit angiotensin-ii zur bindung von b-arrestin an den at r, die bei bdl ratten verstärkt ist. diskussion: bei leberzirrhose kommt es in extrahepatischen gefäßen zu Änderungen in der kopplung von vasokonstriktor-rezeptoren an ihre signalwege. anstatt g-protein abhängiger signalwege unter normalen umständen (z.b. kontraktions-kaskaden) werden bei leberzirrhose b-arrestin -abhängige signalwege aktiviert. the prediabetic and diabetic in vivo modification of circulating low density lipoproteins decreases their potential to stimulate adrenal steroidogenesis context: biochemical modification of low density lipoprotein (ldl) has been implicated in the pathogenesis of impaired glucose tolerance (igt) and type diabetes mellitus (dm ) and its related micro-and macrovascular disease. objective: since ldl serves as a major cholesterol source for adrenal steroidogenesis we investigated whether oxidative and glycoxidative modifications of ldl isolated from igt and dm subjects influence aldosterone and cortisol release from human adrenocortical cells. in a cross-sectional study ldl were isolated from subjects with normal glucose tolerance (ngt), subjects with igt, and patients with dm . individual oxidation and glycoxidation characteristics of ldl apolipoprotein b- were assessed by gas chromatography-mass spectrometry (gc-ms) analysis. human adrenocortical cells (nci-h r) were incubated for h with μg/ml of isolated ldl and after removal of supernatants the cells were further stimulated for next h with angiotensin ii. aldosterone and cortisol release were then quantified in the supernatants after h and h, respectively. results: circulating ldl from igt and dm subjects were substantially more oxidized and glycoxidized than the ldl samples from ngt subjects. each of the five measured oxidation/glycoxidation markers was significantly positively associated with glycemic control, measured as hba c. ldl stimulated aldosterone and cortisol release from adrenocortical cells. however, hormone secretion was inversely related to the degree of ldl modification. conclusion: prediabetic and diabetic biochemical ldl modifications may promote impaired adrenocortical aldosterone and cortisol synthesis. die modulation der interaktion von nephrin und β-arrestin durch protein-kinase c alpha steuert die nephrin-vermittelte endozytose und signaltransduktion zielsetzung: hepatische sternzellen (hscs) sind die primären fibrogenen zellen in der fibrotischen leber. selektive apoptose von hscs stellt einen neuen antifibrotischen therapieansatz dar. wir haben kürzlich gezeigt, dass das endocannabinoid -arachidonoylglycerol ( -ag) die proliferation von hscs blockieren sowie apoptose induzieren kann. hepatozyten sind jedoch gegenüber diesen mechanismen resistent. der exakte molekulare mechanismus dieser selektiven zelltod-induktion in hscs ist noch nicht geklärt. ziel dieser arbeit war es, eine mögliche rolle von cox- im differentiellen zelltod-verhalten primärer hscs und hepatozyten durch -ag-metabolisierung in pro-apoptotischen prostaglandin-d -glycerolester (pgd -ge) zu überprüfen. methoden: primäre hepatozyten und hscs wurden durch collagenaseperfusion aus lebern gesunder ratten isoliert. -ag-oder pgd -ge-induzierte apoptose wurde mittels ldh-assay und western blot für caspase -und parp-cleavage analysiert. bildung von reaktiven sauerstoffspezies (ros) wurde durch dcfda-fluoreszenz ermittelt. cox- -expression wurde mittels western blot bestimmt. ergebnisse: hscs zeigten eine signifikante cox- -proteinexpression, wohingegen hepatocyten keine wesentliche cox- -expression aufwiesen. behandlung von primären in kultur aktivierten hscs mit -ag induzierte einen dosisabhängigen zelltod ( % nach h bei μm), begleitet von parp-und caspase -cleavage. pgd -ge induzierte in primären hscs ebenfalls dosisabhän-gig apoptose. dagegen verursachte -ag in primären hepatozyten sogar bis zu konzentrationen von μm keinen zelltod, pgd -ge jedoch führte ab konzentrationen von μm zum zelltod. -ag und pgd -ge führten zur bildung von ros in hscs, was auf gemeinsame ros-abhängige apoptose-signalwege hinweist. schlussfolgerung: hscs und hepatozyten weisen einen signifikanten unterschied in der expression von cox- auf, was eine unterschiedliche metabolisierung des endocannabinoids -ag bedingt. die produktion von pgd -ge durch cox- in hscs trägt möglicherweise zur disparaten empfindlichkeit von hepatozyten und hscs gegenüber -ag-induzierter apoptose bei. die rolle des hypoxie-induzierbaren-faktors a in der strahlentherapieresistenz von humanen adenokarzinomzellen der lunge (a ) zielsetzung: hypoxie hat einen entscheidenden einfluss auf die chemo-und strahlentherapieresistenz von soliden tumoren und somit auf die prognose von tumorerkrankungen. die hypoxie-induzierbaren-faktoren (hif- a und hif- a) sind transkriptionsfaktoren, die über spezifische zielgenaktivierung adaptive prozesse in den hypoxischen tumorarealen steuern. in dieser studie wurde die strahlensensibilität der adenokarzinomzelllinie der lunge (a ) unter dem einfluss von hif- und hif- untersucht. methoden: im ersten schritt wurde die auswirkung von bestrahlung gy ( x, x, x) auf die a zellen untersucht (zellzählung, kolonie-assay, immunzytofluoreszenz: caspase- , kerngrößen, multinuklei). unter normoxischen und hypoxischen bedingungen wurde die hif- a und hif- a expression mittels western blot und pcr mit und ohne radiatio untersucht. die strahlentherapieresistenz unter inhibition von hif (sirna) wurde in vitro (kolonie-assay, zellzählung) und in vivo (tumor growth delay assay) untersucht. ergebnisse: bestrahlung mit gy führte zu einer reduktion der proliferation von a zellen ohne beeinflussung der apoptose. gleichzeitig änderte sich die kernmorphologie mit einem anstieg der kerngröße und vermehrten multinuklei unter fraktionierter bestrahlung. dies konnte auf einen anstieg der "seneszenten" zellen zurückgeführt werden. ein direkter einfluss einmaliger radiatio auf die expression von hif- oder hif- wurde nicht beobachtet. allerdings konnte über die inhibition von hif- a mittels sirna eine erhöhung der strahlensensibilität in den a zellen in vitro erreicht werden. dies wurde in vivo mittels tumor growth delay assay bestätigt. schlussfolgerung: die aktivierung von hif- a in hypoxischen tumorarealen hat einen entscheidenden einfluss auf die sensibilität von a zellen gegenüber bestrahlung. conditional overexpression of neuronal nitric oxide synthase is cardioprotective in ischemia-reperfusion introduction: we previously demonstrated that conditional overexpression of the neuronal nitric oxide synthase (nnos, nos ) inhibited l-type ca + -channels. we now hypothesize that nnos overexpression has an impact on myocardial contractility and acts cardioprotective after ischemia-reperfusion. we assessed cardiac function in the newly established transgenic mouse model with conditional, myocardial nnos overexpression. nos-activity ( ± . vs. ± μm/sec, n= , p< . ) was significantly enhanced after nnos overexpression. co-immunoprecipitation experiments indicated interaction of nnos with sr ca + atpase and additionally with l-type ca + -channels in nnos overexpressing animals. ica,l (reduction of ± rel.%, n= , p< . ) as well as intracellular ca + -transients and fractional shortening in cardiomyocytes were clearly impaired in nnos overexpressing mice ( . ± . f/f vs. . ± . f/f , n= , p< . and . ± . % vs. . ± . %, n= , p< . ). in vivo examinations of the nnos overexpressing mice showed a decrease of +dp/dtmax (reduction for ± %, n= , p< . ) as well as a reduced ejection fraction ( ± % vs. ± %, n= , p< . ). ischemia-reperfusion experiments showed a cardioprotective effect of nnos overexpression ( min post-ischemia, lvdp ± in non-induced animals vs. ± mmhg in nnos overexpressing animals, n= , p< . ). discussion: in summary, we demonstrated that under basline conditions, conditional transgenic overexpression of nnos resulted in a mild reduction of myocardial contractility, mainly due to inhibition of the l-type ca + -channel. in contrast, under pathophysiological conditions (i.e. ischemia-reperfusion) nnos overexpression acts cardioprotective. these effects might be caused by a reduction of myocardial ca + -overload after reperfusion. hintergrund und ziele: die pathogenese der erhöhten apoptose in der hepatitis c virus (hcv)-infizierten leber ist weitestgehend unbekannt. für zahlreiche hcv proteine ist eine modulation von zellulären apoptosemechanismen beschrieben worden. trail, der tnf-related apoptosis-inducing ligand, wurde kürzlich als zytotoxisch für hcv-infizierte hepatozyten beschrieben. um die zugrunde liegenden molekularen mechanismen einer potentiellen rolle von trail bei der hcv-induzierten hepatitis und der viralen clearance zu studieren, untersuchten wir die effekte der hcv replikation auf die trail-induzierte apoptose in humanen hepatomazellen. methoden: huh . zellen wurden mit dem in zellkultur infektiösen hcv stamm jfh- sowie den mutanten jfh- /Δe e , jfh- /gnd und sgr-jfh- hcv rna transfiziert bzw. infiziert. apoptose wurde durch bestimmung von parp, caspase- und - spaltprodukten sowie mittels tunel-reaktion untersucht. ergebnisse: transfektion von huh . zellen mit replikations-kompetenter jfh- , nicht jedoch mit replikations-defizienter jfh- /gnd rna, führte zu einer apoptose von huh . zellen, nachgewiesen mittels parp spaltung und positiver tunel reaktion. die verstärkte trail apoptose war in mit jfh- /Δe e und sgr-jfh- subgenomischer hcv rna transfizierten huh . zellen ähnlich stark wie beim vollständigen jfh- virus, was auf eine unabhängigkeit der apoptose-sensibilisierung von strukturproteinen sowie die notwendigkeit der viralen replikation hindeutet. untersuchungen zur signaltransduktion der jfh- -bedingten apoptose-sensibilisierung zeigen eine deutliche abhängigkeit von caspase- aktivierung sowie eine jfh- -induzierte suppression von bcl- und bcl-xl. dies deutet auf eine zentrale bedeutung des mitochondrialen signaltransduktionsweges bei der jfh- -induzierten apoptose hin. schlussfolgerung: unsere daten zeigen eine apoptosesensibilisierung von huh . hepatomazellen durch hcv replikation, welche unabhängig von hcv strukturproteinen und wahrscheinlich mitochondrial beding ist. diese ergebnisse geben wichtige hinweise für die pathogenese der hepatitis c und die mechanismen der viralen clearance. bier, jedoch nicht ethanol stimuliert in vitro die enzymsekretion der pankreasazinuszelllinie ar - j und frisch isolierter pankreasazinuszellen der ratte a. gerloff , m. v. singer , p. feick ii. medizinische klinik, universitätsklinikum mannheim, mannheim hintergrund: da der pankreasazinuszelle in der frühen phase der entwicklung der alkoholischen pankreatitis eine wichtige rolle zugesprochen wird, wurde die wirkung von alkohol (ethanol) auf die funktion dieser zelle bereits seit jahren ( ) intensiv untersucht. allerdings wird alkohol meist in form von schmackhaften getränken konsumiert, die viele nichtalkoholische inhaltsstoffe enthalten, welche ebenfalls einen pathophysiologischen einfluss auf die funktion des pankreas haben können. ziel: vergleich der wirkung von bier und adäquaten ethanollösungen auf die proteinsekretion und signaltransduktion der pankreasazinuszelllinie ar - j (ratte) und frisch isolierter ratten-azinuszellen. methoden: ar - j-zellen wurden für h mit dexamethason differenziert und frisch isolierte azinuszellen durch collagenase-verdau des pankreas von spraque-dawley-ratten gewonnen. nach inkubation der zellen für min. wurde die amylasefreisetzung als maß der proteinsekretion mit hilfe eines kommerziellen testkits bestimmt. ergebnisse: die inkubation von ar - j-zellen mit bier ( - % (v/v)) bewirkte eine dosisabhängige stimulation der basalen amylasesekretion. reiner ethanol in vergleichbarer konzentration wie im bier hatte keinen effekt auf die amylasefreisetzung. die bestimmung von ldh nach h inkubation der ar - j-zellen zeigte, dass die bierinduzierte amylasefreisetzung nicht auf einer membranschädigung beruhte. die verwendung selektiver inhibitoren und des ca-indikators fura- /am ergab, dass die bierinduzierte amylasesekretion vorwiegend durch die aktivierung von phospholipase c und der anschließenden kalzium-freisetzung aus intrazellulären speichern vermittelt wird. der stimulatorische effekt von bier auf die proteinsekretion war in frisch isolierten azinuszellen reproduzierbar. schlussfolgerung: unsere daten zeigen, dass der effekt von bier auf die sekretion von pankreasazinuszellen auf nichtalkoholische inhaltsstoffe zurückzuführen ist. diese inhaltsstoffe müssen bei der untersuchung von alkoholinduzierten pathologischen und funktionellen veränderungen berücksichtigt werden. pharmakokinetik und pharmakodynamik einer fckw-freien, fixen kombination aus beclometason-dipropionat und formoterol (- , , , und - , mm hg) erreichte keine statistische signifikanz. bei den mit liraglutid behandelten patienten zeigte sich im vergleich zu placebo eine senkung der triglyceride (adjustiert) von - % ( , mg, p= , ), - % ( , mg, p= , ) und - % ( , mg, p= , ) . es gab keine klinisch relevanten und konsistenten unterschiede bezüglich der gesamtcholesterin-, hdl-, ldl-, apob-, il- -, tnf-α-, leptin-und adiponectin-konzentrationen zwischen den liraglutid-behandlungsgruppen und placebo, eine signifikante reduktion des ldl-cholesterins wurde unter placebo im vergleich zu den aktiven behandlungs-gruppen beobachtet, das galt aber nicht für apob, bei dem lediglich in der , mg behandlungsgruppe ein unterschied zu verzeichnen war. es konnte ein ausgeprägter effekt von liraglutid auf die pai- -konzentrationen mit reduktionen von - % ( , mg, p= , ), - % ( , mg, p= , ) bzw. - % ( , mg, p= , ) gegenüber placebo beobachtet werden. das galt auch für die bnp-konzentrationen mit dosisabhängigen senkungen von - % (p= , ), - % (p= , ) und - % (p= , ) gegenüber placebo für die entsprechenden liraglutid-behandlungsgruppen. die beobachteten dosisabhängigen senkungen des crp gegenüber placebo (- %, - % und - %) erreichten keine statistische signifikanz. schlussfolgerung: die behandlung mit liraglutid war mit einer blutdrucksenkung und einer abnahme der triglycerid-, pai- -und bnp-konzentrationen verbunden, die beobachteten effekte müssen in langzeitstudien bestätigt werden und bedürfen weiterer untersuchungen. objectives: the majority of treated hypertensive patients do not achieve target blood pressure levels < / mmhg. one key reason is inadequate adherence with the prescribed drug regimen. dosing regimens are either not executed as prescribed (non compliance) or patients stop taking the medication (non persistence). it has been demonstrated that drug adherence with angiotensin receptor antagonists like valsartan is superior in comparison to other drug classes. the present study was designed to evaluate whether drug adherence could be further improved by the use of supportive tools. design and methods: centers were randomized to provide pharmacological treatment with or without a set of supportive measures (e.g. structured physicianpatient interaction, printed information about hypertension, reminder stickers, pill box with alarm, home blood pressure measurement). newly diagnosed patients or patients with stage hypertension (blood pressure at baseline . ± . / . ± . mmhg) who had not been treated for at least year were included in this trial. all patients entered the -week treatment phase with valsartan mg. titration to valsartan mg/hctz . mg was allowed if necessary. drug adherence was assessed by electronic monitors (medication event monitoring system -mems). results: patients treated with a valsartan-based therapy in combination with supportive measures demonstrated lower rates of treatment discontinuation. per-sistence at the end of the months observation period was % in the control patients and % in patients receiving supportive measures. in addition, better execution of the dosing regimen (compliance) could be observed in patients receiving supportive measures but this effect tended to fade with time. bp control improved more in patients with the supportive measures. conclusions: drug adherence can be improved with the use of supportive measures. in this study, while the effect on compliance decreased over time, the effect of chosen supportive measures on persistence seems to be long lasting. randomized, double blind parallel group study to evaluate the reduction of the urinary albumin/ creatinine ratio by valsartan plus lisinopril versus lisinopril or valsartan alone in hypertensive patients with microalbuminuria -the valeria trial objectives: microalbuminuria is known as an independent predictor for stroke, myocardial infarction and death and has a higher prevalence in hypertensive subjects than in the general population. intensified inhibition of the renin-angiotensin-aldosterone system seems to be an efficient treatment option and might be achieved by dual blockage with arbs and aceis. it was the aim of the study to compare the efficiency and safety of a combination therapy comprising valsartan and lisinopril with valsartan and lisinopril monotherapy in patients with hypertension and microalbuminuria. methods: this was a randomized, double blind parallel group study. patients with hypertension (mean sitting diastolic blood pressure = mmhg and < mmhg) and microalbumiuria (urinary albumin/creatinine ratio (uacr) = . mg/mmol and = . mg/mmol for men and uacr = . mg/mmol and = . mg/mmol for women in the first morning urine sample on at least two of three visits) were eligible for participation. after a wash-out/placebo-run-in phase of max. weeks, patient were randomized to treatment ( : : ) with either lisinopril - mg (lis), valsartan - mg (val) or the combination of valsartan/linisopril / - / mg (com) for weeks. results: median uacr at baseline in the com, val, and lis group was . mg/mmol, . mg/mmol, and . mg/mmol, respectively. at baseline, systolic/ diastolic bp for com, val, and lis was . / . mmhg, . / . mmhg, and . / . mmhg. treatment with com, val, and lis resulted in an uacr reduction of %, %, and % (median) after weeks of treatment. the reduction achieved with com was significantly greater than with lis (p= . ). normalization of microalbuminuria (uacr < . mg/mmol for men and < . mg for women) with com, val, and lis was achieved in %, % and % of the patients (p= . for com vs. lis). blood pressure differences between the groups were not statistically significant. all treatments were well tolerated. conclusion: in patients with hypertension and microalbuminuria the combination of valsartan and lisinopril provided a significantly better reduction of uacr and doubled the rate of patients with normalized uacr compared to lisinopril alone. analyse der mikrozirkulation der oberen extremitäten unter ldl-apherese the baseline ppg of the rebleeder (group b) was . +- . mmhg before tips and , +- . mmhg after tips insertion, giving an overall ppg reduction of , %. patients who underwent tips for variceal bleeding never had a bleeding episode again after tips (group a) . the baseline ppg of these patients was . +- . mmhg before tips and , +- , mmhg after tips, giving an overall ppg reduction of , % statistical analysis showed that the ppg difference between , mmhg in rebleeder (group b) and mmhg in non-rebleeder (group a) was statistically significant (p-value < . ). patients with refractory ascites never suffered from an episode of bleeding after tips independently from their initial ppg reduction. conclusion: patients who underwent tips for recurrent variceal bleeding do have a significant higher risk for rebleeding with an initial ppg reduction to , mmhg whereas those patients in which the ppg reduction to mmhg can be achieved a rebleeding episode is unlikely. we conclude that a ppg reduction to at least mmhg should be aimed to prevent further bleeding episodes. these findings differ from previous reports in which ppg of less than mmhg is thought to prevent rebleeding. ergebnisse: tgr männchen jedoch nicht tgr-weibchen entwickeln eine albuminurie (tgr-c: mg/ h) und glomerulosklerose, die durch flutamid zu > % unterdrückt wird (tgr-fl: mg/ h). testosterongabe induziert in tgr-weibchen jedoch nicht in wt-weibchen albuminurie (tgr-ovt: mg/ h, wt-ovt: . mg/ h) und glomerulosklerose (schadensindex-tgr-ovt: , wt-ovt: , ). ovariektomie induziert keine albuminurie. testosteron stimuliert in tgr-und wt-weibchen das renale wachstum und die glomeruläre angiotensinogenexpression, das glomeruläre wachstum wird jedoch nur in tgr-weibchen erhöht. androgenrezeptoren werden im glomerulus geschlechtsunabhängig exprimiert. schlussfolgerung: at rezeptorüberexpression in podozyten transgener ratten induziert albuminurie und glomerulosklerose. testosteron ist ein entscheidender kofaktor, der möglicherweise über eine direkte stimulation der glomerulären angii bildung wirkt. hohe prävalenz der ass-non-responder bei acvb-patienten in den multiplen testanalysen hintergrund und zielsetzung: in der therapie der hiv-infektion sind wechselwirkungen und unverträglichkeit der mittlerweile zahlreich zur verfügung stehenden antiretoviralen medikamente ein grosses problem. mehr als % der antiretroviralen medikamente und begleitenden antiinfektiva gegen opportunistische infektionen weisen positiv geladene reste (kationen) auf. die ausscheidung organischer kationen über die leber und niere erfolgt u.a. über die organischen kationentransporter (oct) und , die eine breite substratspezifität aufweisen. ziel der studie war die charakterisierung kationischer antiretroviraler medikamente und antiinfektiva als inhibitoren und substrate von oct / . methoden: hek- zellen wurden stabil transfiziert mit humanem oct und kloniert im eukaryotischen expressionsvektor pcdna . die ic -werte wurden durch messungen der spezifischen aufnahme von h-gelabeltem -methyl- -phenylpyridinium (mpp+) bestimmt. die quantitative bestimmung der spezifischen substrate erfolgte mittels liquid chromatography electrospray-ionizationtandem mass spectrometry (lc-esi-ms/ms). km und vmax wurden mit der michaelis menten gleichung bestimmt. ergebnisse: pentamidine (ic (hoct ) = . ( ) μmol/l (mittelwert (standardfehler); (oct ) . ( )), nelfinavir ( ( ); ( )), ritonavir ( ( ); ( )), lamivudine ( ( ); ( )), trimethoprim ( ( ); ( )), zalcitabine ( ( ); ( )), saquinavir ( ( ); ( )) und indinavir ( ( ); ( )) zeigten eine signifikante hemmung von oct / . lamivudine und zalcitabine sind substrate von oct / (lamivudine: oct (vmax= . ( . ) nmol/mg/min; km= ( ) μmol/l) oct (vmax= . ( . ); km= ( )) zalcitabine: oct (vmax= . ( . ); km= ( ); oct (vmax= . ( . ); km= ( ) ergebnisse: der endogene ligand s p, der selektive s p -rezeptor agonist sew und der unselektive s p-rezeptor agonist fty transaktivieren den ebenfalls bereits als vermittler protektiver intrazellulärer signalwege beschriebenen egf-rezeptor. damit assoziert ist die aktivierung von akt in ratten-kardiomyozyten. im rattenmyokard verbessert bei applikation beginnend mit reperfusion fty , nicht jedoch der selektive s p rezeptor agonist sew die erholung der mechanischen funktion signifikant. in humanen myokardpräparaten zeigt fty ex vivo einen analogen effekt bei applikation unter reperfusion. zusammenfassung: stimulation von s p und s p -rezeptoren induziert die aktivierung von akt in kardiomyozyten. eine verbesserung der erholung der mechanischen funktion lässt sich nur über stimulation des s p rezeptors unter reperfusion erzielen. s p-rezeptoren scheinen daher ein klinisch interessanter angriffspunkt für eine behandlung im sinne einer pharmakologischen postkonditionierung. systemic effects of glucose content in pd fluids on life span and neuronal function in c. elegans background: glucose content in peritoneal dialysis fluids has a decisive role in the formation of glucose degradation products (gdps) during sterilization and is thus associated with the degradation of peritoneal membrane integrity. absorption of gdps from the peritoneal cavity in peritoneal dialysis patients results in rising plasma age levels. one precursor in age formation is methylglyoxal, which is metabolized by glyoxalase- . to further investigate systemic effects of glucose content in pd fluids on life span and neuronal function, wild type and glyoxalase- overexpressing c. elegans were used as model system. homologies to % of all human genes are preserved in c. elegans. thus, this model system allows not only performance of life span assays, but also analysis of neuronal function. methods: c. elegans were cultivated under normal conditions and in the presence of . % and % glucose derived from peritoneal dialysis fluids to perform life span assays. simultaneously locomotive patterns as parameter of neuronal function and neuronal gfp expression were evaluated for each group. results: wild type c. elegans showed a significant reduction of life span of % under the influence of glucose , % and of % using % glucose. in contrast, glyoxalase- overexpression led to an increased resistance towards glucose expo-sure with no significant reduction of life span under . % or % glucose. additionally, with glucose exposure an impairment of neuronal function was observed in wild type c. elegans displaying severe changes in locomotive patterns, which were not present in transgenic c. elegans. neuronal gfp expression in wild type c. elegans declined significantly with glucose exposure without a dose dependant effect. conclusion:. glucose derived from pd fluids has a significant impact on life span and neuronal impairment in c. elegans. whether potential systemic toxicity and neurotoxicity in pd patients can be attributed to pd fluids has to be further evaluated. common pathways in endogenous major depression and depression during ifn-α therapy for chronic hepatitis c background: a major complication of combination therapy with pegylated interferon-alpha (ifn-α) and ribavirin in patients with chronic hepatitis c is the induction of depressive side effects. methods: to elucidate the underlying pathophysiological mechanisms, a total of caucasian patients with histologically proven chronic hepatitis c were treated with standard combination therapy consisting of pegylated ifn-α a and ribavirin. the transcriptional profile h before and h after the first injection of ifn-α was analysed using human genomic microarrays (affymetrix, hg u a) and quantitative real-time rt-pcr. class prediction analysis was performed to identify genes which are differentially regulated in patients with or without ifninduced depression. furthermore, pbmc from patients hospitalized for se vere major depression and controls were cultivated in vitro with pegylated ifn-α a to validate the data in this cohort. results: / hcv patients ( %) developed clinically relevant ifn-induced depression. using class prediction analysis, the development of depression could be predicted with % accuracy by genes including dynlt , gch , tor b, disc , mef a and st gal that were previously described to be relevant for recurrent major depression or neuronal development in the brain. in accordance with this data, increased endogenous ifn-production and selective hyper-responsiveness of these genes to ifn stimulation were observed in hcvnegative patients with severe major depression. conclusions: these data suggest that selective hyper-responsiveness to exogenous (ifn therapy) or endogenous (endogenous depression) type i ifns may lead to the development of depressive symptoms. these data could lead to the discovery of novel therapeutic approaches to treat ifn-induced and major endogenous depression. renal toxicity of glucose degradation products in peritoneal dialysis purpose (zielsetzung): in peritoneal dialysis (pd) residual renal function contributes to improved patient survival and quality of life. glucose degradation products (gdp) impair not only the peritoneal membrane, but also appear in the systemic circulation with the potential for organ toxicity. here we show that in a model of advanced renal failure, gdp affect the structure and function of the remnant kidney. sprague-dawley rats were randomly assigned to a twostage subtotal nephrectomized (snx) or sham operation and were left untreated for weeks. the snx+gdp group received chemically defined gdp intravenously for weeks; the snx and the sham-operated rats remained without gdp. the complete follow-up for all groups was weeks post-operatively. we analyzed renal damage using a semiquantitative score for glomerulosclerosis and tubulointerstitial damage as well as for immunohistochemical analyses. the snx+gdp rats developed significantly more albuminuria ( . ± . mg/ h vs. snx . ± . mg/ h; p ≤ . ) and showed a significantly higher score of glomerulosclerosis index ( . ± . vs. . ± . ; p ≤ . ) and tubulointerstitial damage index ( . ± . vs. . ± . ; p ≤ . ). in the snx+gdp group the expression of carboxymethyllysin and methylglyoxal was significantly higher in the tubulointerstitium and the glomeruli compared to the snx rats. caspase staining and tunel assay were more pronounced in the tubulointerstitium and the glomeruli of the snx+gdp group. in snx+gdp animals, the expression of the slit diaphragm protein nephrin, was significantly lower compared to snx and sham operated animals. in subtotally nephrectomized rats, administration of gdp increased albuminuria, indices of glomerular and tubulointerstitial damage significantly, specifically with a perturbation of podocytes. it is likely that gdp-free pd solutions maintain and stabilize residual renal function in pd. non-parenchymal liver cells (wu et al., hepatology, in press ). therefore, the aim of this study was to investigate whether hbv has the ability to suppress tlr-induced antiviral responses in parenchymal and non-parenchymal liver cells. we have isolated murine kc by counterflow elutriation as well as murine lsec by anti-lsec microbeads. in addition, primary murine hepatocytes were isolated by a two-step perfusion method. npcs and hepatocytes were cultivated in the presence or absence of hbsag, hbeag, hbv virions or supernatants from hbv-producing hbv-met cells, and were stimulated by tlr - ligands. supernatants were collected and tested for antiviral cytokines by viral protection assay or co-cultured with differentiated hbv-met cells. primary hepatocytes, undifferentiated hbv-met cells and highly differentiated hbv-met cells were stimulated by tlr - ligands. results: pretreatment of hepatocytes and npcs with hbv-met cell supernatants (contain hbsag, hbeag and virions), hbsag, hbeag and hbv virions almost completely abrogated tlr and tlr induced antiviral activity which correlated with suppressed isgs induction by poly i:c and lps on the transcriptional level. tlr - stimulation had no direct antiviral effect on hbv-met cells in contrast to primary hepatocytes. in addition, expression of pro-inflammatory cytokines (tnf-a, il- ) after tlr - , - , - , - and - stimulation were significantly reduced in highly differentiated hbv-met cells compared with undifferentiated hbv-met cells. our data indicate that integrated hbv can downregulate the tlr-mediated activation of hepatocytes. furthermore, hbv can almost completely abrogate the antiviral activity of hepatocytes and npcs induced by tlr and tlr stimulation. this has major implications for the interaction between hbv and the immune system. schimäre im pankreas: ein glukagon-produzierendes neuroendokrines karzinom assoziiert mit hypoglykämien conclusions: dbe-ercp is an alternative method for diagnostic as well as therapeutic interventions in the biliary as well pancreatic system in the operated patient. however, it should be limited to selected patients, e.g. with contraindications for ptc, as it is a time consuming as well as a cost intensive procedure. undifferenziertes embryonales sarkom der leber -eine seltene primäre leberneoplasie beim erwachsenen patients with metabolic syndrome (ms) and type diabetes are at increased risk for coronary artery disease. lipoprotein metabolism is characterized by elevated triglycerides (tg), low hdl cholesterol (hdl-c) and a predominance of atherogenic dense ldl (dldl). this is also denoted as atherogenic lipoprotein phenotype (alp). methods: multicenter, randomized, open-label cross-over study investigating the effect of combination therapy of fluvastatin/fenofibrate ( / mg) (ff) on the ldl-subfraction distribution compared to combination therapy of simvastatin/ ezetimibe ( / mg) (se) in patients with ms and type diabetes. at baseline and after weeks of combination therapies ldl-subfractions were measured by endpoint gradient ultracentrifugation. results: patients were randomized, completed the study. blood lipid samples of patients could be analysed. out of patients ( %) showed a profile dominated by dldl. in these patients, tg were elevated and hdl-c was lower compared to non dldl patients. in all patients reduction of total and ldl cholesterol by se was stronger than by ff. the increase of hdl-c was stronger with se in the non dldl group, whereas in the dldl group there was no difference between treatments. in non dldl patients there was no difference with regard to tg reduction and no effect on calculated ldl-radius. however, in the dldl group ff was more efficient in reducing tg (p= . between treatments) and se reduced ldl radius even further, whereas ff increased ldl radius to larger ldl particles (p< . between treatments). conclusions: simvastatin/ezetimibe combination is more efficient in reducing total and ldl cholesterol. this is also true for hdl-c increase in patients without dldl. however, in ms patients with dldl fluvastatin/fenofibrate were more efficient in reducing tg and increasing ldl radius. overall, the number of patients with aes and myalgia was . % hintergrund: bnp und nt-probnp sind molekulare biomarker, welche ebenso für die diagnostik und prognose der chronischen herzinsuffizienz genutzt werden können wie für die optimierung einer medikamentösen therapie und das langzeit-management dieser erkrankung. erstmalig untersuchten wir den einfluss eines strukturierten, multi-modalen, stationären interventionsprogramms -wie es in deutschland in vielen kardiovaskulären rehabilitationskliniken angeboten wird -auf den zeitlichen verlauf der nt-probnp-werte während und sechs monate nach einer solchen intervention. methodik: diese studie wurde in sechs deutschen kardiovaskulären rehabilitationszentren durchgeführt mit folgenden vier interventionsmodalitäten: ( ) optimierung der medikamentösen therapie, ( ) erziehung zu einem und durchführung eines krankheits-bezogenen ausdauertrainings-programms, ( ) intensive information über art und verlauf der erkrankung und erziehung zu einem adäquaten ernährungsverhalten, und ( ) teilnahme an den physischen und psychischen entspannungsübungen. zur steigerung der motivation und dokumentation wurden speziell für diese studie entwickelte patienten-tagebücher ausgegeben, welche den gesamten sechsmonatigen studienverlauf abdeckten. nt-probnp-analysen erfolgten am anfang (r ), während (r ) und am ende der stationären rehabilitationsperiode (r ), ebenso -auf ambulanter basis -nach drei monaten (r ) und sechs monaten r ) nach entlassung. zum vergleich wurde eine gleichaltrige kontrollgruppe von patienten herangezogen, welche nicht an einem solchen intensiven programm teilnahmen, sondern in einer herzinsuffizienz-ambulanz medizinisch betreut wurden. hypothese: wir erwarteten einen positiven effekt unseres interventions-programms auf die nt-probnp-spiegel während des stationären aufenthaltes, und entweder eine konstanz dieser werte nach der entlassung oder -auf grund von mangelhafter compliance -sogar einen wiedereinstieg der werte bis zu sechs monaten nach entlassung. ergebnisse: komplette nt-probnp-werte lagen für patienten vor, welche an einer chronischen herzinsuffizienz im stadium nyha ii -iii litten, eine klinisch relevante dekompensation erlitten hatten und eine lvef = % aufwiesen: der medianwert der nt-probnp-spiegel betrug pg/ml am anfang (r ), war geringfügig aber nicht signifikant reduziert auf (r ) und pg/dl (r ) während des stationären aufenthaltes, fiel jedoch signifikant und klinisch relevant auf pg/ml nach drei monaten (r ; p< , ) und auf pg/ml nach sechs monaten ab (r ; p< , ). der medianwert der kontrollgruppe änderte sich nicht während der sechsmonatigen beobachtungsperiode ( pg/ml und pg/ml; ns). schlussfolgerungen: im gegensatz zu unserer hypothese hat eine modernes, strukturiertes, stationäres rehabilitationsprogramm keinen kurzzeit-effekt auf die nt-probnp-spiegel innerhalb von wochen, sondern vielmehr einen signifikanten und größenordnungsmäßig klinisch relevanten langzeit-effekt innerhalb von monaten. dies könnte in der tatsache begründet liegen, dass die molekularen und strukturellen umbauprozesse des linken ventrikels im sinne eines umgekehrten "left ventricular remodelling" entsprechend zeit benötigen. da die nt-probnp-spiegel mit der prognose der erkrankung assoziiert sind, kann indirekt geschlossen werden, dass unsere stationäre rehabilitationsstrategie langfristig positive effekte auf hospitalisierungsrate und Überlebensprognose hat. cancer fatigue und gestörte ruhe/aktivitätsregulation bei rezidivfreien mammakarzinom-patientinnen, ergebnisse einer prospektiven studie r= , , p= , ) . vergleichbare beziehungen bestanden zwischen dem pap und ahi (r= , , p= , ), dem ai (r= , , p= , ) und dem zai (r= , , p= , ). das hzv war bei patienten mit csa ( , ± , l/ min/m ) signifikant niedriger als bei osa ( , ± , l/min/m ) oder patienten ohne sas ( , ± , l/min/m ). das hzv korrelierte negativ mit dem zai (r= - , , p= , ), dem ai (r= - , , p= , ) und dem ahi (r= - , , p< , ). bei pat mit einer osa waren solche beziehungen nicht nachweisbar. die vorliegenden befunde unterstützen die these, wonach das auftreten einer zsa ein parameter für die schwere einer hi sein kann und erhöhte pa-und pcw-drücke über pulmonal-vagale j-rezeptoren zur hyperventilation und damit entstehung und unterhaltung einer zsa beitragen können. der quotient aus serum-natrium/ urin-natrium-konzentrationen zu den serum-kalium/ urin-kalium-konzentrationen (sus:pup) im screening auf einen primären aldosteronismus hintergrund: das herzzeitvolumen (hzv) ist ein wichtiger parameter in der diagnostik und therapie kardialer erkrankungen. die aktuellen standardmethoden zur bestimmung des hzv sind jedoch entweder invasiv (rechtsherzkatheter, picco) oder technisch aufwändig bzw. teuer (magnetresonanztomographie, mrt). die bisherigen nicht-invasiven methoden zur messung des hzv mittels rückatmung von kohlendioxid oder thorakaler bioimpedanz sind zwar einfach durchzuführen, weisen aber methodisch bedingte ungenauigkeiten auf. ziel der vorliegenden prospektiven studie war es daher, eine neue methode zur bestimmung des hzv mittels cw-doppler ultraschall (cwd) zu evaluieren. methodik: bei konsekutiven patienten wurde unmittelbar vor oder nach einer kardialen mrt das herzzeitvolumen (hzv) mittels cwd im liegen bestimmt (uscom, uscom ltd, sydney australia). als referenzwerte dienten die in der mrt bestimmten werte, die mit dem arithmetischen mittel aus zwei aufeinanderfolgenden cwd messungen verglichen wurden. der statistische vergleich der methoden erfolgte mittels bland-altman-analyse. ergebnisse: das patientenkollektiv bestand aus männern (alter - jahre, median jahre) und frauen (alter - jahre, median jahre). insgesamt konnte bei von konsekutiven patienten im untersuchungszeitraum das hzv und sv mittels cwd bestimmt werden, bei patienten ( %) konnte kein ausreichendes ultraschallsignal empfangen werden. das hzv mittels mrt lag bei , +/- , l/min (mittelwert +/-sd, minimum , l/min, maximum , l/min), das hzv mittels cwd bei , +/- , l/min (minimum , l/min, maximum , l/min). die bland-altman-analyse ergab eine gute Übereinstimmung der beiden methoden für das hzv von , +/- , l/min (mittlere abweichung +/-sd) und für die bestimmung des schlagvolumens von , +/- ml. die methode zeigte eine gute reproduzierbarkeit mit einer mittleren abweichung von , +/- , l/min für das hzv und , +/- , ml für das sv. schlussfolgerung: der cw-doppler ultraschall erlaubt eine zuverlässige nichtinvasive bestimmung des hzv mit guter reproduzierbarkeit. der zukünftige stellenwert der methode in der diagnostik und therapiesteuerung muss durch weitere untersuchungen belegt werden. nichtinvasive bestimmung des herzzeitvolumens mittels inertgas-rückatmung und cw-doppler-ultraschall -sind die ergebnisse vergleichbar? bei patienten ( %) war die messung mittels cwd nicht möglich, bei patienten ( %) konnte die igr-messung nicht durchgeführt werden. das hzv mittels igr lag bei , +/- , l/min (mittelwert+/-sd, , bis , l/min) und bei , +/- , l/min ( , bis , l/min) mittels cwd. die bland-altman-analyse ergab eine gute Übereinstimmung für das hzv von , +/- , l/min (mittlere abweichung+/-sd) und für das sv von +/- ml. es zeigte sich eine gute reproduzierbarkeit mit einer mittleren abweichung von , +/- , l/min für das hzv mittels igr bzw. , +/- , l/min mittels cwd. schlussfolgerung: die bestimmung des hzv und sv mittels igr und cwd ergab eine gute Übereinstimmung und reproduzierbarkeit, so dass die methoden als vergleichbar einzuschätzen sind. zur identifikation des optimalen patientenkollektives für jede der beiden methoden sind weitere untersuchungen zur erarbeitung eines score-systems geplant. validierung einer einfach anzuwendenden neuartigen oszillometrischen methode zur bestimmung der pulswellen-geschwindigkeit obesity in humans is mainly due to high-fat intake and associated with an increased incidence of glomerular injury that is associated with low-grade inflammation and increased production of reactive oxygen species (ros). lauric acid (c : ), a major constituent of coconut oil, has anti-inflammatory activities, however its effects on pro-inflammatory gene expression in the kidney during obesity are unknown. the aim of the study was to quantify and compare renal cortical gene expression of pro-and antioxidant enzymes and icam- in c bl/ j mice fed with standard chow diet (sd), or diets rich in animal fat (afd) or vegetable fat (lauric acid, vfd) for weeks. changes in body weight and glucose tolerance (ip ggt) were also determined. both high-fat diets caused weight gain and glucose intolerance to a similar degree (n.s., afd vs. vfd, both p< . vs. sd). expression levels of proinflammatory genes (no synthase , nos ; catalytic subunit of the phagocytic nadph oxidase, nox ; intracellular adhesion molecule- , icam- ) were more than -fold lower compared to antioxidant genes (gluthatione peroxidase, gpx and cu/zn superoxide dismutase, sod ) and the regulatory subunit of the nadph oxidase complex p phox. only vfd-treatment reduced renal expression of p phox. treatment with afd but not with vfd increased mrna expression of nos , nox , and icam- . vfd, but not afd treatment was associated with upregulation of the antioxidant genes sod and gpx. these data suggest that fat diets rich in lauric acid, in contrast to diets containing animal fats, have distinct and possibly beneficial effects on expression of enzymes regulating cellular redox state in the kidney. contractility in the carotid artery obesity is a risk factor for diseases such as diabetes mellitus and atherosclerosis and has been associated with increased carotid artery intima thickness in obese patients. leptin has been implicated in formation of reactive oxygen species (ros), and functional leptin deficiency or impairment of leptin receptor activity result in obesity. endothelin- (et- ), ros and reduced no bioactivity are involved in vascular changes in obesity and diabetes. the aim of this study was to investigate the role of ros in et- mediated vasoreactivity in the carotid artery of obese leptin-deficient ob/ob and lean c bl/ j wild-type mice. rings were preincubated for minutes with the no synthase inhibitor, l-nitro-argininemethylester (l-name, μmol/l) to block etb receptor-mediated no release. rings were exposed to et- ( . - nmol/l) in the presence or absence of the ros antioxidant epck- (a combination of vitamin c and vitamin e, . mg/ml). endothelium-dependent relaxation to acetylcholine (ach) was in-vestigated in the presence of non-specific cyclooxygenase (cox) inhibitiors. maximal et- -mediated contraction in control mice was similar to obese mice. antioxidant treatment with epc-k reduced et- -induced contractions in control (from . ± . % to . ± . %, p< . ), but not in obese mice ( . ± . % vs. . ± . %, n.s.) endothelium-dependent relaxation to ach remained unchanged in both groups. in conclusion, these results indicate that ros contribute to et- -induced contractions in the carotid artery of healthy mice, an effect that disappears during obesity. leptin may modulate contraction to et- in the carotid artery by mechanisms that are independent of ros. high dietary intake is a major cause of obesity, a risk factor for the development of hypertension, diabetes, and atherosclerosis. the aim of this study was to investigate whether high dietary fat intake-induced vascular changes are reversible upon dietary fat restriction. body weight, glucose tolerance and vascular reactivity were measured in c bl/ j mice, which were fed either with standard chow or with high-fat diet (hfd, % kcal from fat) for months or with hfd for months followed by fat restriction for months. vascular reactivity was analyzed in the carotid artery in response to the α-adrenergic receptor agonist phenylephrine (pe; x - m), and to acetylcholine (ach; - - - m) in the presence or absence of ng-nitro-l-arginine methyl ester (l-name; x - m), a nitric oxide (no) synthase inhibitor. high-fat diet increased body weight and impaired glucose tolerance (p< . vs. control). vascular contractions in response to pe ( x - m) and ach ( - m) were increased both in the absence or presence of no (p< . vs. control), while the endothelium-dependent relaxation was unaffected. in contrast, dietary fat restriction normalized the body weight similar to control animals and also the relative insulin resistance (p< . vs. hfd). in parallel, vascular contraction to ach was attenuated in the absence of l-name (p< . vs. hfd). unexpectedly, dietary fat restriction did not reverse pe-and ach-mediated contraction in the absence of no induced by hfd. in conclusion, intake of high dietary fat has a "memory" effect on vascular reactivity, which cannot completely be reversed even upon dietary fat restriction. this may contribute to an increased risk of vascular injury seen in patients even after normalization of body weight and insulin resistance. die phagozytotische aufnahme von hcv-infizierten apoptose-körperchen durch hsc führt zur induktion der fibrogenese a minority of neuroendocrine tumors (net) present functional syndromes by uncontrolled secretion of peptide hormones or messengers. peptide receptor radiotherapy (prrt) and transcatheter arterial chemoembolisation (tace) have demonstrated efficacy as monotherapy in the treatment of functional nets. prrt has a slower and longer acting mechanism of action whereas tace can be immediately effective. however, there are no reports that both therapies have been applied in combination. we treated three consecutive patients with severe functional syndromes caused by hepatic metastasis of nets by sequential prrt and tace. the first patient (female years) suffered from a pancreatic net metastasized into the liver. severe hypercalcemia with levels of . mmol/l developed leading to fatique and somnolence despite medical treatment.the patient received one course of mci y- dota-tate prrt and days later, tace was performed at the right liver lobe due to persistent hypercalcemia. after days, calcium levels were normalized and the patients was able to walk with support. after demission, the patients died of progressive disease weeks later. the second patient (male, years) presented with somnolence and hypoglycaemia down to mmol/l by disseminated insulinoma of the pancreas. the patient received two courses of prrt and three coursed of tace involving both liver lobes. he tolerated the combined treatment well and is off intravenous glucose since the first tace. the third patient (male, years) presented with severe hypoglycaemia due to hyperinsulinemia caused by disseminated net of unknown origin with prominent liver metastases in both liver lobes. initially, the patient was treated by tace followed by courses of prrt with -lu. the treatment was well tolerated, the patient is off intravenous glucose. a second tace was intended but was not be performed due to remission of the liver metastases. initial experience with combined treatment of prrt and tace demonstrated that hepatic failure did not evolve in our patients and that the treatment was well tolerated. however, prognosis remains poor due to disseminated and progressive disease. die zahl zirkulierender endothelialer progenitorzellen steigt mit zunehmendem schweregrad der peripheren arteriellen verschlusskrankheit these data indicate that the key mirna-processing enzymes dicer and drosha and consequently the overall expression of mirnas are downregulated during neointima formation in vivo. moreover, the augmented proliferative-and attenuated apoptotic response observed following knock down of drosha and dicer in vsmc implicate an important role of these enzymes and of mirnas for vsmc function during the development of vascular proliferative disease. ( ) hochaktives endokrines organ. das fettgewebe produziert nämlich zahlreiche adipokinine, welche möglicherweise einen autokrinen und parakrinen einfluss auf den fettstoffwechsel, sowie eine endokrine wirkung auf andere organe haben. die entdeckung solcher faktoren eröffnet die frage, ob die adipokinine die gesuchte verbindung zwischen adipositas und den damit assoziierten erkrankungen sein könnten. im diesen zusammenhang haben wir untersucht, ob das fettgewebe die herzfunktion direkt mittels sekretion von kardioaktiven faktoren beeinflusst. wir isolierten deshalb adipozyten aus humanem fettgewebe und untersuchten anschließend den effekt ihrer sekretionsprodukte auf kardiomyozyten in zellkultur sowie mittels langendorff system auf isoliert-perfundierte rattenherzen. wie wir feststellen konnten, hatten die sekretionsprodukte der adipozyten die kontraktion der kardiomyozyten durch eine verminderung des intrazellullären kalziumstroms stark gehemmt. durch erhitzen oder trypsin-behandlung wurden die kardiodepressiven faktoren inaktiv. mittels filtration nach molekulargewicht konnten die kardiodepressiven faktoren zwischen und kd isolierten werden. auf ähnliche weise führte die perfusion von isoliertem herz mit adipozytenfaktoren zu einer starken reduktion der kraftentwicklung sowie zu einer reduktion des koronarflusses durch eine kontraktion der koronararterien. zusammenfassend, die ermittelten daten lassen auf einen bislang unbekannten, negativen einfluss des fettgewebes auf die herzkontraktion schließen. dies geschieht einerseits direkt durch die verminderung des intrazellullären kalziumstroms in die kardiomyozyten, andererseits indirekt durch die reduktion des koronarflusses. diese daten liefern somit eine neue erklärung für den zusammenhang zwischen adipositas und herzinsuffizienz. a comparison of serum fractalkine in patients with coronary heart disease, insulin dependent diabetes, and healthy controls background: atherosclerosis is a multifactorial process that involves inflammation of the vessel wall arising from interactions of leukocytes with vascular endothelial and other local cells. these interactions are regulated by cytokines, including chemokines, as well as adhesion molecules. the chemokine fractalkine (fkn) and its receptor, cx cr , have emerged as interesting intermediaries in atherosclerosis. fkn is a unique chemokine because it exists in soluble and membrane-anchored forms. membrane-bound fkn contributes to adhesion of cx cr -expressing leukocytes, thus providing a novel pathway for leukocyte activation. soluble fkn has leukocyte chemotactic activity. therefore, serum fractalkine levels may indicate the inflammatory situation in chd patients. we investigated the serum concentrations of fkn in non-diabetic patients with coronary heart disease (chd), and insulin dependent diabetics (iddm) in patients attending for rehabilitation in the curschmann-clinic, timmendorfer strand. serum fkn was also determined in healthy controls. fkn was analyzed by elisa. results: soluble fkn was slightly, but not significantly increased in patients with chd in comparison to healthy controls (mean ± standard deviation: ± pg/ml in chd-patients versus ± pg/ml in controls, p= . ). surprisingly, soluble fkn was decreased in insulin dependent diabetics (mean±sd ± pg/ml thus, iddm versus controls p= . , and iddm versus chd p= . ). conclusion: previous studies described that soluble fkn is increased in chd patients compared with healthy controls and that membrane-bound fkn was increased in human carotid arteries and animal models. the decreased soluble fkn in insulin dependent diabetics compared with chd-patients and controls in this study may partly be explained by the quiescent disease state predominant in our rehabilitation patients. hintergrund: neben der jeweiligen bilddokumentation und dem deskriptiven befund des interventionalisten exsistieren keine objektiven daten zur veränderung der hämodynamik während peripheren arteriellen gefässinterventionen und dem unmittelbaren hämodynamischen erfolg nach der intervention. versuche der etablierung eines monitorings durch dopplerultraschall scheiterten in der vergangenheit offensichtlich u.a. an der aufwändigen fixierung der dopplersonden. methode: wir verwenden zur kodomo die doppler x-plore sonde ( , mhz, firma medi-stim, deisenhofen), welche durch einen saugnapf mit ultraschallgeldepot über einer zuvor dopplersonographisch detektierten fussarterie fixiert wird. es können so während der intervention kontinuierliche dopplersignale dokumentiert werden. ergebnisse: phänomene die mittels kodomo bei infrainguinalen eingriffen periinterventionell erfasst werden können sind: verbesserung des dopplerflusses postinterventionell, artefakte durch kontrastmittel, periphere embolien, veränderungen des dopplersignals während der angioplastie. beispielhaft wird ein fall mit filiformer superficialisstenose links demonstriert (abb. ). schlussfolgerung: kodomo gibt dem interventionalisten über die bildgebung hinausgehende hämodynamische zusatzinformationen und ist ein verfahren zur unmittelbaren dokumentation des hämodynamischen erfolges der interventionellen therapie. es spart in konsequenter anwendung möglicherweise kontrast-mittel, durchleuchtungs-und untersuchungszeit. durch den artefiziellen gefäßverschluss zum zeitpunkt der angioplastie kann der kollateralfluss der zielläsion zukünftig gemessen werden. einfluss von shunt-verkalkungen auf das Überleben von hämodialysepatienten introduction: two-dimensional strain ( ds) is a novel method to measure strain from standard two-dimensional echocardiographic images. strain imaging has been proposed as a sensitive tool for the assessment of the left ventricular myocardial function. the aim of our study was to characterize global and regional function abnormalities using this technique in patients (pts.) with constrictive pericarditis (cp) and restrictive cardiomyopathy (rcm). methods: we studied consecutive patients (pts.) with heart failure of either proven pericardial (cp) or myocardial origin (rcm; biopsy proven cardiac amy-loidosis). global longitudinal strain (gls) and regional peak systolic strain (pss) was assessed by ds in the apical four-chamber-view using a dedicated software package (vivid , ge healthcare). all pts. underwent a complete echocardiographic and hemodynamic assessment. results: out of the pts. (mean age: ± years) had cp, and rcm. the thickness of the interventricular septum (ivsd) was significant increased in pts. with rcm ( ± vs. ± mm). mean gls was - . ± . % in the cp-group and - . ± . % in the rcmgroup (p< . ). pts. with rcm showed a significant decreased longitudinal pss in the septal segments (basal: - . ± . % vs. - . ± . %, p< . , mid: - . ± . % vs. - . ± . %, p< . ; apical: - . ± . % vs. - . ± . %; p< . ) and a decreased pss in the lateral segments (basal: - . ± . % vs. - . ± . %, n.s.; mid: - . ± . % vs. - . ± . %, n.s.; apical: - . ± . % vs. - . ± . %, n.s.). conclusion: two-dimensional strain is a simple and rapid method to measure gls and pss. this technique might be used as new helpful tool for the differentiation between pts. with rcm and cp, and for the detection of early regional myocardial dysfunction before the onset of congestive heart failure (chf) in patients with cardiac amyloidosis. nichtinvasive koronare plaquedifferenzierung: mehrschicht-computertomographie validiert mit intravaskulärem ultraschall Über die klassischen risiken hinaus sind auch lokale faktoren an der initiation und progression von atherosklerotischen ablagerungen beteiligt. diese faktoren beinhalten multiple variablen wie wechselwirkungen zwischen gewebe und flüssigkeiten, wandspannung und flussgeschwindigkeit . die direkte messung dieser faktoren ist in-vivo nur im tierversuch möglich. entsprechende parameter können jedoch über den einsatz moderner bildgebungstechniken (cardio-cta) unter verwendung von flusssimulationen (computational fluid dynamics=cfd) berechnet werden. das ziel dieser studie war daher ) die durchführbarkeit der in-vivo cfd-berechnung an humanen koronararterien zu demonstrieren und ) die ergebnisse mit radiofrequenzmessungen im intravaskulären ultraschall (ivus) zu korrelieren. methoden und ergebnisse: zehn patienten mit suspektem koronarem befund wurden prospektiv in die studie einbezogen. diese erhielten innerhalb von vier wochen eine ct-gestützte koronarangiographie (dual source slice ct) und eine invasive koronarangiographie. bei diesen patienten wurde die intravaskuläre ultraschalluntersuchung in allen drei hauptstammgefäßen durchgeführt, die erhebung der radiofrequenzdaten geschah simultan. mit hilfe der axialen cta-schnitte wurde ein gittermodell der jeweiligen gefäße erstellt. dieses konnte genutzt werde um die gewebeflüssigkeitinteraktionen, flussgeschwindigkeit und wandspannungsverhältnisse zu simulieren. die berechnung der cfd-parameter konnte in / herzkranzarterien erfolgreich durchgeführt werden. siebzehn koronararterien wurden zusätzlich durch ivus beurteilt. die wandspannung der gefäße korrelierte umgekehrt zu vorhandenen durch ivus ermittelte plaques(p< , ). ein zusammenhang zwischen den erhobenen cfd-daten und den radiofrequenzdaten der gewebe konnte bisher nicht nachgewiesen werden. zusammenfassung: die ergebnisse dieser studie demonstriert die möglichkeit gewebeflüssigkeitsinteraktionen in menschlichen koronararterien, mit hilfe von modernen computertomographischer bildgebung, nichtinvasiv abzuschätzen. die bedeutung dieser zusätzlichen informationen muss prospektiv weiter untersucht werden. welchen einfluss hat die myokardiale fibrose und funktion bei patienten mit hochgradiger aortenklappenstenose auf den klinischen langzeitverlauf nach aortenklappenersatzoperation? ziel dieser studie ist es, die entwicklung des linksventrikulären remodellings sowie der regionalen myokardialen funktion bei patienten mit hochgradiger aortenklappenstenose sowohl vor als auch im verlauf nach aortenklappenersatzoperation zu untersuchen. methodik: bei patienten mit hochgradiger aortenklappenstenose wurde sowohl präoperativ als auch monate postoperativ eine konventionelle echokardiographie und eine strain-rate-imaging studie mit bestimmung der longitudinalen systolischen spitzen-strain-rate durchgeführt. zur beurteilung der myokardialen fibrose wurde eine magnetresonanz tomographie (mrt) mittels gadolinium late enhancement technik durchgeführt und eine biopsie aus dem linksventrikulären septum intraoperativ entnommen. ergebnisse: die patienten wurden entsprechend der nyha-klasse monate nach der operation in drei gruppen aufgeteilt. gruppe (gutes outcome=nyha-klasse i; n= ), gruppe (mittleres outcome, nyha-klasse ii; n= ), gruppe (=schlechtes outcome, nyha -klasse iii bzw. iv; n= ). präoperativ zeigten sich bei allen drei gruppen eine vergleichbare ejektions-fraktion (gruppe = ± %; gruppe = ± %; gruppe = ± %). wohingegen bei der longitudinalen strain rate signifikant höhere werte in gruppe als in gruppe gemessen werden konnten (gruppe = , ± , s - ; gruppe = , ± , s - ; gruppe = , ± , s - ). interessanterweise stieg die longitudinale strain rate erst mit der abnahme der wandstärken nach monaten an. außerdem konnte ein signifikant höherer grad an interstitieller fibrose mittels biopsie-score (gruppe = , ± , ; gruppe = , ± , ; gruppe = , ± , ) sowie auch ein häufigeres late enhancement in der mrt bei patienten mit schlechtem klinischem outcome nachgewiesen werden (segmente mit fibrose: gruppe = , ± , ; gruppe = , ± , ; gruppe = , ± , ). zusammenfassung: diese daten lassen vermuten, dass bei patienten mit hochgradiger aortenklappenstenose sowohl eine myokardiale fibrose als auch eine reduzierte regionale myokardiale funktion präoperativ entscheidene hinweise auf den klinischen langzeit-verlauf nach aortenklappenersatzoperation geben können. indikationen und limitationen der ultraschall-elastographie bei patienten mit erkrankungen des pankreas vor dem hintergrund immer wirksamerer, aber auch sehr kostenintensiver behandlungsoptionen einerseits und dem bewusstsein der früh einsetzenden strukturellen schädigung im arthritischen prozess ("window of opportunity") andererseits, ist ein monitoring des therapieansprechens bei patienten mit rheumatoider arthritis von besonderer bedeutung. eine exakte darstellung des entzündlichen substrates gelingt besonders mit der sonographie, wodurch therapieversager frühzeitig identifiziert werden können. die arthrosonographie erlaubt eine sehr gute differenzierung zwischen exsudativen und proliferativen synovialisveränderungen sowie die beurteilung der perfusion durch die doppler-verfahren. voraussetzung für eine sonographische beurteilung des verlaufes des arthritischen prozesses ist eine quantifizierung der entzündungsaktivität. in anlehnung an den von scheel und kollegen publizierten synovitisscore, wurden patienten mit aktiver rheumatoider arthritis, die durch eine biologikatherapie (tnf-inhibitoren, rituximab, abatacept) in klinische remission gebracht werden konnten (das : < , ), standardisiert sonographisch nachuntersucht. bei der mehrheit der ra-patienten konnte trotz klinischer remission durch die sonographischen nachuntersuchungen und monate nach initiierung der biologikatherapie eine persistierende entzündungaktivität nachgewiesen werden. während eine deutliche therapiebedingte abnahme der hyperperfusion zu beobachten war, konnte nur eine geringgradige abnahme der synovialen proliferationen und ergussbildung festgestellt werden. diese ergebnisse zeigen, dass bei der mehrzahl der ra-patienten trotz klinischer remission eine sonographisch nachweisbare snovitis persistiert, was möglicherweise die ursache für den radiologischen krankheitsprogress bei einem teil dieser patienten darstellt. zielsetzung/aims: granulocyte-colony stimulating factor (g-csf) was shown to improve cardiac function after myocardial infarction (mi). recently, we histologically demonstrated enhanced arteriogenesis and reduced infarct size by g-csf treatment after mi in mice. in this study, we non-invasively, repetitively, and quantitatively investigated g-csf effects on perfusion by a pinhole single-photon emission computed tomography (spect) system after mi in mice. methoden/methods: mi was induced by lad ligation in wt mice (c bl/ j). g-csf ( μg/kg; n= ) or saline (n= ) was daily injected for days. extent of left ventricular (lv) perfusion defects was determined with a [ mtc]-sestamibi triple headed pinhole spect system at days (baseline) and days after lad occlusion. polar maps were normalized by mean of a standardized reference region of interest (roi) in the septal region ( %). best threshold value for identifying infarcted areas was determined by comparing perfusion defects with the histological infarct sizes in these animals. defect size was indicated as% of lv myocardium. primary endpoint was change of defect size from baseline to days after mi. ergebnisse/results: best threshold for identifying infarcted areas compared to histology was less than % of the septal reference roi. mean infarct size was similar in saline ( , % ± , ) and g-csf group ( , % ± , ) at baseline. at day after mi, a slight difference (p= , ) was found between saline ( , ± , ) and g-csf ( , % ± , ) groups. however, change of defect size was significantly different (p= , ) between g-csf (- , % ± , ) and control animals (- , ± , ) . schlussfolgerung/conclusions: this is the first study, demonstrating the suitability of triple headed pinhole spect system for repetitive infarct size measurement in mice, offering a new non-invasive imaging technique for cardiovascular therapy monitoring. in this context, we showed that g-csf administration significantly reduces lv perfusion defects indicating positive effects on ventricular remodelling after mi in mice. endoscopy in patients with acute leukemia after intensive chemotherapy conclusions: endoscopy can be performed relatively safely in patients who received myelosuppressive chemotherapy. the procedure may induce fever and bacteremia. the percentage of patients in whom endoscopic hemostasis was applicable and effective was low. we recommend conservative treatment first. endoscopy should be reserved for patients who are unstable or refractory. impact of pregnancy on prevalence of goitre and nodular thyroid disease in women living in a region of borderline iodine deficiency purpose: an interplay of genetic, epigenetic and environmental factors contributes to thyroid disease. in a cross-sectional study we aimed to determine the actual influence of parity on goitre and nodular thyroid disease (ntd) in women living in a region with borderline iodine deficiency. methods: thyroid ultrasonography ( . mhz; merck thyromobil) was performed by the same investigator in women living in thuringia and saxony. furthermore, age and bmi were documented and all women were asked about the number of previous pregnancies, family history of thyroid disease and past or present smoking. results: goitre prevalence was . %. solitary thyroid nodules were detected in . %, multiple nodules in . % of the study population. age was positively correlated with goitre prevalence and ntd (due to multiple but not solitary nodules). no association was found between parity and goitre prevalence. in contrast, a significant increase in both, solitary ( . %) and multinodular thyroid disease ( . %) was observed in women with at least one pregnancy compared to nullipara ( . % and . %, respectively). bmi in women with goitre ( . kg/ m ) was significantly higher than in women without ( . kg/m ). in addition, a significant correlation was detected between bmi and presence of multinodular disease ( . kg/m ). , % of women with goitre reported a positive family history for thyroid disease, as opposed to % of women with normal size thyroid gland. neither goitre nor ntd were associated with a present or past history of smoking. conclusion: ntd and/or goitre are present in up to % of woman in an area with borderline iodine deficiency. besides age, bmi and family history, parity is positively correlated with presence of ntd, whereas smoking was not associated with goitre/ntd. defizite in der medizinischen versorgung von patienten mit nebennierenkarzinom in deutschland kardiologie, universität heidelberg, heidelberg; koordinierungszentrum für klinische studien leipzig, universität leipzig, leipzig; kardiologie, universität würzburg, würzburg; institut für herzkreislaufforschung, dortmund; kardiologie, universität marburg, marburg; kardiologie, berlin; institut für frauenspezifische gesundheitsforschung, deutsches herzzentrum berlin, berlin; kardiologie, universität göttingen, göttingen; kardiologie, essen einleitung: Ältere patienten und frauen sind in großen klinischen studien zur chronischen herzinsuffizienz regelhaft unterrepräsentiert. die vorliegende arbeit analysiert daher die geschlechts-und altersabhängige leitlinien-Ädhärenz im rahmen der erhebungen des kompetenznetzwerkes herzinsuffizienz. methodik: wir evaluierten die datensätze aller patienten mit systolischer herzinsuffizienz (lvef echokardiographisch ≤ %) die zwischen / und / in das kompetenznetzwerk herzinsuffizienz eingeschlossen wurden: n= ; mittleres alter , ± , jahre; , % frauen. die nyha-angepasste, leitliniengerechte verschreibung der pharmakotherapie (=adhärenz) wurde über die einnahmehäufigkeit der herzinsuffizienzmedikation unter berücksichtigung von kontraindikationen erfasst. der guideline-adherence-indicator (gai) wurde für betablocker, ace-hemmer/at -blocker und aldosteron-rezeptorblocker berechnet (=gai- ), sowie für die zusätzliche indikation für diuretikum und glykosid (=gai- ). ergebnisse: der gai- betrug in den nyha klassen i/ii/iii/iv / / / % und der gai- / / / % (p für trend jeweils < . ). die therapie-adhärenz (gai- und gai- ) war bei männern höher als bei frauen (p= . und p< . ). in der multivariablen ordinalen regression waren zwar alter (or pro dekade , , p= , ; und or , , p< , ) und nyha stadium iii-iv (or , und , , p jeweils < , ), nicht jedoch das geschlecht (p= , und p= , ) prädiktoren des gai- und gai- . zusammenfassung: aktuelle daten des kompetenznetz herzinsuffizienz zeigen bei der therapie der chronischen herzinsuffizienz im vergleich zu früheren studien in deutschland hinsichtlich der verordneten substanzklassen eine klinisch hoch relevante zunahme der leitlinien-adhärenz. in zukünftigen studien wird zu klären sein, ob auch leitliniengerechte dosierungen in der klinischen praxis sinnvoll umsetzbar sind. effekt von nt-probnp purpose: the endothelial specific angiopoietin (ang)-tie ligand-receptor system has a key role in regulating vascular integrity and quiescence. the antagonistic ligands ang- and ang- control the expression of endothelial adhesion molecules and intercellular tight junctions. the role of ang- in anca-associated vasculitis (aav) has not been investigated yet. methods: we measured serum ang- in healthy controls and patients with aav ( patients with active aav at initial presentation and during follow-up, patients in stable long-term remission, patients prior to systemic relapse, and patients with active "limited" wg (ent). the disease activity was monitored by the birmingham vasculitis activity score (bvas) and the enumeration of circulating endothelial cells (cecs). for statistical analysis we used one-way anova with dunn´s correction, friedmann and wilcoxon tests, spearman´s rho test and linear regression. results: ang- was elevated in active aav (mean . ± . ng/ml sem) compared to controls ( . ± . p< . ). most notably, ang- was also elevated in archival serum samples of patients in long-term remission just before systemic vasculitic relapse ( . ± . ng/ml p< . ). in contrast, ang- was normal in patients with stable remission of aav ( . ± . ng/ml and "limited" granulomatous ent disease ( . ± . ng/ml). ang- was elevated at initial presentation ( . ± . ng/ml sem) and declined rapidly after immunosuppessive therapy at month ( . ± . ng/ml), - months ( . ± . ng/ml) and months ( . ± . ng/ml). linear regression analysis demonstrated a strong association of ang- with bvas (rs = . p< . ) and cecs and (rs = . p< . ). these results indicate that ang- might be a useful early marker to discriminate systemic vasculitic activity in aav from limited granulomatous ent disease and remission. moreover, ang- might also be a mediator of endothelial inflammation and detachment. further investigation of the ang/tie-system in vasculitis is crucial since pharmacologic inhibitors of ang- will become available shortly. warfarin-induzierte kalzifikation in mäuseneine neues modell zur untersuchung der interaktion von verkalkungsinhibitoren aim of the study: although hypercholesterolemia, a predominant risk factor of coronary heart disease, is related to cholesterol metabolism, the association between cholesterol metabolism and coronary heart disease is not well known. therefore, this study investigates effects of cholesterol metabolism on coronary heart disease and family history of cardiovascular diseases. methods: in addition to conventional coronary risk factors (age, sex, bmi, arterial hypertension, diabetes mellitus, smoking, family history of cardiovascular diseases, plasma cholesterol) campesterol and sitosterol (indicators of cholesterol absorption) and lathosterol (indicator of cholesterol synthesis) were determined in consecutive men and women with severe aortic stenosis. on coronary angiograms prior to aortic valve replacement extend of coronary heart disease was determined ( , , , -vessel disease). furthermore, a semiquantitative score of the extend of coronary atherosclerotic lesions was determined. results: in patients with a positive history of cardiovascular diseases the ratio of campesterol to lathosterol was significantly increased (p< . ). there was a significant increase in campesterol to lathosterol ratio in plasma with increasing extend of coronary heart disease ( , , , -vessel disease; p< . ). furthermore, there was a positive correlation of coronary vessel score with the ratio of campesterol to lathosterol in plasma (r= . ; p< . ) and in aortic valve cusps (r= . ; p< . ), indicating that enhanced absorption and reduced synthesis is related to the extend of coronary heart disease. logistic regression analysis revealed that of all coronary risk factors tested the ratio of campesterol to lathosterol was the sole, significant predictor of coronary heart disease in this subset of patients (p< . ). the results of this study suggest that in patients with severe aortic stenosis elevated ratios of campesterol to lathosterol are directly related to concomitant coronary heart disease and may serve as a predictor for coronary heart disease in humans. bei postmenopausalen frauen und bei männern mit rheumatoider arthritis und glukokortikoid-therapie besteht zu über prozent die indikation für eine bisphosphonat-therapie nach dvo-leitlinie hintergrund: ein geringes ansprechen auf clopidogrel nach koronarer stentimplantation ist assoziiert mit einem erhöhten risiko für das auftreten von stentthrombosen. die cytochrom p (cyp) isoenzyme cyp c and cyp a / sind bei der bioaktivierung von clopidogrel beteiligt. das ziel der vorliegenden studie ist es, den zusammenhang zwischen dem ansprechen auf clopidogrel und genetischer varianten der cyp isoenzyme bei patienten mit symptomatischer koronarer herzerkrankung zu untersuchen. methoden und ergebnisse: genotypisierungen für cyp c (* , * , * ), cyp a * b und cyp a * polymorphismen wurden bei konsekutiv eingeschlossenen patienten (n= ), die einer koronare stentimplantation aufgrund einer symptomatischen koronaren herzerkrankung erhielten, durchgeführt. die adenosin diphosphat (adp)-induzierte thrombozytenaggregation wurde frühestens stunden nach erstgabe von mg clopidogrel gemesen. träger der cyp c * variante zeigten eine signifikant höhere residuelle thrombozytenaggregation (rta) (chi-quadrat ; p< . ). die übrigen untersuchten polymorphismen zeigten keinen signifikanten einfluß auf die rta. auf der grundlage des predict-score zur vorhersage einer erhöhten rta wurden der cyp c * genotyp und zuvor identifizierte nicht-genetische einflussvariablen (alter, akutes koronarsyndrom, typ diabetes, reduzierte linksventrikuläre funktion, und niereninsuffizienz) untersucht. die logistische regressionsanalyse ergab eine signifikante korrelation der nicht-genetischen risikofaktoren (chi-quadrat , ; p= , ) und des cyp c * polymorphismus (chi-quadrat , ; p< , ) mit einer erhöhten rta, und einen kumulative assoziation zwischen rta und der kombination aus genetischen und nicht-genetischen faktoren (chi-quadrat , ; p < , ). diskussion: träger von zumindest einem cyp c * allel haben ein ungefähr -faches risiko, eine erhöhte rta zu entwickeln. genotypisierung der funktionsverlust-variante cyp c * könnte zu einer verbesserten prädiktion des ansprechens auf die antithrombozytäre therapie mit clopidogrel beitragen. the shape of the glucose curve during an oral glucose tolerance test correlates with changes in glucose tolerance m. reimann , j. li , s. bornstein , j. schulze , p. schwarz medical clinic iii, endocrinology, medical faculty carl gustav carus, technical university dresden, dresden; sächsische landesärztekammer, dresden introduction and objectives: the shape of the glucose curve during an oral glucose tolerance test (ogtt) can be categorized in monophasic and biphasic. the latter has been associated with normal glucose tolerance. the aim of the study was to explore the association between the shape of the glucose curve and changes of glucose tolerance. research design and methods: ogtt data from subjects with different stages of glucose tolerance were analyzed at baseline and three years follow up. the shape of the glucose curve at baseline was classified as monophasic, biphasic and unclassified. the shape index was calculated as the difference between glucose at min and at min and treated as continuous variable in correlation analyses. in the biphasic group, there was a significantly higher proportion of subjects with normal glucose tolerance and a lower proportion of subjects with impaired glucose tolerance and type diabetes. subjects with a biphasic shape had a significant lower bmi and a better profile of carbohydrate metabolism. the shape index correlated significantly with changes of plasma glucose at baseline and at min and insulinauc after adjustment for glucose tolerance state. the prevalence ratio for disease regression was significantly higher in subjects with biphasic glucose curve shape. the shape index gives additional information regarding the stage of glucose tolerance beyond the who classification. a biphasic shape is associated with improvements in plasma glucose and may predict regression of disease irrespective of glucose tolerance. die residuelle aggregation unter dualer thrombozytenhemmung beeinflusst die inzidenz schwerer kardiovaskulärer ereignisse unabhängig vom einsatz medikamentenbeschichteter stents kritisch kranke patienten weisen häufig eine charakteristische konstellation des thyreotropen regelkreises auf, die als non-thyroidal-illness-syndrom (ntis) bezeichnet wird und mit einer erhöhten morbidität und mortalität verbunden ist. trotz langjähriger forschung zu details dieses komplexes sind wichtige fragen noch immer offen. so ist bislang noch unbekannt, inwiefern vorbestehende schilddrüsenerkrankungen zur entstehung und ausprägung eines ntis beitragen. im rahmen der prospektiven aqua-fontis-studie untersuchten wir daher patienten, die auf einer internistischen, einer chirurgischen und einer herzchirurgischen intensivstation des universitätsklinikums bergmannsheil versorgt wurden, bezüglich des auftretens von autoantikörpern gegen thyreoglobulin (tgak), schilddrüsen-peroxidase (tpo-ak) oder tsh-rezeptoren (trak). die antikörpertiter, die wir stunden nach aufnahme auf die intensivstation bestimmten, korrelierten wir mit parametern der schilddrüsenhomöostase, der dauer des stationären aufenthaltes und dem Überleben der patienten. Über % der untersuchten patienten wiesen negative oder intermediäre tgakoder tpo-ak-titer auf, weniger als ein prozent zeigten positive antikörpertiter. ausnahmslos alle untersuchten patienten waren trak-negativ. die höhe sämtlicher antikörpertiter korrelierte weder mit dem auftreten einer thyreotropen insuffizienz noch mit der dauer des stationären aufenthaltes oder der intensivbehandlung und auch nicht mit dem Überleben der betroffenen patienten. auch in der subgruppe der überlebenden patienten korrelierten die antikörpertiter nicht mit der dauer der behandlung. es kann daher festgestellt werden, dass bei kritisch kranken personen schilddrüsenautoantikörper mit annähernd der gleichen prävalenz wie bei einer normalpopulation auftreten. darüber hinaus haben autoimmunthyreopathien keinen stellenwert in der prognose von patienten mit ntis. typ- -diabetes mittels ekg identification of markers for prediction of the clinical course remains a major challenge in the management of diabetic nephropathy. we established a proteomics approach for identification of diabetic nephropathy related biomarkers in urine. we used seldi-tof mass spectrometry and sax protein arrays to compare protein profiles from urine of four defined patient groups. samples from patients with type diabetes (dm) (n = ) without nephropathy and without microalbuminuria (dm-wnp), dm patients with macro-or microalbuminuria (dm-np) (n = ), patients with proteinuria due to non-diabetic renal disease (n = ), and healthy controls (n = ) were analysed. anionic exchange, reversephase fractionation, gel electrophoresis, and mass spectrometry were used to isolate and identify proteins with high discriminatory power. a protein with m/z (p < . ) was strongly released in the urine of healthy controls, patients with proteinuria due to non-diabetic disease, and dm-wnp in contrast to dm-np-patients. a m/z protein (p < . ) was selectively excreted in the urine of dm-np patients, whereas the protein with m/z (p < . ) was significantly excreted by patients with proteinuria and dm-np. the m/z and m/z mass peaks were identified as beta- -microglobulin and uba , an ubiquitin ribosomal fusion protein respectively. the protein with m/z was identified as a processed form of ubiquitin. moreover the ubiquitin degradation assay confirmed the potential role of a urinary protease whose absence was specific for diabetic nephropathy. the release of high amounts of uba in urine of dm-np patients could serve as a diagnostic marker. the identification of the protease and longitudinal studies in larger patient groups will determine the usefulness of the short form of ubiquitin as a marker for predicting the clinical course and the potential role of the protease in the pathophysiology of diabetic renal involvement. rolle von freien fettsäuren und fettsäuretransportproteinexpression in der apoptosevermittelten pathogenese der fettlebererkrankung extravascular lung water index (elwi) has been demonstrated to predict mortality and to correlate to pao /fio -ratio and compliance in patients with sepsis and ards. however, with an increasing number of obese patients, there is the question which body weight should be used for indexation of extravascular lung charité -universitätsmedizin berlin, campus virchow-klinikum, berlin; koordinierungszentrum für klinische studien würzburg therefore it was the aim, to investigate the correlation of elwi to pao / fio -ratio and oxygenation index (mean airway pressure* /pao ) using different weight parameters for indexation. methods: in patients of a medical icu with a body mass index > kg/m , measurements of extravascular lung water were performed using the picco system elwi correlated to the functional parameters with high significance (p< . ) independently of the the index used: correlation to pao /fio -ratio conclusions: .) in obese patients, extravascular lung water and its indices adjusted to abw, pbw, ibw and adpw significantly (p< . ) correlate to pao / fio -ratio and oxygenation index. .) the highest correlation to pao /fio -ratio was found using adbw unsere fallsammlung zeigt den facettenreichtum seltener pilzinfektionen. weitere zentren sind eingeladen, ihre fälle unter www.fungiscope.net zu registrieren ps , ps , ps ps , ps , ps ps , ps , ps ps , ps , ps , ps ps trinkmann, frederik . . ps , ps ps van den elsen antithrombozytäre effekte von ace-hemmern und at -blockern: modifizierte ex-vivo-plättchenaggregation bei kardiovaskulären patienten a. viktor , i. tuleta , m. steinmetz , g. bauriedel , g. nickenig , d. skowasch abteilung für kardiologie und pulmologie, zentrum für innere medizin, stuttgart; innere medizin (kardiologie/pneumologie), universitätsklinikum bonn, medizinische klinik ii, bonn; innere medizin (kardiologie), klinikum meiningen, medizinische klinik i, meiningen hintergrund: ace-hemmer und at -rezeptorblocker (arbs) sind eckpfeiler in der therapie kardiovaskulärer patienten. in mehreren randomisierten und plazebo-kontrollierten studien konnte eine signifikante verminderung von kardiovaskulärer mortalität, myokardinfarkt-und schlaganfallrate nachgewiesen werden. vor diesem hintergrund untersucht diese studie mögliche antithrombozytäre effekte von ace-hemmern und arbs; vergleichskollektive sind patienten mit ass/clopidogrel und unbehandelte patienten. methoden: proben von insgesamt kardiovaskulären patienten wurden mittels vollblutaggregometrie analysiert. dabei war die agonisten-induzierte plättchenaggregation (adp, kollagen) durch die zunahme der impedanz (ohm) quantifiziert. die daten wurden mit vorliegen bzw. fehlen der medikation korreliert. ergebnisse: als zentraler befund war die plättchenaggregation bei studienteilnehmern mit ace-hemmern, arbs, ass und clopidogrel vermindert. die kollagen-induzierte plättchenaggregation wurde unter ace-hemmern ( , %, p< , ) und arbs ( , %, p< , ) signifikant reduziert; unter therapie ass ( , %, p< , ) bzw. ass/clopidogrel ( , %, p= , ) nahm sie ebenfalls ab. nach adp-induktion war die plättchenaggregation unter therapie mit ace-hemmern ( , %, p= , ) und arbs ( , %, p= , ) signifikant reduziert; unter ass ( , %, p= , ) und ass/clopidogrel ( , %, p= , ) zeigte sich ebenfalls eine reduktion. eine zusätzliche verlaufsbeobachtung nach neueinstellung mit dem wirkstoff valsartan zeigte nach tagen eine signifikant reduzierte kollagen-induzierte plättchenaggregation ( %, p= , ). eine in vitro-messreihe mit der rohsubstanz valsartan konnte für äquivalente therapeutische dosen keine signifikante beeinflussung zeigen. folgerung: die therapie mit arbs und ace-hemmern führt zu einer signifikanten verminderung der plättchenaggregation ex vivo. das antithrombotische potential der arbs und ace-hemmer könnte für die reduktion der konsekutiven thrombosen und der progression atherosklerotischer organleiden mitverantwortlich sein und so die reduktion kardiovaskulärer endpunkte zumindest miterklären. kombination einer dendritischen zellvakzine mit gemcitabin im murinen pankreaskarzinom:charakterisierung der immunantwort und wirksamkeit m. dauer with or without the tlr- ligand cpg before (prophylactic) or after (therapeutic) tumor induction. cd + t cell responses were analyzed in peripheral blood. antigen uptake, activation and cytokine production of dendritic cells (dc) in lymph nodes was analyzed by flow cytometry. data: vaccine draining lymph nodes increased in size and cellularity. the iscom vaccine was effectively taken up by dc, resulting in upregulation of activation markers, production of il- and potent t cell stimulation. key: cord- -ajmj hyj authors: ellegren, h.; mikk, s.; wallin, k.; andersson, l. title: limited polymorphism at major histocompatibility complex (mhc) loci in the swedish moose a. alces date: - - journal: mol ecol doi: . /j. - x. .tb .x sha: doc_id: cord_uid: ajmj hyj the swedish moose was analysed for genetic variability at major histocompatibility complex (mhc) class i and class ii dqa, dqb and drb loci using restriction fragment length polymorphism (rflp) and single strand conformation polymorphism (sscp) techniques. both methods revealed limited amounts of polymorphism. since the sscp analysis concerned an expressed drb gene it can be concluded that the level of functional mhc class ii polymorphism, at least at the drb locus, is low in swedish moose. dna fingerprinting was used to determine if the unusual pattern of low mhc variability could be explained by a low degree of genome‐wide genetic diversity. hybridizations with two minisatellite probes gave similarity indices somewhat higher than the average for other natural population, but the data suggest that the low mhc variability cannot be explained by a recent population bottleneck. however, since minisatellite sequences evolve more rapidly than mhc sequences, the low levels of mhc diversity may be attributed to a bottleneck of more ancient origin. the selection pressure for mhc variability in moose may also be reduced and we discuss the possibility that its solitary life style may reduce lateral transmission of pathogens in the population. the gene products of the major histocompatibility complex (mhc) system are recognized as key molecules in the immunological recognition of self and nonself (klein ). there are two distinct classes of mhc molecules, class i and class ii, which are encoded by separate but tightly linked loci. both are cellsurface glycoproteins which function as presenters of intracellulary processed peptides. in a majority of the species so far studied, mhc represents the most polymorphic gene system found in the genome. unlike most other polymorphic genes, mhc variability is characterized by the presence of a large number of alleles that occur at intermediate frequencies (klein ). this is a pattern expected for loci under balancing selection (hedrick & thomson ) and it is generally believed that the high degree of genetic variability at mhc correspondence: dr hans ellegren. fax: + . e-mail: hans.ellegren@bmc.uu.se is maintained by overdominant or frequency-dependent selection (klein et al. ) . many investigators argue that this relates to the immunological defence against pathogen-induced diseases (bodmer ; doherty & zinkemagel ) , although other selective forces such as mating preference and maternal-fetal interactions have been suggested as well (hedrick & thomson ; potts & wakeland ) . it has been suggested that populations which have experienced extensive bottlenecks, leading-to educed mix variability, would be particularly vulnerable to infectious disease (o'brien & evermann ) . preserving mhc polymorphism has in fact been put forward as being a prime objective in all conservation programs (hughes ) . in line with this, it has been proposed that the extremely low level of mhc variability in the african cheetah (o'brien et al. ; yuhki & o'brien ) may be related to a marked susceptibility to a feline coronavirus infection in a group of captive individuals (o'brien et al. ) . severe outbreaks of infectious disease in some other species speculated to possess reduced mhc variability may represent similar situations as well (obrien & evermann ) . we are involved in a study of the possible association between genetic variation at mhc loci and susceptibility to a lethal disease syndrome, still of unknown origin, among swedish moose a. alces (stken et nl. ) . we have now used various molecular techniques to assess the amount and characteristics of mhc class i and class i variability in swedish moose and in parallel analysed their genome-wide variability by means of dna fingerprinting. we show here that these animals exhibit low levels of mhc polymorphism concurrent with a fairly high degree of genomic diversity. two sets of animals were analysed. first, liver samples were obtained from free-living moose collected in four different areas spread from southern to northern sweden. second, liver samples were also derived from free-living individuals shot during the autumn hunt in a kmz area in central sweden. apparent relatives were avoided in the latter case by only collecting a single specimen from each shooting location. pieces of liver were transferred to - "c not later than six hours after shooting. we have previously made extensive genetic studies of the mhc system of cattle (e.g. sigurdardottir et al. ). we will make reference to cattle several times in this study since this species represents the closest relative to the moose for which mhc variation previously has been well characterized. all cattle data cited have been obtained from the swedish red and white breed. gnomic dna was prepared from blood and liver as previously described (ellegren et al. ) . southern blots of tiqi, pvuii, ecori or hindiii digests ( pg) were prepared foilowing standard procedures, i.e. electrophoretic separation of fragments in . % agarose gels and subsequent transfer to nylon filters (biodyne b) at aibaline conditions. hybridization was carried out according to mariani et al. ( ) with a final washing stringency of . x ssc; . % sds at + "c. we used four human mhc cdna clones as hybridization probes: (gustafsson et al. ) . the three latter probes correspond to h hc class ii genes. filters were prepared as above except that the dna was digested with hiiifl and that . % agarose gels were used. we employed two commonly used minisatelhe probes, ms (jeffreys et al. ) and a bp bstni fragment from phage m (vassart et nl. ) . hybridization was performed in the absence of competitor dna and filters were washed at a final stringency of x ssc; . % sds at + "c. exon two of the mhc drb gene was amplified by pcr using the cattle specific primers la and la (sigurdardottir et al. ) . the -pl reactions contained . mm dntp, . p~ of each primer, ng of genomic dna and u taq polymerase. a total of cycles was performed, the three first had + "c for s, + "c for s and + "c for s, the following had + "c for s, + "c for s and + "c for s. the pcr was performed on a perkin-elmer-cetus instrument. the sscp analysis essentially followed maekawa et al. ( ) . briefly, pcr products were mixed with a . volume denaturing solution containing % formamide, -m~ edta, . % bfb and . % xylene cyanol, and were incubated at + "c for min. samples were subsequently applied to phastsystem gradient gels - (previously prerunned at v, ma and . w for vh) and electrophoresed with native buffer strips at + "c in a phastsystem device (pharmacia, uppsala) for vh (using the same settings as above). after separation, the gels were silver-stained with the phastsystem silver staining kit. human cdna clones encoding hla class i (locus not determined), hla-dqa, hla-dqb and hl -drb genes were hybridized to moose tqi, pvuii, ecori and hindiii digests. the screening was performed on unrelated individuals sampled at various sites in sweden. the outcome of the experiments is summarized in table . clearly, the levels of polymorphism were very limited. completely monomorphic hybridization patterns were revealed in seven out of probe/enzyme combinations. furthermore, the seven polymorphic class il probe/enzyme combinations did most likely only detect two different d q haplotypes. this conclusion was reached on the basis of that the polymorphic fragments, only one or two in each case, consistently were present in the same @ blackwell science ltd, molecular ecology, , - table summary of the moose mhc rflp analysis. the number of polymorphic fragments are indicated for each probe/ enzyme combination with the total number of fragments in parentheses. obviously cross-hybridizing fragments were excluded from the total numbers enzyme locus raqr paun class i (ii) four individuals in all seven combinations. the variability seen with the class i probe was similarly low, i.e. monomorphic pattern with two enzymes and one polymorphc fragment with two other enzymes. the latter polymorphisms represented different haplotypes as compared to the class i polymorphisms (the polymorphic tuqi fragment was present in five individuals whereas the polymorphic ecori fragment only occurred in two individuals). in an extended screening we analysed additiocal moose sampled in central sweden. the screening was restricted to dqb and drb probes hybridized to puuii digests. as for the first individuals, a completely monomorphic pattern was revealed with the drb probe. four out of five hybridization fragments specifically detected by the dqb probe were polymorphic in this material but could be interpreted as a simple two-allele polymorphism (fig. la) . one allele was composed of . -highly significant difference (xz = . , p < . ). furthermore, in a study of bovine mhc class i polymorphism, lindberg q distinguished no less than pvuii haplotypes in a sample of o unrelated individuals. there were no more than two class i haplotypes seen with any enzyme in moose. as an illustration of the varying levels of mhc polymorphism in cattle and moose, a blot with bovine pvuii digests hybridized with the same human dqb probe as employed in the present study is shown in fig. i@) . clearly, the degree of polymorphism differs considerably between the two species. the primers la and la specifically amplify exon two of the bovine drb gene, the most predominantly expressed and the most polymorphic drb gene in cattle. the primer pair was used in amplifications with moose dna and a single distinct pcr product was revealed for each individual. the size of the amplified product exactly corresponded to that obtained for the bovine drb gene ( bp) and subsequent sequence analysis made evident that the product represented a moose mhc drb gene, denoted drbl (mikko c andersson ) . it can be concluded that this locus constitutes an mhc gene of func- tional importance since it is the pmiominantly expressed drb locus in moose according to rt-pcr analysis (mikko & anderson ) . sscp analysis of the drbl gene amplified from moose identified three different alleles (fig. a) . one allele was obviously rare since it was only found in one heterozygous animal. the frequencies of the other two alleles were and . , respectively (h-= . ). again this indicates that the moose has limited mhc class il poly-morphism. a sample of cattle analysed with the same sscp methodology (i.e. using the same primers) is shown in fig. b . a characteristic feature of the mhc is the presence of strong linkage disequilibrium between loci. this was also evident from the present data. using the method of hill ( ), a significant linkage disequilibrium between the moose dqb rflp and the drb sscp alleles was observed (d = . ; p c . ). the relative amount of disequilibrium (hedrick et al. ) was estimated at . . we carried out dna fingerprinting using two minisatellite probes and hinff digested dna to analyse the genomewide variability of swedish moose. statistics derived from these experiments are given in table and a representative autoradiograph is shown in fig. . similarity indices (d) calculated from hybridizations with ms and mi were . and . , respectively. these values are somewhat higher than that previously =ported for a number of out- and is more similar to that obtained for several domestic animals (e.g. georges et al. ). we have investigated the genetic variability of moose mhc class i and class ii genes by means of (i) rflp analy- technique that is one of the most sensitive methods for detecting point mutations. sscp analysis relies on the fact that the mobility rate of a single-stranded dna molecule in a nondenaturing gel is not only determined by its size but also by its nudeotide sequence, which, in turn, govems its threedm * ensional structure (orita ef al. ) . even a single nudeotide substitution may slightly alter this structure leading to a differential migration pattern. it has been estimated that sscp can distinguish between and % of all possible mutations in pcr products blackwell science ltd, molecular ecology, , - < bp and about % for those < bp (michaud et al. ; sheffield et al. ). the finding of low levels of mhc variability in swedish moose is thus supported by the inherent sensitivity of the sscp technique. the fact that an expressed moose mhc gene was analysed rules out the possibility that the limited variability could be explained by a reduced selection pressure at a locus of no functional significance. the sequence analysed by sscp, exon two of drb, encodes the antigen recognition site in the first extracellular domain of dass ii / chain genes. in other species, the genetic polymorphism of class n gene products is concentrated to particular amino acids involved in antigen binding encoded by this exon (brown et al. ) . a characteristic feature of this polymorphism, as known from various species, is that there is a considerable amino acid divergence between alleles (klein ; andersson & davies ) . in an accompanying study we have shown that the swedish as well as the canadian moose does not only possess limited amounts of drb poly&orphism but also has minor sequence variations between alleles (mikko & andersson ) . what is the causal basis for the low levels of mhc polymorphism among the moose analysed in this study? a possible explanation would be that a previous bottleneck could have reduced the genetic variability. similar to the situation for the scandinavian beaver population (eiiegren et nl. ! ), the size of the swedish moose population was reduced due to over-hunting in the last century. the pop ulation has subsequently recovered and has increased dramatically during the th century, currently harbouring some ooo individuals. however, the extent of the bottleneck in the th century is uncertain (cf. ryman et al. ) and our data strongly argue against a severe reduction since dna fingerprinting patterns were far from monomorphic. the observed band-sharing probabilities ( . . ) are somewhat higher than that previously found among outbred natural populations (e.g. reeve et al. ), but do not indicate a dramatic loss of genetic variability. similarly, ryman et al. ( ) analysed allozyme variability in a large number of moose sampled at various localities in scandinavia and found a mean heterozygosity, within each popdation, of . . again cheetah, menotti-raymond & o'brien ( ) calculated that the moderate dna fingerprinting variability found among modem cheetahs could have been reconstituted (from complete homozygosity) in the order of ooo years. although the dna fingerprinting variability in our moose sample was higher than among the cheetahs, calculations similar to those made by menotti-raymond and obrien are consistent with that the swedish moose may have experienced a severe bottleneck in the last oocl ooo years. if so, extensive mhc variability is not to be expected among contemporary moose since the mutation rate at mhc loci is significantly lower than that at minisatellite loci. as evident from interspecific comparisons, extensive mhc polymorphism reflects the accumulation of mutations during millions of years of evolution (klein et al. ) . besides the possible effects caused by an ancient bottleneck, the low levels of moose mhc variability could also be due to a relatively weak selection pressure for p l ymorphism at these loci. there is a clear difference between the moose and cattle as regards their degrees of mhc polymorphism in relation to their genome-wide genetic variability measured by dna fingerprinting. the dna fingerprinting variability of moose is similar to that among several domestic animals, including cattle (georges et nl. ), and is significantly lower than that in humans. in contrast, the degree of mhc polymorphism in moose is much lower than that in both cattle and humans. differences in the intensity of pathogen-driven, balancing selection affecting moose and cattle may contribute to the marked differences in their degree of mhc diversity. domestic cattle are kept in dense herds which obviously facilitates the transmission of pathogens between animals. the moose, on the other hand, has a rather solitary life style. males' close contacts with other animals are probably restricted to the reproduction period, a situation that would reduce the lateral transmission of pathogens in the population. the swedish moose consequently provides an additional example of a viable population with restricted mhc polymorphism. slade ( ) noticed that three marine mammals from two different orders (the elephant seal, the sei whale and the fin whale) exhibit low degrees of mhc variability despite no apparent loss of allozyme heterozygosity. he proposed that this could be due to a reduced balancing selection pressure in the marine environment, a possible consequence of decreased exposure to pathogens. trowsdale et nl. ( ) , who analysed the two whale species, argued that the aquatic environment would impose less contact between the animals compared to a terrestrial environment and that this would hamper the spread of pathogens in the population. a similar but controversial idea has been invoked to explain the apparent lack of mhc class i polymorphism in syrian hamsters (darden & streilein ) ; the solitary life style of this species could act as a barrier for pathogen transmission as well (mcguire et nl. ) . in this context it could be noted that beavers, of which at least three european populations appear to be devoid of mhc polymorphism (ellegren et nl. ), also have a rather isolated life style. although they live in family groups and dispersal may occur in natal drainage systems, large land areas separating different river systems can be seen as barriers for long-range dispersal and hence also for lateral transfer of pathogens. most authors, including us, have interpreted the presence of reduced levels of mhc diversity in certain species to be due to previous bottlenecks or relaxed pathogen-driven selection. however, it should be noted that the hypothesis of pathogen-driven selection for mhc polymorphism has not yet been confirmed by experimental data, nor has it been shown that the level of mhc diversity influence the long term survival of natural populations. it is dear that data from a broad range of species will be required to shed light on this important question. the major histocompatibility cornplex analysis of class i genes of the chicken mhc (b) by use of human dna probes evidence for balancing selection at hla maternal-fetal interactions and the maintenance of hla polymorphism multilocus systems in evolution estimation of linkage disequilibrium in randomly mating population mhc polymorphism and the design of captive breeding programs hypervariable 'minisatellite' regions in human dna spontaneous mutation rates to new length alleles at tandem-repetitive hypervariable loci in human dna natural history of the major histocompatibdify complex the molecular descent of the major histocompatibility complex complete amino acid sequence of an hla-dr antigen-like chain as predicted from the nucleotide sequence: similarities with immunoglobulins and hla-a, -b, and -c antigens close association between dna polymorphism of bovine major histocompatibility complex class i genes and serological bola-a specificities detection of genetic mutations in ldh-a and ldh-b genes by pcr-sscp analysis on phastsystem multiple rflps in the porcine calcium release channel gene (crc): assignment to the halothane (hal) linkage group syrian hamster dna shows limited polymorphism at class i-like loci dating the genetic bottleneck of the african cheetah strand-separating conformational polymorphism analysis: efficacy of detection of point mutations in the human omithine d-aminotransferase gene low major histocompatibility complex class i diversity in european and north-american moose genetic variation in natural populations: patterns and theory interactive influence of infectious disease and genetic diversity in natural populations genetic basis for species vulnerability in the cheetah rapid and sensitive detection of point mutations and dna polymorphisms using the polymerase chain reaction evolution of mhc genetic diversity: a tale of incest, pestilence and sexual preference dna 'fingerprinting' reveals high levels of inbreeding in colonies of the eusocial naked mole-rat genetic variation and differentiation in scandinavian moose (alces alces): are large mammals monomorphic? evolution both a and fi chains of hla-dc class i histocompatibility antigens display extensive polymorphism in their amineterminal domains analysis ofthe eficiency of single base substitution detection by sscp restriction fragment length polymorphism of dq and dr class i genes of the bovine major histocompatibility complex cloning and sequence analysis of drb alleles of the bovine mhc by using the polymerase chain reaction limited mhc polymorphism in the southern elephant seal: implications for mhc evolution and marine mammal population biology an erosivelulcerative alimentary disease of undetermined etiology in swedish moose limited mhc polymorphism in whales a sequence in m phage detects hypervariable minisatellites in human and animal dna large mammals are genetically less variable dna variation of the mammalian major histocompatibility complex reflects genomic diversity and population history this work is part of long-term project aimed at understanding the evolution and polymorphism of mhc in different mammals. hans ellegren is associate professor, sofia mikko is phd student and leif andersson is professor in molecular and disease genetics at the swedish university of agricultural sciences in uppsala. this particular study arose from a collaboration with dr kjell wallin at the same university who has a keen interest in the biology of moose. key: cord- -ml pbewf authors: manczinger, máté; boross, gábor; kemény, lajos; müller, viktor; lenz, tobias l.; papp, balázs; pál, csaba title: pathogen diversity drives the evolution of generalist mhc-ii alleles in human populations date: - - journal: plos biol doi: . /journal.pbio. sha: doc_id: cord_uid: ml pbewf central players of the adaptive immune system are the groups of proteins encoded in the major histocompatibility complex (mhc), which shape the immune response against pathogens and tolerance to self-peptides. the corresponding genomic region is of particular interest, as it harbors more disease associations than any other region in the human genome, including associations with infectious diseases, autoimmune disorders, cancers, and neuropsychiatric diseases. certain mhc molecules can bind to a much wider range of epitopes than others, but the functional implication of such an elevated epitope-binding repertoire has remained largely unclear. it has been suggested that by recognizing more peptide segments, such promiscuous mhc molecules promote immune response against a broader range of pathogens. if so, the geographical distribution of mhc promiscuity level should be shaped by pathogen diversity. three lines of evidence support the hypothesis. first, we found that in pathogen-rich geographical regions, humans are more likely to carry highly promiscuous mhc class ii drb alleles. second, the switch between specialist and generalist antigen presentation has occurred repeatedly and in a rapid manner during human evolution. third, molecular positions that define promiscuity level of mhc class ii molecules are especially diverse and are under positive selection in human populations. taken together, our work indicates that pathogen load maintains generalist adaptive immune recognition, with implications for medical genetics and epidemiology. a a a a a the major histocompatibility complex (mhc) genes in vertebrates encode cell surface proteins and are essential components of adaptive immune recognition [ ] . mhc proteins are endowed with highly variable peptide-binding domains that bind short protein fragments. the mhc region is one of the most polymorphic gene clusters in vertebrate genomes [ ] . co-evolutionary arms race with pathogens is considered largely responsible for the observed exceptionally high levels of genetic diversity [ ] [ ] [ ] [ ] , yet it cannot fully account for the observed geographic differences in human mhc genetic diversity [ , ] . this indicates that, beyond mhc allelic diversity, other mhc-related factors contribute to the capacity of human populations to withstand pathogens. in this paper, we argue that peptide-binding repertoire size (or, shortly, promiscuity) of mhc alleles is one important factor. recent empirical studies demonstrated that there is a substantial variation in the size of the bound and presented antigen repertoire across mhc class i alleles. certain mhc class i alleles appear to be promiscuous and are capable of binding an exceptionally large set of epitope peptide segments [ , ] . for example, paul and colleagues carried out bioinformatics analysis to predict the binding capacity of common hla-a and hla-b alleles to a set of , dengue virus-derived peptides [ ] . the analysis revealed over -fold variation in the number of peptides bound by the different alleles, indicating significant variation in epitope repertoire size across hla molecules. the authors selected three alleles for further study in an in vivo transgenic mouse model. immunization of the corresponding hla transgenic mouse strains with a set of dengue virus-derived peptides revealed a positive relationship between epitope repertoire size and immunogenicity. similarly, kosmrlj and colleagues computed the fraction of self-peptides that bind to various hla-b molecules, and found that this fraction varies extensively across four hla-b alleles [ ] . the authors then demonstrated that the self-peptidebinding repertoire of hla-b shapes the native repertoire of t-cell clones developed in the thymus, with implications for recognizing human immunodeficiency virus (hiv) epitopes. their results could explain why individuals carrying hla-b � alleles can maintain low hiv rna without therapy. remarkably, analogous mhc class i alleles with the hla-b � superfamily is widespread in chinese rhesus macaques, animals which show especially slow progression of simian immunodeficiency virus (siv)/hiv [ ] . finally, by focusing on seven chicken mhc class i haplotypes and four human hla-b alleles, chappel and colleagues demonstrated that mhc class i molecules that can bind a wide range of viral epitopes show lowered expression on the cellular surfaces of immune cells, such as monocytes and lymphocytes [ ] . the authors suggested that the breadth of epitope-binding repertoire shapes genetic susceptibility to marek's disease virus in chickens and hiv disease progression in humans. more generally, by recognizing more peptide segments, promiscuous mhc molecules may promote immune response against a broader range of pathogens and are hence generalists [ , ] . prior case studies in chicken indicate that this may be so [ ] [ ] [ ] [ ] . however, it remains to be established whether this relationship generally holds across mhc class i and ii alleles and a wide range of infectious diseases. specifically, we propose that in regions of high pathogen diversity, human populations should carry promiscuous mhc alleles. moreover, as migrating human populations have been exposed to changing sets of pathogens [ ] , shifts in mhc promiscuity level should have occurred repeatedly and in a rapid manner during the course of human evolution. to test these predictions, we first focused on the human hla class ii drb gene, for several reasons. first, drb is the most variable hla class ii locus, with over , registered alleles [ ] . together with hla-dra, hla-drb encodes the heterodimeric hla-dr protein complex, but hla-dra is basically invariant. second, drb shows the strongest general signature of selection among hla class ii loci [ ] , while at the same time showing the weakest evidence for divergent allele advantage, an alternative mechanism at the genotype level for presenting a broader set epitopes [ ] . third, drb has diversified very rapidly in the human lineage [ ] . many of the drb alleles appear to be human specific and most likely evolved after the migration of ancestral human populations out of africa [ ] . these periods have been associated with human populations encountering numerous new pathogens [ , ] . for other hla class ii loci, the level of genetic diversity is lower [ , ] , probably driven by selection for functions partly unrelated to pathogens. notably, hla-dq has a fundamental role in the development of immune tolerance [ , ] , while hla-dp contributes to the presentation of epitopes of intracellular origins [ ] [ ] [ ] . fourth, epitope-binding prediction algorithms show higher accuracy for drb than for other hla class ii loci [ , ] . finally, the abundance of drb on the cell surface is especially high compared with other hla class ii molecules [ ] [ ] [ ] . subsequently, we also evaluated promiscuity patterns of hla class i molecules. estimates on epitope-binding promiscuity were derived from two sources: experimental assays that measured individual peptide-mhc interactions in vitro and systematic computational predictions. in a series of analyses, we show that predictions of our hypothesis are upheld, regardless of how hla-drb promiscuity level is estimated. given that large-scale experimental assays to measure individual peptide-mhc interactions are extremely tedious, we first employed established bioinformatics tools to predict the binding affinities of experimentally verified epitope peptides for a panel of nonsynonymous hla-drb alleles, all of which are present at detectable frequencies in at least one human population [ ] [ ] [ ] . the set of investigated epitopes was derived from the immune epitope database (iedb) and contains , peptide epitopes of pathogens known to be bound by certain hla class ii alleles [ ] (s data). epitopes showing high levels of amino acid similarity to each other were excluded from the analysis (see methods). most included epitopes are to amino acids long and are found in only one of the pathogens (s fig, s data) . the netmhciipan algorithm was used to predict individual epitope-mhc interactions [ ] , not least because it outperforms other prediction algorithms [ ] . the breadth of epitope-binding repertoire or, shortly, the level of promiscuity of individual hla-drb alleles was estimated as the fraction of epitopes with a binding affinity stronger than nm to the given mhc molecule. this threshold corresponds to high-affinity binding, which is frequently observed in mhc molecules displaying immunodominance [ ] . we found large variation in promiscuity levels across hla-drb alleles (s fig, s data) . using a smaller dataset with information from both approaches, we show that the computationally predicted and the empirically estimated promiscuity values are strongly correlated with each other (spearman's rho: . , taking advantage of the confirmed reliability of computational predictions, we next investigated the geographic distribution of hla-drb alleles. we first collected high-quality hla-drb allele prevalence data of human populations residing in countries from two databases and an article [ ] [ ] [ ] . the weighted average of promiscuity level in each population was calculated based on the promiscuity values and allele frequencies of individual alleles in the population (see methods). the analysis revealed a large variation in mean promiscuity across geographical regions and the corresponding human populations (s table) . importantly, several distantly related but highly promiscuous alleles contribute to this pattern (s table) . notably, an especially high allelic promiscuity level was found in southeast asia, an important hot spot of emerging infectious diseases [ ] . to minimize any potential confounding effect of high genetic relatedness between neighboring populations, we merged populations with similar hla allele compositions for all further analyses (see methods). using the global infectious diseases and epidemiology network (gideon), we compiled a dataset on pathogen richness in the corresponding geographic regions [ ] . it consists of diseases caused by extracellular pathogens, including diverse bacterial species, fungi, protozoa, and helminthes. using the same protocol, we additionally compiled a dataset on the prevalence of diseases in the same regions caused by viral and other obligate intracellular pathogens. the dataset and methodology employed for the analysis are standardized and have been used previously in similar contexts [ , , ] . we report a strong positive correlation between extracellular pathogen diversity and mean promiscuity: hla-drb alleles that can bind epitopes from a broader range of pathogens are more likely to be found in regions of elevated pathogen diversity ( fig a) . this pattern is unlikely to be explained by confounding factors, such as country size or hla-drb genetic diversity across countries (s table) . by contrast, we found no significant association between hla-drb promiscuity level and diversity of intracellular pathogens (fig b) . we conclude that the geographical distribution of promiscuous hla-drb alleles has been mainly shaped by the diversity of extracellular pathogens. the above results hold-and are even stronger-when estimates on promiscuity were derived from empirical in vitro mhc binding data (shortly, in vitro promiscuity), downloaded from the iedb database [ ] (fig c and d , s table and s data). however, these results do not exclude the possibility that the geographical link between pathogen diversity and promiscuity is indirect. more direct support on the causal relationship between the two variables comes from analysis of prior human genetic studies. to investigate this issue, we focused on two geographically widespread allelic groups with exceptionally high (hla-drb � ) and exceptionally low (hla-drb � ) promiscuity values, respectively, and conducted literature mining on their reported associations with infectious diseases (s table) . as expected, hla-drb � was associated with protection against at least five infectious diseases, while hla-drb � was associated with susceptibilities to eight infectious diseases, which is highly unlikely by chance (fisher test, p = . ) (s table, s data). the data also indicate local adaptation towards elevated promiscuity under diverse pathogen pressure. the hla-drb � : allele is prevalent in specific regions of southeast asia. compared with other alleles detected in this region, hla-drb � : has a relatively high promiscuity value (top . indeed, this allele is associated with protection from recurrent pulmonary tuberculosis, recurrent typhoid fever, and hepatosplenic schistomiasis (s data), all of which are endemic diseases in southeast asia [ ] [ ] [ ] . remarkably, the frequency of this allele increases with extracellular pathogen diversity in this region ( fig b) . together, these observations support the hypothesis that promiscuous epitope binding of hla-drb alleles is favored by selection when extracellular pathogen diversity is high. an important unresolved issue is how promiscuity has changed during the course of human evolution. under the assumption that local pathogen diversity drives the evolution of epitope recognition of hla class ii alleles, promiscuity as a molecular trait should have evolved rapidly as human populations expanded into new territories. to investigate this issue, we combined an established phylogeny of hla-drb alleles [ ] with predicted epitope-binding promiscuity values. we found that alleles with a high promiscuity level have a patchy distribution across the tree (s fig), indicating that high promiscuity has multiple independent origins. to investigate this observation further, we selected a set of hla-drb alleles with a detectable frequency in at least one human population and appropriate sequence data (see methods). a comparison of all pairs of these alleles revealed that even very closely related alleles show major differences in promiscuity levels ( fig a) . for example, alleles belonging to the hla-drb � group show over % amino acid sequence identity to each other, but display as much as -fold variation in the predicted promiscuity levels. we conclude that the switch between high and low promiscuity levels has occurred repeatedly and in a rapid manner during the allelic diversification of the hla-drb locus. we next asked how selection on promiscuity has shaped the genetic diversity along the epitope-binding region of hla molecules. to quantify protein sequence variability at each amino acid position, we calculated the shannon entropy index based on the alignment of the selected hla-drb alleles from above. for each position, we also calculated promiscuity fragility, that is, the median impact of single amino acid substitutions on promiscuity (see methods). a strong positive correlation was found between shannon entropy and promiscuity fragility ( the above data suggest a link between allele promiscuity and hla-drb diversification, probably as an outcome of selection for locally optimized promiscuity levels. finally, we note that several variable molecular sites in the binding region of hla-drb affect epitope-binding characteristics without any major impact on promiscuity per se. for example, our computational analysis indicates that mutations at amino acid site numbers and do not seriously affect promiscuity level ( fig b) . however, several mutations at these sites are associated with binding self-peptides and thereby shape vulnerability to specific autoimmune diseases [ , ] . for all pairs of selected alleles, the predicted promiscuity difference between two hla-drb alleles is shown as a function of amino acid distance measured after excluding the epitope-binding region. large differences in promiscuity can be observed even between closely related pairs of alleles (e.g., at zero amino acid distance). as a result, there is no correlation between amino acid distance and promiscuity fold difference (spearman's rho = . , p = . ). amino acid distances were binned as shown on the figure (n = , , , , , , ). violin plots show the density function of promiscuity fold difference values for allele pairs in the given bin. white circles show median values; bold black lines show the interquartile range. (b) sequence variability of an amino acid site in the epitope-binding region of hla-drb (measured as shannon entropy) correlates positively with the site's promiscuity fragility, measured as the median predicted promiscuity fold difference caused by a random amino acid change at the given site (see inset, spearman's rho: . , p = . ). sites that have a larger impact on promiscuity are more diverse in human populations. line in inset represents linear regression between the two variables. the same result was obtained when promiscuity fragility was calculated based on nucleotide substitutions instead of amino acid substitutions (spearman's rho: . , p = . , see methods) or when sequence variability was measured as nonsynonymous nucleotide diversity (π a ) instead of sequence entropy (s fig). sites under positive selection as identified by furlong and colleagues [ ] show significantly higher promiscuity fragility (wilcoxon rank sum test, p = . ) and are marked with asterisks (see also s fig) . the underlying data for this figure can be found in s data. the relationship between pathogen diversity and epitope-binding promiscuity may be more general, as similar results hold for the hla-a locus. hla-a is one of the three types of classical human mhc class i molecules and is mainly involved in the presentation of epitopes from intracellular pathogens [ ] . in agreement with expectation, we report a positive correlation between local intracellular pathogen diversity and the hla-a promiscuity level of the corresponding human populations (s a and s b fig, s data) . no marked positive correlation was found for two other mhc class i genes (hla-b and hla-c, see s c to s f fig, and s data) . therefore, other unrelated evolutionary forces may shape the geographical distribution of promiscuous hla-b and hla-c alleles (s text). central players of the adaptive immune system are the groups of proteins encoded in the mhc. by binding short peptide segments (epitopes), mhc molecules guide both immune response against pathogens and tolerance to self-peptides. the genomic region encoding these mhc molecules is of special interest, for two reasons. it harbors more disease associations than any other regions in the human genome, including associations with infectious diseases, autoimmune disorders, tumors, and neuropsychiatric diseases [ , ] . a growing body of literature is now revealing that certain mhc class i alleles can bind a wider range of epitopes than others, but the functional implications of this variation remain largely unknown [ ] . by recognizing a larger variety of epitopes, such promiscuous mhc alleles promote immune response against a broader range of pathogens at the individual level. therefore, promiscuous epitope binding of mhc molecules should be favored by selection in geographic regions where extracellular pathogen diversity is high. importantly, this mechanism is conceptually distinct from the well-established concept of heterozygote advantage at the mhc [ ] , as it concerns individual alleles and not allele combinations or genotypes. to test this hypothesis, we combined data on the geographic distribution of human mhc class ii alleles and prevalence of extracellular pathogens, empirical/computational estimates of epitope-binding promiscuity, and phylogenetic analyses. our main findings, strongly supporting our hypothesis, are as follows. first, in geographical regions of high extracellular pathogen diversity, human hla-drb alleles have exceptionally high epitope-binding repertoires. this suggests that the geographical distribution of promiscuous hla-drb alleles has been shaped by the diversity of extracellular pathogens. the hla-drb � : allele highlights this point. hla-drb � : is a promiscuous allele that has been associated with protection from certain infectious diseases (s data). as expected, this allele is especially prevalent in regions of southeast asia with elevated pathogen load (fig b) . it is well established that antigens presented by hla class ii molecules derive mainly from extracellular proteins [ ] . however, hla class ii molecules have well-established roles in controlling immune response against viruses [ , ] . additionally, viral peptides are reported to be processed and presented also by the hla class ii pathway [ ] . therefore, it remains to be established why intracellular pathogen diversity has no major impact on the global distribution of hla-drb alleles. notably, the relationship between pathogen load and epitope-binding promiscuity may be more general, as similar results hold for the hla-a locus: we found a positive correlation between local intracellular pathogen diversity and the hla-a promiscuity level of the corresponding human populations (s a and s b fig, s data) . second, a phylogenetic analysis revealed major differences in promiscuity levels of very closely related hla-drb alleles. this suggests that high promiscuity level in hla-drb has evolved rapidly and repeatedly during human evolution. finally, amino acid positions with a prominent role in shaping hla-drb promiscuity level are especially variable in human populations and tend to be under positive selection. in sum, we conclude that hla promiscuity level is a human trait with paramount importance during adaptation to local pathogens. our work has important ramifications for future studies. mhc is the most variable region of the human genome, and the variation is associated with numerous infectious and immunemediated diseases [ , , [ ] [ ] [ ] [ ] [ ] . the impact of mhc promiscuity level on population allelic diversity is an interesting area for future research. in a similar vein, mhc allelic diversity is associated with olfaction-based mating preferences in human and other animals [ ] . the roles of mhc promiscuity in mating success and mating preferences are a terra incognita. we note that the most promiscuous hla-drb alleles are rare in certain human populations (s table; s data). this suggest that these alleles are not particularly favored by natural selection in these areas. why should it be so? first, high promiscuity may not be able to cope with the rise of novel and highly virulent pathogens. in such cases, displaying a particular epitope might be the most efficient way to achieve resistance, and high promiscuity might be suboptimal due to a reduced specificity [ , ] . second, high promiscuity level may elevate the risk of immune reactions against host tissues and non-harmful proteins [ , ] . clearly, future work should elucidate the evolutionary trade-offs between protection from pathogens and genetic susceptibility to autoimmune diseases. this will require high-throughput experimental methods to determine epitope-binding repertoire [ ] , and hla transgenic mice studies on the role of promiscuity in immune response [ ] . finally, genetic variation within particular mhc genes influences vaccine efficacy [ ] , rejection rates of transplanted organs [ ] , susceptibility to autoimmune diseases [ ] , and antitumor immunity [ , , ] . our work raises the possibility that, by altering the maturation and functionality of the immune system, the size of the epitope-binding repertoire of mhc alleles itself could have an impact on these processes. the exact role of mhc promiscuity in these crucial public health issues is an exciting future research area. the iedb has collected the results of individual and systematic studies on epitope binding by mhc alleles [ ] . the experimental studies include hla-binding assays, t-cell activation assays, and immunopeptidomic studies as well. epitopes of all available viral, bacterial, and eukaryotic pathogens known to be bound by at least one hla-i or hla-ii allele were extracted from iedb. reference proteomes of pathogenic species that carry at least one of the collected epitope sequences were retrieved from the uniprot database ( for hla-i and for hla-ii epitopes) [ ] . only epitopes of these species were analyzed further. all proteomes were scanned for each epitope sequence, and epitope sequences found in only one proteome (i.e., species-specific epitopes) were kept for further analysis. highly similar epitope sequences were identified using clustal omega [ ] and excluded as follows. a protein distance matrix was created and epitopes were discarded iteratively. in each iteration, the epitope pairs with the lowest k-tuple distance were identified. then, the epitope with the highest average similarity to all other sequences was excluded. iterations were repeated until distance values less than . (corresponding to greater than approximately % sequence identity) were eliminated from the matrix [ ] . note that this filtering procedure was carried out separately for epitope sequences bound by hla-i and hla-ii. binding affinities of the remaining , hla-i epitope sequences to hla-a, hla-b and hla-c alleles were predicted with the netmhcpan- . algorithm [ ] . the binding of , hla-ii epitope sequences to hla-drb alleles was predicted using the netmhciipan- . algorithm [ ] . all alleles are present in at least one of the human populations studied here (see below). the "pep" sequence input format was used for both hla-i and hla-ii epitope-binding prediction. a binding affinity threshold of nm was applied, yielding peptides that are likely to be immunodominant [ ] . for alternative binding threshold definitions, see s fig. for each binding threshold, epitope-binding promiscuity was defined as the fraction of the epitope set bound by a given allele. to determine the epitope-binding promiscuity of hla-drb alleles based on previously published experimental data, we used the iedb database [ ] . specifically, we downloaded all mhc ligand and t-cell assay data, which was available for hla-drb alleles. binding data of alleles screened for at least ligands each were further analyzed. the epitope set of each allele was filtered for highly similar sequences, as described above. as the majority of in vitro assay data were available in a binary format (i.e., presence or absence of binding), promiscuity was calculated as the fraction of positive binding assays for a given allele. to calculate population-level promiscuity values, we obtained hla allele frequency data from the allele frequency net database (afnd) and the international histocompatibility working group (ihwg) populations [ , ] . haplotype-level data of the th international hla and immunogenetics workshop (ihiw) populations were downloaded from dbmhc (national center for biotechnology information [ncbi]; ftp://ftp.ncbi.nlm.nih.gov/pub/mhc/mhc/final % archive). additionally, allele frequency data of the th and th ihiw populations, as published by riccio and colleagues [ ] , and populations in the afnd were used in the analyses. to avoid potential confounding effects of recent genetic admixture and migration, we focused on native populations, similarly to previous studies (s table) [ , ] . we excluded ihwg populations reported to deviate from hardy-weinberg equilibrium [ ]. among the afnd populations and ihwg populations without haplotype-resolution data ( th and th ihiw), those comprising less than genotyped individuals or those in which the sum of allele frequencies deviated from by more than % were excluded. populations reported in multiple databases were included only once in the analysis. for each hla loci, we calculated mean population promiscuity by averaging promiscuity values of alleles weighted by their relative frequencies in the populations. in all of these calculations, we used standardized (i.e., z-score) promiscuity values to make the in silico and in vitro values more easily comparable. finally, when calculating population-level promiscuity based on in vitro promiscuity data, we excluded populations for which in vitro promiscuity values could be assigned to less than % cumulative allele frequency. to tackle the issue of nonindependence of data points, we focused on populations instead of countries and grouped those populations that have highly similar hla allele compositions, based on standard measures of genetic distance (see below). we merged populations with highly similar hla allele compositions, allowing us to avoid pseudoreplication of data points while retaining informative allele frequency differences between populations residing in the same broad geographical areas. to this end, we first generated a genetic distance matrix between populations with the adegenet r library using allele frequency data of the examined locus. we used the rogers' genetic distance measure [ ] because it does not assume that allele frequency changes are driven by genetic drift only, an unlikely scenario for hla genes. next, populations were merged using a network-based approach. populations were treated as nodes and two nodes were connected if their genetic distance was under a cutoff value. populations were grouped in an iterative manner. in each iteration, all maximal cliques (i.e., subsets of nodes that are fully connected to each other) in the network were identified. maximal cliques represent groups of populations in which all populations have similar allele compositions to each other. then, mean genetic distance within each clique was calculated. the clique with the lowest average distance was selected and its populations were grouped together. then, this clique was deleted from the network. iterations were repeated until no maximal cliques remained in the network. grouping of populations was carried out using different distance value cutoffs ( st, th, th, and th rank percentile of all distance values). the resulting population groups and the individual populations that remained in the network were treated as independent data points in subsequent statistical analyses. mean promiscuity level in population groups was calculated by averaging population promiscuity values. unless otherwise indicated, all figures are based on population groups using the th percentile genetic distance cutoff value. importantly, using different cutoffs has no impact on our results (s data). finally, we note that genetic differences among human populations mostly come from gradations in allele frequencies rather than from the presence of distinctive alleles [ ] . therefore, traditional clustering of populations based on hla composition would have been ill-suited for our purposes. data on infectious diseases were collected from gideon [ ] . for each disease, the number of causative species or genera (when species were not listed for the genus) was determined using disease information in the gideon database, as described previously [ ] . causative agents were classified into obligate intracellular and extracellular pathogen groups based on a previous study [ ] and literature information. putative facultative intracellular pathogens were excluded from the analysis. diseases caused by agents that could not be clearly classified were also excluded from the analysis. extracellular and intracellular pathogen diversity (richness) of each country was approximated by the number of identified endemic extracellular and intracellular species, respectively. finally, we assigned country-level measures of pathogen and hla diversity to population groups as follows. for each population group, extracellular and intracellular pathogen counts were calculated by averaging the corresponding country-level values across the populations within the group. for example, if a population group contained two populations residing in neighboring countries, then we assigned the average pathogen diversity of the two countries to it. to examine associations between selected hla allele groups and infectious diseases, we carried out a systematic literature search on pubmed database using the following terms: "assoc � drb ", "assoc � drb ", "assoc � drb ", "assoc � drb ", "assoc � dr ", "assoc � drb � ", "assoc � drb ", "assoc � drb ", "assoc � dr ", "assoc � drb � ", "assoc � dr ", "infect � drb ", "infect � drb ", "infect � drb ", "infect � drb ", "infect � dr ", "infect � drb � ", "infect � drb ", "infect � drb ", "infect � dr ", "infect � drb � ", and "infect � dr ". "assoc � " and "infect � " refer to any word beginning with these letters. each resulting paper containing hla association data was examined, and statistically significant associations between allele groups (drb � or drb � ) or common alleles in allele groups (drb � : , drb � : , drb � : ) and infectious diseases were collected. associations with diseases caused by intracellular pathogens were excluded from the analysis. hla-disease associations were classified as beneficial or detrimental, if all related studies supported the beneficial or detrimental role of hla allele/allele group in the development or course of the given disease. otherwise, the association was classified as controversial. the results were summarized (s table) , and statistical association between beneficial/detrimental effects and high/low promiscuity across allele groups was determined by a fisher's exact test. we used amino acid distance as a proxy for phylogenetic distance between pairs of drb alleles. to this end, nucleotide sequences of drb alleles that contained full exon and regions were downloaded from the ipd-imgt/hla database [ ] . to limit our analyses to alleles that have an impact on the inferred promiscuity level of a population, we considered only those sequences that had a nonzero frequency in at least one human population (see above). from allele groups that code for the same protein sequence (synonymous differences, differentiated by the third set of digits in the hla nomenclature), one of the alleles was randomly chosen. this selection procedure resulted in alleles. multiple alignment of nucleotide sequences was performed using the muscle algorithm as implemented in the mega software [ ] and converted to protein sequence alignments. amino acid distance was calculated using the jones-taylor-thornton substitution model in mega [ ] (fig a) . epitope-binding region sites-as defined previously [ ]-were excluded when calculating amino acid distance. the rationale behind this exclusion is that these sites are known to be under positive selection [ , ] and are therefore less informative on evolutionary distance. additionally, by removing these sites, the amino acid distance remains independent of promiscuity predictions. finally, as intragenic recombination may distort the inference of evolutionary distance, we identified such events across all alleles following the protocol of satta and colleagues [ ] using gene-conv [ ] and rdp algorithms [ ] as implemented in the rdp software [ ] . recombinant alleles were removed when calculating amino acid distance. we first defined the epitope-binding region of hla-drb alleles, as previously [ ] . to estimate sequence diversity along the epitope-binding region, we employed two measures: standard shannon entropy [ ] and nucleotide diversity (π), a widely employed measure of genetic variation [ ] . using the protein sequence alignment of the alleles defined above, we calculated amino acid sequence variability as the shannon entropy of the given amino acid site as follows: where p i is the fraction of residues of amino acid type i at a given site, and m is the number of amino acid types observed at that site. nonsynonymous nucleotide diversity (π a ) measures the average number of nonsynonymous nucleotide differences per nonsynonymous site between two randomly chosen protein coding dna sequences from the same population [ , ] . π a was calculated for each amino acid site in the epitope-binding region for each population using dnasp software [ ] and custom-written r scripts. nucleotide sequences of drb alleles were downloaded from the ipd-imgt/hla database [ ] . the calculation is based on the work of nei and colleagues [ ] using the equation where x i and x j are the frequencies of the ith and jth alleles in the population, respectively, and p a ij is the number of nonsynonymous nucleotide differences per nonsynonymous nucleotide site between the two codon sequences of the given amino acid site in the ith and jth alleles. to calculate π a for each population, allele frequency data of human populations were obtained, as described earlier (see above). an overall nucleotide diversity index was calculated by averaging π a across populations. to calculate each amino acid site's impact on epitope-binding promiscuity (promiscuity fragility), promiscuity was predicted for each possible amino acid change along the epitopebinding region of each of alleles. the fold difference in promiscuity resulting from each amino acid substitution was calculated. the median promiscuity fold difference of each possible allele and amino acid change combination ( × ) was used to estimate promiscuity fragility at each amino acid position. as some of the possible amino acid changes are not accessible via a single nucleotide mutation, and the accessible amino acid changes can have different likelihoods based on the codon sequence of the site and the genetic code, we also calculated promiscuity fragility based on each nonsynonymous nucleotide substitution of the codon instead of each amino acid substitution of the site. all statistical analyses were carried out in r version . . [ ] . smooth curves were fitted using the cubic smoothing spline method [ ] . . we selected (i) epitope sequences from this dataset, for which binding affinity data to all the alleles were available, and (ii) , epitopes used for calculating in silico promiscuity throughout the paper, which were not used for the training of the netmhciipan algorithm. in vitro and predicted promiscuity of the alleles was determined at a nm binding threshold using the selected and , epitopes, respectively. we used standardized (i.e., z-score) allele promiscuity values for the comparisons. we report a strong correlation between the in silico and in vitro promiscuity . one might speculate that there might be no selection for elevated hla-b promiscuity level due to a dominant balancing selection on this locus (see s text) [ , ] . similarly, (e) the promiscuity level of hla-c molecules showed no significant correlation with intracellular pathogen diversity (spearman's rho: − . , p = . ). finally, (f) hla-c promiscuity level showed a marginally significant positive correlation with extracellular pathogen diversity (spearman's rho: . , p = . ). this is surprising, but this preliminary result needs to be considered with caution, and studied further in future works. for detailed explanation of these results, see s text. population groups were created using the th percentile genetic distance cutoff (see methods). for a list of populations assigned to each group, see s data. for results of multivariate models and obtained upon using alternative distance cutoff values, see s data. red curves indicate smooth curves fitted using cubic smoothing spline method in r (see methods). the underlying data for this figure can be found in s data. (tif) s table. list of populations and their mean hla-drb allele promiscuity. table. the relationship between promiscuity and pathogen diversity is independent of hla diversity and country size. table. results of hla association studies suggest a protective role of high allele promiscuity in infectious diseases. 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transgenic mice as models of human diseases genetic variants within the mhc region are associated with immune responsiveness to childhood vaccinations indirect recognition of donor hla-dr peptides in organ allograft rejection mhc-i genotype restricts the oncogenic mutational landscape evolutionary pressure against mhc class ii binding cancer mutations uniprot: the universal protein knowledgebase fast, scalable generation of high-quality protein multiple sequence alignments using clustal omega performance comparison between k-tuple distance and four model-based distances in phylogenetic tree reconstruction netmhcpan- . : improved peptide-mhc class i interaction predictions integrating eluted ligand and peptide binding affinity data deriving phylogenetic trees from allele frequencies: a comparison of nine genetic distances genetic structure of human populations mega : molecular evolutionary genetics analysis version . for bigger datasets nucleotide substitution at major histocompatibility complex class ii loci: evidence for overdominant selection natural selection at major histocompatibility complex loci of vertebrates. annual review of genetics mhc class ii dqb diversity in the japanese black bear, ursus thibetanus japonicus statistical tests for detecting gene conversion rdp: detection of recombination amongst aligned sequences rdp : detection and analysis of recombination patterns in virus genomes bio d: an r package for the comparative analysis of protein structures mathematical model for studying genetic variation in terms of restriction endonucleases synonymous and nonsynonymous polymorphisms versus divergences in bacterial genomes dnasp v : a software for comprehensive analysis of dna polymorphism data r: a language for data analysis and graphics pathogen diversity drives the evolution of generalist mhc-ii alleles in human populations we wish to thank jim kaufman for the insightful comments we have received on an earlier version of the manuscript. we are also grateful to károly kovács for his suggestions on data analysis. key: cord- -tp o fxx authors: oliveira, cláudia c.; van hall, thorbald title: alternative antigen processing for mhc class i: multiple roads lead to rome date: - - journal: front immunol doi: . /fimmu. . sha: doc_id: cord_uid: tp o fxx the well described conventional antigen-processing pathway is accountable for most peptides that end up in mhc class i molecules at the cell surface. these peptides experienced liberation by the proteasome and transport by the peptide transporter tap. however, there are multiple roads that lead to rome, illustrated by the increasing number of alternative processing pathways that have been reported during last years. interestingly, tap-deficient individuals do not succumb to viral infections, suggesting that cd t cell immunity is sufficiently supported by alternative tap-independent processing pathways. to date, a diversity of viral and endogenous tap-independent peptides have been identified in the grooves of different mhc class i alleles. some of these peptides are not displayed by normal tap-positive cells and we therefore called them teipp, for “t-cell epitopes associated with impaired peptide processing.” teipps are hidden self-antigens, are derived from normal housekeeping proteins, and are processed via unconventional processing pathways. per definition, teipps are presented via tap-independent pathways, but recent data suggest that part of this repertoire still depend on proteasome and metalloprotease activity. an exception is the c-terminal peptide of the endoplasmic reticulum (er)-membrane-spanning ceramide synthase trh that is surprisingly liberated by the signal peptide peptidase (spp), the proteolytic enzyme involved in cleaving leader sequences. the intramembrane cleaving spp is thereby an important contributor of tap-independent peptides. its family members, like the alzheimer’s related presenilins, might contribute as well, according to our preliminary data. finally, alternative peptide routing is an emerging field and includes processes like the unfolded protein response, the er-associated degradation, and autophagy-associated vesicular pathways. these data convince us that there is a world to be discovered in the field of unconventional antigen processing. defective ribosomal product (drip) proteins generates small peptides. potentially, a multitude of proteolytic systems may generate antigenic peptides, but the proteasome is responsible for the liberation of majority of them. inhibition of proteasome activity strongly decreased the pool of mhc class i-binding peptides ( ) . proteasomal cleavage typically creates a peptide's c-terminus compatible with mhc class i binding, and peptides are typically extended at their n-terminus ( , ) . peptides generated in the cytosol are translocated into the endoplasmic reticulum (er) by the tap /tap peptide transporter, where they have access to the peptide loading complex (plc), which is located within the er. tap is a heterodimeric member of the atp-binding cassette (abc) family of transporters, and peptide binding induces atp hydrolysis and transport across the er membrane. once in contact with the plc, the er-amino peptidase eraap (also known as erap ) trims n-extended peptides to a length appropriate for mhc class i binding ( ) ( ) ( ) . chaperone tapasin promotes the formation of stable mhc class i/peptide complexes and acts as an editor. additionally, calnexin facilitates the early folding of mhc class i heavy chains, whereas calreticulin and erp are involved in peptide loading ( ) ( ) ( ) . this pathway is also known as the proteasome-tap pathway and is considered as the conventional processing route because it is the mainstream pathway operating in cells under normal conditions ( ) ( ) ( ) . however, cells are equipped with alternative routes leading to liberation and loading of peptides into mhc class i molecules. these routes are independent of one or more molecules from the conventional pathway such as the proteasome, tapasin, or tap. this has become apparent from studies on cells with deficiencies in the conventional processing pathway. in this review, we will discuss what is known to date regarding alternative enzymes and routes to peptide loading compartments of endogenously generated peptides that feed the direct mhc class i pathway, especially important in cases of failure of the conventional route. we have not included interesting literature on cross-presentation pathways for mhc class i peptides. more than years ago, several papers made the important discovery that a large oligopeptidase, called tripeptidyl peptidase ii (tppii), participates in endoproteolytic activity in the cytosol and partially compensates for a deficient proteasomal activity ( ) ( ) ( ) ( ) . increased tppii activity even allowed for cell survival in lethal conditions of proteasome inhibition ( , ) . in these conditions, tppii activity also partially restored peptide presentation in mhc class i molecules and it was speculated that it could account for the generation of some epitopes independently or in cooperation with the proteasome ( ) . in fact, a paper from seifert et al. showed that tppii was involved in the generation of an epitope from the human immunodeficiency virus (hiv) protein negative factor (nef) ( ) . after that, an increasing number of proteolytic enzymes have been implicated in the generation of peptide-epitopes independently of the proteasome. insulindegrading enzyme (ide) generates an epitope from the human melanoma antigen mage-a ( ) . thimet oligopeptidase (top) and nardilysin are required for the generation of three other clinically relevant ctl epitopes: the tumor-antigen prame, an epitope from epstein-barr virus (ebv) protein ebna c, and an epitope from the melanoma protein mart- ( ) . these enzymes are part of an array of cytosolic endo-and exo-proteases that complement proteasomal activity and degrade proteasome products ultimately into amino acids. importantly, the process of peptide liberation from the protein context is inevitably coupled to the destruction pathway and all proteases mentioned above also destroy some antigenic peptides ( ) ( ) ( ) ( ) . peptides that are "rescued" from total destruction are transported by tap into the er and can potentially bind mhc class i molecules. in eukaryotic cells, secretory and membrane proteins contain a signal sequence essential for protein targeting to the er, the entrance for the secretory pathway ( , ) . these signal sequences are typically composed of three domains: a hydrophobic core (h region) of - amino acids, a polar c-terminal end (c region) with small uncharged amino acids, and a polar nterminal region (n region) with a positive net charge ( ). after insertion into the protein-conduction channel, signal peptides are usually cleaved from the preprotein by signal peptidase (sp) ( ). thereafter, signal peptides, which are small domains and trapped in the er membrane, can undergo intramembrane proteolysis by cleavage within their transmembrane region by the presenilintype aspartic protease signal peptide peptidase (spp) ( , ). peptide ligands suitable for mhc class i binding are thought to be generated after the intramembrane proteolysis by spp that promotes the release of signal peptide fragments from the er membrane ( , ). the spp-cleaved fragments in the vicinity of the cytosol can get access to the cytosol again and be further processed by the proteasome and transported by tap into the er. most hla class i molecules donate their leader sequences for binding to the non-classical hla-e, and the cleavage of these signal sequences is mediated by sp and spp ( - ). these leader peptides are even the most dominant source of peptides for hla-e. proper surface expression of hla-e prevents cytotoxic action of natural killer cells that continuously sense the presence of peptide/hla-e complexes through their cd /nkg receptors ( - ). the absence of these complexes at cell surface results in failure of interaction with cd /nkg a receptors, which activate nk cells for killing their targets. pioneering studies by peter cresswell and victor engelhard in revealed that most peptides presented at the surface of tap-deficient cells were derived from signal sequences, specific protein regions at the n-terminus of proteins ( , ). indeed, the parts of the leader peptide within the er membrane that are closest to the er lumen are released there and can get access to mhc class i grooves independent of proteasomes or tap (figure ) . after the cleavage of the transmembrane sequence by spp, the peptide fragments are released and can associate with mhc class i/β m nascent molecules. this intramembrane proteolysis by spp is thought to be important for the clearance of the er membrane by removing small protein remnants anchored at figure | classical and alternative pathways for mhc class i presentation. cells with deficiencies in components of the mhc class i processing pathway, such as tap, can present a repertoire of peptides derived from alternative processing pathways. different "housekeeping" cell functions such as signal peptide cleavage, protein maturation in the golgi, and protein/organelle disposal via autophagy can provide peptide ligands for mhc class i loading. the membrane rendering them susceptible for subsequent degradation. the intramembrane cleavage by spp is favored by spp topology that conceals the catalytic center within the plane of the membrane. the two aspartic residues required for the proteolytic activity of spp are located within conserved (y/f)d and g(l/i/f)gd amino acid motifs in two adjacent transmembrane domains ( , ). regarding a cleavage motif for spp, no consensus cleavage site has been described. however, spp demonstrates a strong preference for substrates with helix-destabilizing residues in their transmembrane domain ( - ). amino acids like asparagine, serine, and cysteine disturb a perfect alfa-helical conformation of the transmembrane domain and are therefore referred to as "helix-bending" or "helix-breaking" residues. signal peptides have been shown to contain amino acids with "helixbreaking" capacity within their h region, which critically influence their proteolytic processing by spp ( , ( ) ( ) ( ) . the disturbance of the α-helix caused by these residues is thought to facilitate intramembrane proteolysis. this and other issues would be clarified with the atomic resolution of this protease but this is still lacking due to its technical difficulties. we can have an approximation of that by looking at the crystal structure of presenilin/spp homologs recently published ( ) ( ) ( ) ( ) . jr is an spp homolog from the archaeon methanoculleus marisnigri that harbors nine transmembrane helices, similar to what is predicted for spp, with tmd and tmd containing the yd and gxgd motif, respectively. the two catalytic aspartate residues are located close to each other and approximately Å into the lipid membrane. proteolytic activity occurs in the presence of water molecules that gain access to the catalytic aspartates through a large cavity between two terminal domains ( ) . the three-dimensional structure of a human presenilin comprised into the γ-secretase complex has also been described ( ) . for the near future, we can expect more information on the catalytic activity of this family of proteases, including spp, which is definitely an important contributor of peptides for mhc class i presentation. our recent data revealed an additional role for intramembrane proteolysis by spp. regardless its name, spp also appeared to liberate a c-terminal peptide, independent of proteasome activity. the processing of c-terminal regions of a type ii protein inserted in the er membrane leads to the presentation of peptides independently of proteasome and tap ( , ) . the cterminal region from the ceramide synthase trh , which is a multiple membrane-spanning protein in the er, contains a mer peptide-epitope that is located at the very c-terminal end of the protein and protrudes into the lumen of the er ( - ). the trh protein has a housekeeping function and is ubiquitously expressed. inhibition of spp activity blocked the generation of the trh peptide. experiments with mutant forms of the trh protein indicated that the intramembrane cleavage by spp occurs at the direct upstream region of the t cell epitope within the lipid bilayer ( ) . we speculated that spp activity in the er membrane is sufficient to liberate the minimal c-terminal -mer peptide and release of this peptide into the er lumen. other proteolytic enzymes, such as amino-peptidases, were dispensable. further carboxy-terminal processing was not needed since the epitope is located at the very c-terminal end of the protein. direct release of the liberated peptide into the er lumen is very likely, due to the type ii transmembrane orientation of the trh protein tail (nterminus to c-terminus orientation). the exact peptide loading mechanism of the trh membrane peptide, however, remains to be determined. what triggers the cleavage of trh by spp is not known yet. in addition to its liberation function of small transmembrane substrates, spp has been shown to associate with misfolded membrane proteins in complexes where spp is represented as a monomer, dimer, or multimer ( ) . it was suggested that such high molecular weight complexes act as chaperones to dispose of membrane aggregates ( , ) . the role of spp in this degradation machinery might be to liberate such aggregates from the er membrane. these discoveries were based on the viral us and us proteins, which successfully labels mhc class i molecules in the er for retrograde transport to push this protein back to the cytosol for degradation by proteasomes ( , ) . interestingly, the spp family member, presenilin , seems to be associated with this membrane proteolysis as well ( ) . since spp and presenilin have opposing preference for type i and type ii transmembrane orientations, such a "degradome" machinery might be responsible to clear the er membranes. this type of machinery is called the erassociated degradation (erad) pathway. erad is an er quality control system, which monitors the integrity of nascent or erresident proteins and targets incorrectly folded or misassembled proteins to degradation. the disposal of misfolded mhc class i heavy chains also occurs through the erad pathway in the absence of viral interference ( ) . clearly, viruses take advantage of these existing pathways and hijack them in order to evade immune recognition by cytotoxic t cells ( , ) . another role for spp in erad has come from a recent paper describing the cleavage of the unfolded protein response (upr) regulator xbp u by spp ( ) . xbp u is a type ii membrane protein and undergoes intramembrane cleavage within a conserved type ii tm domain while integrated in a complex containing spp, the protease derlin- , and the e ligase trc , which prime spp for xbp u cleavage. an ectodomain shedding of spp substrates prior to spp cleavage is thought to be required and normally performed by sp in signal sequences but such cleavage was unnecessary in the case of xbp u, similarly to what we have described for trh ( , ) . in general, the upr induces a strong downregulation of mhc class i molecules at the cell surface ( , ) . thus, spp activity seems to have a direct impact on mhc class i peptide presentation by cleavage of leader sequences from nascent proteins and the liberation of some c-termini for mhc class i loading. a more indirect role that impacts on mhc class i presentation has been revealed and occurs through an erad pathway. this is also supported by a recent study using a systemslevel strategy reporting the involvement of spp in the network that coordinates the erad response ( ). the group of yewdell showed more than a decade ago that peptides located at the c-terminus of er-targeted proteins are very efficiently generated and presented on mhc class i ( ) ( ) ( ) ( ) . the location of the peptide was essential to be at the very end of the c-terminus of the protein, not requiring c-terminal trimming, in line with the fact that there is poor carboxypeptidase activity in the er ( ) . they described the presentation of tap-independent peptides from one er-resident protein, jaw , and proteins in the secretory pathway, like ovalbumin and cd . in each case, the peptides were efficiently liberated from the very c-terminus by the activity of yet unidentified endo-proteases to be generated as class i ligands. based on this pathway of peptide liberation, the authors provided the term "c-end rule" to highlight the capacity of er-resident proteases to liberate class i ligands from the cterminal ends of er-targeted proteins. the liberation of peptides without the intervention of the proteasome can also occur in the trans-golgi network ( , ) . the main proteolytic enzyme involved was shown to be furin, a known protease of the trans-golgi network normally required for the maturation of secreted proteins (e.g., growth factors and neurotransmitters) by cleaving at precise stretches of three to four basic residues ( ) . furin is part of a family of proprotein convertases that comprises nine members ( ) . three members (pc / , pace , and pc ) including furin are widely expressed and together they take part in a variety of processes occurring in the trans-golgi network, cell surface, or endosomes. this leads to the activation or inactivation of receptors, ligands, enzymes, viral glycoproteins, or growth factors ( ) . furin also has important functions during development by processing substrates like bone morphogenetic protein (bmp ), a member of the tgf-β superfamily that plays a critical role in heart development ( ) . furin processes a wide variety of precursor proteins after the c-terminal arginine residue in the preferred consensus motif -arg-x-arg/lys-arg↓-x-(x is any amino acid and "↓" indicates the cleavage position) ( ) . initially, this pathway was studied with the use of a model peptide at the cterminus of the secreted hepatitis hbe protein. furin-processed antigens targeted to the secretory route were presented by mhc class i at the cell surface and could elicit functional cd t-cell responses in vivo in a tap-independent fashion ( , ) . the ctermini of secretory or er-localized proteins thus appear to be processed for presentation to cd t cells (figure ). the generation of mhc class i ligands described above defines several alternative ways to generate peptide ligands without the intervention of the proteasome. these represent unusual pathways for peptide generation. now, we will discuss a different constraint in the conventional antigen presentation pathway related to the blockade of peptide entrance in the er due to tap deficiency. in human beings, tap-deficiency syndrome has been described in several independent families and results from mutations in either one of the subunits of the peptide transporter, tap and tap ( , ) . interestingly, these tap-deficient individuals do not succumb to viral infections, suggesting that cd t cell immunity is sufficiently supported by alternative tap-independent processing pathways. to date, a diversity of viral and endogenous tapindependent peptides have been identified in the grooves of different mch class i alleles. importantly, these tap-deficient patients harbor a polyclonal cd t-cell repertoire that is capable of recognizing peptides from the ebv virus, like protein lmp , presented on tap-deficient cells ( ) . the tap-independent processing pathway is capable of generating enough mhc class i/peptide complexes in order to keep immunosurveillance and control of viral infections. studies with tap -knockout mice have shown that surface expression levels of mhc class i are indeed lower, but the remaining complexes do induce a broad and polyclonal repertoire of cd t-cells ( ) ( ) ( ) . the tcr usage was shown to be very comparable to that of wild-type mice and tap -knockout mice were capable of mounting anti-viral cd t cell responses. together, these data show that, although crippled, the mhc class i-presented peptide repertoire in the absence of tap is sufficient to support cd t cell immunity. interestingly, peptides emerging from alternative tapindependent routes appeared to be immunogenic. following immunizations in mice with tap-deficient tumor cells, specific cd t-cells were induced that recognize tap-deficient cells, but not normal cells ( , ) . these t-cell epitopes seemed to be selectively presented by tap-deficient cells but not under normal conditions. this alternative peptide repertoire emerges due to processing defects and therefore these peptides were named "t cell epitopes associated with impaired peptide processing" (teipp). the molecular identification of some teipp peptides revealed that they can be diverse in length (from -mer to -mer), amino acid composition, and mhc class i binding, as some are presented by classical mhc class i molecules and others by the non-classical mhc molecule hla-e and the mouse homolog qa- b ( table ) ( , [ ] [ ] [ ] [ ] . they are derived from normal housekeeping proteins with ubiquitous expression, but are surprisingly not loaded on mhc class i in cells with an intact antigen-processing machinery. they constitute normal self-peptides (non-mutated, not pathogen-or tumor-specific) and can be regarded as real neo-antigens. the immunogenicity of teipp peptides exists because they are not presented by normal cells including the thymus. during thymic development, t cells are subjected to two subsequent processes called positive and negative selection ( , ) . negative selection is necessary for the maintenance of self-tolerance as it induces the deletion or inactivation of potentially autoreactive thymocytes ( ) . we recently demonstrated that teipp-specific cd t-cells indeed do not undergo negative selection and are thereby available for therapeutic exploitation. since the peptides recognized by teipp-specific ctl are derived from housekeeping proteins, we tried to understand why teipps are not presented by processing intact cells. collectively, our data show that teipp peptides are actually produced within processing proficient cells, but somehow are not or not sufficiently presented by their surface mhc class i molecules. taking the trh -derived teipp peptide as a model, we have analyzed the expression of the trh gene in several epithelial populations isolated from wild-type and tap -ko mice ( ) . this analysis revealed the same level of rna transcripts between the normal and knockout populations, suggesting comparable protein levels in both cell types. the liberation of the trh peptide is performed by spp, which is active in tap-positive as well as tap-negative targets ( ) . overexpression of the trh gene in tap-positive cells leads to surface presentation in mhc class i ( ), still in a proteasome-independent way. moreover, proteasome inhibition in tap-positive cells results in presentation of the endogenous trh peptide ( ) , indicating that, indeed, this teipp peptide is generated in all cells but loses competition with the overwhelming amount of tap-imported peptides in tappositive cells. some alternative peptides, like the ones derived from ebv proteins were shown to be presented on tap-positive cells to comparable extent, although using alternative pathways. moreover, our data suggest that the limited quantity of the trh peptide-epitope in the er is the main cause of selective presentation in tap-deficient cells. interestingly, gradual increase of overexpression correlated with the degree of recognition by the teipp-specific ctl clone, implying that tap transport actually constitutes a strong barrier for teipp peptides. the study of human teipp antigens corroborates these findings. one antigenic peptide is encoded by the human calca gene and derives from the signal sequence of preprocalcitonin (ppct) protein. this peptide is liberated in the er lumen by sequential cleavage with sp and spp, independently from proteasomes ( table ) ( ) . the presentation of the ppct peptide to specific ctl was found in human lung and medullary thyroid carcinomas that had very low expression of tap. presentation of the ppct peptide occurred also in normal non-transformed cells, such as dendritic cells (dcs), after knockdown of tap. overexpression of the calca gene in dcs and tap-positive tumor cells resulted in recognition by the specific ctl clone ( ) . identification of additional human teipp antigens at the molecular level will enable cd t cell targeting of otherwise ctl-resistance tap-negative tumor variants ( , ) . together, these findings support the model of peptide competition in the er as a factor that prevents presentation of peptides from alternative sources, and shape a picture of alternative processing pathways that emerge upon defects in the conventional one (figure ). the precise loading mechanism of tap-independent signal peptides into mhc class i molecules is not known, since processing by sp and spp is thought to take place outside of the plc. this sophisticated machinery for optimizing ligand length and quality and facilitating peptide loading onto nascent mhc class i molecules greatly facilitates peptide loading by physical bridging transporters to chaperones for loading and also "edits" the repertoire of bound peptides to maximize their affinity ( ) . the plc molecule tapasin tethers mhc class i molecules to the peptide transporter acting together with the chaperone calreticulin and the oxidoreductase erp ( ). tapasin can sense the quality of peptide bound by mhc class i complexes, and allows successive rounds of peptide binding until a certain affinity threshold is met. trimming of incoming peptides by eraap can be necessary for obtaining a good peptide length before selection by a defined mhc class i allele. a recent paper states that mhc class i molecules initially bind a variety of peptides, including some lowaffinity or n-terminally extended ones but then quickly dissociate from the molecule followed by the selection of the best fitting candidates ( ) . however, tap-independent leader peptides do not arrive in the er via tap and thus lack these editing and optimizing chaperones. in the absence of tap transporters, the loading of peptides into mhc class i occurs without a fully functional plc at hand. in that respect, inefficient loading of leader peptides in tap-positive cells can be expected, whereas in cells devoid of tap, this alternative er entrance mechanism allows emergence of these peptides. so here, the plc floats in the er membrane and is dispatched from the entry site of peptides. indeed, differential mass spectrometry analysis showed an enhanced presentation of leader peptides in cells lacking the peptide transporter ( ) . after all, mhc class i molecules get loaded with the available repertoire: from conventional or unconventional sources. but how do these "untapped" peptides find their way to empty mhc class i molecules at all? it has been shown that loading of peptides in mhc class i can occur with the minimal components of tapasin-erp -mhc class i complexes ( ), but we have to assume that the chances of a peptide to find the plc machinery in the er are scarce. on the other hand, tap-deficient cells harbor more peptide-receptive class i molecules compared to normal cells ( ) . these peptides might actually be actively chaperoned toward these open grooves. chaperones with high peptide binding capacity in the er are heat shock proteins (e.g., hsp ) and pdi, an isomerase that efficiently binds free peptide ( ) . it is possible that tap-independent peptides are captured by these molecules and chaperoned to mhc class i. recent studies suggest that the tapbpr molecule ("tapasin-like") binds those mhc class i proteins that are not bound to tapasin in the plc, so we might hypothesize that this pool of peptide-receptive grooves may function to load leader peptides ( ) . however, this still needs to be investigated. other ways to access peptide-receptive mhc has been proposed and are related to the intracellular traffic of hydrophobic peptides. studies with several epitopes from the lmp protein of the ebv showed that peptides, which possess a high hydrophobicity index, were presented in a tap-independent manner ( ) . the proposed mechanism describes the generation of these peptides outside the er, since proteasome activity is needed for presentation, and subsequently free diffusion across the er membrane possible due to their high hydrophobicity. these peptides might transgress membranes spontaneously or via alternative membrane transporters. a recent study by tey et al. showed that the processing of a peptide antigen from the human cytomegalovirus (hcmv) latency associated protein, pul , occurs entirely in the vesicular pathway and is mediated by autophagy ( ) . also other examples of autophagy enhanced mhc class i presentation of viral antigens were reported ( , ) . during autophagy, large portions of cytoplasmic content, including proteins and organelles, are encapsulated in double membrane vesicles called autophagosomes ( ) ( ) ( ) ( ) . the autophagosomal membrane has been proposed to originate from the er ( , ) and peptide-receptive mhc class i molecules might be present in autophagosomes, allowing for loading of peptides in these vesicular compartments ( ) . in addition, recirculating mhc class i molecules from the cell membrane end up in endosomes and can have contact with autophagosomes before returning to the endocytic network. transit for membrane-associated proteins between autophagosomes and endosomes has been observed by live cell imaging ( ) . moreover, peptides generated by the proteasome seem to get access to the endocytic vesicular pathway as well. we recently found at least one example for this in our teipp repertoire proportions and combinations. the mhc class i peptide repertoire can therefore be compared to a painter's palette where the different "colors" (peptides) are mixed and used to create a colorful and complex "picture." that is generated by the proteasome but is tap-independently presented ( table ) ( ) . the presentation of this peptide was surprisingly enhanced by blockade of the proton pump in endosomes, indicating that this antigen most likely crosses the vesicular pathway ( ) . finally, mass spectrometry analysis of the tapindependent peptide repertoire pointed at proteins located in the vesicular compartment as an important source ( ) ( ) ( ) . though these alternative processing and loading compartments have not fully been unraveled, these data strongly support the notion that the vesicular pathway and autophagy contributes to antigen presentation by mhc class i molecules. on summarizing, we can conclude that, even though the conventional proteasome-tap pathway represents the major source of the mhc class i peptide repertoire, alternative processing pathways clearly complement the total pool. this multitude of mhc class i processing pathways can be compared to a colorful palette where the painter combines the different colors that will end up in different proportions and combinations in the final picture (figure ) . in situations of viral infections, cellular transformation, or other initiators of stress, the contribution of these alternative pathways might gain importance. the spp and maybe their family members are convincing examples of alternative proteolytic systems that feed the alternative routes of antigen presentation. the precise molecular identification of alternative loading mechanisms unto peptide-receptive mhc class i molecules, whether it be in the er or in autophagosomes, still needs indepth investigation. in that respect, peptides can walk on multiple different paths before ending up in the grooves of mhc class i and therefore illustrate the old expression that multiple roads lead to rome. the protease inhibitor, n-acetyl--leucyl--leucyl-leucyl--norleucinal, decreases the pool of major histocompatibility complex class i-binding peptides and inhibits peptide trimming in the endoplasmic reticulum s proteasomes and immunoproteasomes produce mainly n-extended versions of an antigenic peptide the sizes of peptides generated from protein by mammalian and s proteasomes. implications for understanding the degradative mechanism and antigen presentation an ifngamma-induced aminopeptidase in the er, erap , trims precursors to mhc class i-presented peptides eraap customizes peptides for mhc class i molecules in the endoplasmic reticulum the er aminopeptidase erap enhances or limits antigen presentation by trimming epitopes to - residues mechanisms of mhc class i-restricted antigen processing and 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aspects of hla class i deficiency human peptide transporter deficiency: importance of hla-b in the presentation of tap-independent ebv antigens positive selection of self-and alloreactive cd + t cells in tap- mutant mice tap -deficient mice select a cd + t cell repertoire that displays both diversity and peptide specificity tap mutant mice are deficient in antigen presentation, surface class i molecules, and cd - + t cells generation of cd + t cells specific for transporter associated with antigen processing deficient cells alternative peptide repertoire of hla-e reveals a binding motif that is strikingly similar to hla-a cd + t cell responses against tap-inhibited cells are readily detected in the human population the nonpolymorphic mhc qa- b mediates cd + t cell surveillance of antigen-processing defects nonclassical mhc class ib-restricted cytotoxic t cells monitor antigen processing in the endoplasmic reticulum antigen presentation in the thymus for positive selection and central tolerance induction selection of the t cell repertoire the thymic medulla: a unique microenvironment for intercellular self-antigen transfer preprocalcitonin signal peptide generates a cytotoxic t lymphocyte-defined tumor epitope processed by a proteasome-independent pathway different expression levels of the tap peptide transporter lead to recognition of different antigenic peptides by tumor-specific ctl strategies to counteract mhc-i defects in tumors a novel category of antigens enabling ctl immunity to tumor escape variants: cinderella antigens tapasin-the keystone of the loading complex optimizing peptide binding by mhc class i molecules in the endoplasmic reticulum the first step of peptide selection in antigen presentation by mhc class i molecules features of tap-independent mhc class i ligands revealed by quantitative mass spectrometry selective loading of high-affinity peptides onto major histocompatibility complex class i molecules by the tapasin-erp heterodimer peptide-receptive class i major histocompatibility complex molecules on tap-deficient and wild-type cells and their roles in the processing of exogenous antigens tap-translocated peptides specifically bind proteins in the endoplasmic reticulum, including gp , protein disulfide isomerase and calreticulin the binding of tapbpr and tapasin to mhc class i is mutually exclusive processing of a multiple membrane spanning epstein-barr virus protein for cd (+) t cell recognition reveals a proteasome-dependent, transporter associated with antigen processing-independent pathway autophagy mediates transporter associated with antigen processing-independent presentation of viral epitopes through mhc class i pathway alternative pathways for mhc class i presentation: a new function for autophagy autophagy enhances the presentation of endogenous viral antigens on mhc class i molecules during hsv- infection autophagy and adaptive immunity when autophagy meets viruses: a double-edged sword with functions in defense and offense unveiling the roles of autophagy in innate and adaptive immunity endoplasmic reticulum and golgi complex: contributions to, and turnover by eating the endoplasmic reticulum: quality control by autophagy generation of mhc class i ligands in the secretory and vesicular pathways the itinerary of autophagosomes: from peripheral formation to kiss-and-run fusion with lysosomes dominant contribution of the proteasome and metalloproteinases to tapindependent mhc-i peptide repertoire allele-dependent processing pathways generate the endogenous human leukocyte antigen (hla) class i peptide repertoire in transporters associated with antigen processing (tap)-deficient cells diversity of natural self-derived ligands presented by different hla class i molecules in transporter antigen processing-deficient cells a transporter associated with antigen-processing independent vacuolar pathway for the mhc class i-mediated presentation of endogenous transmembrane proteins financial support was received from the portuguese foundation for science and technology (grant sfrh/bd/ / to co). gamblin artists colors (portland, or, usa) is acknowledged for the free use of figure . the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord- -rk pwoa authors: schuller-levis, georgia; kozlowski, piotr b.; kascsak, richard j. title: central nervous system: viral infection and immune-mediated inflammation date: - - journal: xenobiotics and inflammation doi: . /b - - - - . - sha: doc_id: cord_uid: rk pwoa nan locally disabled for long periods of time (reese and karnovsky, ; hirano etal, ) . a peculiarity of the bbb is that it excludes macromolecules from entering the cns but allows megastructures such as entire hematogenous inflammatory cells to cross the barrier without opening the tight junctions (hirano et al, ; azarelli et al, ; lossinsky et al, ; raine et al, ; powell, ) . nonetheless, the entry of hematogenous inflammatory cells into the cns appears to be strictly controlled by the ecs. an initial event in the inflammatory reaction in the cns is the egress of hematogenous cells into the extravascular milieu of the cns parenchyma ( figure ). only the previously sensitized inflammatory cells, i.e., lympho cytes, can invade the cns (raine et al, ; powell, ) . the prerequisite for transendothelial passage is the attachment of sensitized cells to the luminal surface of endothelial cells. a growing body of evidence suggests that lymphocytes and monocytes (but not neutrophils) attach to parajunctional endothelial cell receptors and subsequently insert pseudopodial pro jections into tubular, channel-like openings in the endothelial cells (lossin sky et al, ; powell et al, ) . these channels serve as eventual conduits for the transendothelial cell passage into the extravascular space. numerous studies have shown a great variety of specific membrane recep tors on endothelial cells that are critical for the binding of hematogenous cells. these highly specific receptors include several distinct classes of adhesion molecules. these receptors include the immunoglobulin super family (icam- and - , lfa- , vcam- , endocam- , and ncam- ); the integrins (lfa- or c d l l a / , mac- , lpam- , , β , β families); the lec-cam family for lectins ; cadherins (Ρ, Ν, e-cadherins), and the hermes group of antigens (cd- ) (harlan, ; dustin and springer, ; lassmann et al, , staunton et al, yednock, ) . the expression of these receptors, also called addressins, on the endothelial surface is under the control of several cytokines, some of which are present only in local inflammatory response and absent under normal conditions. neutrophils, which also require addressins for adherence, are able to enter the cns by opening and passing through a tight junction between adjacent endothelial cells (broadwell, ) . second, there are structural cells of the cns that, in addition to their "main" roles, may, under certain conditions, play a role in antigen presenta tion and/or processing. these cells include astrocytes, endothelial cells, and microglial cells (fontana et al, ) . under normal conditions, only a small portion of meningeal, perivascular dendritic cells and resident mi croglial cells may express low levels of class ii major histocompatibility complex (mhc) antigens. the expression of these antigens on various cell types seems to be related to the severity of the inflammatory response. in mild inflammation, class ii mhc antigen expression is upregulated mostly on microglia cells; only in severe inflammation can some expression of these antigens be seen on astrocytes. gamma interferon (ifn-γ) is known to stimulate in vitro the expression of class ii mhc antigens on endothelial cells, microglial cells, and astrocytes (wong et al., ; hirsch et al., ; fierz et al., ) . the nonhematogenous cells that are native to the cns have a very limited role in cns inflammation. neurons, oligodendroglia cells, and ependymal cells, if not affected directly by an infectious agent, appear to be inert bystanders, even in the midst of severe inflammation. astrocytes perform a variety of functions within the cns. astrocytic processes are a vital part of the bbb, and the cells themselves maintain the homeostasis of the neuropil (figure ). astrocytic cells are the main cells involved in scar formation following cns injury. they are now considered an accessory cell to the immune system of the cns (hertz etal., ; yong et al., ; rodriguez et al., ) . they express mhc antigens, may act as antigen-presenting cells (apcs), and secrete cytokines. in inflammation, astrocytic cells (especially type ) may be stimulated by laf-and icam- , by cytokines from Τ cells, macrophages, microglial cells, and/or other astrocytes. il- stimulates proliferation of astroglia in experimental brain injury. in vitro, il- , tumor necrosis factor (tnf), and ifn-γ stimulate proliferation of astrocytes. ifn primes astrocytic cells to the effect of il- by secretion of tnf, il- , il- , il- , and lymphotoxin. the endothelial cells are not only an anatomical and functional barrier between the blood and the brain; they are also pivotal cells in inflammation. ecs not only regulate the influx of cells and solutes, but they may also act as apcs, and the luminal surface of ecs may be the site of antigen presenta tion and recognition. class i mhc molecules necessary for antigen recogni tion can be found on all cells constituting the bbb, whereas class ii mhc can be induced by ifn-γ stimulation. class ii mhc antigens expressed on ecs increase with the increased severity of inflammation, suggesting that the mhc expression is progressively turned on and may, under certain conditions, lead to amplification of local inflammation. ecs activation in chronic (and not necessarily severe) inflammatory processes may not be a secondary consequence of a local process but may, in fact, initiate and perpetuate the inflammation (hertz et al., ; gimborne and beviaqua, ; montovani and dejana, ; cotran, ) . it also appears that the endothelial cell may be a pivotal cell in autoim mune inflammatory reaction (with local intrathecal antibody formation) without the involvement of extraneuronal antigens or infectious agents. ecs may present antigens that are specific for the cns and attract sensitized Τ cells. Τ cells may attach to cns antigens and perpetuate inflammation by secreting lymphokines (ifn, il- ) and attract even more lymphocytes. this critical mass of lymphocytes and lymphokines may open the bbb, which allows Τ cells to enter the cns parenchyma to recruit Β cells, which then locally produce antibodies against the cns antigens presented by endothelial cells. microglia cells are considered immune-competent cells that are native to the cns but whose origin is still unclear (graeber and streit, ) . they are found throughout the cns and constitute from to % of all cns cells (figure ). microglia cells share common surface antigens with mono cytes and macrophages but not with neuroectodermal cells. the functions of microglial cells include phagocytosis, presentation of antigens, produc tion of il- , and stripping of synaptic buttons (deafferentation) from dis- the antigen is also present on brain resident microglial cells. note the cell surface staining and presence of the dichotomically branching processes. cells are evenly spaced, and do not form a connection with each other. anti-ln- monoclonal antibody, avidin-biotin technique, -/ltm-thick vibratome section; nuclei were counterstained with hematoxylin, x . tressed neurons. there is a low level of expression of class ii mhc antigens on microglial cells under normal conditions, and this expression can be dramatically increased with infusion of ifn-γ (graeber and streit, ; tribolet de et al, ; hayes et al, ; hayes et al, ; delisle et al, ; hickey and kimura, ; mcgeer et al, ; mcgeer et al, ) . microglia cells also express the cd molecule, which is considered an entry point for the human immunodeficiency virus (hiv) (watkins et al, ; bourdial et al, ; levy, ; michaels et al, ). yet another unique feature of the cns is the presence of cns-specific antigens such as myelin basic protein (mbp), glial fibrillary acidic protein (gfap), or neuron-specific enolase (nse). such proteins can be the focus of autoimmune responses within the cns. this autoimmune response can be a major contributing factor to pathology and clinical disease (see later discussions on viral-induced autoimmune response and experimental aller gic encephalomyelitis). a strong body of evidence now suggests that some diseases of the cns have an immunologic pathogenesis (paterson, (paterson, , (paterson, , (paterson, , . ex amples include experimental allergic encephalomyelitis (eae), acute dis seminated encephalomyelitis (adem), and multiple sclerosis (ms). acute and chronic relapsing eae can be induced in laboratory animals by an injection of cns tissue, cns myelin, myelin basic protein, or more recently, t-cell lines specific for nervous system antigens. adem occurs in humans and animals after an antecedent virus infection (see later discussion on viral induced autoimmunity) or vaccination with a living virus or a vaccine containing nervous tissue. ms is an acute or chronic progressive remitting disorder of humans characterized by inflammatory cell infiltration and demyelination. eae is a useful autoimmune disease model for the study of mechanisms of cellular immune reactions in the cns and has been considered an experi mental model of ms since the earliest descriptions by rivers et al ( ) . in recent years, reproducible small animal models of chronic eae have become available (stone and lerner, ; wisniewski and keith, ; massanari, ; lubin et al, ; brown et al, ) . these small animal models cover the full spectrum of ms pathology (wisniewski et al, ; lassmann and wisniewski, ) and allow the study of the pathogenesis of chronic inflammatory demyelinating plaque formation. progress on the pathogenesis and treatment of eae has been advanced by the selection and maintenance of permanent autoantigen mbp-specific t-cell lines. Τ cells can now be isolated from animals with eae that will mediate eae in the recipient animal. this isolation is possible as a result of progress in the characterization of immune cells subsets, using monoclonal antibodies, and the definition of their growth requirements (i.e., il- ) (ben-nun et al, ) . it is not clear how the sensitized Τ cells present in the circulation of eae animals, cross the bbb and recognize an antigen or antigens that are located in the major dense lines of the myelin sheath. one possible explanation of this phenomenon is that the low rate of physiological ex change of lymphocytes that appears to exist between the cns and the circulation allows the entry of the sensitized Τ cells (lassmann et al, ; wekerle et al, ; wekerle et al, ) . interaction between inflammatory cells and the endothelial cells lining the bbb is an important initial event during inflammation of the cns. the expression of la and of the endothelial leukocyte adhesion molecule (elam- ) on endothelial cells in the cns are some of the earliest changes detected to date in brains of animals with eae (rose et al, ) . although the etiology of ms is still unknown, the similarity of ms to remitting-relapsing eae suggests that ms, like eae, might be the conse quence of either a direct or indirect autosensitization to myelin antigens (i.e., mbp and proteolipid apoprotein) (rose et al, ) . as a result of the application of advances in molecular biology, such as the polymerase chain reaction, and developments in cellular immunology, such as the ability to grow t-cell clones, great progress has been made in the pathogenesis and treatment of ms (steinman, ) . recent reports that most encephalitogenic (bp-specific) t-cell clones derived from mice or rats use common variable region genes of the rearranged t-cell receptor (tcr) have intensified the search for analogous associations in humans (burns et al, ; urban et al, ) . steinman ( ) has recently reviewed the strong parallels between t-cell receptor (tcr) usage in the pathogenesis of eae and tcr usage in mbp, and specific Τ cells in ms peripheral blood and Τ cells in demyelinated plaques in ms brains. the development of strategies for selective immunotherapy in eae was based on these similarit ies. this therapy uses monoclonal antibodies on either class ii molecules of the mhc or tcr-variable regions or peptides that compete with hla class ii molecules or vaccination against tcr-v regions. for example, vaccination of mice with appropriate tcr peptides is highly effective at inducing both cellular and humoral response to tcr and at inhibiting eae (howell et al, ; vanderbark et al, ) . monoclonal antibodies that bind only the complex of bp and i-as inhibited eae in h- s mice when injected within - to + days of disease induction (aharoni et al, ) . sriram and carroll (sriram and carroll, ) have shown that in vivo treatment with i-a antibodies at the earliest sign of eae results in decreased homing of radiolabeled cells to the brain. these data suggest that class ii antigens play an important role in cellular migration across the bbb. other potential therapies for eae include the use of cytokines (see cyto kine section) and suppressor Τ cells. although mechanisms involved in eae remission are unclear, suppressor Τ cells have been postulated to play a role in the prevention of autoimmunity (ofosu-appiah and mokhtarian, ) . an adaptively transferred suppressor t-cell line, obtained from spleens of mice that recovered from eae, was able to downgrade eae in mice subsequently challenged with mbp-activated Τ cells. immunologically important cytokines are produced chiefly by lympho cytes and macrophages. inflammation in the nervous system is likely to be the same as in other organ systems, with a few modifications. because of the bbb and the macromolecular nature of cytokines, some of these important inflammatory molecules are likely to be cns resident derived. recent studies have also indicated astrocytes, microglia, and endothelial cells as possible additional sources of cns-derived cytokines (fontana et al, ; frei et al, ; giulian et al, ; frei et al, ; sawada et al, ) . immunocytochemical techniques have demonstrated the presence of a variety of cytokines (see earlier discussion) (i.e., mif, ifn-γ, tnfa, il- , , ) during chronic relapsing eae . il- , il- , tnfa, and il- have also been shown in the serum and/or csf of eae animals and/or ms patients (merril et al, ; gallo et al, ; gijbels et al, ) . a. tgf-/ and il- transforming growth factor β (tgf-β) has pleotrophic effects. evidence to date includes the possible role of down-regulation of the ifn-γ induction of class ii mhc expression on the inductive phase of the immune response, as well as inhibition of cytotoxic t-cell development and antagonism of tnf at the effector end of the immune response (racke et al, ; wahl et al, ; rook et al, ; ranges et al, ; czarniecki et al, ; schluesener, ; shalaby and ammann, ) . when administered dur ing eae induction, tgf-/ slightly delays the onset of disease (kuruvilla et al, ) . however, when given during remission, tgf-/ prevents the occurrence of relapses in relapsing eae, suggesting an anti-inflammatory effect of tgf-/ . when tgf-j was preincubated in vitro, activation and proliferation of myelin basic protein-specific lymph node cells in vitro were decreased and severity of the clinical course was reduced (racke et al, ) . recent data indicate that il- may promote cns inflammation, and that blocking il- activity may prove beneficial in the treatment of cns inflammatory disease. for example, eae in the lewis rat has been shown to be exacerbated by il-Ια. in addition, the disease was significantly delayed and attenuated by il- receptor antagonist (il-lra) (jacobs et al, ) . il- receptor antagonists have been used to ameliorate other inflamma tory diseases such as rheumatoid arthritis and septic shock, and colitis in a rabbit model (cominelli et al, ; ohlsson et al, ; wakabayashi et al, ; arend and dayer, ; higgins and postlethwaite, ) . several structural variants of il-lra have been described (arend, ) that might be of interest in exploring the therapeutic potential of il-lra. administration of il-lra into the lateral ventricle of rabbit brains has been reported to block il-l-induced non-rapid-eye movement sleep as well as il-l-induced fever (opp and krueger, ) . the similarities of macrophages and microglia are striking (merz et al, ) . for example, il- , il- , and tnfa are known to be produced by monocytes and macrophages. in the cns, microglia have been implicated as a major contributor to the production of il- , il- , and tnf (woodroofe et al., ) . using a continuous microdialysis probe, woodroofe et al. ( ) showed a -fold increase in il- over a to hr period and a slight increase in il- at day , as a result of mechanical trauma to the brain. another potential cellular source of il- is the astrocyte. however, the authors have immunocytochemical evidence that in this model the astrocyte response appears much later-at day . an increase in peripheral benzodi azepine receptors after local injection of il- and tnfa has been demon strated on glial cells, but not on neurons (bourdial et al, ) , raising the possibility of a sequential mechanism involving the activation of microglia, the release of il- and tnfa, and the promotion by these cytokines of the astroglial reaction. administration of human serum amyloid a to mice inhibited fever in duced by ril- / or rtnfa in vivo, whereas the addition of human serum amyloid a to murine hypothalamic slices inhibited il- / -or tnfa-induced prostaglandin e production. these data suggest a possible feedback rela tionship between serum amyloid a and cytokines (shainkin-kestenbaum et al, ) . injection of il- results in an increased release of corticotropin-releasing factor (ohgo et al, ; besedovsky et al, ; sapolsky et al, ; uehara et al, ) . in addition, il-Ια injected into the third ventricle of castrated rats inhibited the pulsatile release of luteinizing hormone (rettori et al, ) . in a recent report, palazzolo et al ( ) suggest that the central and neuroendocrine effects of il- are most likely produced through changes in neurotransmitter metabolism in the brain. in another report, mohankumai et al. ( ) provide evidence that il- stimulates the release of catecholamines (e.g., dopamine and its metabolite, dihydroxyphenylacetic acid (dopac) from discrete hypothalamic nuclei of conscious, freely mov ing rats. the precise role that il- may play in neuroimmunomodulation is still not clear. the important known role of the hypothalamicpituitary-adrenocortical (ΗΡΑ) axis in regulating inflammation mandates further studies of the role of il- as a neuroimmunomodulator. another potential interaction of the nervous system and the immune system is the involvement of cytokines in neurogenic inflammation (kim ball, ) . in the kimball model, substance p, a neuropeptide, is at the center, interacting with fibroblasts, mast cells, Β cells, and macrophages. in addition to substance p, other neuropeptides, neurotransmitters, and other vasoactive amines act in concert with cytokines to affect immunologic mechanisms. b. il- , il- , tnfa, and ifn-γ the roles of established monokines (il- and tnfa) and t-cell products (ifn-γ and il- ) on cns pathology and physiology are stimulating areas of ongoing research. tnf appears to be a major cytokine involved in cellular injury. axon and myelin abnormalities have been demonstrated in vitro by tnf (selmaj and raine, ) . after il- perfusion in rats, tnf was noted to coincide with myelin damage. interestingly, il- perfusion has been shown to compromise the bbb (ellison and merchant, ) . several struc tural variants of il-lra have been described (arend, ) that might be of interest in exploring the therapeutic potential of il-lra. several phosphodi esterase inhibitors (e.g., theophylline, pentoxifylline, and -isobutyl-lmethylxanthine) have been shown to suppress tnfa synthesis (endres et al., ) . high tnf levels have been reported to be associated with several manifestations of malaria (kremsner et al, ; shaffer et al, ) , and decreasing tnf, levels with recovery. in a murine model of cerebral malaria, pentoxifylline, which reduces tnf, given for days after infection, pro tected the mice from development of cerebral malaria (kremsner et al, ) . one of the pleotrophic effects of il- is a neurotrophic effect that has been described in viral diseases (frei, ) and improved survival of mes encephalic catachoaminergic and septal cholinergic neurons (hama et al., ) . mice infected with lethal lymphocytic choriomeningitis (lcm) virus as well as lcm carrier mice have been shown to be correlated with high levels of secretion of il- (maskophidis et al., ) . of interest is the possible role of il- in the formation of alzheimer amyloid plaque (baurer and strauss, ) . bauer et al. have shown a potent human proteinase inhibitor, a- macroglobulin (a m), after stimulation with il- (ganter et al, ) . subsequently, they examined whether a m and il- could be detected in alzheimer disease (ad) brain. they report that ad cortical senile plaques display strong a m and il- immunoreactivity, with no such immunoreactivity found in age-matched control brains. the data indicate that neuronal cells are the site of a m synthesis in ad brains. among the best known pleotrophic effects of ifn-γ is its ability to induce the expression of class ii mhc molecules on several cell types. class ii mhc antigens are known to be essential in antigen presentation. studies have shown that ifn-γ can induce expression of class ii mhc molecules on astroglia (sedgwick et al., ) . however, sedgewick et al. ( ) were unable to show that astroglial cells act as stimulators of c d + Τ cells and therefore postulated another cell type as the major antigen-presenting cell in cns inflammation. infection with mouse hepatitis virus (mhv), has been shown to block expression of mhc molecules on murine cerebral endothelial cells (see later discussion) (joseph et al., ) . joseph et al. postulate possible release of cytokines by endothelial cells as a mechanism for blocking ifn-induced class ii antigens. several additional cytokines have been reported that may play a role in cns pathology and physiology. growth factors and, to some extent, il- / and tnfa appear to increase nerve growth factor synthesis and secretion by astrocytes (yoshida and gage, ) . cultured astrocytes have been reported to express macrophage colony-stimulating factor activity at a high level, which may be important in the replication of microglial precursors (alliot et al, ) . growth differentiation factor- , a recently described member of the trans forming growth factor β superfamily, has been reported to be restricted almost exclusively to the cns (lee, ) . this extracellular signaling factor is postulated to play a general role in nervous system maintenance and function. of interest is one of two platelet-activating factor (paf) antago nists, which has been shown to also inhibit il-l/ -induced acth secretion (rougeot et al, ) . this suggests that il- ΗΡΑ secretion may be medi ated, at least in part, by the production of paf. virus infection of the cns can result in a wide range of pathogenic outcomes: a rapid, severely acute form; a chronic, progressive form; a reversible, nonproductive, latent type; or various intermediate stages. dis ease is a direct result of interaction between host and infecting virus. host parameters include age, genetic makeup, and immune competency, whereas viral factors include class and strain of agent, target specificity, and genomic variation. the phenotypic expression of disease is a complex multifactorial interaction among these parameters. the first step in the disease process involves entry of the virus into the cns. various host protective barriers prevent direct viral entry into the cns and force indirect routes of infection. agents that cause viremia can enter by replication in the endothelial cells that line the blood vessels. viruses known to enter by this route include the toga viruses, enteroviruses, and certain retroviruses. viruses can also invade from the bloodstream by entering the stroma and the choroid plexus through the fenestrated capillary and endothelium and either infecting or being transported across the cho roid plexus into the csf. recently, it has been demonstrated that fibroblastlike cells isolated from the human choroid plexus can be infected with hiv (harouse et al, ) . once in the csf, the virus can infect ependymal cells lining the ventricles and invade the underlying cns tissue. the virus can also enter through "trojan horse" mechanisms, being transported within circulating leukocytes such as lymphocytes or macrophages/mono cytes, which can contain such viruses as measles, mumps, canine distem per, lentiviruses, or togaviruses. viruses can also infect peripheral neurons at such sites as the neuromuscular junction and can be axonally transported toward the cns. rabies and herpes simplex may utilize such a route (john son, ) . herpes simplex may also gain entrance to the cns by means of the sensory neurons of the olfactory system. once within the cns, viruses encounter a differentiated cell population with complex, functionally integrated cell-to-cell interactions. the highly specialized cytoplasmic membranes of this cell population allow for great variation in virual receptor sites and in the abilities of cells to support viral replication. viral tropism to cells within the cns involves both cell and viral receptor molecules . polioviruses display a particular affinity for anterior horn motor neurons, whereas the rabies virus normally prefers neurons of the limbic system (johnson, ) . infections with orthomyxoviruses or paramyxoviruses usually involve the selective infection of ependymal cells (wolinsky et al, ) . the polyomavirus, which is the causative agent of progressive multifocal leukoencephalopathy (see later discussion), produces a lytic infection of oligodendrocytes and a nonpermissive infection of astrocytes (richardson, ) . the la antigen receptors present on a variety of cell types have been implicated as viral receptors (inada and mims, ) . cellular cd protein interacts with hiv gp and is the receptor for this virus on lymphocytes and monocytes (wigdahl and kunsch, ) . several studies suggest the presence of the cd receptor on cells within the cns (dewhurst et al, ) . the specific affinity of rabies virus to acetylcholine receptors serves to facilitate uptake and transfer of virus to the cns and to determine neuronal specificity (lentz and burrage, ) . as stated earlier, one potential outcome of cns viral infection is rapid acute disease, usually including encephalitis and/or meningoencephalitis. this can be accompanied by perivascular inflammation involving polymor phonuclear cells followed later by macrophages, lymphocytes, and microg lia. during infection by certain viruses, i.e., poliovirus, the inflammatory response consists of a meningeal reaction, perivascular cuffing, and paren chymal infiltration. polymorphonuclear leukocytes are seen early, with a later shift to predominantly mononuclear cells. microglial cell infiltration and neuronophagia are also seen (johnson, ) . pathogenesis is normally a two-step process consisting of viral-mediated cell destruction as well as immune-mediated, viral-induced cellular destruc tion. viral antigens are normally good immunogens, and host immune surveillance directs a t-cell-dependent immune response (burns, ) . such a response is directed primarily against infected cells that express viral or altered cell surface proteins. antibodies can function as intermediaries in the destruction of infected cells by means of complement-mediated cytotox icity or antibody-dependent cellular cytotoxicity or by serving as opsinizing factors. cytotoxic Τ cells can destroy such cells directly or release lympho kines to recruit, activate, and/or sensitize other lymphoreticular elements. cytolytic Τ lymphocytes (ctls) are an important effector in antiviral immu nity (zinkernagel and doherty, ) . such cells can recognize foreign viral antigens on cell surfaces only in the context of self mhc structures. until recently, the hla class i molecules were thought to be the primary, if not the only, hla recognition structure for ctls. studies of measles and epstein-barr virus (ebv) infection suggest that hla class ii molecules can also serve as recognition sites, further expanding the potential action of ctls. viral antigens recognized by ctls have also been expanded beyond the traditional cell surface molecules (braciale and braciale, ) . prelimi nary data indicate that recognition of internal viral polypeptides and per haps even nonstructural gene products may be a common feature of antivi ral ctls. coronavirus mhv- can selectively block gamma-interferoninduced class ii antigen expression on cerebral endothelial cells (joseph etal, ) . studies by other investigators have shown class i modulation on mouse glial cells by other related coronaviruses (suzumura etal., ) . in such a manner, viruses may be able to evade immune-mediated events that occur at the level of the bbb. the virus is able to avoid host immune surveillance and to enter the cns. such events probably also prevent late immune-mediated demyelinating disease, which is absent in mhv- virus infection (see later discussion). immunodeficiency states, such as aids or iatrogenic immunosuppres sion in transplant recipients, may sometimes modify the inflammatory response of the cns to pathogens. in aids, especially at the terminal stage of disease, the picture of cns inflammation, i.e., in toxoplasma gondii encephalitis or in progressive multifocal leukoencephalopathy caused by jc virus, can differ from that seen in nonimmuno-suppressed individuals. the inflammation seems muted, and the destructive component of the process (fulminant necrosis) may dominate. this may serve as another example that the inflammatory response within the cns is partly depen dent on the peripheral immune system. as discussed earlier, immune surveillance in response to viral infections can lead to virus-induced immune response to normal host components. during acute infections, this response can lead to increased tissue damage. during chronic or persistent infections, such responses can perpetuate or be the primary cause of the disease process. involvement can take the form of humoral response (autoantibodies) as well as cell-mediated autoimmune (cmai) response (ter meulen, ) . such autoimmunity may involve a phenomenon known as molecular mimicry (srinvasappa et al, ) , an immune process in which molecules coded for by dissimilar genes share similar structures. in such a manner, antiviral antibodies may bind to host antigens as well as to antigens of the virus. many examples of such mimicry have now been identified: measles virus phosphoprotein and keratin ; vaccinia virus hemagglutin and vimentin (dales et al., ) ; fusion protein of measles and heat shock protein (shesheberadarin and norby, ) ; hiv gpl and neuroleukin (mizarachi, ) . in an analysis of monoclonal antibodies to different viruses, % of such anti bodies cross-reacted with host cell determinants (srinvasappa et al., ) . generation of cytotoxic cross-reactive effector lymphocytes or antibodies would recognize "self proteins" located at target cells. the virus need not be present for such events to take place. myelin basic protein exhibits a significant degree of homology with several viral proteins, including hepati tis Β virus polymerase. inoculation of hepatitis Β virus polymerase peptide into rabbits caused perivascular infiltration localized to the cns, reminis cent of eae (fujinami and oldstone, ) . several other mechanisms are potentially operational in virus-induced autoimmune disease. viruses may disrupt normal immune regulation by direct interaction with cells of the reticuloendothelial system (res). as discussed earlier, many viruses can replicate in these cell types, leading to destruction of lymphocyte subpopulations or stimulation of autoreactive clones. viruses may incorporate host molecules into their envelope or coat, or they may insert, modify, or expose other cellular components on the cell surface. immune pathological events may also be triggered by induction of class ii mhc antigens on the surface of infected cns cells. mhc antigens are expressed only at low levels or not at all on the majority of cns cells (hart and fabre, ). astrocytes exposed in vitro to measles or corona virus jhm begin to express mhc class ii antigens, which are further enhanced in the presence of tnf (massa et al, ). an analogous situation in vivo would certainly facilitate ctl-mediated autoimmune response. postinfectious encephalomyelitis has been associated with a wide range of virus infections including measles, mumps, rubella, vaccinia, and herpes zoster and certain respiratory viral infections (johnson and griffin, ) . neuropathological changes resemble those seen in eae and appear to be a direct result of an mbp-directed immune response (ter meulen, ) . coronavirus jhm causes acute demyelinating encephalitis by selective in fection of oligodendrocytes. lymphocytes from sick animals that are pas sively transferred to syngeneic animals produce lesions in the recipients that resemble those in individuals with allergic encephalomyelitis (ter meulen, ) . viral infection of oligodendrocytes appears capable of inducing an autoimmune response against the myelin proteins produced by these cells. in measles-induced encephalomyelitis, there is little evidence that virus invades the cns. deregulation of autoreactive cells may occur secondarily to viral infection of lymphoid cells. demyelinating disease caused by theiler's murine encephalomyelitis virus (tmev), a nonbudding enterovirus, may result from a virus-specific, delayed-type hypersensitivity (dth) (clatch et al., ) . lymphokines produced by mhc class ii-restricted, tmev-specific, dth Τ cells primed by interaction with tmev-infected macrophages would recruit and activate additional macrophages, leading to nonspecific macrophage-induced damage. in contrast to the autoimmune mechanisms described above, persistent viral infections can be a consequence of the ability of the virus to avoid immune surveillance. in the face of excessive viral antigen, both virusspecific antibodies and ctls are suppressed or rendered ineffective. vi ruses may be able to down regulate appropriate recognition molecules on the surface of immune cells. expression of virus surface proteins involved in immune recognition may also be reduced. reduced expression of glyco protein has been observed during persistent infections with arenaviruses, paramyxoviruses, retroviruses, and rhabdoviruses (oldstone, ) . antibody-induced antigenic modulation can lead to measles virus persistent infection in the cns (fujinami and oldstone, ) . presumably, this mod-ulation can occur in body compartments that are devoid of the effector molecules of complement such as the cns. subacute sclerosing panenceph alitis (sspe) results from a persistent cns infection with defective measles virus (wechsler and meissner, ) . a defect in viral envelope genes prevents expression of viral antigen on cell surfaces. infection is propagated by cell-to-cell transmission of virus, allowing the agent to avoid immune surveillance. viruses can also replicate in the cellular constituents of the immune system and in such a way disable specific immune responsiveness. viruses thus persist within cells that are ordinarily utilized to provide clearance. viruses such as measles and hiv can infect lymphocytes and/ or macrophages, resulting in generalized immunosuppression. such immu nosuppression leaves the host susceptible to infection by a wide range of other microorganisms, leading to the opportunistic infections often associ ated with hiv and other such viruses. viruses can also avoid immune surveillance through latency, a state of reversible, nonproductive infection. viral genetic information can remain within the host in either an integrated or an episomal state. retroviruses such as hiv use reverse transcriptase to insert their dna proviral form into host-cell dna; this integration step is a prerequisite for the replication of these rna viruses (mahry, ) . herpes simplex virus (hsv), which resides in sensory ganglion cells, integrates its dna directly into host nuclear dna. the dna of other dna viruses such as ebv or jc agent, the polyomavirus that causes progressive multifocal leukoencephalopathy, remains latent in a nonintegrated episomal form. latent infections estab lished by these viruses may result from a lack of host factors that are critical for the expression of viral early gene products (garcia-blanco and cullen, ) . the subsequent activation of specific cellular transcription factors in response to extracellular stimuli can induce the expression of virus and lead to cns disease. herpesviruses such as hsv- , hsv- , or vzv cause initial acute peripheral infection followed by a latent infection of neurons. in this environment, sheltered from immune surveillance, the virus can remain for the life of the host. hsv- can be activated by a number of seemingly unrelated stimuli such as physical or emotional stress or damage to adjacent tissue. reactivation probably results from a signal transduction event that directly or indirectly induces hsv- gene transcription (leib et ah, ) . latently ebv-infected Β cells can also persist for the life of the individual. if immediate early viral gene expression does not occur, ebv may establish a latent infection (miller, ) . both activated Τ lymphocytes and nondividing macrophages are able to support the synthesis and integra tion of hiv- proviruses (fauci, ) . resting Τ cells are nonpermissive and do not support replication or integration. resultant unintegrated viral intermediates may persist in a viable yet transcriptionally inert form for extended periods of time. in quiescent macrophages or in Τ cells in a resting state, cellular factors critical for proviral transcription may not be active. postintegration latency in hiv- may result from inefficient proviral tran scription and from a suboptimal amount of rev, a viral-coded transactivation regulatory protein (pomerantz et al, ) . stimulation of these cells in some manner could induce cellular transcription factors increasing rev which leads to productive virus replication. the rare demyelinating disor der, progressive multifocal leukoencephalopathy (pml) (see earlier discus sion), is caused by the reactivation of latent polyomavirus ( j c v ) in the oligodendroglia cells of affected individuals (mazlo and tariska, ) . this disease is usually associated with an underlying disorder of the res system in which immune responsiveness is impaired. loss of immune surveillance allows reexpression of virus and cytocidal effects on oligodendrocytes, lead ing to demyelination. the final type of interaction of virus within the cns is a chronic persistent infection that does not elicit an immune response. wild mouse ecotropic immune leukemia virus (mulv) induces a progressive form of hind limb paralysis in a natural population of mice as well as in laboratory animals (gardner et al., ) . pathologically, the disease is characterized by the absence of host inflammatory response at the site of tissue destruction and by a picture of neuronal degeneration, spongiform gray matter lesions, and gliosis. paralysis is a direct result of viral replication, most notably in anterior horn motor neurons, but virus can also infect endothelial and glial cells. there is no virus-mediated ctl or antibody response. the cns is devoid of inflammatory infiltrates or deposits of igg or complement. in genetically susceptible laboratory strains of mice, ability to induce disease is age depen dent and involves tolerance to the virus. passive immunization with anti bodies to mulv virus can prevent paralysis. neuronal destruction and spongiosis may be a direct result of the interaction of viral gene expression in the neuron. recent evidence suggests that a posttranscriptional step in virus envelope protein synthesis is impaired and that neurological disease may be a consequence of abortive replication of virus within neurons (sharpe et al, ) . such abortive infections can make the presence of virus difficult to detect. analogies to other retrovirus cns infections, notably hiv and htlv- , suggest that similar mechanisms may be operational in cns disease caused by these agents. another group of infectious agents that establish cns disease in the absence of inflammatory response are the agents of the transmissible spon giform encephalopathies (tses) or prion diseases (carp et al, ) . unlike the retroviral disease described above, in which immune response to virus is age dependent and the disease process may include tolerance, there is no evidence for the ability of the host to generate any immune response to the agents of tse. these agents cause creutzfeldt-jakob disease (cjd), gerstmann-straussler-scheinker syndrome (gss), and kuru in humans and are associated with scrapie, bovine spongiform encephalopathy (bse), chronic wasting disease, and transmissible mink encephalopathy (tme) in animals. the agents that cause these diseases are poorly characterized, but it is certain that they do not exhibit properties of known viruses or virus like agents. these agents replicate peripherally in the res system, notably in spleen and lymph nodes. agent crosses into the cns, leading to spon giosis, gliosis, and neuronal destruction in the absence of any inflammatory response, similar to that described above for the retroviruses. modulation of the immune system only affects the agent given peripherally. immuno logical compounds, given close to the time of peripheral infection, that stimulate the immune response shorten incubation periods, whereas com pounds that suppress the immune response extend the incubation period. treatment with dextran sulfate (farkuhar and dickinson, ) extends disease and may mediate its effect through interaction with macrophages. recent studies suggest the importance of follicular dendritic cells (kitamoto et al., ) in agent peripheral replication. similar dendritic cells are in volved in antigen presentation and virus replication in infections caused by other viruses. similar to evidence described for the neurotropic retroviruses, pathology within the cns may include abnormal protein processing, in this case, the production of an abnormal host-coded protein termed prp (prusiner, ) . both agent replication and spongiform change are associ ated with the presence of this protein within the cns. in conclusion, the inflammatory response within the cns appears to have several aspects. first, the cns itself is unique from other organs in the presence of bbb with specialized endothelial cells, the presence of cnsspecific cells (e.g., microglia or astrocytes), and the presence of cns-specific antigens. second, infectious agents such as viruses comprise a wide spec trum of agents with varied neural tissue virulence and varied specificity of agents to certain cns cell types. some of these agents may produce fulmi nant disease with severe, if not lethal, cns damage, whereas others may persist for decades, resulting in chronic or latent effects. the interactions among the cells and environment of the cns, infectious agents, and the immune response are quite 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and secretion by astrocytes mhc restricted cytotoxic Τ cells: studies on the biological role of polymorphic major transplantation antigens determining t-cell restriction specificity, function and responsiveness key: cord- -eq obbte authors: kaur, manpreet; rai, anant; bhatnagar, rakesh title: rabies dna vaccine: no impact of mhc class i and class ii targeting sequences on immune response and protection against lethal challenge date: - - journal: vaccine doi: . /j.vaccine. . . sha: doc_id: cord_uid: eq obbte rabies is progressive fatal encephalitis. who estimates , rabies deaths and more than million pep every year world-wide. a variety of cell-culture derived vaccines are available for prophylaxis against rabies. however, their high cost restricts their usage in developing countries, where such cases are most often encountered. this is driving the quest for newer vaccine formulations; dna vaccines being most promising amongst them. here, we explored strategies of antigen trafficking to various cellular compartments aiming at improving both humoral and cellular immunity. these strategies include use of signal sequences namely tissue plasminogen activator (tpa), ubiquitin (uq) and lysosomal-associated membrane protein- (lamp- ). tpa, lamp- and their combination were aimed at enhancing the cd (+) t cell and antibody response. in contrast, the uq tag was utilized for enhancing cd (+) response. the potency of modified dna vaccines assessed by total antibody response, antibody isotypes, cytokine profile, neutralizing antibody titer and protection conferred against in vivo challenge; was enhanced in comparison to native unmodified vaccine, but the response elicited did not pertain to the type of target sequence and the directed arm of immunity. interestingly, the dna vaccines that had been designed to generate different type of immune responses yielded in effect similar response. in conclusion, our data indicate that the directing target sequence is not the exclusive deciding factor for type and extent of immune response elicited and emphasizes on the antigen dependence of immune enhancement strategies. rabies, progressive fatal encephalitis [ ] is caused by rabies virus of genus lyssavirus. majority of rabies cases reported from developed countries involve wild animals like raccoons, skunks, bats, and foxes. however, it is a major health concern in developing countries which account for more than % of all human deaths from rabies [ ] . exposure to rabid dogs is the cause of bulk (> %) of human rabies deaths world-wide [ ] . india has a particularly severe problem, with as many as , human deaths and million people requiring post-exposure vaccination yearly. stray and community dogs cause vast majority of human cases. though potent and safe cell-culture derived inactivated vaccines are available, their efficacy may be compromised by disruption of cold chain storage, poor general health status of the subject, poor vaccination techniques. as a consequence, several approaches are currently being investigated experimentally; out of which dna vaccines appear to be particularly promising as they can induce persistent, cell-mediated and humoral immune responses to antigens isolated from a variety of viral, bacterial, and parasitic pathogens. besides their immunogenicity, dna vaccines offer several other practical advantages. dna vaccines being free from foreign proteins may not cause the various side reactions, which may be observed for conventional vaccines. in addition to the safety, they may have benefits of being inexpensive, overcome the need of time consuming procedures that are needed for purification of subunit proteins, are stable, can be stored and transported at room temperature. in animal models of human disease, dna vaccines have been shown to induce protective responses against hiv, influenza, bovine herpes virus, rabies, leishmaniasis, malaria, herpes simplex virus, and tuberculosis [ ] [ ] [ ] . in addition, human clinical trials have established their safety and potency, further encouraging studies in this direction [ ] [ ] [ ] [ ] [ ] . in this regard, various dna vaccination strategies have shown to provide protection against lethal rabies virus challenge. these strategies relied on the usage of adjuvants like cationic-lipids [ , ] , intradermal injection using gene gun [ ] [ ] [ ] , repeated dna vaccination [ , ] or dna vaccine in association with a single dose of anti-rabies immune serum [ ] for immune enhancement. prophylactic immunization was found to be effective in preventing canine rabies [ , , ] . rabies dna vaccine has also been found to be highly efficient in large size mammals [ ] . for post-exposure treatment, single dose of rabies dna vaccine was found to be as potent as dose regimen of cell-culture vaccine in balb/c mice [ ] . considering these studies, we attempted further improvement in humoral and cell-mediated immune response elicited by dna vaccination by antigen trafficking to various cellular compartments. efficient delivery of antigens to both mhc class i and class ii processing and presentation pathways is required for generating an ideal immune response comprising of both cd + and cd + (cellmediated) and antibody (humoral) immune response. accordingly, this study investigates strategies for targeting glycoprotein antigen to mhc class i and class ii pathways for improving its antigenicity, immunogenicity and protective efficacy. for targeting mhc class ii pathway, we utilized tissue plasminogen activator (tpa) and human lysosomal-associated membrane protein- (lamp- ) signal sequences. tpa-fused antigens are highly expressed secreted proteins with elevated uptake by antigenpresenting cells; and thus, bring about a more generalized activation of the immune system. they have been shown to induce significant humoral and cell-mediated responses [ ] . lamp- is a type of transmembrane protein localized predominantly to lysosomes and late endosomes. antigen trafficking of lamp- -fused antigens to the cellular site of mhc class ii processing and presentation pathway could enhance its presentation to mhc class ii restricted cd + t cells [ ] and thus augment the humoral response. on the other hand, for directing the antigen to mhc class i, signal sequence of ubiquitin a- (uq) was employed. uq-conjugated antigens are trafficked through the proteasome, an organelle that generates short peptides for presentation via the mhc class i pathway [ ] . such uq-conjugated antigens are expected to enhance the cellular immune response. uq-conjugated proteins have been shown to generally undergo rapid intracellular degradation and can elicit cytokine responses in the absence of specific antibody production [ ] . thereby, we explored the potential of targeted dna vaccine encoding the glycoprotein antigen fused to tpa, lamp- or uq to elicit superior immune response in comparison to the unmodified (without target sequence) dna vaccine. baby hamster kidney (bhk)- cells; procured from national centre for cell science (nccs), pune, india were maintained in dulbecco's modified eagle's medium (dmem, sigma) supplemented with % of heat-inactivated fetal bovine serum (fbs, biological industries) and u/ml penicillin (amersham) and g/ml streptomycin (amersham), in a humidified % co incubator at • c. virus pitman-moore (pv- ) strain of rabies virus was propagated on bhk- cells. virus was purified, inactivated with beta-propionolactone (bpl) and used for in vitro re-stimulation assay. the challenge virus standard (cvs- ) strain was propagated and maintained in mice brain. it was titrated on bhk- cells to determine the optimal dose for rapid fluorescence focus inhibition test (rffit) to determine virus neutralization antibodies and for intracerebral rabies virus challenge to determine protection conferred. plasmid ptarget-rab-g [ ] was used as the parental plasmid for construction of all the clones used in this study. the glycoprotein ( . kb) gene was amplified by pcr from ptarget.rabgp plasmid using sequence-specific primers (supplementary table ) and cloned in eukaryotic plasmids bearing address tags; pdnavacc vectors (nature technology corporation, nebraska). the sequences of clones bearing the address tags, pgp-native, ptpa.gp.lamp- (genbank accession number eu ), ptpa.gp (genbank accession number eu ), puq.gp (genbank accession number eu ), pgp.lamp- (genbank accession number eu ) were confirmed by sequencing using abi prism, model , version . (sequencing primers listed in supplementary table ) and designated as represented in the fig. . the respective constructs were processed for the purification of plasmid dna using the endofree plasmid isolation maxi kit (qiagen) according to the manufacturer's instructions. the purified plasmid dna ( - mg/ml) was dissolved in autoclaved milli quartz (mq) water and stored at − • c, until further use. the glycoprotein gene was similarly pcr amplified (see supplementary table for primer sequences) using ptarget.rabgp as template and cloned in pqe expression vector (t expression system). rgp was expressed as a fusion protein with × histidine tag in e. coli sg (prep- ) strain and was purified on a ni + -nta column to more than % homogeneity under native conditions. the rgp was dialyzed against mm hepes overnight and stored in aliquots at − • c. the ability of vaccine constructs to express glycoprotein antigen was studied in vitro in a mammalian cell-culture system. briefly, bhk- cells were cultured and seeded at a density of × cells/ml in a -well tissue culture plate, a day prior to transfection. bhk- cells were subsequently transfected with ng dna complexed with l of lipofectamine (invitrogen) and l of plus buffer (invitrogen). the analysis of expression and localization was carried out h post-transfection. for assessing expression, flow cytometric analysis was carried out, in which bhk- cells were transfected with various dna vaccine combinations. transfected cells were fixed with % paraformaldehyde (pfa) and then permeabilized in . % triton x- in pbs. the cells were then probed with mouse anti-rabies polyclonal sera, diluted in pbs containing . % bsa; followed by staining with alexa fluor labeled secondary antibody (molecular probes) diluted in pbs containing . % bsa and analyzed for fluorescence using cell lab quanta tm sc mpl flow cytometer (beckman coulter inc.). control healthy bhk- cells were also stained. ten thousand cells per sample were analyzed using fl filter ( nm) and percent green fluorescent cells were recorded using quanta sc mpl analysis software version . (beckman coulter inc.). for immunoblotting, total cell lysate was prepared. transfected cells were solubilized using radioimmunoprecipitation assay (ripa) buffer [ mm tris(hydroxymethyl)aminomethane (tris), ph . , mm sodium chloride (nacl), . % sodium dodecyl sulphate (sds), % triton x- , % sodium deoxycholate, mm phenyl methyl sulphonyl fluoride (pmsf)] supplemented with protease inhibitor cocktail (sigma). lysosomal fraction was extracted using lysosome enrichment kit for tissue and cultured cells (pierce) according to the manufacturer's protocol. the presence of lysosomes in different fractions was determined by analyzing the activity of ␤-hexosaminidase [ ] . cell membrane protein fraction was prepared by qproteome membrane protein kit (qiagen) according to the manufacturer's protocol. the presence of cell membrane in the fractions was determined by the associated nadh oxidase activity [ ] . culture supernatant proteins were precipitated by ice-cold acetone. solubilized proteins from the total cell lysate, lysosomes, membranes and cell-culture supernatants were subjected to % sds-page, blotted on to nitrocellulose membrane and probed with mouse anti-rabies polyclonal sera, followed by incubation with goat anti-mouse immunoglobulins conjugated with alkaline phosphatase (sigma) and visualized with nbt/bcip substrate (sigma). all animal experiments were conducted in compliance with the animal ethics committee. four to six weeks old female balb/c mice were used to verify the immunogenicity of the constructs. mice were purchased from nin, hyderabad; and maintained in pathogen free environment at the animal house facility. each group comprised of ten mice. mice were vaccinated intramuscularly (i.m.) with g endotoxin-free plasmid dna in l pbs/animal in the individual groups (dna vaccine or vector control), thrice at three-week intervals. control mice were immunized with only pbs. the mice from each group were bled at days, , and ; sera were prepared and stored at − • c. antigen-specific antibody (igg total) and isotypes (igg , igg a) levels were determined by elisa in the serum from immunized mice. recombinant glycoprotein, expressed in bacterial system ( ng/well) in l of . m pbs was coated overnight at • c [ ] . plates were then blocked with % bsa in pbs for h at • c followed by three washings with pbs-tween ( . %). this was followed by incubation with sera samples for h at • c and washing with pbst. secondary antibodies, anti-mouse igg or its isotypes conjugated with horseradish peroxidase; raised in sheep (santa-cruz) were incubated for h at • c. estimation of the enzymatic activity was carried out using tmb as the substrate. the reaction was stopped with l of m h po and the absorbance was measured at nm, with nm as the reference filter using microplate reader (bio rad). the antibody response generated in a group of vaccinated mice was represented as the geometric mean of the absorbance obtained by pooled serum samples of the animals; the reaction being carried out in triplicates. mouse sera were tested in vitro for the presence of virusneutralizing antibodies with rffit, as described previously [ ] . briefly, sera from mice were heat inactivated at • c for min. l of various sera dilutions were mixed with l of the cvs- strain of rabies virus (containing ffd ) in -well tissue culture plate and incubated at • c, % co for min. after the incubation period, bhk- cells ( × ) were added to each well and the plates were incubated for h, following which they were fixed with chilled acetone and stained with fitc-conjugated antirabies monoclonal antibody (vmrd, usa) for min. the wells were washed thrice with pbs, mounted in glycerol: pbs ( : ), and visualized under fluorescence microscope (nikon, japan). data were expressed as the neutralizing antibody titer that is the mean of the serum resulting in a % reduction in the number of the virus-infected cell foci in the presence of the test serum. rabies reference antiserum of known international units (iu/ml) of rabies virus-neutralizing antibody was included as positive control in the assay. splenic cells were prepared by grinding spleens between frosted slides. erythrocytes were lysed with . m ammonium chloride. remaining spleen cells were washed twice with dmem medium and then were suspended in complete dmem medium supplemented with % heat-inactivated fetal bovine serum and − m -mercaptoethanol. viability was determined by trypan blue exclusion test. splenocytes were cultured in triplicate ( × cells/well) in a -well culture plate (costar), stimulated without antigen or with g/ml of bpl inactivated pv- virus or concanavalin a (cona) ( g/ml; sigma), and incubated at • c under % co and % humidity. supernatants were harvested after , and h and the levels of cytokines were determined. levels of il- , il- , il- , and ifn-␥ were determined using bd opt eia tm kits according to manufacturer's protocol (pharmingen). briefly, -well microtiter elisa plate was coated with capture antibody of the respective cytokines and incubated overnight at • c. plate was aspirated and washed thrice and blocked with l subsequently, the protein samples were resolved on % sds-page under reducing conditions and transferred onto nitrocellulose membrane. presence of rabies glycoprotein was detected using mouse polyclonal anti-rabies hyperimmune sera followed by alkaline phosphatase-conjugated anti-mouse igg. the blot was developed using bcip/nbt as substrate. of % bsa for h at • c. after the incubation period, plate was aspirated and washed thrice and incubated with the harvested supernatants for h at rt. the plate was then aspirated and washed five times; plate was incubated with detector (anti-mouse igg-hrp) for h at rt. following this, plate was aspirated and washed times and incubated with l substrate solution for min in dark at rt. reaction was stopped by adding l stop solution to each well. the absorbance was read at nm using a microplate reader (bio rad) within min of stopping the reaction. the concentrations of cytokines in the culture supernatants were calculated using a linear regression equation obtained from the absorbance values of the standards provided by the manufacturer. each vaccine construct was tested in two independent experiments. for challenge, immunized mice were inoculated with ld of rabies virus cvs- strain intracerebrally days postimmunization. the challenged mice were observed for days for symptoms indicative of rabies virus infection. mice that developed complete bilateral hind leg paralysis, characteristic of the terminal stage of rabies, were euthanized for humanitarian reasons. upon challenge, pbs or vector vaccinated mice died within - days. surviving mice were kept and observed for an additional two to three weeks to ensure that they survived the infection. survivorship rates obtained with the different vaccine constructs were compared. the experimental data were analyzed by sigma plot . and were expressed as means ± standard deviations (s.d.). comparisons between individual data points were made using a student's t-test and levels of significance (p value) were determined. p value < . was considered statistically significant. rv-g based dna vaccine constructs were made wherein the glycoprotein gene was fused to various signal sequences (fig. ) to analyze the influence of signal sequences on immunogenicity and generation of rvna titers. the sequences of insert in native dna vaccine construct (pgp) and modified constructs bearing the target sequences, pt.gp.l, pt.gp, pu.gp, pgp.l were confirmed by sequencing. for assessing the expression of dna vaccine constructs, transiently transfected bhk- cells were subjected to flow cytometric analysis. the percentages of cells stained with the antibody are shown in the figure (fig. a) . the transfected cells were expressing the rabies glycoprotein as indicated by fluorescence recorded as . %, . %, . %, . %, . % for pgp, pt.gp.l, pt.gp, pu.gp and pgp.l respectively; whereas the control cells revealed a low fluorescence signal ( . %) (fig. a) . as the majority of cells showed expression of the rabies glycoprotein, it can be inferred that all the constructs were capable of expressing the protein efficiently in transfected cells. for assessing the localization of dna vaccine constructs, transiently transfected bhk- cells were subjected to subcellular fractionation and subsequently visualization by western blotting. for the same, total cell lysate, lysosomal fraction, membrane fraction and cell-culture supernatant of tranfected cells were resolved on sds-page followed by probing with hyperimmune polyclonal serum from mice immunized with rabies virus. prominent immunoreactive protein bands were observed on the blot corresponding to cell lysate of the all the dna vaccine constructs (fig. b, topmost panel) . there was no corresponding band in cell lysate from vector-transfected or mock-transfected bhk- cells (data not shown). the observed molecular weight of approximately kda was consistent with the expected sizes of glycosylated glycoprotein. analysis of subcellular fraction revealed that the pgp.l construct encoded lysosomal form of glycoprotein (second panel). the pt.gp construct encoded secreted form of glycoprotein (third panel). further, dual tagged construct pt.gp.l expressed both secreted and lysosomal form of glycoprotein (second and third panels). pu.gp construct was exclusively expressed in cell associated form (topmost panel). the native construct encoded membrane associated glycoprotein (fourth panel). expression of glycoprotein in various subcellular fractions was comparable to that from cell lysate. to address the issue if these vaccines could induce efficient humoral response was assessed. groups of balb/c mice were vaccinated intramuscularly with dna encoding either the unmodified or modified antigen. anti-glycoprotein antibody response was estimated by elisa with recombinant glycoprotein (expressed in bacterial system, unpublished data). all the mice sero-converted after priming, however, maximum titers were obtained after second booster both for unmodified as well as modified dna vaccine. for clarity, only the means and standard deviations for each group are shown (fig. ) . all vaccine groups mounted antibody response higher than the unmodified vaccine. the highest antibody response was generated in the pgp.l immunized group (p value < . ), closely followed by pt.gp.l. there was insignificant antibody response in vector and pbs immunized mice. trafficking of glycoprotein through different pathways may affect the type of immune response elicited against it. like pt.gp mediated trafficking of glycoprotein from cytoplasm to secretion pathway, which targets molecules through the endoplasmic reticulum and golgi; may lead to higher induction of th type of immune response. likewise, pgp.l mediated trafficking may drive the glycoprotein through trans-golgi network directly to . the isotype profile of the rv-g-specific igg (black bars) and igg a (gray bars) titers in mice immunized by different protocols. each group of mice (n = ) was immunized respectively by dna, vector or pbs. mice were bled at three weeks after the last immunization and glycoprotein-specific igg and igg a titers were detected by elisa. optical density was measured at nm. data shown represent geometric mean titers and standard deviations for each group of animals. endosomes and then to lysosomes, again influencing the th type response. pt.gp.l may channelize the glycoprotein through either of the above pathways, to affect the th type of immune response. on the contrary, pu.gp is expected to enhance the proteolysis of conjugated glycoprotein mediated by the ubiquitin-proteasome pathway for enhancing the processing and presentation for th type of immune response. to examine such a possibility, serum from mice immunized with pgp, pt.gp, pt.gp.l, pu.gp and pgp.l was assayed by probing with isotype specific secondary antibodies. immunization with all the constructs led to an igg -dominated response (fig. ) indicating the th bias. thus, our results show that addition of signal sequence did not affect the isotype inclination following the immunization and there was convergence of the antibody response, in spite of differential targeting. further, we explored the possibility of enhancement in neutralizing antibodies against glycoprotein when modified antigens were employed for the immunization experiments. rabies virusneutralizing antibody (rvna) titers were assessed by rffit; three weeks post the last immunization corresponding to the time of lethal challenge. the rvna titer in all the groups of immunized mice was > . iu/ml; the minimum titer against rabies as recommended by who. as shown in fig. , the highest geometric mean rvna titer was observed for pgp.l ( iu/ml, p value < . ), followed by pt.gp.l and pu.gp with titer of iu/ml. the neutralizing antibody potential of tpa tagged vaccine was found be the lowest, equivalent to the unmodified antigen based dna vaccine ( iu/ml). in comparison, vector or pbs immunized group did not induce significant neutralizing antibodies. t helper cells (th /th ) play an important role in eliciting both humoral and cellular responses via expansion of antigenstimulated b cells and cd + t cells or ctls respectively. the levels of some cytokines which may play key roles in the induction of protective immune responses against rabies virus were studied as parameters of polarization of immune response. th cytokines (il- , il- , and ifn-␥) and th cytokine (il- ) were measured from splenocytes from immunized mice by elisa at , and h after re-stimulation with inactivated pv- virus. il- production substantially increased on immunization with pgp.lamp- ; . pg/ml i.e., ∼ fold higher as compared to the response from control (splenocytes from pbs immunized mice) was observed (p value < . ). all the constructs exhibited significant il- and ifn-␥ production. there was no significant increase in the cytokine levels of mice immunized with vector or pbs. il- production also strongly increased in case of pgp.lamp- , ∼ fold superior than the control group (p value < . ). the cytokine profile is summarized in fig. . the ability of these dna vaccines to elicit protective responses in immunized mice was assessed by intracerebral challenge with ld of virulent rabies virus cvs strain. for controls, vector and pbs immunized mice were also challenged. the lethality of the challenge was confirmed by death of all the mice in the vector and pbs immunized group within - days post-challenge. groups of vaccinated mice that developed significant levels of virus-neutralizing antibodies also survived rabies virus challenge. the protection conferred by dna vaccines was found to be significant (p value < . ). all modified antigen groups apart from pt.gp conferred higher protection than the unmodified dna vaccine, with % and % protection levels conferred respectively. surviving mice did not show any signs of rabies virus infection. kaplan-meier curves for survival of dna vaccine immunized mice are summarized in fig. . a variety of cell-culture derived vaccines are available for prophylaxis against rabies [ ] . however, the high cost of the vaccination therapy along with the risk of developing anaphylactic, neuroparalytic or encephalitic side reactions limit their therapeutic application. these facts indicate the need of more faithful candidate vaccines which must be capable of inducing strong immune response to protect from infection. more importantly, the candidate immunogen must be able to induce a strong th immunity as it has been established that th ; that is, the humoral immune response plays a predominant role in induction of protective immunity against rabies virus [ ] [ ] [ ] . rabies virus glycoprotein is the main antigen responsible for inducing the production of rabies virus-neutralizing antibodies and for conferring immunity against lethal rabies infection. out of the various strategies being employed for enhancing the immunoprophylactic potential of vaccination strategy, dna vaccines have been the most promising. in an effort to develop an optimal dna vaccine against rabies virus, this study was aimed at evaluating the immune enhancement potential of different antigen targeting strategies to selectively improve responses mediated by cd + and cd + t lymphocytes and by antibodies, induced after intramuscular immunization with dna plasmids. addition of target sequences like tpa, lamp- , uq have been employed for vaccination against various pathogens including sars coronavirus [ ] ; dengue virus [ ] ; orthopox virus [ ]; influenza a [ ] ; mycobacterium [ ] . the signal sequences would target the heterologous protein to different sites targeting the model; for (i) high expression and secretion by fusing with tpa and a more generalized activation of the immune system for induction of significant humoral and cell-mediated responses [ ] (ii) lysosomal degradation by fusing with lamp- and class ii presentation [ , ] ; (iii) wider and enhanced immune response by fusion with tpa and lamp- and (iv) cytoplasmic degradation by the proteasome by fusing with ubiquitin and class i presentation [ ] . classically, the transmembrane (tm) region is excised from the dna vaccine immunogen, such that it can be secreted into extracellular milieu. targeted dna vaccines based on immunogens with deleted tm have been successfully employed for vaccination against tumours [ ] . nevertheless, wang et al. found that hemagglutinin (ha) proteins from different serotypes of influenza a virus elicits contrasting response to full length and truncated transmembrane forms [ ] . further, rath et al. reported that tm domain along with a secretion signal of rv glycoprotein was required for eliciting highest level of neutralizing antibodies. they inferred that tm domain is critical for proper folding of protein otherwise the critical epitopes may get disrupted [ ] . gupta et al. also reported that dna vaccine encoding rabies virus glycoprotein lacking transmembrane domain though enhances antibody response but does not confer protection [ ] . therefore, we retained the tm domain in our dna vaccine constructs and utilized full gene for targeting strategies. thus, different plasmid dna constructs were made-pgp, the unmodified constructs and modified constructs including p.gp.l (n terminal tpa and c terminal lamp- ), pt.gp (n terminal tpa), pu.gp (n terminal uq), and pgp.l (c terminal lamp- ). transient transfection of bhk- cells with all the plasmid dna constructs revealed expression of rabies glycoprotein by flow cytometric analysis. majority of the cells were found to express glycoprotein as seen by the fluorescence monitored by cell sorter. thus, dna vaccine constructs were capable of efficiently expressing the glycoprotein. distribution of chimeras was analyzed by subcellular fractionation and immunoblotting. total cell lysate of transfected bhk- cells of all the constructs expressed glycoprotein at approximately kda. the observed high molecular weight of rv-g expressed in bhk- cells could be due to the influence of host factors on glycosylation [ ] . morimoto et al. showed both bhk and murine neuroblastoma (mna) cell lines, transformed with the same retroviral expression vector encoding rv-g cdna, show different patterns of glycosylation of the expressed rv-g [ ] . rrv-g expressed by bhk cells was highly glycosylated and sialylated in comparison to mna expressed rrv-g, indicating that the glycosylation and sialylation of rv g is dependent on the cellular conditions in which rv-g is produced. analysis of subcellular fractions indicated that glycoprotein along with the targeting sequences was suitably recognized by mammalian cells and directed towards the respective pathway. from flow cytometric and immunoblotting analysis of transfected cells, it can be inferred that there was efficient recognition and expression of dna vaccine immunogens in the mammalian system. the signal sequences successfully directed the glycoprotein to respective cellular locations, with comparable levels of expression as of total cell lysate. vaccination of mice with all rv-g plasmid dna constructs led to the generation of anti-rv-g antibodies. all the modified vaccines elicited higher anti-rv-g antibody levels than the unmodified one. the highest antibody response was observed with pgp.lamp- . the generation of rvna is the most important adaptive immune system response for conferring protection against rabies. therefore, to compare the utility of the rv-g plasmid dna constructs, rvna response elicited by each construct was determined by rffit. the neutralizing antibodies were more than . iu/ml, which is the minimum titer recommended by who. the highest rvna titer was elicited by pgp.lamp- which is also supported by an enhanced antibody response by elisa, in comparison to other rv-g constructs. the effectiveness of the constructs to induce th /th type of immune response was indirectly evaluated by determining th (igg a) and th (igg ) antibody isotypes. we found a strong igg response in all the dna constructs. even though igg a antibodies were produced, the ratios of igg /igg a were consistently more than one, thus emphasizing on the th bias. presence of both types of immune responses may be due to the presence of more than one type of antigenic sites in the glycoprotein immunogen. it is worth noting that differential targeting for enhancing th and th responses yielded in effect a similar response. the increase in antibodies to dna vaccine may reflect an effect of the antigen on the t helper cell response needed to promote differentiation of naïve b cells into antibody secreting plasma cells. this was assessed by cytokine profiling of splenocytes immunized with signal sequence tagged glycoprotein based vaccine or only vector; upon in vitro stimulation with inactivated pv- virus. we found that all the cytokines analyzed could be detected from the splenocytes of dna vaccine immunized mice, with a pronounced enhancement in the level of il- and il- in the pgp.lamp- immunized group. for other cytokines, namely il- and ifn-␥, similar levels of cytokines were observed for all the four groups, with the level being several folds in comparison to the splenocytes from control group. antigen binding to the t cell receptor (tcr) stimulates the secretion of il- , and the expression of il- receptors il- r. the il- /il- r interaction then stimulates the growth, differentiation and survival of antigen-selected cytotoxic t cells via the activation of the expression of specific genes. il- facilitates production of immunoglobulins made by b cells and induces the differentiation and proliferation of natural killer cells. il- , produced mainly by macrophages and dendritic cells, is quickly induced by viral infections or by vaccination stimuli. il- strengthens the non-specific immune responses by activating nk cells to produce ifn-␥ and in synergy with ifn-␥, drives the differentiation of cd + t cells into th cells, more adapted to the control of viral infections. various groups of immunized mice when challenged with cvs virus showed higher protection as compared to a vehicle control. high titers of rvna and protection conferred in dna vaccines might be due to the possibility that modified immunogens led to the expression of rv-g with appropriate folding and better accessibility of epitopes to immune system, critical for generating rvna titers. in spite of similar magnitude of immune response generated, protective efficacies against viral challenge varied. the unmodified and secreted forms of vaccines were found to be inferior in inducing protection against viral challenge. xiang et al. also reported that secreted form of vaccine did not confer significant protective immunity [ ] . protection against rabies virus is mainly mediated by neutralizing antibodies [ ] ; subtle differences in the conformation of the secreted protein, not readily detectable by conventional biochemical methods, might select a different repertoire of neutralizing antibodies with lower avidity to the full length g protein present on the surface of viral particles, thus being less able to prevent the spread of virus [ ] . interestingly, pt.gp.l, pt.gp, pu.gp, pgp.l dna vaccine combinations designed to generate different types of immune responses yielded in effect similar data. a probable explanation for this could be that the tagged antigens evoke similar levels of immunity and act to enhance survival via the same primary protective mechanism. we observed that ubiquitination of antigen for mhc class i targeting also enhanced the igg antibody and cd + mediated cytokine response. thus, we infer that the peptides generated by proteasomal degradation could also be presented by mhc-ii. while, there is no specific information of how protein processing in transfected cells occurs in vivo, different mechanisms have been postulated. they include direct priming by somatic cells, direct priming by antigen-presenting cells, or cross priming of antigen-presenting cells. activation by cross priming appears to be the most probable immune mechanism which occurs following intramuscular vaccination that could be shared by the tpa, lamp- and uq vaccines [ , [ ] [ ] [ ] [ ] . cross priming may occur via exit of exogenous antigens from the endocytic compartments and its processing in the cytosol, recycling of mhc-i molecules through endosomal/lysosomal pathway and transfer of processed peptides to the endosomal compartments. it is well known that cd + t-cell stimulation can result from endocytosis of exogenous peptides or proteins followed by antigenic processing via mhc class ii pathways [ ] . lamp- targeting of antigen has been reported to increase the number of immunogenic peptide epitopes that activate cd + t cells, thus inducing a broadened immune response in comparison to untargeted antigen [ ] . recent studies have also demonstrated that exogenous proteins or peptides, possibly complexed to heat shock proteins, can be taken up by antigen processing cells, processed through the mhc class i pathway, and ultimately stimulate naïve cd cells [ , ] . thus, via cross-priming mechanisms, secreted fusion proteins expressed from tpa plasmids, membrane bound fused proteins expressed from lamp- or peptides released from cells transfected with the uq constructs could induce both cd + and cd + t-cell populations. several researchers have applied targeting strategies and reported conflicting results with different antigens and different infectious systems. successful targeting was demonstrated for several pathogens including human papillomavirus [ ] , influenza a [ ] ; mycobacterium [ ] ; but not for all the constructs tested against malaria [ ] . thus, a tagged dna vaccine may represent an 'ideal' immunogen for generating protective immune response, nevertheless; the antigen dependence of immune strategies has to be considered for successful vaccination against any pathogen. further, optimization of immunization doses, routes, schedule, adjuvant supplementation and a greater understanding of the immune mechanisms responsible for producing protective immunity in response to dna vaccination should facilitate the creation of further improved rabies dna vaccination strategies. rabies re-examined world survey of rabies: no. for the year . geneva, world health organization rabies: a new look at an old disease genetic immunization: a new era in vaccines and immunotherapy first human trial of a dna based vaccine for treatment of human immunodeficiency virus type infection: safety and host responses cellular cytotoxic response induced by dna vaccination in hiv- -infected patients induction of antigen-specific cytotoxic t lymphocytes in humans by a malaria dna vaccine excellent safety and tolerability of the human immunodeficiency virus type pga /js plasmid dna priming vector vaccine in hiv type uninfected adults phase i clinical trial safety of dna-and modified virus ankara-vectored human immunodeficiency virus type (hiv- ) vaccines administered alone and in a prime-boost regime to healthy hiv- -uninfected volunteers sustained protective rabies neutralizing antibody titers after administration of cationic lipid-formulated pdna vaccine dna vaccination of mice against rabies virus: effects of the route of administration and the adjuvant monophosphoryl lipid a (mpl r ) gene gun particle-mediated vaccination with plasmid dna confers protective immunity against rabies virus infection rabies vaccination: comparison of neutralizing antibody responses after priming and boosting with different combinations of dna, inactivated virus, or recombinant vaccinia virus vaccines rabies dna vaccination of non-human primates: post-exposure studies using gene gun methodology that accelerates induction of neutralizing antibody and enhances neutralizing antibody titers immunization of dogs and cats with a dna vaccine against rabies virus immunization of dogs with a dna vaccine induces protection against rabies virus post-exposure dna vaccination protects mice against rabies virus canine rabies dna vaccination: a single-dose intradermal injection into ear pinnae elicits elevated and persistent levels of neutralizing antibody field trials of a very potent rabies dna vaccine which induced long lasting virus neutralizing antibodies and protection in dogs in experimental conditions rabies dna vaccine in the horse: strategies to improve serological responses postexposure therapy in mice against experimental rabies: a single injection of dna vaccine is as effective as five injections of cell culture-derived vaccine immunogenicity of dna vaccines expressing tuberculosis proteins fused to tissue plasminogen activator signal sequences dna vaccine encoding human immunodeficiency virus- gag, targeted to the major histocompatibility complex ii compartment by lysosomal-associated membrane protein, elicits enhanced long-term memory response enhancing dna immunization dna vaccination against tuberculosis: expression of a ubiquitin-conjugated tuberculosis protein enhances antimycobacterial immunity development of rabies dna vaccine using a recombinant plasmid cortisone-evoked decrease of acid-galactosidase, glucuronidase, n-acetyl-glucosaminidase and arylsulphatase in the ileum of suckling rats breaking down the wall: fractionation of mycobacteria dna vaccine for rabies: relevance of the trans-membrane domain of the glycoprotein in generating an antibody response a rapid fluorescent focus inhibition test (rffit) for determining rabies virus-neutralizing antibody rabies virus glycoprotein. ii. biological and serological characterization oral immunization and protection of raccoons (procyon lotor) with a vaccinia-rabies glycoprotein recombinant virus vaccine experimental immunization of cats with a recombinant rabies-canine adenovirus vaccine elicits a long-lasting neutralizing antibody response against rabies sars coronavirus nucleocapsid immunodominant t-cell epitope cluster is common to both exogenous recombinant and endogenous dna-encoded immunogens dna vaccines against dengue virus based on the ns gene: the influence of different signal sequences on the protein expression and its correlation to the immune response elicited in mice targeting the vaccinia virus l protein to the cell surface enhances production of neutralizing antibodies immunization with plasmid dna encoding influenza a virus nucleoprotein fused to a tissue plasminogen activator signal sequence elicits strong immune responses and protection against h n challenge in mice evaluation of the anti-tuberculosis activity generated by different multigene dna vaccine constructs the enhanced immune response to the hiv gp /lamp chimeric gene product targeted to the lysosome membrane protein trafficking pathway hiv- p gag encoded in the lysosome-associated membrane protein- as a dna plasmid vaccine chimera is highly expressed, traffics to the major histocompatibility class ii compartment, and elicits enhanced immune responses regulating protein degradation by ubiquitination dna vaccines encoding full-length or truncated neu induce protective immunity against neu-expressing mammary tumors hemagglutinin (ha) proteins from h and h serotypes of influenza a viruses require different antigen designs for the induction of optimal protective antibody responses as studied by codon-optimized ha dna vaccines comparison of rabies virus g proteins produced by cdna-transfected animal cells that display either inducible or constitutive expression of the gene immune responses to nucleic acid vaccines to rabies virus use of mouse anti-rabies monoclonal antibodies in post-exposure treatment of rabies antibodies i post exposure treatment of rabies in vivo priming by dna injection occurs predominantly by antigen transfer mhc-i class restricted ctl responses to exogenous antigens priming of cytotoxic t lymphocytes by dna vaccines: requirement for professional antigen presenting cells and evidence for antigen transfer from myocytes dna vaccines: immunology, application, and optimization alternative processing of endogenous or exogenous antigens extends the immunogenic, h- class-i restricted peptide repertoire capture and processing of exogenous antigens for presentation on mhc molecules characterization of mhc class ii-presented peptides generated from an antigen targeted to different endocytic compartments receptor-mediated uptake of antigen/heat shock protein complexes results in major histocompatibility complex class i antigen presentation via two distinct processing pathways multiple antigen-specific processing pathways for activating naive cd + t cells in vivo characterization of hpv- e dna vaccines employing intracellular targeting and intercellular spreading strategies targeting antigen to mhc class i and class ii antigen presentation pathways for malaria dna vaccines inactivated pv viral antigen and rabies reference antiserum were kindly provided by dr. v. srinivasan, indian immunological ltd. the authors acknowledge dr. shardul solanke (national biotechnology center, ivri, india) for transfection and anuj kumar sharma (school of biotechnology, jnu, india) for flow cytometry studies. special thanks are extended to dr. subhash chandra (cornell university, ny) for vital inputs in the study. this work was supported by department of biotechnology, government of india. manpreet kaur is recipient of senior research fellowship from csir, government of india. supplementary data associated with this article can be found, in the online version, at doi: . /j.vaccine. . . . key: cord- - k hc ww authors: bien, christian g.; bauer, jan title: t-cells in human encephalitis date: journal: neuromolecular med doi: . /nmm: : : sha: doc_id: cord_uid: k hc ww encephalitis literally means inflammation of the brain. in general, this inflammation can result from a viral or bacterial infection in the brain itself or alternatively from a secondary autoimmune reaction against an infection or a tumor in the rest of the body. besides this, encephalitis is present in (believed autoimmune) diseases with unknown etiology, such as multiple sclerosis or rasmussen encephalitis (re). this article summarizes the existing data on the role of t-cells in the pathogenesis of three types of human encephalitis: re, paraneoplastic encephalomyelitis, and virus encephalitis. in all of them, t-cells play a major role in disease pathogenesis, mainly mediated by major histocompatiblity complex class i-restricted cd (+) t-lymphocytes. inflammatory processes within the central nervous system (cns) are mediated by three main effector systems: macrophages and microglial cells; b-lymphocytes and plasma cells and their effector products, antibodies; and t-lymphocytes. the t-cell system is the most strictly regulated and probably the most complex one among these. to perform an effective immune reaction, t-cells must patrol the cns regularly and, on contact with a specific antigen in the context of the appropriate molecules, must expand clonally and enter the cns in large numbers. within the cns, they have to perform their specific functions. cd + lymphocytes contribute to the activation of microglia/macrophages and b-cells, whereas cd + lymphocytes can act as cytotoxic effector cells, but also as regulatory cells. the brain milieu, on the other hand, does not explicitly favor these actions as evident from rapid apoptosis of t-cells in some disorders (bauer et al., ) . in encephalitides, t-cells have been viewed primarily as "detrimental" for the cns. however, they do also exert "beneficial" effects. this is particularly evident in cases of viral encephalitis. t-cells contribute to the clearance of infected cells (hodgson et al., ; liu and chambers, ) . on the other hand, people with numerically reduced or functionally impaired t-cells have an increased risk of succumbing to viral encephalitides. also beyond viral diseases, t-cells may (down) regulate inflammatory processes and thereby prevent excessive damage to the cns. in this review, we attempt to summarize the existing knowledge on t-cell effects and-if availablepotential ways of its therapeutic modification in rasmussen encephalitis (re), paraneoplastic encephalomyelitis (pem), and virus encephalitides. where appropriate, data from animal models of these human encephalitic diseases will be discussed. re is a chronic inflammatory brain disorder characterized by unihemispheric brain destruction, progressive impairment of neurological functions, and intractable seizures (rasmussen et al., ; bien et al., b; ) . the disorder mainly affects children with a peak of incidence at the age of - yr (oguni et al., ) . however, adolescent and adult patients with re have been reported and probably account for about % of all cases (hart et al., ) . re is an example of a disorder in which a humoral autoimmune genesis was proposed before the pathogenesis was linked to t-cells instead. the hypothesis of humoral autoimmunity at the core of re pathogenesis was developed starting from a serendipitous observation: when four rabbits were immunized to raise antibodies against the subunit of the ionotropic glutamate receptor (glur ), two developed seizures. histopathological examination of their brains showed bihemispheric inflammatory changes that were thought to mimic those of re. three out of four subsequently studied patients with re, but none of the controls, were found to harbor those glur antibodies in their sera. one of the glur antibody-positive patients was treated by plasma exchange and improved transiently (rogers et al., ) . however, recent studies of larger re and control groups either did not find a difference between the number of glur antibody-positive patients in re and noninflammatory epilepsy groups (wiendl et al., ; mantegazza et al., ) or even concluded (on the basis of five different tests for glur antibodies) that none of the tested patients with re really harbored functional glur antibodies (watson et al., ) . the most relevant data regarding t-cell pathology in re have been obtained by studies on brain specimens that had been collected during epilepsy surgery procedures or diagnostic open brain biopsies. the first histopathological study using immunhistochemistry techniques showed that most of the inflammatory cells, apart from activated microglial cells, were t-lymphocytes (farrell et al., ) . in a subsequent study, re brain tissue was analyzed by means of quantitative polymerase chain reaction (pcr) assessment of t-cell receptor (tcr) gene transcripts. a restricted (i.e., oligoclonal) bv family usage was not observed. however, the tcr vβ families that were predominantly expressed, displayed a limited size heterogeneity and extensive repetition of inframe cdr nucleotide motifs compared with controls. the authors concluded that the local lymphocytic immune process in re includes restricted t-cell populations that have likely expanded from a few precursor t-cells responding to discrete antigenic epitopes (li et al., ) . another immunohistochemical study of re brain specimens provided evidence of a granzyme b (grb)-mediated cytotoxic t-lymphocyte (ctl) attack against neurons. the authors were able to document several relevant elements of such a reaction within the diseased brains: cd + cd + t-cells containing grb granules attached to neurons that expressed major histocompatibility complex class i (mhc i) as well as neurons dying by apoptosis (bien et al., a) . confirmatory observations have been provided by another recent histopathological study on a large hemidecortication series (pardo et al., ) . further support for the t-cell concept of re pathogenesis comes from a recent report on elevated numbers of cd + t-cells and increased levels of interleukin (il)- in the blood of a group of untreated patients with re that was compared to a healthy age-matched control cohort (tekgul et al., ) . the only well-established way of re therapy is functional hemispherectomy or deafferentation of the affected side of the cerebrum for treatment of the severe epilepsy. the results are excellent regarding seizure freedom, however, at the price of complete loss of all cortical functions of the affected hemisphere. that means that after this operation, spastic hemiplegia of the contralateral extremities, homonymous hemianopia, and-if performed on the language dominant side-aphasia will be inevitably present. therefore, it has been common practice (bien et al., ) to reserve hemispherectomy for nonseizure-free patients in whom further relevant deterioration of everyday functions is unlikely (because severe deficits have been brought about by the natural course of the disease and no relevant language functions reside on the affected brain hemisphere). during the last decade, case reports and small patient series have been published reporting on beneficial effects of different kinds of immunotherapies in patients not eligible for hemispherectomy. a recent open trial of the drug tacrolimus (a calcineurin inhibitor known to suppress the activation of t-lymphocytes) in comparison with a historical untreated control cohort showed a superior outcome of the treated patients regarding motor function and progression of hemiatrophy. the seizure outcome, however, was not superior in the tacrolimus group. these results were interpreted as suggesting a brain tissue and function protective effect of this drug (bien et al., ) . they provide indirect evidence for the concept of a t-cell-mediated tissue destruction in re. the above-described ctl mechanism in re is suitable to explain the progressive brain tissue loss in this condition. however, it cannot directly account for the high-epileptic activity in re brains. the most important limitation in the understanding of the pathophysiology of re, however, is that the antigen/ antigens against which the ctls are directed is/are unknown. the solution of this issue, in other words is the elucidation of the etiology of re will probably provide an explanation for the most awkward feature of this condition: the limitation of the inflammation to one of the cerebral hemispheres. in the s, a series of clinico-pathological studies reported on the coexistence of encephalitis and myelitis with malignant diseases (brierley et al., ; henson et al., ; corsellis et al., ) . later on, the term pem was coined for this constellation. besides this, paraneoplastic peripheral neuropathies were encountered. the predilection sites for the inflammation in the cns (and the resulting clinical syndromes) are cerebellum (resulting in ataxia); mediotemporal structures (resulting in "limbic" symptoms like disturbance of recent memory, temporal lobe seizures, and psychiatric abnormalities); brainstem (bulbar dysfunction); anterior horn cells in the spinal cord (resulting in motor impairment). the above quoted early pathological studies on autopsy tissue (brierley et al., ; henson et al., ; corsellis et al., ) revealed encephalitis with round cell (lymphocytic) perivascular cuffs and parenchymal infiltration plus microglial activation (but relatively low numbers of macrophages). later on, immunohistochemical histopathological studies showed that the infiltrating lymphocytes mainly are cd + cd + t-cells (graus et al., b; panegyres et al., ; bernal et al., ) . a major breakthrough was the identification and antigenic characterization of serum autoantibodies in those patients during the s and s (to mention only the earlier reports: jaeckle et al., ; cunningham et al., ; graus et al., ; dalmau et al., ; . this led to the proposal of antibody-defined neurological syndromes. even though there is no absolute correlation of antibody, type of neurological syndrome and type of underlying malignancy, several typical constellations have been observed over the years (table ) . further research led to the demonstration that these autoantibodies do not only react with distinct structures of human and rodent brains (one of the standard test procedures for these antibodies does make use of this property) but also with cells from the underlying tumor. based on this observation, it was hypothesized that the neurological syndromes result from those antibodies, which were thought to be primarily directed against the tumor and ("unfortunately") get access to the brain in which they crossreact with brain epitopes . however, any further attempt to prove this hypothesis failed: most of the antigens were found to reside mainly within the cytoplasma or even the nuclei of brain cells (thereby being "out of reach" of antibodies); removal of antibodies from patients' circulation by plasma exchange or immunoabsorption did not consistently improve these patients' clinical symptoms (graus et al., a; cher et al., ; gultekin et al., ) ; and passive transfer and immunization studies failed to induce disease in animals (sillevis smitt et al., ; tanaka et al., ) . today, pem is viewed as t-cell mediated. this assumption is based on the neuropathological findings of elements of a cytotoxic t-cell attack within the brains of affected people (bernal et al., ) but also on some other observations regarding anti-yo, anti-hu, and anti-ma syndromes and their respective antigens, cerebellar degenerationrelated protein (cdr ), hud, and pnma . the most prominent evidence for a pathogenetically relevant contribution of t-cells to pem comes from studies on patients with anti-yo positive paraneoplastic cerebellar degeneration. the related antigen, called cdr , is present in the cytoplasm of purkinje cells and binds to c-myc thereby downregulating its activity. this antigen is also expressed in some gynecological cancers (corradi et al., ; okano et al., ; darnell et al., ) . in blood and cerebral spinal fluid of anti-yo-positive patients, circulating cdr -specific ctls were detected that were directed against the tumor and in addition against purkinje cells (albert et al., ; . the results from these studies suggest that cdr -specific ctls are the pathogenetically relevant, "crossreactive" link between tumor immunity and the disease process within the nervous system. it must be stressed that despite earlier observations of a lack of purkinje cells in the absence of inflammatory infiltrates, such infiltrates have been readily observed within the cerebellum of an anti-yo-positive patient (verschuuren et al., ) . it appears likely that the t-cells disappear after the destruction of the purkinje cells and are not seen during later disease stages. the protein, hud, is normally restricted to neurons, but it is ectopically expressed in small cell lung cancer cells. it is an intranuclear rna-binding protein, probably involved in the regulation of the cell cycle (szabo et al., ) . two anti-hu antibody positive patients but not control patients harbored t-cells reacting to stimulation with hud in their blood (benyahia et al., ) . activated cd + t-cells from a patient with paraneoplastic anti-hu positive neuropathy lysed her own fibroblasts after incubation with interferon-γ(for mhc i induction) and after injection of recombinant hud protein into the cells (tanaka et al., ) . as in the case of cdr , these reports are highly suggestive of a t-cell response against hud. the results of an immunohistochemical and pcr anna, antineuronal nuclear antibody; cdr , cerebellar degeneration-related protein ; crmp, collapsin response mediator protein; lems, lambert eaton myasthenic syndrome; pca, purkinje cell antibody; pcd, paraneoplastic cerebellar degeneration; ple, paraneoplastic limbic encephalitis; poma, paraneoplastic opsoclonus myoclonus ataxia syndrome; sclc, small cell lung cancer. study on tcr vβ families of t-lymphocytes within the brains of anti-hu positive patients (autopsy specimens) providing evidence for an oligoclonal expansion of cd + t-cells are concordant with this concept (voltz et al., ) . the induction of a (subclinical) brain inflammation by adoptive transfer of t-cells specific for the autologous onconeuronal antigen pnma (the antigen of anti-ma antibodies) in da rats (pellkofer et al., ) provides further, at least provisional, evidence for a t-cell mediated pathogenesis for this subform of pem. the results of therapeutic efforts directed against the inflammatory brain attack in pem have in general been rather disappointing. usually, patients deteriorate or remain on a low level of function despite immunosuppressive or immunomodulatory treatment. most patients die rapidly as a consequence of the neurological disorder rather than by the tumor itself. only an effective tumor treatment seems to be associated with an improved neurological outcome (keime-guibert et al., ; . for further information on pem, see the recent review literature (voltz, ; bataller and dalmau, ; roberts and darnell, ) . recently anti-inflammatory "tysabri" trials (natalizumab, given in combination with β-interferon) in patients with multiple sclerosis (ms) were stopped as a result of development of progressive multifocal leukoencephalopathy (pml) in several cases (sheridan, ; kleinschmidt-demasters and tyler, ; langer-gold et al., ) . natalizumab is a selective adhesion-molecule inhibitor that acts by binding to cell surface receptors known as α β (vla- ) and α β integrins. α- integrins are important in mediating the migration of lymphocytes from the bloodstream to sites of inflammation in the tissues. it is likely that blockage of lymphocyte migration into the brain in combination with the immunosuppressive action of β-interferon, has predisposed those patients for the opportunistic infection. this unfortunate development of pml shows that a certain degree of immune surveillance by tlymphocytes is needed to counteract virus proliferation in brain. most of what we know about the role of t-lymphocytes in virus encephalitis comes from experi-mental models. susceptible (immunological knockout) mouse and rat strains have been infected with a large number of viruses such as measles, polio, herpes simplex, and theilers's (picorna). the immunological mechanisms induced on virus infections are extremely complex since both the properties encoded by the virus as well as the hosts immunological reaction, which may differ strongly between different strains of a certain species, determine the outcome of the disease (liebert and ter meulen, ; watanabe et al., ) . nevertheless, a number of basic processes have been delineated. after infection of cns cells, surveilling t-cells may enter the brain and initiate various processes such as upregulation of cytokines like il- a, il- , il- , tumor necrosis factor-α, and interferon (ifn)-γ (frei et al., ; schneider-schaulies et al., ; morris et al., ) , upregulation of adhesion molecules (soilu-hanninen et al., ) , and upregulation of mhc expression (brankin et al., ) . this is the start of inflammation, which in susceptible animals and depending on the type of virus used, may lead to chronic demyelination or neuronal damage. cd + lymphocytes may play an important role in virusencephalitis by elimination of the virus. in this respect, one study showed that elimination of borna disease virus by virus-specific cd + t-cells is achieved via induction of cytotoxic cd + t-cells (noske et al., ) . as for demyelination, in theilers's virus encephalomyelitis (tmev) this may not be cd -mediated (pullen et al., ; murray et al., b) but seems to depend strongly on the balance between persistent virus infection and the immune cells (rodriguez et al., ) . here, the extent to which cd + or cd + cells are responsible for the neurological deficits is unclear. studies of tmev induction in β -microglobulin-deficient transgenic mice, which therefore lack functional cd + t-cells, develop a high-level virus-specific cd -mediated inflammation. although clinical deficits in these animals were absent, they could be induced after further subcutaneous immunization with the virus. this suggested that cd + t-cells appear to be primarily involved in downregulation of a potentially damaging cd + t-cell response in resistant animals (pullen et al. ). on the other hand, studies by murray et al. ( a,b) , which induced theilers' encephalitis in cd and cd knockout animals, showed that in the absence of cd + cells neurological deficits were lacking, whereas the absence volume , of cd + cells resulted in severe neurological deficits. a comparable situation is found in acute cns infection of mice by the neurotropic jhm strain of mouse hepatitis virus (jhmv). although demyelination in jhmv correlates well with t-cell infiltration (houtman and fleming, ) , contrary results regarding the role of cd and cd t-cells are found (wang et al., ; wu et al., ; pewe and perlman, ) . in herpes simplex virus (hsv), influenza a, and borna virus-induced demyelination, an important role of cd + cells has been consistently shown (hudson and streilein, ; stevenson et al., ) . amore recent paper (van der most et al., ) shows that cd + t-lymphocytes play a role beyond the immediate cytotoxic function. dengue virus-specific cd + cells, displaying an effector-memory phenotype, remained in the cns for a long time (up to d) after infection. unfortunately, the authors did not present data demonstrating that persistence of inflammation here was accompanied by perseverance of virus antigen such as demonstrated in human hsv encephalitis (see the following paragraph). these experimental studies may give a general idea how in virus encephalitis these immunological mechanisms function. the situation in human encephalitis may, however, differ, depending on the human virus type and specific human genetic/ immunological factors. although virus encephalitis is not uncommon, the knowledge about the contribution of cellular immunity in eliminating virus and to the cns damage is remarkably fragmented (booss and esiri, ) . overall, there are several lines of evidence that suggest that especially mhc i-restricted cd + t-lymphocytes also play an important role in human virus encephalitis. first of all, in virus encephalitis as in pem, re and in ms (booss et al., ; woodroofe et al., ; hayashi et al., ; gay et al., ) , detailed quantification of inflammatory cells show that cd + cells generally outnumber cd + cells in the tissue infiltrates. furthermore, cd + cells are more restricted to the perivascular inflammatory cuffs, whereas cd + cells tend to diffusely infiltrate the parenchyma of the lesions (gay et al., ; babbe et al., ; anlar et al., ) , although some studies result the opposite (nagano et al., ) . more specific studies reveal a role of ctl in virus encephalitis. as an example, in pml, jc-specific t-lymphocytes can be found in the blood of affected patients. these specific ctls were detected in out of pml patients who survived vs only out of pml patients who progressed. these data suggest that the presence of jcspecific cytototoxic t-lymphocytes is indicative for a favorable outcome (du pasquier et al., a,b) . a role for t-lymphocytes in virus elimination was also suggested in hiv encephalitis; immunopathological studies reveal the presence of both cd + as well as cd + t-lymphocytes surrounding hippocampal neurons suggesting that these cells are in the process of eliminating these virus-infected neurons. aproblem with these specific studies is that, in situ, neurons have not been found to be infected by hiv and thus a hiv-specific t-cell-mediated response against these neurons cannot be proven (petito et al., ) . also in west nile fever (wnf) encephalitis a role of cytotoxic t-cells has been shown. primary or memory cd + t-cells from patients with wnf that were generated in vivo efficiently killed target cells that displayed west nile viral antigens in a class i mhc-restricted manner suggesting that cd + t-cells have an important function in clearing infection from tissues and preventing viral persistence (shrestha and diamond, ) . case studies of patients with hsv encephalitis reveal that infiltrating lymphocytes can remain in the brain for a very long time ( - yr) after clinical recovery. persistent hsv dna could be recognized by pcr even though immunohistochemical stainings for hsv proteins were negative (nicoll et al., a,b; . as mentioned earlier, one important aspect in t-cell cytotoxicity, but also in the cellular defense against viruses, is the presence of mhc i and ii expression in neural (virus-infected) cells of the cns. the expression of mhc ii antigens in brain, predominantly on microglia, has been shown in a large number of studies (kennedy et al., ; achim et al., ; nagano et al., ; an et al., ) . detection of mhc i antigens have been shown only in a very small number of publications (achim and wiley, ; gogate et al., ) . interestingly in pml, mhc i expression was seen on jc virus-infected oligodendrocytes, suggesting that these oligodendrocytes can be targets for a mhc i-dependent cytotoxic t-cell response (achim and wiley, ) . in subacute sclerosing panencephalitis, mhc i upregulation was found in neurons of which a small number were double-labeled for measles virus, suggesting that here virus-infected cells could be the target for a cytotoxic cellular response, too (gogate et al., ) . it is known that ifn-γ is an important factor in upregulation of mhc i (linda et al., ; parra et al., ; bergmann et al., ; . to what extent neural mhc class i expression is upregulated by the virus infection itself is unclear. it is, however, well known that viruses can actively downregulate this mhc class i expression, a function that is one of the many ways in which viruses limit the immune response, something that is also called immune evasion (ploegh, ; lieberman et al., ; basta and bennink, ) . in the described forms of human encephalitis, most of the existing (still highly fragmentary) knowledge about the role of t-cells basically comes from studies of human brain tissue obtained by autopsies, diagnostic brain biopsies, or epilepsy surgery procedures and from studies of peripheral blood lymphocytes and soluble factors of the immune system. animal studies have provided additional insights into t-cell function in some instances. it is obvious that most often cd + t-cells play a role as mhc i restricted cytotoxic effector cells directed against brain cells. the antigens involved in this process are only rarely known. regulatory functions of cd + cells are, however, also conceivable. up to now, t-cell studies have not become diagnostic routine procedures in those disorders. at least in re, insights into the t-cell pathogenesis of this disorder have guided the first promising anti-t-cell treatment approaches. expression of major histocompatibility complex and hiv antigens within the brains of aids patients expression of major histocompatibility complex antigens in the brains of patients with progressive multifocal leukoencephalopathy detection and treatment of activated t cells in the cerebrospinal fluid of patients with paraneoplastic cerebellar degeneration tumor-specific killer cells in paraneoplastic cerebellar degeneration investigation on the expression of major histocompatibility complex class ii and cytokines and detection of hiv- dna within brains of asymptomatic and symptomatic hiv- -positive patients tissue inflammatory response in subacute sclerosing panencephalitis (sspe) clonal expansions of cd (+) t cells dominate the t cell infiltrate in active multiple sclerosis lesions as shown by micromanipulation and single cell polymerase chain reaction a survival game of hide and seek: cytomegaloviruses and mhc class i antigen presentation pathways paraneoplastic disorders of the central nervous system: update on diagnostic criteria and treatment apoptosis of t lymphocytes in acute disseminated encephalomyelitis cell-mediated autoimmunity in paraneoplastic neurological syndromes with anti-hu antibodies perforinmediated effector function within the central nervous system requires ifn-gamma-mediated mhc up-regulation perforin and gamma interferon-mediated control of coronavirus central nervous system infection by cd t cells in the absence of cd t cells immunohistochemical analysis of anti-hu-associated paraneoplastic encephalomyelitis destruction of neurons by cytotoxic t cells: a new pathogenic mechanism in rasmussen's encephalitis an open study of tacrolimus therapy in rasmussen encephalitis pathogenesis, diagnosis and treatment of rasmussen encephalitis: a european consensus statement the natural history of rasmussen's encephalitis viral encephalitis in humans immunohistological analysis of t lymphocyte subsets in the central nervous system in chronic progressive multiple sclerosis adhesion molecule expression and lymphocyte adhesion to cerebral endothelium: effects of measles virus and herpes simplex virus subacute encephalitis of later adult life mainly affecting the limbic areas therapy for paraneoplastic neurologic syndromes in six patients with protein a column immunoadsorption a post-transcriptional regulatory mechanism restricts expression of the paraneoplastic cerebellar degeneration antigen cdr to immune privileged tissues limbic 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encephalitis. evaluation in a murine model infection and in patients selective expression of purkinje-cell antigens in tumor tissue from patients with paraneoplastic cerebellar degeneration the application of multifactorial cluster analysis in the staging of plaques in early multiple sclerosis. identification and characterization of the primary demyelinating lesion major histocompatibility complex class i expression on neurons in subacute sclerosing panencephalitis and experimental subacute measles encephalitis effect of plasmapheresis on serum and csf autoantibody levels in cns paraneoplastic syndromes sensory neuronopathy and small cell lung cancer. antineuronal antibody that also reacts with the tumor immunohistochemical analysis of the immune reaction in the nervous system in paraneoplastic encephalomyelitis paraneoplastic limbic encephalitis: neurological symptoms, immunological findings and tumour association in patients chronic encephalitis and epilepsy in adults and adolescents: a variant of rasmussen's syndrome? dual-label immunocytochemistry of the active multiple sclerosis lesion: major histocompatibility complex and activation antigens encephalomyelitis with carcinoma perforin and fas/fas ligand-mediated cytotoxicity in acute and chronic woodchuck viral hepatitis pathogenesis of mouse hepatitis virus-induced demyelination functional cytotoxic t cells are associated with focal lesions in the brains of sjl mice with experimental herpes simplex encephalitis autoimmune response of patients with paraneoplastic cerebellar degeneration to a purkinje cell cytoplasmic protein antigen clinical outcome of patients with anti-hu-associated encephalomyelitis after treatment of the tumor treatment of paraneoplastic neurological syndromes with antineuronal antibodies (anti-hu, anti-yo) with a combination of immunoglobulins, cyclophosphamide, and methylprednisolone studies on the pathogenesis of neurological diseases associated with varicella-zoster virus progressive multifocal leukoencephalopathy complicating treatment with natalizumab and interferon beta- a for multiple sclerosis progressive multifocal leukoencephalopathy in a patient treated with natalizumab local-clonal expansion of infiltrating t lymphocytes in chronic encephalitis of rasmussen avoiding the kiss of death: how hiv and other chronic viruses survive virological aspects of measles virus-induced encephalomyelitis in lewis and bn rats expression of mhc class i and beta -microglobulin in rat spinal motoneurons: regulatory influences by ifngamma and axotomy yellow fever virus encephalitis: properties of the brain-associated t-cell response during virus clearance in normal and gamma interferon-deficient mice and requirement for cd + lymphocytes antibodies against glur peptides are not specific for rasmussen's encephalitis but are also present in epilepsy patients with severe, early onset disease and intractable seizures characterization of the cellular and cytokine response in the central nervous system following semliki forest virus infection perforin-dependent neurologic injury in a viral model of multiple sclerosis cd (+) and cd (+) t cells make discrete contributions to demyelination and neurologic disease in a viral model of multiple sclerosis immunocytochemical analysis of the cellular infiltrate in brain lesions in subacute sclerosing panencephalitis distribution of herpes simplex virus dna in the brains of human long-term survivors of encephalitis autopsy neuropathological findings in burnt out' herpes simplex encephalitis and use of the polymerase chain reaction to detect viral dna use of the polymerase chain reaction to detect herpes simplex virus dnain paraffin sections of human brain at necropsy virusspecific cd + t cells eliminate borna disease virus from the brain via induction of cytotoxic cd + t cells the syndrome of chronic encephalitis and epilepsy. a study based on the mni series of cases the cytoplasmic purkinje onconeural antigen cdr down-regulates c-myc function: implications for neuronal and tumor cell survival the inflammatory reaction of paraneoplastic ganglionitis and encephalitis: an immunohistochemical study the pathology of rasmussen syndrome: stages of cortical involvement and neuropathological studies in hemispherectomies ifngamma is required for viral clearance from central nervous system oligodendroglia modelling paraneoplastic cns disease: t-cells specific for the onconeuronal antigen pnma mediate autoimmune encephalomyelitis in the rat cd + and cd + cells accumulate in the brains of acquired immunodeficiency syndrome patients with human immunodeficiency virus encephalitis cutting edge: cd t cell-mediated demyelination is ifn-gamma dependent in mice infected with a neurotropic coronavirus immunopathogenic role of t-cell subsets in borna disease virusinduced progressive encephalitis viral strategies of immune evasion class i-deficient resistant mice intracerebrally inoculated with theiler's virus show an increased t cell response to viral antigens and susceptibility to demyelination focal seizures due to chronic localized encephalitis neuroimmunology of the paraneoplastic neurological degenerations the balance between persistent virus infection and immune cells determines demyelination autoantibodies to glutamate receptor glur in rasmussen's encephalitis differential induction of cytokines by primary and persistent measles virus infections in human glial cells tysabri raises alarm bells on drug class role of cd + t cells in control of west nile virus infection immunization with the paraneoplastic encephalomyelitis antigen hud does not cause neurologic disease in mice semliki forest virus infection leads to increased expression of adhesion molecules on splenic t-cells and on brain vascular endothelium protection against lethal influenza virus encephalitis by intranasally primed cd + memory t cells hud, a paraneoplastic encephalomyelitis antigen, contains rna-binding domains and is homologous to elav and sex-lethal cytotoxic t-lymphocyte-mediated cell death in paraneoplastic sensory neuronopathy with anti-hu antibody trial to establish an animal model of paraneoplastic cerebellar degeneration with anti-yo antibody prolonged presence of effector-memory cd t cells in the central nervous system after dengue virus encephalitis inflammatory infiltrates and complete absence of purkinje cells in anti-yo-associated paraneoplastic cerebellar degeneration paraneoplastic neurological syndromes: an update on diagnosis, pathogenesis, and therapy t-cell receptor analysis in anti-hu associated paraneoplastic encephalomyelitis demyelination induced by murine hepatitis virus jhm strain (mhv- ) is immunologically mediated comparative analysis of coronavirus jhm-induced demyelinating encephalomyelitis in lewis and brown norway rats absence of antibodies to glutamate receptor type (glur ) in rasmussen encephalitis glur antibodies: prevalence in focal epilepsy but no specificity for rasmussen's encephalitis immunocytochemical characterisation of the immune reaction in the central nervous system in multiple sclerosis. possible role for microglia in lesion growth cd and cd t-cells have redundant but not identical roles in virus-induced demyelination key: cord- - at rhqk authors: cann, alan j. title: infection date: - - journal: principles of molecular virology doi: . /b - - - - . - sha: doc_id: cord_uid: at rhqk virus infection of higher organisms is the cumulative result of all the processes of replication and gene expression described in the previous chapters. together, these determine the overall course of each infection. infections range in complexity and duration from a very brief, superficial interaction between the virus and its host to infections that may span the entire life of the host organism, from before birth to its eventual death. a common misconception is that virus infection inevitably results in disease. in reality, the reverse is true—only a small minority of virus infections gives rise to any disease symptoms. this chapter provides an overview of the numerous patterns of virus infection and forms an introduction to the discussion of virus pathogenesis in chapter . unlike previous and subsequent chapters, this chapter deals primarily with the interaction of viruses with intact organisms rather than with the molecular biologist’s usual concern about the interaction between a virus and the cell. i explain how the immune responses to viruses enables the body to resist infection, and how viruses respond to this pressure. i describe and understand how virus infections are prevented and treated. life on earth depends on the primary productivity of plants-the production of organic molecules from inorganic molecules such as co -(with some an additional contribution from some bacteria). from the smallest single-celled alga in the ocean to the largest forest giant tree (and everything in between, such as broccoli), they are vitally important. photosynthetic algae in the oceans play a major role in controlling the atmosphere and the climate, and interaction with viruses is one of the major mechanisms which in turn control the algae. all higher animals depend on the primary productivity of plants for their food. so plants are a big deal, and anything which affects plant growth is of great importance. in purely economic terms, viruses are only of importance if it is likely that they will affect crops during their commercial lifetime, a likelihood that varies greatly between very short extremes in horticultural production and very long extremes in forestry. some estimates have put total worldwide cost of plant virus infections as high as us$ per year. by which plant viruses are transmitted between hosts is therefore of great importance. there are a number of routes by which plant viruses may be transmitted: i seeds: these may transmit virus infection either by external contamination of the seed with virus particles or by infection of the living tissues of the embryo. transmission by this route leads to early outbreaks of disease in new crops which are usually initially focal in distribution but may subsequently be transmitted to the remainder of the crop by other mechanisms. i vegetative propagation/grafting: these techniques are inexpensive and easy methods of plant propagation but provide the ideal opportunity for viruses to spread to new plants. i vectors: many different groups of living organisms can act as vectors and spread viruses from one plant to another: i bacteria (e.g., agrobacterium tumefaciens-the ti plasmid of this organism has been used experimentally to transmit virus genomes between plants) i fungi i nematodes i arthropods: insects (e.g., aphids, leafhoppers, planthoppers, beetles, thrips) i arachnids (e.g., mites) i mechanical: mechanical transmission of viruses is the most widely used method for experimental infection of plants and is usually achieved by rubbing virus-containing preparations into the leaves, which in most plant species are particularly susceptible to infection. however, this is also an important natural method of transmission. virus particles may contaminate soil for long periods and be transmitted to the leaves of new host plants as wind-blown dust or as rain-splashed mud. some of the most original experimental biology currently being done involves plant science. biologists can do experiments with plants that they can only dream of being able to perform with animals. and yet the idea persists among many that botany is boring. much of the most exciting plant science involves plant viruses, either as experimental tools or in terms of finding ways to prevent infection. and as this section describes, the biology of plant viruses has some striking differences from that of animal viruses. so if you think botany is boring, you probably need to find out more about plant viruses. the problems plant viruses face in initiating infections of host cells have already been described (chapter ), as has the fact that no known plant virus employs a specific cellular receptor of the types that animal and bacterial viruses use to attach to cells. transmission of plant viruses by insects is of particular agricultural importance. huge areas of monoculture and the inappropriate use of pesticides that kill natural predators can result in massive population booms of pest insects such as aphids. plant viruses rely on a mechanical breach of the integrity of a cell wall to directly introduce a virus particle into a cell. this is achieved either by the vector associated with transmission of the virus or simply by mechanical damage to cells. transfer by insect vectors is a particularly efficient means of virus transmission. in some instances, viruses are transmitted mechanically from one plant to the next by the vector and the insect is only a means of distribution, through flying or being carried on the wind for long distances (sometimes hundreds of miles). insects that bite or suck plant tissues are the ideal means of transmitting viruses to new hosts-a process known as nonpropagative transmission. however, in other cases (e.g., many plant rhabdoviruses), the virus may also infect and multiply in the tissues of the insect (propagative transmission) as well as those of host plants. in these cases, the vector serves as a means not only of distributing the virus but also of amplifying the infection. initially, most plant viruses multiply at the site of infection, giving rise to localized symptoms such as necrotic spots on the leaves. the virus may subsequently be distributed to all parts of the plant either by direct cell-to-cell spread or by the vascular system, resulting in a systemic infection involving the whole plant. however, the problem these viruses face in reinfection and recruitment of new cells is the same as the one they faced initially-how to cross the barrier of the plant cell wall. plant cell walls necessarily contain channels called "plasmodesmata" which allow plant cells to communicate with each other and to pass metabolites between them. however, these channels are too small to allow the passage of virus particles or genomic nucleic acids. many (if not most) plant viruses have evolved specialized movement proteins that modify the plasmodesmata. one of the best known examples of this is the -k protein of tobacco mosaic virus (tmv). this protein is expressed from a subgenomic mrna (figure . ), and its function is to modify plasmodesmata causing genomic rna coated with -k protein to be transported from the infected cell to neighboring cells (figure . ). other viruses, such as cowpea mosaic virus (cpmv; comoviridae) have a similar strategy but employ a different molecular mechanism. in cpmv, the -/ -k proteins form tubular structures allowing the passage of intact virus particles to pass from one cell to another (figure . ). typically, virus infections of plants might result in effects such as growth retardation, distortion, mosaic patterning on the leaves, yellowing, wilting, etc. these macroscopic symptoms result from: i necrosis of cells, caused by direct damage due to virus replication, i hypoplasia-localized retarded growth frequently leading to mosaicism (the appearance of thinner, yellow areas on the leaves), i hyperplasia-excessive cell division or the growth of abnormally large cells, resulting in the production of swollen or distorted areas of the plant. plants might be seen as sitting targets for virus infection-unlike animals, they cannot run away. however, plants exhibit a sophisticated range of responses to virus infections designed to minimize harmful effects. plants fight virus infections in a number of ways. first, they need to detect the infection, which they do by means of sensing virus signature molecules (so-called pathogen-associated molecular patterns or pamps, e.g., particular proteins) via dedicated receptors. when this happens, the production of resistance proteins that activate highly specific resistance mechanisms is triggered. in response, plant viruses attempt to evade these defense mechanisms by altering protein structures where possible and by producing proteins which bind to and hide small rnas which would trigger rna silencing. infection results in a "hypersensitive response," manifested as: i the synthesis of a range of new proteins, the pathogenesis-related ("pr") proteins, i an increase in the production of cell wall phenolic substances, i the release of active oxygen species, i the production of phytoalexins, i the accumulation of salicylic acid-amazingly, plants can even warn each other that viruses are coming by airborne signaling with volatile compounds such as methyl salicylate. the hypersensitive response involves synthesis of a wide range of different molecules. some of these pr proteins are proteases, which presumably destroy virus proteins, limiting the spread of the infection. there is some similarity here between the design of this response and the production of interferons (ifns) by animals. systemic resistance to virus infection is a naturally occurring phenomenon in some strains of plant. this is clearly a highly desirable characteristic that is prized by plant breeders, who try to spread this attribute to economically valuable crop strains. there are probably many different mechanisms involved in systemic resistance, but in general terms there is a tendency of these processes to increase local necrosis when substances such as proteases and peroxidases are produced by the plant to destroy the virus and to prevent its spread and subsequent systemic infection. an example of this is the tobacco n gene, which encodes a cytoplasmic protein with a nucleotide-binding site which interferes with the tmv replicase. when present in plants, this gene causes tmv to produce a localized, necrotic infection rather than the systemic mosaic symptoms normally seen. there are many different mechanisms involved in systemic resistance, but in general terms there is a tendency toward increased local necrosis as substances such as proteases and peroxidases are produced by the plant to destroy the virus and to prevent its spread and subsequent systemic disease. virus-resistant plants have been created by the production of transgenic plants expressing recombinant virus proteins or nucleic acids which interfere with virus replication without producing the pathogenic consequences of infection, for example: i virus coat proteins, which have a variety of complex effects, including inhibition of virus uncoating and interference of expression of the virus at the level of rna ("gene silencing" by "untranslatable" rnas), i intact or partial virus replicases which interfere with genome replication, i antisense rnas, i defective virus genomes, i satellite sequences (see chapter ), i catalytic rna sequences (ribozymes), i modified movement proteins. this is a very promising technology that offers the possibility of substantial increases in agricultural production without the use of expensive, toxic, and ecologically damaging chemicals (fertilizers, herbicides, or pesticides). in some countries, notably in europe, public resistance to genetically engineered plants has so far prevented the widespread adoption of new varieties produced by genetic manipulation without considering the environmental cost of not utilizing these new approaches to plant breeding. the most significant response to virus infection in vertebrates is activation of both the cellular and humoral parts of the immune system. a complete description of all the events involved in the immune response to the presence of foreign antigens is beyond the scope of this book, so you should refer to the books mentioned in the further reading at the end of this chapter to ensure that you are familiar with all the immune mechanisms (and jargon!) described below. a brief summary of some of the more important aspects is worth considering however, beginning with the humoral immune response, which results in the production of antibodies. the major impact of the humoral immune response is the eventual clearance of virus from the body. serum neutralization stops the spread of virus to uninfected cells and allows other defense mechanisms to mop up the infection. figure . shows a very simplified version of the mammalian humoral response to infection. virus infection induces at least three classes of antibody: immunoglobulin g (igg), igm, and iga. igm is a large, multivalent molecule that is most effective at cross-linking large targets (e.g., bacterial cell walls or flagella) but is probably less important in combating virus infections. in contrast, the production of iga is very important for initial protection from virus infection. secretory iga is produced at mucosal surfaces and results in "mucosal immunity," an important factor in preventing infection from occurring. induction of mucosal immunity depends to a large extent on the way in which antigens are presented to and recognized by the immune system. similar antigens incorporated into different vaccine delivery systems (see "prevention and therapy of virus infection") can lead to very different results in this respect, and mucosal immunity is such an important factor that similar vaccines may vary considerably in their efficacy. igg is probably the most important class of antibody for direct neutralization of virus particles in serum and other body fluids (into which it diffuses). direct virus neutralization by antibodies results from a number of mechanisms, including conformational changes in the virus capsid caused by antibody binding, or blocking of the function of the virus target molecule (e.g., receptor binding) by steric hindrance. a secondary consequence of antibody binding is phagocytosis of antibody-coated ("opsonized") target molecules by mononuclear cells or polymorphonuclear leukocytes. this results from the presence of the fc receptor on the surface of these cells, but as has already been noted in chapter , in some cases opsonization of virus by the binding of nonneutralizing antibodies can result in enhanced virus uptake. this has been shown to occur with rabies virus, and in the case of human immunodeficiency virus (hiv) may promote uptake of the virus by macrophages. nonphagocytic cells can also destroy antibody-coated viruses via an intracellular pathway involving the trim protein. antibody binding also leads to the activation of the complement cascade, which assists in the neutralization of virus particles. structural alteration of virus particles by complement binding can sometimes be visualized directly by electron microscopy. complement is particularly important early in virus infection when limited amounts of low-affinity antibody are made-complement enhances the action of these early responses to infection. despite all the above mechanisms, in overall terms cell-mediated immunity is probably more important than humoral immunity in the control of virus infections. this is demonstrated by the following observations: i congenital defects in cell-mediated immunity tend to result in predisposition to virus (and parasitic) infections, rather than to bacterial infections. i the functional defect in acquired immune deficiency syndrome (aids) is a reduction in the ratio of t-helper (cd ), which may have been present before the onset of aids but were previously suppressed by the intact immune system. cell-mediated immunity depends on three main effects ( figure . ). these all act via molecular mechanisms that will be explained later in this chapter (see "viruses and apoptosis," below): i nonspecific cell killing (mediated by "natural killer" [nk] cells), i specific cell killing (mediated by cytotoxic t-lymphocytes [ctls]), i antibody-dependent cellular cytotoxicity (adcc). nk cells carry out cell lysis independently of conventional immunological specificity, that is, they do not depend on clonal antigen recognition for their action. they are not major histocompatibility complex (mhc) restricted. in other works, nk cells are able to recognize virus-infected cells without being presented with a specific antigen by a macromolecular complex consisting of mhc antigens plus the t-cell receptor/cd complex. the advantage of this is that nk cells have broad specificity (many antigens rather than a single epitope) and are also active without the requirement for sensitizing antibodies. they are therefore the first line of defense against virus infection. nk cells are most active in the early stages of infection (i.e., in the first few days), and their activity is stimulated by ifn-α/β. nk cells are not directly induced by virus infection-they exist even in immunologically naive individuals and are "revealed" in the presence of ifn-α/β. they are thus part of the "innate" rather than the "adaptive" immune response. their function is complementary to and is later taken over by ctls which are part of the "adaptive" immune response. not all of the targets for nk cells on the surface of infected cells are known, but they are inhibited by mhc class i antigens (which are present on all nucleated cells), allowing recognition of "self" (i.e., uninfected cells) and preventing total destruction of the body. it is well known that some virus infections disturb normal cellular mhc-i expression and this is one mechanism by which nk cells recognize virus-infected cells. nk cell cytotoxicity is activated by ifn-α/β, directly linking nk cell activity to virus infection. unlike nk cells which may be either cd or cd , ctls are usually of cd (suppressor) phenotype, that is, they express cd molecules on their surface. ctls are the major cell-mediated immune response to virus infections and are mhc restricted-clones of cells recognize a specific antigen only when presented by mhc-i antigen on the target cell to the t-cell receptor/ cd complex on the surface of the ctl. (mhc-i antigens are expressed on all nucleated cells in the body; mhc class ii antigens are expressed only on the surface of the antigen-presenting cells of the immune system-t-cells, b-cells, and macrophages.) ctl activity requires "help" (i.e., cytokine production) from t-helper cells. the ctls themselves recognize foreign antigens through the t-cell receptor/cd complex, which "docks" with antigen presented by mhc-i on the surface of the target cell ( figure . ). the mechanism of cell killing by ctl is similar to that of nk cells (explained below). the induction of a ctl response also results in the release of many different cytokines from t-helper cells, some of which result in clonal proliferation of antigen-specific ctl and others that have direct antiviral effects-for example, ifns. the kinetics of the ctl response (peaking at about days after infection) is somewhat slower than the nk response (e.g., À days cf. . À days)-so nk cells and ctls are complementary systems. the induction of a ctl response is dependent on recognition of specific t-cell epitopes by the immune system. these are distinct from the b-cell epitopes recognized by the humoral arm of the immune system. t-cell epitopes are more highly conserved (less variable) than b-cell epitopes, which are more able to mutate quickly to escape immune pressure. these are important considerations in the design of antiviral vaccines. the specificity of cell killing by ctls is not absolute. although they are better "behaved" than nk cells, diffusion of perforin and local cytokine production frequently results in inflammation and bystander cell damage. this is a contributory cause of the pathology of many virus diseases (see chapter ), but the less attractive alternative is to allow virus replication to proceed unchecked. adcc is less well understood than either of the two mechanisms mentioned above. adcc can be carried out by nk cells or by ctls. the mechanism of cell killing is the same as that described in the next section, although complement may also be involved in adcc. the distinguishing feature of adcc is that this mechanism is dependent on the recognition of antigen on the surface of the target cell by means of antibody on the surface of the effector cell. the antibody involved is usually igg, which is bound to fc receptors on the surface of the t-cell. adcc therefore requires a preexisting antibody response and hence does not occur early during primary virus infections-it is part of the adaptive immune response. the overall contribution of adcc to the control of virus infections is not clear, although it is now believed that it plays a significant part in their control. apoptosis, or "programmed cell death," is a critical mechanism in tissue remodeling during development and in cell killing by the immune system. there are two ways in which a cell can die: necrosis or apoptosis. i necrosis is the normal response of cells to injury caused by toxins or environmental stress. necrosis is marked by nonspecific changes such as disruption of the plasma membrane and nuclear envelope, rupture of membrane-bounded organelles such as mitochondria and lysosomes, cell swelling, random fragmentation of dna/rna, influx of calcium ions into the cell, and loss of membrane electrical potential. the release of cellular components from the dying cell causes a localized inflammatory response by the cells of the immune system. this frequently leads to damage to adjacent cells/tissue-"bystander" cell damage. i apoptosis is, in contrast, a tightly regulated process that relies on complex molecular cascades for its control. it is marked by cell shrinkage, condensation, and clumping of chromatin, a regular pattern of dna fragmentation, and "bubbling off" of cellular contents into small membrane-bounded vesicles ("blebbing") which are subsequently phagocytosed by macrophages, preventing inflammation. when triggered by the appropriate signals, immune effector cells such as ctls and nk cells release previously manufactured lytic granules stored in their cytoplasm. these act on the target cell and induce apoptosis by two mechanisms: i release of cytotoxins such as: ( ) perforin (aka cytolysin), a peptide related to complement component c which, on release, polymerizes to form polyperforin, which forms transmembrane channels, resulting in permeability of the target cell membrane; and ( ) serine proteases related to trypsin. these two effectors act collaboratively, the membrane pores allowing the entry of granzymes into the target cell. the membrane channels also allow the release of intracellular calcium from the target cell, which also acts to trigger apoptotic pathways. i in addition, ctls (but not nk cells) express fas ligand on their surface which binds to fas on the surface of the target cell, triggering apoptosis. binding of fas ligand on the effector cell to fas (cd ) on the target cell results in activation of cellular proteases known as "caspases," which in turn trigger a cascade of events leading to apoptosis. the process of induction and repression of apoptosis during virus infection has received much attention during the last few years. it is now recognized that this is an important innate response to virus infection. the regulation of apoptosis is a complex issue that cannot be described fully here (see further reading and figure . for a summary), but virus infections disturb normal cellular biochemistry and frequently trigger an apoptotic response, for example: the pathways controlling apoptosis are very complex. this diagram represents only a simple summary of some of the mechanisms of major significance in virus infections. i pkr activation: the ifn effector pkr (rna-activated protein kinase may be activated by some viruses (e.g., hiv, reovirus). i p activation: viruses that interact with p (chapter ) may cause either growth arrest or apoptosis (e.g., adenoviruses, sv , papillomaviruses). i transcriptional disregulation: viruses that encode transcriptional regulatory proteins may trigger an apoptotic response (e.g., htlv tax). i foreign protein expression: overexpression of virus proteins at late stages of the replication cycle can also cause apoptosis by a variety of mechanisms. in response to this cellular alarm system, many if not most viruses have evolved mechanisms to counteract this effect and repress apoptosis: i bcl- homologues: a number of viruses encode bcl- (a negative regulator of apoptosis) homologues (e.g., adenovirus e b- k, human herpesvirus [hhv- ] ksbcl- ). i caspase inhibition: caspases are a family of cysteine proteases that are important inducers of apoptosis. inhibiting these enzymes is an effective way of preventing apoptosis (e.g., baculovirus p , serpins, viaps-"inhibitors of apoptosis"). i fas/tnf inhibition: viruses have evolved several mechanisms to block the effects of fas/tnf, including blocking signaling through the plasma membrane (e.g., adenovirus e ), tumor necrosis factor receptor (tnfr) mimics (e.g., poxvirus crma), mimics of death signaling factors (vflips), and interactions with signaling factors such as fas-associated death domain (fadd) and tnfr-associated death domain (tradd) (e.g., hhv- [ebv] lmp- ). i p inhibition: a number of viruses that interact with p have evolved proteins to counteract possible triggering of apoptosis (e.g., adenovirus e b- k and e , sv t-antigen, papillomavirus e ). i miscellaneous: many other apoptosis-avoidance mechanisms have been described in a wide variety of viruses. without such inhibitory mechanisms, most viruses would simply not be able to replicate due to the death of the host cell before the replication cycle was complete. however, there is evidence that at least some viruses use apoptosis to their benefit. positive-sense rna viruses such as poliovirus, hepatitis a virus, and sindbis virus with lytic replication cycles appear to be able to regulate apoptosis, initially repressing it to allow replication to take place, then inducing it to allow the release of virus particles from the cell. by the s, interference (i.e., the blocking of a virus infection by a competing virus) was a well-known phenomenon in virology. in some cases, the mechanism responsible is quite simple. for example, avian retroviruses are grouped into nine interference groups (a through i), based on their ability to infect various strains of chickens, pheasants, partridges, quail, etc., or cell lines derived from these species. in this case, the inability of particular viruses to infect the cells of some strains is due to the expression of the envelope glycoprotein of an endogenous provirus present in the cells which sequesters the cellular receptor needed by the exogenous virus for infection. in other cases, the mechanism of virus interference is less clear. in , alick issacs and jean lindenmann were studying this phenomenon and performed the following experiment. pieces of chick chorioallantoic membrane were exposed to ultraviolet (uv)-inactivated (noninfectious) influenza virus in tissue culture. the "conditioned" medium from these experiments (which did not contain infectious virus) was found to inhibit the infection of fresh pieces of chick chorioallantoic membrane by (infectious) influenza virus in separate cultures ( figure . ). their conclusion was that a soluble factor, which they called "interferon," was produced by cells as a result of virus infection and that this factor could prevent the infection of other cells. as a result of this provocative observation, ifn became the great hope for virology and was thought to be directly equivalent to the use of antibiotics to treat bacterial infections. the true situation has turned out to be far more complex than was first thought. ifns do have antiviral properties, but by and large their effects are exerted indirectly via their major function as cellular regulatory proteins. ifns are immensely potent; less than molecules per cell show evidence of antiviral activity. hence, following isaacs and lindenmann's initial discovery, many fairly fruitless years were spent trying to purify minute amounts of naturally produced ifn. a b figure . discovery of ifns. in , alick issacs and jean lindenmann discovered ifns by performing the following experiment. (a) pieces of chick chorioallantoic membrane were exposed to uv-inactivated (noninfectious) influenza virus in tissue culture. (b) the "conditioned" medium from these experiments (which did not contain infectious virus) was found to inhibit the infection of fresh pieces of chick chorioallantoic membrane by (infectious) influenza virus in separate cultures. they called inhibitory substance in the condition medium "interferon." this situation changed with the development of molecular biology and the cloning and expression of ifn genes, which has led to rapid advances in our understanding over the last years. there are a number of different types of ifns: i ifn-α: there are at least molecular species of ifn-α, all of which are closely related; some species differ by only one amino acid. they are synthesized predominantly by lymphocytes. the mature proteins contain amino acids, with a minimum homology of % between the different types. all the genes encoding ifn-α are located on human chromosome , and gene duplication is thought to be responsible for this proliferation of genes. i ifn-β: the single gene for ifn-β is also located on human chromosome . the mature protein contains amino acids and, unlike ifn-α, is glycosylated, with approximately % homology to other ifns. it is synthesized predominantly by fibroblasts. i other ifns: the single gene for ifn-γ is located on human chromosome . the mature protein contains amino acids, is glycosylated, and has very low sequence homology to other ifns. it is synthesized predominantly by lymphocytes. other ifns, such as ifn-γ, -δ, -k, -τ, etc., play a variety of roles in cellular regulation but are not directly involved in controlling virus infection. because there are clear biological differences between the two main types of ifn, ifn-α and -β are known as type i ifn, and ifn-γ as type ii ifn. induction of ifn synthesis results from upregulation of transcription from the ifn gene promoters. there are three main mechanisms involved: i virus infection: this mechanism is thought to act by the inhibition of cellular protein synthesis that occurs during many virus infections, resulting in a reduction in the concentration of intracellular repressor proteins and hence in increased ifn gene transcription. in general, rna viruses are potent inducers of ifn while dna viruses are relatively poor inducers; however, there are exceptions to this rule (e.g., poxviruses are very potent inducers). the molecular events in the induction of ifn synthesis by virus infection are not clear. in some cases (e.g., influenza virus), uv-inactivated virus is a potent inducer; therefore, virus replication is not necessarily required. induction by viruses might involve perturbation of the normal cellular environment and/or production of small amounts of double-stranded rna. i double-stranded (ds) rna: all naturally occurring double-stranded rnas (e.g., reovirus genomes) are potent inducers of ifn, as are synthetic molecules (e.g., poly i:c); therefore, this process is independent of nucleotide sequence. single-stranded rna and doublestranded dna are not inducers. this mechanism of induction is thought to depend on the secondary structure of the rna rather than any particular nucleotide sequence. i metabolic inhibitors: compounds that inhibit transcription (e.g., actinomycin d) or translation (e.g., cycloheximide) result in induction of ifn. tumor promoters such as tetradecanoyl phorbol acetate or dimethyl sulfoxide are also inducers. their mechanism of action remains unknown but they almost certainly act at the level of transcription. the effects of ifns are exerted via specific receptors that are ubiquitous on nearly all cell types (therefore, nearly all cells are potentially ifn responsive). there are distinct receptors for type i and type ii ifn, each of which consists of two polypeptide chains. binding of ifn to the type i receptor activates a specific cytoplasmic tyrosine kinase (janus kinase, or jak ), which phosphorylates another cellular protein, signal transducer and activator of transcription (stat ). this is transported to the nucleus and turns on transcriptional activation of ifn-responsive genes (including ifn, resulting in amplification of the original signal). binding of ifn to the type ii receptor activates a different cytoplasmic tyrosine kinase (jak ), which phosphorylates the cellular protein stat , leading to transcriptional activation of a different set of genes. the main action of ifns is on cellular regulatory activities and is rather complex. ifn affects both cellular proliferation and immunomodulation. these effects result from the induction of transcription of a wide variety of cellular genes, including other cytokines. the net result is complex regulation of the ability of a cell to proliferate, differentiate, and communicate. this cellregulatory activity itself has indirect effects on virus replication. type i ifn is the major antiviral mechanism-other ifns act as potent cellular regulators, which may have indirect antiviral effects in some circumstances. the effect of ifns on virus infections in vivo is extremely important. animals experimentally infected with viruses and injected with anti-ifn antibodies experience much more severe infections than control animals infected with the same virus. this is because ifns protect cells from damage and death. however, they do not appear to play a major role in the clearance of virus infections-the other parts of the immune response are necessary for this. ifn is a "firebreak" that inhibits virus replication in its earliest stages by several mechanisms. two of these are understood in some detail, but a number of others (in some cases specific to certain viruses) are less well understood. ifns induce transcription of a cellular gene for the enzyme , -oligo a synthetase ( figure . ). there are at least four molecular species of , -oligo a, induced by different forms of ifn. this compound activates an rna-digesting enzyme, rnase l, which digests virus genomic rnas, virus and cellular mrnas, and cellular ribosomal rnas. the end result of this mechanism is a reduction in protein synthesis (due to the degradation of mrnas and rrnas)-therefore the cell is protected from virus damage. the second method relies on the activation of a -kda protein called pkr (figure . ). pkr phosphorylates a cellular factor, eif α, which is required by ribosomes for the initiation of translation. the net result of this mechanism is also the inhibition of protein synthesis and this reinforces the , -oligo a mechanism. a third, well-established mechanism depends on the m x gene, a single-copy gene located on human chromosome , the transcription of which is induced by type i ifn. the product of this gene inhibits the primary transcription of influenza virus but not of other viruses. its method of action is unknown. in addition to these three mechanisms, there met-trna met .elf .gtp (initiation of translation) gdp + "gef" the modified nucleic acid , -oligo a is involved in one of the major mechanism by which ifns counteract virus infections. are many additional recorded effects of ifns. they inhibit the penetration and uncoating of sv and some other viruses, possibly by altering the composition or structure of the cell membrane; they inhibit the primary transcription of many virus genomes (e.g., sv , hsv) and also cell transformation by retroviruses. none of the molecular mechanisms by which these effects are mediated has been fully explained. ifns are a powerful weapon against virus infection, but they act as a blunderbuss rather than a "magic bullet." the severe side effects (fever, nausea, malaise) that result from the powerful cell-regulatory action of ifns means that they will never be widely used for the treatment of trivial virus infections-they are not the cure for the common cold. however, as the cell-regulatory potential of ifns is becoming better understood, they are finding increasing use as a treatment for certain cancers (e.g., the use of ifn-α in the treatment of hairy cell leukemia). current therapeutic uses of ifns are summarized in table . . the longterm prospects for their use as antiviral compounds are less certain, except for possibly in life-threatening infections where there is no alternative therapy (e.g., chronic viral hepatitis). in total, the many innate and adaptive components of the immune system present a powerful barrier to virus replication. simply by virtue of their continued existence, it is obvious that viruses have, over millennia, evolved effective "counter-surveillance" mechanisms in this molecular arms race. as described above, ctls can only respond to foreign antigens presented by mhc-i complexes on the target cell. a number of viruses interfere with mhc-i expression or function to disrupt this process and evade the ctl response. such mechanisms include downregulation of mhc-i expression by adenoviruses and interference with the antigen processing required to form an mhc-iÀantigen complex by herpesviruses. the mhc-ii antigens are essential in the adaptive immune response in order to stimulate the development of antigen-responsive clones of effector cells. again, herpesviruses and papillomaviruses interfere with the processing and surface expression of mhc-iiÀantigen complexes, inhibiting the ctl response. the poxvirus molluscum contagiosum encodes a homologue of mhc-i that is expressed on the surface of infected cells but is unable to bind an antigenic peptide, thus avoiding killing by nk cells that would be triggered by the absence of mhc-i on the cell surface. similar proteins are made by other viruses, such as hhv- (cmv), and herpesviruses in general appear to have a number of sophisticated mechanisms to avoid nk cell killing. see viruses and apoptosis earlier in this chapter. cytokines are secreted polypeptides that coordinate important aspects of the immune response, including inflammation, cellular activation, proliferation, differentiation, and chemotaxis. some viruses are able to inhibit the expression of certain chemokines directly. alternatively, herpesviruses and poxviruses encode "viroceptors"-virus homologues of host cytokine receptors that compete with cellular receptors for cytokine binding but fail to give transmembrane signals. high-affinity binding molecules may also neutralize cytokines directly, and molecules known as "virokines" block cytokine receptors again without activating the intracellular signaling cascade. ifns are cytokines which act as an effective means of curbing the worst effects of virus infections. part of their wide-ranging efficacy results from their generalized, nonspecific effects (e.g., the inhibition of protein synthesis in although direct humoral immunity is less significant than cell-mediated immunity, the antiviral action of adcc and complement make this a worthwhile target to inhibit. the most frequent means of subverting the humoral response is by high-frequency genetic variation of the b-cell epitopes on antigens to which antibodies bind. this is only possible for viruses that are genetically variable (e.g., influenza virus and hiv). herpesviruses use alternative strategies such as encoding viral fc receptors to prevent fc-dependent immune activation. poxviruses, herpesviruses, and some retroviruses encode mimics of normal regulators of complement activation proteins (e.g., secreted proteins that block c convertase assembly and accelerate its decay). poxviruses can also inhibit c polymerization, preventing membrane permeabilization. viruses do not set out to kill their hosts. virus pathogenesis is an abnormal situation of no value to the virus-the vast majority of virus infections are asymptomatic. however, for pathogenic viruses, a number of critical stages in replication determine the nature of the disease they produce. for all viruses, pathogenic or nonpathogenic, the first factor that influences the course of infection is the mechanism and site of entry into the body (figure . ): i the skin: mammalian skin is a highly effective barrier against viruses. the outer layer (epidermis) consists of dead cells and therefore does not support virus replication. very few viruses infect directly by this route unless there is prior injury such as minor trauma or puncture of the barrier, such as insect or animal bites or subcutaneous injections. some viruses that do use this route include hsv and papillomaviruses, although these viruses probably still require some form of disruption of the skin such as small abrasions or eczema. i mucosal membranes: the mucosal membranes of the eye and genitourinary (gu) tract are much more favorable routes of access for viruses to the tissues of the body. this is reflected by the number of viruses that can be sexually transmitted; virus infections of the eye are also quite common (table . ). i alimentary canal: viruses may infect the alimentary canal via the mouth, oropharynx, gut, or rectum, although viruses that infect the gut via the oral route must survive passage through the stomach, an extremely hostile environment with a very low ph and high concentrations of digestive enzymes. nevertheless, the gut is a highly valued prize for viruses-the intestinal epithelium is constantly replicating and a good deal of lymphoid tissue is associated with the gut which provides many opportunities for virus replication. moreover, the constant intake of food and fluids provides ample opportunity for viruses to infect these tissues (table . ). to counteract this problem, the gut has many specific (e.g., secretory antibodies) and nonspecific (e.g., stomach acids and bile salts) defense mechanisms. i respiratory tract: the respiratory tract is probably the most frequent site of virus infection. as with the gut, it is constantly in contact with external virus particles which are taken in during respiration. as a result, the respiratory tract also has defenses aimed at virus infection-filtering of particulate matter in the sinuses and the presence of cells and antibodies of the immune system in the lower regions. viruses that infect the respiratory tract usually come directly from the respiratory tract of others, as aerosol spread is very efficient: "coughs and sneezes spread diseases" (table . ). the natural environment is a considerable barrier to virus infection. most viruses are relatively sensitive to heat, drying, uv light (sunlight), etc., although a few types are quite resistant to these factors. this is particularly important for viruses that are spread via contaminated water or foodstuffsnot only must they be able to survive in the environment until they are ingested by another host, but, as most are spread by the fecalÀoral route, they must also be able to pass through the stomach to infect the gut before being shed in the feces. one way of overcoming environmental stress is to take advantage of a secondary vector for transmission between the primary hosts ( figure . ). as with plant viruses, the virus may or may not replicate while in the vector. viruses without a secondary vector must rely on continued host-to-host transmission and have evolved various strategies to do this (table . ): i horizontal transmission: the direct host-to-host transmission of viruses. this strategy relies on a high rate of infection to maintain the virus population. i vertical transmission: the transmission of the virus from one generation of hosts to the next. this may occur by infection of the fetus before, during, or shortly after birth (e.g., during breastfeeding). more rarely, it may involve direct transfer of the virus via the germ line itself (e.g., retroviruses). in contrast to horizontal transmission, this strategy relies on long-term persistence of the virus in the host rather than rapid propagation and dissemination of the virus. having gained entry to a potential host, the virus must initiate an infection by entering a susceptible cell (primary replication). this initial interaction frequently determines whether the infection will remain localized at the site of entry or spread to become a systemic infection (table . ). in some cases, virus spread is controlled by infection of polarized epithelial cells and the preferential release of virus from either the apical (e.g., influenza virus-a localized infection in the upper respiratory tract) or basolateral (e.g., rhabdoviruses-a systemic infection) surface of the cells (figure . ). following primary replication at the site of infection, the next stage may be spread throughout the host. in addition to direct cellÀcell contact, there are two main mechanisms for spread throughout the host: i via the bloodstream: viruses may get into the bloodstream by direct inoculation-for example, by arthropod vectors, blood transfusion, or intravenous drug abuse (sharing of nonsterilized needles). the virus may travel free in the plasma (e.g., togaviruses, enteroviruses) or in association with red cells (orbiviruses), platelets (hsv), lymphocytes (ebv, cmv), or monocytes (lentiviruses). primary viremia usually precedes and is necessary for the spread of virus to other parts of the body via the bloodstream and is followed by a more generalized, higher titer secondary viremia as the virus reaches the other target tissues or replicates directly in blood cells. i via the nervous system: as above, spread of virus to the nervous system is usually preceded by primary viremia. in some cases, spread occurs directly by contact with neurones at the primary site of infection; in other cases, it occurs via the bloodstream. once in peripheral nerves, the virus can spread to the central nervous system (cns) by axonal transport along neurones. the classic example of this is hsv (see "latent infection," below). viruses can cross synaptic junctions as these frequently contain virus receptors, allowing the virus to jump from one cell to another. the spread of the virus to various parts of the body is controlled to a large extent by its cell or tissue tropism. tissue tropism is controlled partly by the route of infection but largely by the interaction of a virus-attachment protein (vap) with a specific receptor molecule on the surface of a cell (as discussed in chapter ) and has considerable effect on pathogenesis. at this stage, following significant virus replication and the production of virus antigens, the host immune response comes into play. this has already been discussed earlier and obviously has a major impact on the outcome of an infection. to a large extent, the efficiency of the immune response determines the amount of secondary replication that occurs and, hence, the spread to other parts of the body. if a virus can be prevented from reaching tissues where secondary replication can occur, generally no disease results, although there are some exceptions to this. the immune response also plays a large part in determining the amount of cell and tissue damage that occurs as a result of virus replication. as described above, the production of ifns is a major factor in preventing virus-induced tissue damage. the immune system is not the only factor that controls cell death, the amount of which varies considerably for different viruses. viruses may replicate widely throughout the body without any disease symptoms if they do not cause significant cell damage or death. retroviruses do not generally cause cell death, being released from the cell by budding rather than by cell lysis, and cause persistent infections, even being passed vertically to the offspring if they infect the germ line. all vertebrate genomes, including humans, are littered with retrovirus genomes that have been with us for millions of years (chapter ). at present, these ancient virus genomes are not known to cause any disease in humans, although there are examples of tumors caused by them in rodents. conversely, picornaviruses cause lysis and death of the cells in which they replicate, leading to fever and increased mucus secretion, in the case of rhinoviruses, and paralysis or death (usually due to respiratory failure due to damage to the cns resulting, in part, from virus replication in these cells) in the case of poliovirus. the eventual outcome of any virus infection depends on a balance between two processes. clearance is mediated by the immune system (as discussed previously); however, the virus is a moving target that responds rapidly to pressure from the immune system by altering its antigenic composition (whenever possible). the classic example of this phenomenon is influenza virus, which displays two genetic mechanisms that allow the virus to alter its antigenic constitution: i antigenic drift: this involves the gradual accumulation of minor mutations (e.g., nucleotide substitutions) in the virus genome which result in subtly altered coding potential and therefore altered antigenicity, leading to decreased recognition by the immune system. this process occurs in all viruses all the time but at greatly different rates; for example, it is much more frequent in rna viruses than in dna viruses. in response, the immune system constantly adapts by recognition of and response to novel antigenic structures-but it is always one step behind. in most cases, however, the immune system is eventually able to overwhelm the virus, resulting in clearance. i antigenic shift: in this process, a sudden and dramatic change in the antigenicity of a virus occurs owing to reassortment of the segmented virus genome with another genome of a different antigenic type (see chapter ). this results initially in the failure of the immune system to recognize a new antigenic type, giving the virus the upper hand ( figure . ). the occurrence of past antigenic shifts in influenza virus populations is recorded by pandemics (worldwide epidemics; figure . ). these events are marked by the sudden introduction of a new antigenic type of hemagglutinin and/or neuraminidase into the circulating virus, overcoming previous immunity in the human population. previous hemagglutinin/neuraminidase types become resurgent when a sufficiently high proportion of the people who have "immunological memory" of that type have died, thus overcoming the effect of "herd immunity." the other side of the relationship that determines the eventual outcome of a virus infection is the ability of the virus to persist in the host. long-term persistence of viruses results from two main mechanisms. the first is the regulation of lytic potential. the strategy followed here is to achieve the continued survival of a critical number of virus-infected cells (i.e., sufficient to continue the infection without killing the host organism). for viruses that do not usually kill the cells in which they replicate, this is not usually a problem; hence, these viruses tend naturally to cause persistent infections (e.g., retroviruses). for viruses that undergo lytic infection (e.g., herpesviruses), it this chart shows the history of influenza pandemics throughout the twentieth century. the first pandemic of the twenty-first century occurred in and was caused by an h n type virus, although this was not as damaging as earlier pandemics. is necessary to develop mechanisms that restrict virus gene expression and, consequently, cell damage. the second aspect of persistence is the evasion of immune surveillance, discussed above. patterns of virus infection can be divided into a number of different types. abortive infection occurs when a virus infects a cell (or host) but cannot complete the full replication cycle, so this is a nonproductive infection. the outcome of such infections is not necessarily insignificant, for example, sv infection of nonpermissive rodent cells sometimes results in transformation of the cells (see chapter ). this pattern is familiar for many common virus infections (e.g., "colds"). in these relatively brief infections, the virus is usually eliminated completely by the immune system. typically, in acute infections, much virus replication occurs before the onset of any symptoms (e.g., fever), which are the result not only of virus replication but also of the activation of the immune system; therefore, acute infections present a serious problem for the epidemiologist and are the pattern most frequently associated with epidemics (e.g., influenza, measles). these are the converse of acute infections (i.e., prolonged and stubborn). to cause this type of infection, the virus must persist in the host for a significant period. to the clinician, there is no clear distinction among chronic, persistent, and latent infections, and the terms are often used interchangeably. they are listed separately here because, to virologists, there are significant differences in the events that occur during these infections. these infections result from a delicate balance between the virus and the host organism, in which ongoing virus replication occurs but the virus adjusts its replication and pathogenicity to avoid killing the host. in chronic infections, the virus is usually eventually cleared by the host (unless the infection proves fatal), but in persistent infections the virus may continue to be present and to replicate in the host for its entire lifetime. the best-studied example of such a system is lymphocytic choriomeningitis virus (lcmv; an arenavirus) infection in mice (figure . ). mice can be experimentally infected with this virus either at a peripheral site (e.g., a footpad or the tail) or by direct inoculation into the brain. adult mice infected in the latter way are killed by the virus, but among those infected by a peripheral route there are two possible outcomes to the infection: some mice die but others survive, having cleared the virus from the body completely. it is not clear what factors determine the survival or death of lcmv-infected mice, but other evidence shows that the outcome is related to the immune response to the virus. in immunosuppressed adult mice infected via the cns route, a persistent infection is established in which the virus is not cleared (due to the nonfunctional immune system), but, remarkably, these mice are not killed by the virus. if, however, syngeneic lcmv-specific t-lymphocytes (i.e., of the same mhc type) are injected into these persistently infected mice, the animals develop the full pathogenic symptoms of lcmv infection and die. when newborn mice, whose immune systems are immature, are infected via the cns route, they also develop a persistent infection, but, in this case, if they are subsequently injected with syngeneic lcmv-specific t-lymphocytes, they clear the virus and survive the infection. the mechanisms that control these events are not completely understood, but evidently there is a delicate balance between the virus and the host animal and the immune response to the virus is partly responsible for the pathology of the disease and the death of the animals. not infrequently, persistent infections may result from the production of defective-interfering (d.i.) particles (see chapter ). such particles contain a partial deletion of the virus genome and are replication defective, but they are maintained and may even tend to accumulate during infections because they can replicate in the presence of replication-competent helper virus. the production of d.i. particles is a common consequence of virus infection of animals, particularly by rna viruses, but also occurs with dna viruses and plant viruses and can be mimicked in vitro by continuous high-titer passage of virus. although not able to replicate themselves independently, d.i. particles are not necessarily genetically inert and may alter the course of an infection by recombination with the genome of a replication-competent virus. the presence of d.i. particles can profoundly influence the course and the outcome of a virus infection. in some cases, they appear to moderate pathogenesis, whereas in others they potentiate it, making the symptoms of the disease much more severe. moreover, as d.i. particles effectively cause restricted gene expression (because they are genetically deleted), they may also result in a persistent infection by a virus that normally causes an acute infection and is rapidly cleared from the body. in a latent state, the virus is able to downregulate its gene expression and enter an inactive state with strictly limited gene expression and without ongoing virus replication. latent virus infections typically persist for the entire life of the host. an example of such an infection in humans is hsv. infection of sensory nerves serving the mucosa results in localized primary replication. subsequently, the virus travels via axon transport mechanisms further into the nervous system. there, it hides in dorsal root ganglia, such as the trigeminal ganglion, establishing a truly latent infection. the nervous system is an immunologically privileged site and is not patrolled by the immune system in the same way as the rest of the body, but the major factor in latency is the ability of the virus to restrict its gene expression. this eliminates the possibility of recognition of infected cells by the immune system. restricted gene expression is achieved by tight regulation of α-gene expression, which is an essential control point in herpesvirus replication (chapter ). in the latent state, hsv makes an . -kb rna transcript called the latent rna or latency-associated transcript (lat). the lat is broken down into even smaller strands called micrornas (mirnas), and these block the production of proteins which reactivate the virus. drugs which block production of these mirnas could in theory "wake up" all the dormant viruses, making them vulnerable to the immune system and to antiviral therapy, and this raises the eventual possibility of a cure for herpes infections. expression of the lat promotes neuronal survival after hsv infection by inhibiting apoptosis. this anti-apoptosis function could promote reactivation by: i providing more latently infected neurons for future reactivations, i protecting neurons in which reactivation occurs, i protecting previously uninfected neurons during a reactivation. when reactivated by some provocative stimulus, hsv travels down the sensory nerves to cause peripheral manifestations such as cold sores or genital ulcers. it is not altogether clear what constitutes a provocative stimulus, but there are many possible alternatives, including psychological and physical factors. periodic reactivation establishes the pattern of infection, with sporadic, sometimes very painful reappearance of disease symptoms for the rest of the host's life. even worse than this, immunosuppression later in life can cause the latent infection to flare up (which indicates that the immune system normally has a role in helping to suppress these latent infections), resulting in a very severe, systemic, and sometimes life-threatening infection. in a manner somewhat similar to herpesviruses, infection by retroviruses may result in a latent infection. integration of the provirus into the host genome certainly results in the persistence of the virus for the lifetime of the host organism and may lead to an episodic pattern of disease. in some ways, acquired immunodeficiency syndrome (aids), which results from hiv infection, shows aspects of this pattern of infection. the pathogenesis of aids is discussed in detail in chapter . there are two aspects of the response to the threat of virus diseases: first, prevention of infection, and second, treatment of the disease. the former strategy relies on two approaches: public and personal hygiene, which perhaps plays the major role in preventing virus infection (e.g., provision of clean drinking water and disposal of sewage; good medical practice such as the sterilization of surgical instruments) and vaccination, which makes use of the immune system to combat virus infections. most of the damage to cells during virus infections occurs very early, often before the clinical symptoms of disease appear. this makes the treatment of virus infection very difficult; therefore, in addition to being less expensive, prevention of virus infection is undoubtedly better than cure. to design effective vaccines, it is important to understand both the immune response to virus infection and the stages of virus replication that are appropriate targets for immune intervention. to be effective, vaccines must stimulate as many of the body's defense mechanisms as possible. in practice, this usually means trying to mimic the disease without causing pathogenesis-for example, the use of live attenuated viruses as vaccines such as nasally administered influenza vaccines and orally administered poliovirus vaccines. to be effective, it is not necessary to get % uptake of vaccine. "herd immunity" results from the break in transmission of a virus that occurs when a sufficiently high proportion of a population has been vaccinated. this strategy is most effective where there is no alternative host for the virus (e.g., measles) and in practice is the situation that usually occurs as it is impossible to achieve % coverage with any vaccine. however, this is a risky business; if protection of the population falls below a critical level, epidemics can easily occur. synthetic vaccines are short, chemically synthesized peptides. the major disadvantage with these molecules is that they are not usually very effective immunogens and are very costly to produce. however, because they can be made to order for any desired sequence, they have great theoretical potential, but none are currently in clinical use. recombinant vaccines are produced by genetic engineering. such vaccines have been already produced and are better than synthetic vaccines because they tend to give rise to a more effective immune response. some practical success has already been achieved with this type of vaccine. for example, vaccination against hepatitis b virus (hbv) used to rely on the use of australian antigen (hbsag) obtained from the serum of chronic hbv carriers. this was a very risky practice indeed (because hbv carriers are often also infected with hiv). a completely safe recombinant hbv vaccine produced in yeast is now used. dna vaccines are the newest type of vaccine and consist of only a dna molecule encoding the antigen(s) of interest and, possibly, costimulatory molecules such as cytokines. the concept behind these vaccines is that the dna component will be expressed in vivo, creating small amounts of antigenic protein that serve to prime the immune response so that a protective response can be rapidly generated when the real antigen is encountered. in theory, these vaccines could be manufactured quickly and should efficiently induce both humoral and cellmediated immunity. initial clinical studies have indicated that there is still some way to go until this experimental technology becomes a practical proposition. subunit vaccines consist of only some components of the virus, sufficient to induce a protective immune response but not enough to allow any danger of infection. in general terms, they are completely safe, except for very rare cases in which adverse immune reactions may occur. unfortunately, they also tend to be the least effective and most expensive type of vaccine. the major technical problems associated with subunit vaccines are their relatively poor antigenicity and the need for new delivery systems, such as improved carriers and adjuvants. virus vectors are recombinant virus genomes genetically manipulated to express protective antigens from (unrelated) pathogenic viruses. the idea here is to utilize the genome of a well-understood, attenuated virus to express and present antigens to the immune system. many different viruses offer possibilities for this type of approach. one of the most highly developed systems so far is based on the vv genome. this virus has been used to vaccinate millions of people worldwide in the campaign to eradicate smallpox and is generally a safe and effective vehicle for antigen delivery. such vaccines are difficult to produce. no human example is clearly successful yet, although many different trials are currently underway, but vvÀrabies recombinants have been used to eradicate rabies in european fox populations. vv-based vaccines have advantages and disadvantages for use in humans-a high percentage of the human population has already been vaccinated during the smallpox eradication campaign, and this lifelong protection may result in poor response to recombinant vaccines. although generally safe, vv is dangerous in immunocompromised hosts, thus it cannot be used in hiv-infected individuals. a possible solution to these problems may be to use avipoxvirus vectors (e.g., fowlpox or canarypox) as "suicide vectors" that can only establish abortive infections of mammalian cells and offer the following advantages: i expression of high levels of foreign proteins, i no danger of pathogenesis (abortive infection), i no natural immunity in humans (avian virus). inactivated vaccines are produced by exposing the virus to a denaturing agent under precisely controlled conditions. the objective is to cause loss of virus infectivity without loss of antigenicity. obviously, this involves a delicate balance. however, inactivated vaccines have certain advantages, such as generally being effective immunogens (if properly inactivated), being relatively stable, and carrying little or no risk of vaccine-associated virus infection (if properly inactivated, but accidents can and do occur). the disadvantage of these vaccines is that it is not possible to produce inactivated vaccines for all viruses, as denaturation of virus proteins may lead to loss of antigenicity (e.g., measles virus). although relatively effective, "killed" vaccines are sometimes not as effective at preventing infection as "live" virus vaccines, often because they fail to stimulate protective mucosal and cell-mediated immunity to the same extent. a more recent concern is that these vaccines contain virus nucleic acids, which may themselves be a source of infection, either of their own accord (e.g., ( )sense rna virus genomes) or after recombination with other viruses. virus vaccines do not have to be based on virion structural proteins. the effectiveness of attenuated vaccines relies on the fact that a complete spectrum of virus proteins, including nonstructural proteins, is expressed and gives rise to cell-mediated immune responses. live attenuated virus vaccines are viruses with reduced pathogenicity used to stimulate an immune response without causing disease. the vaccine strain may be a naturally occurring virus (e.g., the use of cowpox virus by edward jenner to vaccinate against smallpox) or artificially attenuated in vitro (e.g., the oral poliomyelitis vaccines produced by albert sabin). the advantage of attenuated vaccines is that they are good immunogens and induce long-lived, appropriate immunity. set against this advantage are their many disadvantages. they are often biochemically and genetically unstable and may either lose infectivity (becoming worthless) or revert to virulence unexpectedly. despite intensive study, it is not possible to produce an attenuated vaccine to order, and there appears to be no general mechanism by which different viruses can be reliably and safely attenuated. contamination of the vaccine stock with other, possibly pathogenic viruses is also possible-this was the way in which sv was first discovered in oral poliovirus vaccine in . inappropriate use of live virus vaccines, for example, in immunocompromised hosts or during pregnancy may lead to vaccineassociated disease, whereas the same vaccine given to a healthy individual may be perfectly safe. despite these difficulties, vaccination against virus infection has been one of the great triumphs of medicine during the twentieth century. most of the success stories result from the use of live attenuated vaccines-for example, the use of vv against smallpox. on may , , the world health organization (who) officially declared smallpox to be completely eradicated, the first virus disease to be eliminated from the world. the who aims to eradicate a number of other virus diseases such as poliomyelitis and measles, but targets for completion of these programs have undergone much slippage due to the formidable difficulties involved in a worldwide undertaking of this nature. although prevention of infection by prophylactic vaccination is much the preferred option, postexposure therapeutic vaccines can be of great value in modifying the course of some virus infections. examples of this include rabies virus, where the course of infection may be very long and there is time for postexposure vaccination to generate an effective immune response and prevent the virus from carrying out the secondary replication in the cns that is responsible for the pathogenesis of rabies. other potential examples can be found in virus-associated tumors, such as hpv-induced cervical carcinoma. most existing virus vaccines are directed against viruses which are relatively antigenically invariant, for example, measles, mumps, and rubella viruses, where this is only one unchanging serotype of the virus. viruses whose antigenicity alters continuously are a major problem in terms of vaccine production, and the classic example of this is influenza virus (see earlier). in response to this problem, new technologies such as reverse genetics could be used to improve and to shorten the lengthy process of preparing vaccines. rna virus genomes can be easily manipulated as dna clones to contain nucleotide sequences which match currently circulating strains of the virus. infectious virus particles are rescued from the dna clones by introducing these into cells. seed viruses for distribution to vaccine manufacturers can be produced in as little as À weeks, a much shorter time than the months this process takes in conventional vaccine manufacture. using the same technology, universal influenza vaccines containing crucial virus antigens expressed as fusion proteins with other antigenic molecules could feasibly be produced, making the requirement for constant production of new influenza vaccines obsolete. although this has not yet been achieved, advances toward these goals are being made. the explosion of molecular techniques described in earlier chapters is now being used to inform vaccine design (as well as the design of antiviral drugs) rather than simply relying on trial-and-error approaches. however, developing safe and effective vaccines remains one of the greatest challenges facing virology. rna interference (rnai) is a posttranscriptional gene silencing process that occurs in organisms from yeast to humans. in mammals, small rnas include small interfering rnas (sirnas) and mirnas. sirnas, with perfect base complementarity to their targets, activate rnai-mediated cleavage of the target mrnas, while mirnas generally induce rna decay and/or translation inhibition of target genes (figure . ). mammals, including humans, encode hundreds or thousands of mirnas. some viruses with eukaryotic hosts also encode mirnas. herpesviruses in particular encode multiple mirnas; most other nuclear dna viruses encode one or two mirnas. rna viruses and cytoplasmic dna viruses appear to lack any mirnas. virus mirnas may serve two major functions. several have been shown to inhibit the expression of cellular factors that play a role in cellular innate or adaptive antiviral immune responses, so reducing the effectiveness of the immune response. alternatively, virus mirnas may downregulate the expression of virus proteins, including key immediate-early or early regulatory proteins. in hsv, mirnas are expressed at high levels during latency, but not during productive replication, so their action is thought to stabilize latency. recently there have been controversial claims that mirna can exert antiviral activity in mice, at least in some circumstances. antiviral sirna activity is only seen in stem cells and in newborn mice and many scientists think sirna is not a major part of the innate immune system in adult animals. there is evidence that sirna may be turned on in responses to virus infection, but rather than acting directly against the virus, it may be used to regulate the ifn response. that still leaves the fact that in mammals mirna is a powerful regulator of gene expression, including virus genes. many viruses use mirna to control their own gene expression and that of their host cells. on infection of a host cell, viruses encounter a range of mirna species, many of which have been shown to restrict virus gene expression. thus they have had to evolve a range of mechanisms to evade mirna restriction is the same way that they have evolved other mechanisms to mitigate the impact of innate immunity. these include: i blocking mirna function i avoiding utr targets complementary to cellular mirnas i evolving very short utrs i evolving structured utrs rnai expression can be induced by dsrna, and this approach has been used to investigate gene function in a variety of organisms including plants and insects. however, this method cannot be applied to mammalian cells as dsrnas longer than nucleotides induces the ifn response (see earlier), which results in the degradation of mrnas and causes a global inhibition of translation. to circumvent this problem, chemically synthesized sirnas or plasmid-vectors manipulated to produce short hairpin rna molecules can be used to investigate gene function in mammals. in the future it may be feasible to treat virus diseases by shutting off gene expression by directing the degradation of specific mrnas, and many clinical trials are currently underway. although rna interference has been used widely in cultured cells to inhibit virus replication and to probe biological pathways, considerable problems must be overcome before it becomes a useful therapy, including the development of suitable delivery and targeting systems and solving the issue of stability in vivo. the natural world is a soup of bacteriophages. so how do bacteria survive against this constant onslaught? with their own form of adaptive immunity. crisprs (clustered regularly interspaced short palindromic repeats) are short, direct repeats of dna base sequences. each crispr contains a series of bases followed by the same series in reverse (a palindrome) and then by or so base pairs known as "spacer" dna. the spacers are short segments of virus or plasmid dna. crisprs are found in the genomes of approximately % of bacteria and % of archaea. crispr loci are typically located on the bacterial chromosome, although some are found on plasmids. bacteria may contain more than one crispr locus-up to in some cases. crisprs function as a sort of prokaryotic immune system, conferring resistance to exogenous genetic elements such as plasmids and bacteriophages. intriguingly, the crispr system provides a form of acquired immunity, allowing the cell to remember and respond to sequences it has encountered before. how do crisprs work? crisprs are often adjacent to cas (crispr-associated) genes. the cas genes encode a large and heterogeneous family of proteins including nucleases, helicases, polymerases, and polynucleotide-binding proteins, forming the crispr/cas system. (note: cas genes, cas the proteins encoded by these genes.) the interesting bits are the unique spacer elements (derived from exogenous sequences such as viruses and plasmids) rather than the repeats themselves. the spacer elements originate from exogenous dna the bacterium (or its ancestors) has previous encountered-they are typically pieces of phage or plasmid dna. this allows the cell to recognize these sequences via base homology if they enter the cell again, for example, if the bacterium is infected with a bacteriophage whose genome contains this sequence: . cas-encoded nucleases cleave invading dna into short pieces. . other cas proteins allow a fragment of the foreign dna to be incorporated as a novel repeat-spacer unit at the leader end of the crispr site. . the crispr array is then transcribed to form a pre-crispr rna (crrna) transcript. . the pre-crrna is cleaved within the repeat sequence by cas proteins to generate small crispr rnas, crrnas. . the crrnas to work in a similar way to rnai in eukaryotic cells, although there are important differences in the machinery by which this happens. crisprs are an important way in which bacteria are able to survive constant attack by bacteriophages in the environment, but phages have been around for a very long time too, so they must have found ways of counteracting the crispr system. eukaryotic viruses may express inhibitors such as dsrnabinding proteins that interfere with the rna silencing machinery, whereas bacteriophages acquire mutations or recombine the sequence corresponding to the crispr spacer to avoid recognition in an analogous way to how viruses of eukaryotes acquire mutations in b-cell and t-cell epitopes in proteins to evade the mammalian immune system. so who (apart from bacteria) cares about crisprs? altering the spacer via genetic manipulation can provide novel phage resistance, whereas spacer deletion results in loss of phage resistance. although crisprs originate in bacteria, they also work in eukaryotic cells if introduced by genetic engineering. this provides a convenient way of targeting genes in cells, including human cells. recent work suggests that crisprs might also be involved in control of bacterial gene expression as well as in immunity. we will undoubtedly see much more widespread use of crisprs in biotechnology over the next few years. phage therapy, the use of bacteriophages to treat or prevent disease, stretches back a century to the earliest days of the discovery of phages. long before the discovery of antibiotics, the thought that viruses which lyse bacteria could be used to treat diseases was highly attractive. yet this idea has never become a widespread practical reality. devotees of phage therapy defend their cherished belief with almost religious fervor, but there are serious obstacles to be overcome, such as the narrow host range of most phages (a few strains of bacteria, not even an entire species) and the speed at which bacteria develop resistance to infection. as the spectrum of clinically useful antibiotics dwindles, phage therapy increases in attractiveness, but is unlikely ever to replace the antibiotic golden era of disease treatment we are now leaving behind. another aspect of "virotherapy" is the growing interest in oncolytic virusesviruses engineered to kill only cancer cells. the usefulness of many different types of virus has been investigated, including adenoviruses, herpesviruses, reoviruses, and poxviruses. although safety is a concern even in patients with terminal illnesses, this is one area of medical research where optimism is considerable. many clinical trials are underway at it seems certain that this approach to cancer treatment will eventually become more common, possibly as an adjunct to other forms of therapy such as surgery, drugs, and radiotherapy. viruses have also developed as gene delivery systems for the treatment of inherited and acquired diseases. gene therapy offers: i delivery of large biomolecules to cells, i the possibility of targeting delivery to a specific cell type, i high potency of action due to replication of the vector, i potential to treat certain diseases (such as head and neck cancers and brain tumors) that respond poorly to other therapies or may be inoperable. the very first retroviral and adenoviral vectors were characterized in the early s. the first human trial to treat children with immunodeficiency resulting from a lack of the enzyme adenosine deaminase began in and showed encouraging although not completely successful results. like most of the initial attempts, this trial used recombinant retrovirus genomes as vectors. in , the first successful gene therapy for motor neurons and skin cells was reported, while the first phase three (widespread) gene therapy trial was begun in . in , the first successful treatment of a patient with severe combined immunodeficiency disease (scid) was reported, but, sadly, the first death due to a virus vector also occurred, and in the occurrence of leukemias due to oncogenic insertion of a retroviral vector was seen in some scid patients undergoing treatment. several different viruses are being tested as potential vectors ( integrate into cellular dna at high frequency to establish a stable latent state; not associated with any known disease; vectors can be constructed that will not express any viral gene products. only b kb of dna can be packaged into the parvovirus capsid, and some virus sequences must be retained for packaging; integration into host-cell dna may potentially have damaging consequences. herpesviruses relatively easy to manipulate in vitro; grows to high titers; long-term persistence in neuronal cells without integration. (long-term) pathogenic consequences? integrate into cell genome, giving long-lasting (lifelong?) expression of recombinant gene. difficult to grow to high titer and purify for direct administration (patient cells must be cultured in vitro); cannot infect nondividing cells-most somatic cells (except lentiviruses?); insertional mutagenesis/activation of cellular oncogenes. can express high levels of foreign proteins. avipoxvirus vectors (e.g., fowlpox or canarypox) are "suicide vectors" that undergo abortive replication in mammalian cells so there is no danger of pathogenesis and no natural immunity in humans. a high proportion of the human population has already been vaccinated-lifelong protection may result in poor response to recombinant vaccines (?). dangerous in immunocompromised hosts. the alternative to vaccination is to attempt to treat virus infections using drugs that block virus replication (table . ). historically, the discovery of antiviral drugs was largely down to luck. spurred on by successes in the treatment of bacterial infections with antibiotics, drug companies launched huge blind-screening programs to identify chemical compounds with antiviral activity, with relatively little success. the key to the success of any antiviral drug lies in its specificity. almost any stage of virus replication can be a target for a drug, but the drug must be more toxic to the virus than the host. this is measured by the chemotherapeutic index, given by: dose of drug that inhibits virus replication dose of drug that is toxic to host the smaller the value of the chemotherapeutic index, the better. in practice, a difference of several orders of magnitude between the two toxicity values is usually required to produce a safe and clinically useful drug. modern technology, including molecular biology and computer-aided design of chemical compounds, allows the deliberate design of drugs, but it is necessary to "know your enemy"-to understand the key steps in virus replication that might be inhibited. any of the stages of virus replication can be a target for antiviral intervention. the only requirements are: i the process targeted must be essential for replication. i the drug is active against the virus but has "acceptable toxicity" to the host organism. what degree of toxicity is "acceptable" clearly varies considerably-for example, between a cure for the common cold, which might be sold over the counter and taken by millions of people, and a drug used to treat fatal virus infections such as aids. the attachment phase of replication can be inhibited in two ways, by agents that mimic the vap and bind to the cellular receptor or by agents that mimic the receptor and bind to the vap. synthetic peptides are the most logical class of compound to use for this purpose. while this is a promising line of research, there are considerable problems with the clinical use of these substances, primarily the high cost of synthetic peptides and the poor pharmacokinetic properties of many of these synthetic molecules. it is difficult to target specifically the penetration/uncoating stages of virus replication as relatively little is known about them. uncoating in particular is largely mediated by cellular enzymes and is therefore a poor target for intervention, although, like penetration, it is often influenced by one or more virus proteins. amantadine and rimantadine are two drugs that are active against influenza a viruses. the action of these closely related agents is to block cellular membrane ion channels. the target for both drugs is the influenza matrix protein (m ), but resistance to the drug may also map to the hemagglutinin gene. this biphasic action results from the inability of drug-treated cells to lower the ph of the endosomal compartment (a function normally controlled by the m gene product), which is essential to induce conformational changes in the ha protein to permit membrane fusion (see chapter ). many viruses have evolved their own specific enzymes to replicate virus nucleic acids preferentially at the expense of cellular molecules. there is often sufficient specificity in virus polymerases to provide a target for an antiviral agent, and this method has produced the majority of the specific antiviral drugs currently in use. the majority of these drugs function as polymerase substrates (i.e., nucleoside/nucleotide) analogues, and their toxicity varies considerably, from some that are well tolerated (e.g., acyclovir) to others that are quite toxic (e.g., azidothymidine or azt). there is a problem with the pharmacokinetics of these nucleoside analogues in that their typical serum half-life is to hours. nucleoside analogues are in fact pro-drugs, as they must be phosphorylated before becoming effective-which is key to their selectivity: i acyclovir is phosphorylated by hsv thymidine kinase times more efficiently than by cellular enzymes. i ganciclovir is times more effective against cmv than acyclovir but must be phosphorylated by a kinase encoded by cmv gene ul before it becomes pharmaceutically active. i other nucleoside analogues derived from these drugs and active against herpesviruses have been developed (e.g., valciclovir and famciclovir). these compounds have improved pharmacokinetic properties, such as better oral bioavailability and longer half-lives. in addition to these there are a number of nonnucleoside analogues that inhibit virus polymerases; for example, foscarnet is an analogue of pyrophosphate that interferes with the binding of incoming nucleotide triphosphates by virus dna polymerases. ribavirin is a compound with a very wide spectrum of activity against many different viruses, especially against many (À)sense rna viruses. this drug acts as an rna mutagen, causing a -fold increase in mutagenesis of rna virus genomes and a % loss in virus infectivity after a single round of virus infection in the presence of ribavirin. ribavirin is thus quite unlike the other nucleoside analogues described above, and its use might become much more widespread in the future if it were not for the frequency of adverse effects associated with this drug. virus gene expression is less amenable to chemical intervention than genome replication, because viruses are much more dependent on the cellular machinery for transcription, mrna splicing, cytoplasmic export, and translation than for replication. to date, no clinically useful drugs that discriminate between virus and cellular gene expression have been developed. as with penetration and uncoating, for the majority of viruses the processes of assembly, maturation, and release are poorly understood and therefore have not yet become targets for antiviral intervention, with the exception of the antiinfluenza drugs oseltamivir and zanamivir, which are inhibitors of influenza virus neuraminidase. neuraminidase is involved in the release of virus particles budding from infected cells, and these drugs are believed to reduce the spread of virus to other cells. the most striking aspect of antiviral chemotherapy is how few clinically useful drugs are available. as if this were not bad enough, there is also the problem of drug resistance to consider. in practice, the speed and frequency with which resistance arises when drugs are used to treat virus infections varies considerably and depends largely on the biology of the virus involved rather than on the chemistry of the compound. to illustrate this, two extreme cases are described here. acyclovir, used to treat hsv infections, is easily the most widely used antiviral drug. this is particularly true in the case of genital herpes, which causes painful recurrent ulcers on the genitals. it is estimated that to million people suffer from this condition in the united states. fortunately, resistance to acyclovir arises infrequently. this is partly due to the high fidelity with which the dna genome of hsv is copied (chapter ). mechanisms that give rise to acyclovir resistance include: i hsv pol gene mutants that do not incorporate acyclovir i hsv thymidine kinase (tk) mutants in which tk activity is absent (tk ) or reduced or shows altered substrate specificity strangely, it is possible to find mutations that give rise to each of these phenotypes with a frequency of to in clinical hsv isolates. the discrepancy between this and the very low frequency with which resistance is recorded clinically is probably explained by the observation that most pol/tk mutants appear to be attenuated (e.g., tk mutants of hsv do not reactivate from the latent state). conversely, azt treatment of hiv infection is much less effective. in untreated hiv-infected individuals, azt produces a rise in the numbers of cd cells within À weeks. however, this beneficial effect is transient; after weeks, cd t-cell counts generally revert to baseline. this is due partly to the development of azt resistance in treated hiv populations and to the toxicity of azt on hematopoiesis, as the chemotherapeutic index of azt is much worse than that of acyclovir. azt resistance is initiated by the acquisition of a mutation in the hiv reverse transcriptase (rt) gene at codon . in conjunction with two to three additional mutations in the rt gene, a fully azt-resistant phenotype develops. after weeks of treatment, À % of azt-treated patients develop at least one of these mutations. this high frequency is due to the error-prone nature of reverse transcription (chapter ). because of the large number of replicating hiv genomes in infected patients (chapter ), many mistakes occur continuously. it has been shown that the mutations that confer resistance already exist in untreated virus populations. thus, treatment with azt does not cause but merely selects these resistant viruses from the total pool. with other anti-rt drugs, such as didanosine (ddi), a resistant phenotype can result from a single base pair change, but ddi has an even lower therapeutic index than azt, and relatively low levels of resistance can potentially render this drug useless. however, some combinations of resistant mutations may make it difficult for hiv to replicate, and resistance to one rt inhibitor may counteract resistance to another. the current strategy for therapy of hiv infection is known as haart (highly active antiretroviral therapy) and employs combinations of different drugs such as a protease inhibitor plus two nucleoside rt inhibitors. molecular mechanisms of resistance and drug interactions are both important to consider when designing combination regimes: i combinations such as azt ddi or azt tc have antagonistic patterns of resistance and are effective. i combinations such as ddc tc that show cross-reactive resistance should be avoided. certain protease inhibitors affect liver function and can favorably affect the pharmacokinetics of rt inhibitors taken in combination. other potential benefits of combination antiviral therapy include lower toxicity profiles and the use of drugs that may have different tissue distributions or cell tropisms. combination therapy may also prevent or delay the development of drug resistance. combinations of drugs that can be employed include not only small synthetic molecules but also "biological response modifiers" such as interleukins and ifns. virus infection is a complex, multistage interaction between the virus and the host organism. the course and eventual outcome of any infection are the result of a balance between host and virus processes. host factors involved include exposure to different routes of virus transmission and the control of virus replication by the immune response. virus processes include the initial infection of the host, spread throughout the host, and regulation of gene expression to evade the immune response. medical intervention against virus infections includes the use of vaccines to stimulate the immune response and drugs to inhibit virus replication. molecular biology is stimulating the production of a new generation of antiviral drugs and vaccines. further reading rna-based viral immunity initiated by the dicer family of host immune receptors viral subversion of apoptotic enzymes: escape from death row understanding hiv- latency provides clues for the eradication of long-term reservoirs implications of high rna virus mutation rates: lethal mutagenesis and the antiviral drug ribavirin five questions about viruses and micrornas how do viruses avoid inhibition by endogenous cellular micrornas? clinical applications of dna vaccines: current progress modulation of natural killer cell activity by viruses pros and cons of phage therapy microrna in the immune system, microrna as an immune system crispr interference: rna-directed adaptive immunity in bacteria and archaea new viruses for cancer therapy: meeting clinical needs how do plant viruses induce disease? interactions and interference with host components interferons and viruses: an interplay between induction, signalling, antiviral responses and virus countermeasures antiviral drugs for viruses other than human immunodeficiency virus plant virus ecology the prospects and challenges of universal vaccines for influenza viral subversion of the immune system viral tricks to grid-lock the type i interferon system oncolytic viruses as anticancer vaccines. front. oncol. , key: cord- -djxoiytq authors: gran, bruno; hemmer, bernhard; vergelli, marco; mcfarland, henry f.; martin, roland title: molecular mimicry and multiple sclerosis: degenerate t‐cell recognition and the induction of autoimmunity date: - - journal: ann neurol doi: . / - ( ) : < ::aid-ana > . .co; -q sha: doc_id: cord_uid: djxoiytq various mechanisms have been proposed for the initiation of autoimmune responses by autoreactive t‐cell clones. one of these, the molecular mimicry hypothesis, postulates that myelin‐reactive t‐cell clones are activated by foreign antigens. until recently, sequence homology between self‐ and foreign antigens was considered necessary for cross‐recognition to occur in multiple sclerosis. this article reviews current progress in t‐cell receptor immunology that led to modify this view and proposes a role for degenerate t‐cell antigen recognition in the induction of autoimmunity. ann neurol ; : – both clinical and experimental evidence supports the hypothesis that immune mechanisms are involved in the pathogenesis of inflammatory demyelination in multiple sclerosis (ms) and that autoreactive t lymphocytes initiate the process of central nervous system (cns) myelin damage. molecular mimicry has been proposed as a way by which an autoimmune response to myelin antigens may be initiated. , according to this model, self-reactive t cells may be activated by cross-reactivity with infectious agents that "mimic" or share immunological epitopes with the autoantigen. sequence homology between a self-antigen such as myelin basic protein (mbp) and a foreign antigen such as a viral protein was initially considered a requirement for such cross-recognition. recent studies on the mechanisms of t-cell activation have shown that, at least for some t-cell clones, antigen recognition is much more "degenerate" than previously appreciated, and that sequence homology is not necessary for cross-reactivity. this article will focus on current advances in basic t-cell receptor immunology that bear on the occurrence of autoimmunity in the cns. various fields of research, such as pathology, epidemiology, immunogenetics, pharmacology, brain imaging, and studies of experimental autoimmune encephalomyelitis (eae), an animal model resembling ms, have contributed complementary evidence that ms is an immune-mediated disease. the presence of perivenous inflammatory infiltrates of cd ϩ , cd ϩ , and ␥/␦ t-cell receptor ϩ (tcr ϩ ) t lymphocytes, , plasma cells, and macrophages suggests that these cell types contribute to myelin damage in ms lesions. the association with certain alleles of mhc class ii genes (ie, dr dw and dqw in whites, dr and dr in japanese, and dr in sardinians ) suggests a role of immunogenetic background in ms susceptibility, and was recently supported by two of three large-scale genome screenings. - this suggests a possible role of major histocompatibility locus (mhc) class iidependent t-cell responses in the pathogenesis of ms. immune system involvement is also suggested by the clinical and biological response to immunomodulatory and immunosuppressive treatments [ ] [ ] [ ] as well as worsening of the disease by interferon-␥. although the combined evidence points to an immunological basis for ms, the origin of autoreactivity has remained uncertain. the molecular mimicry hypothesis, an important conceptual framework for how autoreactivity could be initiated, was first tested in experimental allergic encephalomyelitis (eae), an animal model of ms that can resemble ms both clinically and pathologically. [ ] [ ] [ ] discussing the immunology of ms is difficult without briefly recapitulating the relevant findings that have emerged from eae research. eae can be induced in susceptible animals by active immunization with brain homogenate, myelin antigens such as mbp or proteolipid protein (plp) or peptides derived from these antigens. because eae resembles ms and is induced by cd ϩ t cells, it has stimulated research efforts to clarify the role of cd ϩ t cells in ms. this disease model has provided insight into the pathogenic "steps" that may be relevant to ms, including ( ) genetic susceptibility, ( ) priming and activation of myelin-specific t cells, ( ) interaction of autoreactive t cells with endothelium and migration into the cns, and ( ) recognition of myelin antigens and initiation of inflammatory or demyelinating damage (fig ) . autoreactive t cells have been shown to be part of the mature immune repertoire of healthy, nonimmunized animals. genetic control of the frequency and function of such t cells in different animal strains may play an important role in disease susceptibility. for potential autoreactivity to become overt autoimmunity, however, myelinreactive t cells must be activated by immunization with myelin antigens or strong unspecific stimuli such as bacterial superantigens. the molecular mimicry hypothesis of autoimmunity proposes that cross-reactive foreign antigens can activate autoimmune t cells, and subsequently mediate pathological and clinical damage (fig ) . once activated in the periphery, autoreactive t cells can cross the blood-brain barrier (bbb), infiltrate the cns, recognize myelin antigens, and damage oligodendrocytes and the myelin sheath by various effector mechanisms. with the exception of active immunization, similar pathogenic steps have been proposed for ms. indeed, the presence of mbp-and plp-reactive t cells has been extensively documented in the mature repertoire of both ms patients and healthy controls. [ ] [ ] [ ] [ ] [ ] based on epidemiological data linking viral infections to ms exacerbations , and possibly also to the cause of the disease, viral antigens are attractive candidates for initiating autoimmune mechanisms through molecular mimicry. although it is as yet unknown how exactly a virus initiates autoimmunity, the molecular mimicry hypothesis provides an elegant conceptual framework for how autoreactivity may be triggered and will therefore be discussed herein. one cannot understand molecular mimicry without describing the mechanisms underlying antigen recognition by t lymphocytes. unlike antibodies, which react with complex protein or polysaccharide structures in particulate form or in solution, the tcr recognizes short peptide fragments derived from larger proteins in the context of self-mhc. t lymphocytes respond to short peptides generated by intracellular proteolytic degradation of antigenic proteins by antigen-presenting cells (apcs). ten-to -amino acid-long peptides are loaded onto mhc molecules and transported to the surface of apcs where they can be recognized as a complex by specific cd ϩ t cells (class ii mhc- restricted antigen recognition) (fig ) . after recognition of antigen/mhc, t cells become activated, undergo clonal expansion, and acquire effector functions such as cytokine production and cytotoxicity. the production of cytokines such as interferon-␥ and tumor necrosis factor-␣ and ␤ by a subset of cd ϩ t cells (t-helper type cells) may be important in mediating part of the immunological damage in ms. after clonal expansion, subpopulations of the expanded cell clones enter the pool of circulating memory t cells, whose requirements for subsequent activation by antigenic challenge appear to be lower than those of naive t cells. does molecular mimicry play a role in activating autoreactive t cells? fujinami and oldstone observed that rabbits immunized with a hepatitis b polymerase peptide (icgygslpqe; one-letter code) that shared six amino acids with the sequence of mbp (tthygslpqk) developed an antibody response to mbp and, in some cases, cns lesions reminiscent of eae. quite recently, gautam and colleagues induced clinical eae by immunizing susceptible mice with a herpesvirus saimiri peptide (aaqrrpsrpfa) that has five amino acids of discontinuous sequence homology to mbp ( - peptide, asqkrpsqrhg). studies conducted in other animal models of autoimmune diseases such as adjuvant arthritis confirmed that mimicry of host antigens by infectious agents could lead to the development of disease by inducing cellular as well as humoral autoimmune responses. , what is the molecular basis of mimicry and what extent of sequence homology is required? the tcr, the antigenic peptide, and the mhc molecule form the trimolecular complex of t-cell antigen recognition (see fig ) . antigenic peptide and mhc molecule form the ligand that is recognized by antigen-specific t cells via their tcr. "pockets" in the mhc peptidebinding groove preferentially "anchor" amino acids with certain chemical properties in specific positions of the antigenic peptides. outside of the pockets, amino acid side chains that do not fit the mhc groove may have a strong negative influence and thus hinder binding. , when peptide-mhc complexes are formed based on such "mhc-binding motifs" and exposed on the surface of apcs, both components will be recognized by a specific tcr (see fig ) . certain amino acid positions in the peptide sequence are more critical than others for the interaction with the tcr. allen and co-workers proposed that one amino acid residue in the antigenic peptide sequence is strictly required (primary tcr contact), so that even a conservative amino acid substitution at this position will abolish recognition. amino acids in other tcr contact positions (secondary tcr contacts) modulate the interaction and can be substituted with amino acids that are the molecular mimicry hypothesis of autoimmunity. immunization with a viral ("mimic") peptide activates cross-reactive t cells that will also recognize a myelin antigen. after activation, clonal expansion, and passage of the bloodbrain barrier (bbb), the t cells recognize the myelin antigen and initiate autoimmune inflammation. part of the expanded, cross-reactive t cells will become part of the memory cell pool and will be more readily activated by new antigenic challenge. cns ϭ central nervous system. similar in charge, polarity, or size. , thus, the antigenic peptide interacts with both mhc and tcr, and certain amino acids may be more important for contact in either direction. the mhc-tcr interface (ie, contacts formed between tcr and mhc directly rather than between tcr and peptide) is also crucial. in fact, the outcome of thymic selection of the mature t-cell repertoire is strongly influenced by the affinity of tcrs for self-mhc displayed on thymic epithelium. this interaction is also relevant to the occurrence of autoimmunity, as will be discussed later. based on this theoretical background, amino acid residues critical for binding to the ms-associated class ii molecules dr and dq (mhc contacts), as well as for recognition by specific tcr (tcr contacts), have been defined for the mbp peptide preferentially recognized in the context of dr . , molecular mimicry motifs that would satisfy both mhc binding and recognition by specific tcr were used by wucherpfennig and strominger to identify microbial peptides that were effective in activating three mbpspecific t-cell clones (tccs) derived from ms patients (table ) . thus, experimental data confirmed the theoretical prediction that sequence homology was not required for cross-recognition of self-and foreign antigens. indeed, only one of the stimulatory peptides could have been identified as a molecular mimic by sequence alignment, as opposed to structural criteria. a further development of the molecular mimicry model originated from studies conducted in our laboratory to systematically dissect t-cell recognition of the above-mentioned immunodominant peptide mbp ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) . single amino acid substitutions in each position of the sequence were used to analyze the response to altered peptide ligands derived from mbp ( - ) that bind mhc with approximately the same strength. as expected, some substitutions were tolerated (they did not change the functional response of the clone) whereas others caused a reduction or abolition of the response. a new and intriguing finding was that certain amino acid substitutions were not only tolerated, but actually generated superagonist peptides that were even more potent stimulators of tcc functions (proliferation, cytokine production, cytotoxicity, and early tcr-signaling events) than the native mbp peptide. thus, at least for some autoreactive tccs, the immunodominant mbp peptide used to grow and expand the tccs was not the optimal ligand. when multiple amino acid substitutions were introduced in the antigenic peptide, their combined effect on t-cell antigen recognition was largely predictable by the additive effect (ie, positive or negative) of single amino acid modifications. the effect of "negative" substitutions in virtually any position of the peptide sequence (those leading to a reduced functional response) could be compensated by "positive" substitutions in other positions (leading to more potent responses). , no amino acid seemed to be strictly necessary for antigen recognition, but rather to independently contribute to its recognition by the tcr. based on these findings, it was even possible to design peptides that differed in all positions from the native antigenic sequence, and were still able to stimulate the tccs. these observations offer a new perspective on the concept of molecular mimicry; ie, sequence homology may not be required at all for cross-recognition. indeed, all amino acid in corresponding positions of the peptides can differ (see table ). the use of synthetic peptide combinatorial libraries , has provided a novel, unbiased tool to further investigate the degeneracy of tcr antigen interactions. synthetic peptide combinatorial libraries are highly complex mixtures of peptides in which the natural amino acids occur in completely randomized order at each position in the sequence. for a given peptide length, they represent the complete set of peptides that can theoretically be built with the naturally occurring amino acids (except cysteine to avoid secondary structures). for example, a completely randomized decapeptide library is made of different individual peptides. for the purpose of analyzing t-cell antigen recognition, the positional scanning approach was chosen. , sets of "sublibraries" were used that are completely randomized, except for one defined position in the sequence. in the example of a decamer library, sublibraries ( amino acids ϫ positions in the se- quence) are synthesized, each with only one fixed amino acid, whereas all other positions are completely randomized. if a clone preferentially responds to a complex mixture of peptides with one amino acid fixed in a defined position, that amino acid is considered optimal in that position of a hypothetical agonist peptide. by using the positional scanning approach, we defined the spectrum of stimulatory ligands for several autoreactive, mbp-specific tccs and searched for cross-reactive, high-potency ligands. for each position of the antigenic peptides, one or more optimal amino acids were defined and used to synthesize a series of high-potency ligands. these were found to be effective agonists at concentrations up to orders of magnitude lower that the immunodominant mbp peptide. in addition, real protein sequences derived from both self-and foreign antigens were identified by database searches based on synthetic peptide combinatorial library predictions. again, such peptides were synthesized and proved to be effective agonist ligands for the tccs, in some cases even at lower concentrations than the mbp peptide. , by extending the observations made with amino acid substitutions to their theoretical limits, the combinatorial chemistry approach supports a previously unrecognized level of tcr cross-reactivity. , it is noteworthy that this new concept is supported by other lines of experimental evidence (for review, see mason ). how does this dissection of t-cell specificities help us to understand the induction of autoimmune diseases? recognition of antigen by cd ϩ t cells is considered a crucial check point for the development of any kind of immune response. this is because of not only the specificity of interaction (a feature that also characterizes b-cell antigen recognition) but also the helper function that cd ϩ t cells exert on other cells of the immune system. the concept of t-cell antigen recognition has evolved from high specificity into high flexibility and, at least in some cases, extreme degeneracy. the affinity between tcr and mhc-peptide ligands is crucial to understanding the implications of their interactions. as demonstrated for some autoreactive tccs, ligands can be found that are several orders of magnitude more potent than those used to expand them in vitro. experimental data clearly show that for the same tccs, ligands with intermediate and low tcr affinity also exist. the full spectrum of recognized ligands seems to be characterized by a continuum of affinity, from the highly specific ligands (high tcr affinity, full agonist activity) to the suboptimal ones (low tcr affinity, weak/partial agonists). , - a model can be proposed in which for each clone an "affinity hierarchy" of recognition exists whereby a set of ligands defines the recognition potential of the clone and eventually the extent of its functional activation. by assigning to each clone a limited set of optimal ligands (ie, those that will activate it at the lowest concentration) and a more extensive set of suboptimal ligands, this model reconciles the new findings on degenerate antigen recognition with the essential specificity that is necessary for immune function (fig ) . degeneracy of tcr recognition may have relevance for the process of thymic selection, which may set the stage for the occurrence of autoimmune responses by shaping the immune repertoire. the process involves the positive selection of t cells that are able to recognize a pool of self-peptides presented by self-mhc molecules. what appears to be crucial at this stage is the capacity to recognize such mhc-peptide ligands with a degree of affinity that is not extreme. t cells with very high affinity for self-mhc-peptide complexes are deleted by programmed cell death (ie, negative selection), whereas t cells with very low affinity do not expand (death by neglect; fig ) . - positive selection seems to occur at intermediate levels of affinity. the nature of the peptides displayed on thymic epithelial cells and their precise role in mediating the positive selection of the mature t-cell repertoire is currently under intense investigation. peptides derived from mhc class ii molecules and other proteins abundantly available in the endocytic compartment have been eluted from thymic epithelial cells as well as other apcs in different organs. , it is noteworthy that a variety of self-antigens involved in the pathogenesis of autoimmune disease that were thought to be only expressed extrathymically, including mbp, - plp, uveitogenic retinal proteins, and other proteins that are not "sequestered" by blood-organ barriers (thyroglobulin, , insulin, and glutamic acid decarboxylase ), have been shown to be expressed at the mrna or protein level by thymic epithelial cells. these find- ings suggest that thymic presentation of peptides derived from these antigens may participate in shaping the mature immune repertoire by both negative and positive selection. in some instances, thymic presentation of autoantigens correlates with resistance to autoimmune disease, suggesting that resistant animal strains effectively establish central tolerance to such antigens. on the other hand, presentation of self-peptides in the thymus may also explain why autoreactive t cells are part of the normal mature immune system. in fact, incomplete clonal deletion may cause the "escape" of autoreactive clones from the thymus. , although the importance of individual peptides in positive selection of t cells in the thymus is not yet completely understood, it is clear that degenerate interactions between tcr and mhc-peptide ligands play an important role. indeed, animals that express a single mhc-peptide ligand select a remarkably diverse t-cell repertoire. conversely, different self-peptides have the capacity to promote the selection of a single tcr. , these mechanisms may ensure that the selected repertoire is diverse enough to respond to virtually any foreign antigen encountered in the periphery, but may also impose a risk for reactivity against self. another potential role for t-cell cross-reactivity is in the maintenance of the mature t-cell repertoire. if a high number of self-antigenic ligands are recognized with low affinity by mature t-cell clones, these ligands may provide the low degree of stimulation that is required for t-cell survival in the periphery. results obtained with the use of peptide combinatorial libraries and other experimental approaches suggest that a certain degree of degeneracy in recognition of antigens by the tcr is a normal feature of the im-mune system. a very important implication is that the potential for autoreactivity is very high, and any concept about autoimmunity must explain not only how autoreactivity is initiated, but also why it is rare. a model for how cross-recognition of foreign and selfpeptides could initiate autoimmune responses is proposed, based on two fundamental requirements, the activation of cross-reactive t cells in the periphery, and the recognition of myelin antigens in the cns. it is clear from the above considerations as well as from data obtained in other autoimmune diseases that autoreactive t cells will always be part of the normal mature repertoire. based on our current understanding of tcr antigen recognition, a subset of such cells is expected, for statistical reasons, to be cross-reactive to foreign antigens. in the course of infections, viral antigens may often activate cross-reactive t cells in the periphery. a fraction of such t cells may then cross the bbb and enter the cns parenchyma. if the viral peptide is a more potent agonist than the myelin antigen peptide (fig ) , several other factors may be required to facilitate recognition of a myelin antigen that would normally be "seen" with lower affinity ( table ) . t-cell factors may include the increased expression of adhesion molecules and coreceptors that characterize the memory/effector phenotype. in addition, upregulation of mhc and costimulatory molecules on apcs in the cns will increase the avidity of interaction with t cells. , it is interesting that the viral infection itself may cause local inflammation that will facilitate autoreactivity in the target organ. this has clearly been shown in a murine model of autoimmune diabetes, in which "bystander activation" of transgenic, ␤-islet antigen-specific t cells by local infection with coxsackievirus was sufficient to initiate pancreatic damage. although the viral infection is eventually cleared, the local release of self-antigen may lead to a selfperpetuating chronic inflammation. another possibility is that peripheral activation of t cells by viral peptides may lead to cross-recognition of myelin antigens with the same or even higher affinity. in the scenario of highly degenerate tcr crossrecognition, this is indeed a possible event. , however, we favor the view that it may occur less frequently in vivo. in fact, consistent with the negative selection of the high-affinity autoreactive t-cell repertoire, myelin-specific tccs generated from both ms patients and healthy subjects are usually characterized by a relatively low affinity for their myelin peptide ligands (martin and colleagues, unpublished data). taken together, these considerations suggest that the concurrent effect of several factors may be required for "physiological cross-recognition" to become "dangerous mimicry" and frank autoimmunity. it is the need for several events to occur simultaneously that may help explain why autoimmune diseases are relatively rare. compared with other target organs, the cns may be protected from immune-mediated damage by the high selectivity of the bbb and the very low expression of mhc molecules. elegant studies of transgenic expression of viral peptides in the ␤-islet cell of the pancreas (viral peptides expressed as self) have shown that infection with the same virus is required to initiate autoimmunity even if most peripheral t cells are specific for "viral self" peptides. , when the same viral antigens were expressed as transgenes on oligodendro-cytes, tissue damage induced by viral infection was less severe than in the pancreas, and only a second infection caused demyelination and obvious motor deficits. this elegant work also showed that the exacerbating effect of the second infection could be caused by an unrelated virus, a situation reminiscent of ms, where different viruses may play a role in disease exacerbations, but no single agent has been consistently associated with the disease. in summary, the data discussed in this article suggest that although the conceptual framework of molecular mimicry remains a valid hypothesis for the occurrence of autoimmunity, the requirements for cross-reactivity are more flexible than previously appreciated. new powerful tools are available for the study of these interactions. the application of these methods to t cells isolated from the cns compartment 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adjuvant arthritis as a model for human rheumatoid arthritis molecular mimicry: can epitope mimicry induce autoimmune disease? promiscuous and allele-specific anchors in hla-dr binding peptides separation of t helper clone cytolysis from proliferation and lymphokine production using analog peptides structural basis for t cell recognition of altered peptide ligands: a single t cell receptor can productively recognize a large continuum of related ligands self-nonself discrimination by t cells ligand motifs of hla-drb * and drb * molecules delineated from self-peptides structural requirements for binding of an immunodominant myelin basic protein peptide to dr isotypes and for its recognition by human t cell clones molecular mimicry in t cell-mediated autoimmunity: viral peptides activate human t cell clones specific for myelin basic protein differential activation of human autoreactive t cell clones by altered peptide ligands derived from myelin basic protein peptide ( - ) modifications of peptide ligands enhancing t cell responsiveness imply large numbers of stimulatory ligands for autoreactive t cells predictable tcr antigen recognition based on peptide scans leads to the identification of agonist ligands with no sequence homology complementary mutations in an antigenic peptide allow for crossreactivity of autoreactive t-cell clones generation and use of synthetic peptide combinatorial libraries for basic research and drug discovery decrypting the structure of major histocompatibility complex class i-restricted cytotoxic t lymphocyte epitopes with complex peptide libraries probing degeneracy in t-cell recognition using combinatorial peptide libraries identification of high potency microbial and self ligands for a human autoreactive class ii-restricted t cell clone a very high level of crossreactivity is an essential feature of the t-cell receptor separation of il- production from th cell proliferation by an altered t cell receptor ligand relationships among tcr 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to antigen presentation the costimulatory molecule b is expressed on human microglia in culture and in multiple sclerosis acute lesions diabetes induced by coxsackie virus: initiation by bystander damage and not molecular mimicry viral infection of transgenic mice expressing a viral protein in oligodendrocytes leads to chronic central nervous system autoimmune disease endogenous myelin basic protein inactivates the high avidity t cell repertoire multiple sclerosis: oligodendrocytes in active lesions do not express class ii major histocompatibility complex molecules ablation of "tolerance" and induction of diabetes by virus infection in viral antigen transgenic mice virus infection triggers insulin-dependent diabetes mellitus in a transgenic model: role of anti-self (virus) immune response the virology of demyelinating diseases the h (cd r) antigen is selectively decreased in multiple sclerosis lesions endothelial-leukocyte adhesion molecules adhesion-related molecules in the central nervous system. upregulation correlates with inflammatory cell influx during relapsing experimental autoimmune encephalomyelitis spreading of t-cell autoimmunity to cryptic determinants of an autoantigen epitope spreading we thank drs b. bielekova, i. cortese, and k. p. wandinger (neuroimmunology branch, ninds, nih) for critical reading of the manuscript. key: cord- - ogs xr authors: giotis, efstathios s. title: inferring the urban transmission potential of bat influenza viruses date: - - journal: front cell infect microbiol doi: . /fcimb. . sha: doc_id: cord_uid: ogs xr bats are considered natural reservoirs of various, potentially zoonotic viruses, exemplified by the influenza a-like viruses h n and h n in asymptomatic neotropical bats. these influenza viruses are evolutionarily distinct, are poorly adapted to laboratory mice and ferrets and cannot reassort in vitro with conventional strains to form new influenza subtypes. however, they have attracted renewed attention following reports that their entry in host cells is mediated by the trans-species conserved mhc-ii proteins, suggesting that they hold zoonotic potential. despite the recent studies, the viruses' epidemiology and public health significance remain incompletely understood. delineating the mechanistic basis of the interactions with their hosts and assessing their global distribution are essential in order to fully assess the zoonotic threat that these strains pose. the severe acute respiratory syndrome (sars), middle east respiratory syndrome (mers), nipah (niv), hendra (hev), and ebola (ebov) viruses' outbreaks confirmed the inextricable nature of human and bat health and disease and highlighted that focusing on the "spillover" potential of known, and novel, bat viruses is critical to predict and prevent pandemics. the remarkable ability of bats to coexist with a wide range of viruses that would be pathogenic in flightless mammals (fl − m) is not yet fully understood but possibly relates to their unique, flight-adapted antiviral immunity (calisher et al., ; hayman et al., ) . intriguingly, genetic material from viruses that resemble influenza type a viruses has been recovered from asymptomatic fruit bats of the neotropic bat family phyllostomidae (sturnira lilium and artibeus planirostris) in several countries of central and south america (figure ) (tong et al., (tong et al., , campos et al., ) . influenza a viruses (iavs) are orthomyxoviruses with eight single-stranded negative-sense viral rnas (vrnas) encapsidated into viral ribonucleoproteins (vrnps). iavs emerge from aquatic birds, via genome reassortment and mutation, and are able to cause epidemics (and sporadic pandemics; simonsen, ) in humans, lower animals and birds (simonsen, ) . the bat influenza viruses (bativs) are phylogenetically distinct from the conventional iavs and they were designated as h n and h n (table ) . bats in latin america, but not in central europe, have been found seropositive for bativs (tong et al., ; fereidouni et al., ) . antibodies against human h n and h n , as well as classical h , have also been found in bats elsewhere (l'vov et al., ; kelkar et al., ; isaeva et al., ) , suggesting they are susceptible to iav infection. the epithelial kidney cells of flying foxes (pteropus alecto) co-express both avian (α , -gal) and human (α , -gal) sialic acid (sa) receptors (chothe et al., ) and are thus susceptible to infection by both avian and human iavs, but more importantly figure | countries of central and south america where bat influenza a viruses have been reported. map showing the global pattern of bat species richness was provided by clinton jenkins (see biodiversitymapping.org) using species data from the iucn red list of threatened species ( ; https://www.iucnredlist.org) (jenkins et al., ) . they allow reassortment between co-infecting influenza viruses (dlugolenski et al., ) . the n-terminal domain of the h n pa subunit of the influenza virus polymerase complex possesses endonuclease activity comparable to that of iavs (tefsen et al., ) . equally intriguingly, in the position of the polymerase gene pb , one of the most commonly identified iav virulence markers, bativs have a serine compared to glutamic acid in avian and lysine in mammalian influenza strains, suggestive of an alternative evolutionary pathway for avian iav's adaptation in mammals (mehle, ) . this all raised the question whether novel iavs could emerge from bats to which human and animal populations would be immunologically "naïve, " causing pandemics. the genetic material of bativs is similar to classic flu viruses, but their surface glycoproteins haemagglutinin (ha) and neuraminidase (na) are evolutionarily and functionally diverged zhu et al., ; sun et al., ; tong et al., ) . bativs cannot be cultured in embryonated chicken eggs and do not agglutinate red blood cells (tong et al., (tong et al., , . initial efforts by researchers to isolate live infectious bativs directly from bats failed, due to unavailability of permissive cell lines (ciminski et al., ) . in addition, research on these viruses was further complicated by the dearth of bat cell lines and the limited bat genomic data. attempts to circumvent these limitations have included: (i) using ha- or ha- pseudotyped vesicular stomatitis virus (vsv) and hiv- based lentiviruses (hoffmann et al., ; maruyama et al., ; carnell et al., ; giotis et al., ) , (ii) engineering bativ/iav chimaeric viruses (juozapaitis et al., ; zhou et al., ) , and (iii) reconstructing authentic bativs using reverse genetics (moreira et al., ; sato et al., ; zhong et al., ) . ha -vsv was able to infect bat cell lines (eidni, hypni, and eponi) but only a few of the common fl − m cell lines, including human u- mg glioblastoma and sk-mel- melanoma cells, canine rie and mdck ii kidney cells (hoffmann et al., ; maruyama et al., ; moreira et al., ) . the identification of mdck ii, in particular, as susceptible cell lines to bativs opened the way for a more comprehensive characterization of these strains (moreira et al., ; giotis et al., ; karakus et al., ) . crystal structure analyses revealed that the bat haemagglutinins display typical ha protein folds but lack any obvious cavity to accommodate sa, which are the conventional receptors of iavs. recently, two independent studies demonstrated that the cell-entry of h n (giotis et al., ) and h n (karakus et al., ) is mediated by mhc-ii receptors that are well-conserved in many species. hence, immortalized cell lines that express mhc-ii receptors on their surface such as several human leukemia and lymphoma cell lines raji, ramos, and bjab b-lymphocytes could be used for the study of the viruses' biology (giotis et al., ) . interestingly, ectopic expression of pig, mice, and chicken mhc-ii have been shown to confer susceptibility to h n in non-susceptible cells (karakus et al., ) implying a potential role for the respective animals as intermediary hosts. it is as yet unclear whether mhc-ii receptors function with other unknown factors to facilitate virus internalization and also whether the viruses remain cell associated following cell-entry and are being passed on by direct cell-cell contact. mhc-ii molecules occur as three highly polymorphic isotypes (hla-dr, hla-dp, and hla-dq). they are selectively expressed on the surface of professional antigen presenting cells (apcs), act as ligands for the t-cell receptor (tcr), and play a key role in the presentation of foreign antigens to cd + t helper cells and immune surveillance (jones et al., ; roche and furuta, ) . bats contain all classical mhc class ii gene families that are responsible for antigen presentation with an extra drb gene copy located outside the mhc-ii region in p. alecto (ng et al., ) . the mhc-ii dependent cell entry suggests that bativs might hijack apcs such as b lymphocytes and dendritic cells for viral dissemination and/or survival perhaps in the early stages of infection. it is unknown to what degree their binding to apcs might influence the global outcome of the host immune responses. a blockade of tcr recognition by steric hindrance as described for epstein-barr virus (ressing et al., (ressing et al., , wiertz et al., ) , could explain the asymptomatic status of the infection in the captured new world bats. the bat neuraminidases (nas) are structurally similar to classical nas but lack conserved amino acids for sa binding or cleavage zhu et al., ) . moreover, unlike classical nas, they display no enzymatic activity (garcia-sastre, ; carnell et al., ) , have a dispensable role in viral entry (hoffmann et al., ; maruyama et al., ; giotis et al., ) and their function is not yet elucidated. recent studies demonstrated that the passage of reverse-genetics-generated h n virus in cell cultures, accumulates mutations in the n protein that increase virus titers in culture and may enhance organ tropism in vivo (zhong et al., ) . ciminski et al. demonstrated by utilizing a chimeric bat influenza virus (pr -h n ) that viruses encoding the full-length n protein exhibited a growth advantage over viruses that encode a truncated protein version and also showed that n is essential for viral transmission (ciminski et al., b) . another study showed that the n protein facilitates heterosubtypic (h and h ) influenza hemagglutinin-bearing pseudotype release in the absence of another source of neuraminidase, indicating a possible role of n in viral release (carnell et al., ) . it has been proposed that n downregulates mhc-ii, thereby facilitating virion release but mechanistic data for such function is as yet missing (ciminski et al., b) . there is no evidence that a functional balance exists between bat has and nas although it is possible that the proteins coordinate their actions. despite the recent progress in our understanding, the exact function of bat nas remains an enticing mystery. it is now becoming more evident that bativs may transmit in a different manner than conventional flu strains. it has recently been shown that the h n virus readily transmits between bats (ciminski et al., b) . following experimental intranasal h n infection, the neotropical phylostomidae bat species artibeus jamaicensis shed high viral loads via the fecal route and were able to infect naïve contact animals (ciminski et al., b) . histopathological analysis of the infected bats indicated that viral replication proceeds in the follicle-associated epithelium of gutassociated lymphoid tissue, suggesting virus uptake from the gastrointestinal lumen (ciminski et al., b) , in line with the rich gut epithelial expression of mhc-ii (wosen et al., ) . furthermore, the researchers detected high loads of h n viral transcripts in rectal swabs and excretions (ciminski et al., b) . collectively, these findings imply that an environmental (fecal-oral) mode of bativs transmission is more likely than an airborne one, albeit the latter is not yet compellingly disproved. other infection routes including subcutaneous, transplacental, vaginal, intracranial infections have not yet been reported. in contrast to bats, h n has been reported to have limited replicative ability in laboratory mice and more interestingly in ferrets which share similar lung physiology and sa distributions to humans (ciminski et al., b; karakus et al., ; zhong et al., ) . whether these observations are animal/infectionroute-dependent or they actually reflect a low zoonotic risk for bativs remains to be seen. in absence of conclusive scientific proof, the question remains as to whether bativs are confined to a sylvatic transmission cycle and perpetuate in neotropical bat populations or are capable of urban adaptation. anthropogenic disruptions of ecological habitats that led to urban transmission of other enzootic bat viruses (i.e., hev, niv) have been extensively described in the scientific literature. despite the increasing disturbances in the fire-prone neotropical forests, new world bats have not yet been implicated in the transmission of zoonotic viruses, other than rabies, to humans (moratelli and calisher, ) . even so, latin america is home to the richest and most diverse bat fauna in the world (figure ) including almost phyllostomidae species (jenkins et al., ) in which bativs have been detected. unlike african and asian bats which are consumed regularly, new world bats are only eaten by few native indigenous people (moratelli and calisher, ) . a possible exposure to infected bat blood and body fluids may hypothetically create a pathway for disease transmission to humans. another scenario may involve the accidental introduction of bativs to the local fauna or other phyllostomidae species such as the widespread bloodeating vampire bat (desmodus rotundus), which thrives in both native and anthropogenically transformed ecosystems (bergner et al., ) . vampire bats have long been suspected of passing on rabies to humans and livestock in latin america by biting and scratching (rupprecht et al., ) . it will be useful to explore in future studies whether haematophagous bats can act as maintenance hosts for bativs and if their biting can form a potential zoonotic transmission route either directly or through mammalian intermediate hosts. bativs, like all viruses, have to compromise with positive and negative genetic factors present in target cells for their survival at each replication stage. little is known regarding the interaction of bativ proteins and rna with the host or viral factors even though such interactions may determine the fate and/or efficiency of infection, transmission, and epidemic potential of the viruses. previous studies revealed that the bat nonstructural ns proteins can act as interferon (ifn) antagonists in human cells, and likely inhibit induction of ifn at a pretranscriptional level (ciminski et al., ) . more recently, it has been shown that the ifn-induced human mxa protein controls the replication of h n , but it is not clear whether sufficient mxa-escape mutations in h n np can be acquired in vivo that could potentially result in full mxa resistance (ciminski et al., a) . nearly all lab work examining host and viral immune-modulating proteins is performed with human/rodent cell lines. the difficulty in interpreting these data is that evolutionarilyoptimized immune factors behave differently in non-natural hosts. certainly, comprehensive kinetic analyses of immuneresponses to bativs using primary or immortalized bat cell lines will be particularly informative. for instance, a comparison of the transcriptome of bativ infected versus uninfected bat cells could help us identify specific immune genes contributing to host resistance and the molecular mechanisms underlying the viral pathogenesis. reassortment of gene segments between co-infecting viruses is a key process mediating the genetic evolution of influenza viruses and the generation of novel epidemic and pandemic strains. bativs are able to reassort between themselves but not with conventional iavs in vitro (juozapaitis et al., ; zhou et al., ) . generation of bat chimeric viruses was only possible when the ha/na coding regions were flanked with the authentic bativ packaging signals demonstrating packaging incompatibilities between iavs and bativs (juozapaitis et al., ; zhou et al., ) . this finding dismisses the scenario of emergence of a new "reassortant" virus with human/avian iavs unless the bat viruses undergo major genetic changes over time. however, the abilities of bativs to (i) reassort between themselves, (ii) to mutate in order to infect and transmit sustainably among their hosts, and (iii) enter human hla-dr + cells, highlight that a zoonotic transmission of bativs is theoretically possible. the documented spillover of other nonreassortant bat-borne rna viruses following continued hostpathogen interaction (i.e., niv and ebov) lends certain credence to this hypothesis, albeit clearly, supporting evidence is lacking. to explore the ecological and evolutionary dynamics of these and possibly other unknown influenza-a-like viruses, further prevalence and serological studies in neotropical bat populations are required coupled with the surveillance of bat-exposed humans and livestock. surveys of bat colonies have previously led to identification of other zoonotic viruses, including hev in pteropus sp. in australia, niv in pteropus lylei in thailand, and marv in rousettus aegyptiacus in uganda (wacharapluesadee et al., ; amman et al., ; field et al., ) . a computational study which used spatial empirical models to trace the steps of emergence of bat viruses and the transmission opportunities to humans pinpointed sub-saharan africa as the top-priority location for pathogen discovery in wildlife (brierley et al., ) . west subsaharan africa, in particular, hosts enormous populations of sedentary and migrating bats living in proximity to human and animal populations. considering the number, the morbidity and mortality of emerging viruses that are hosted in african bats as well as the serological evidence against iavs (freidl et al., ) , future surveillance and serological studies could lead to the identification of novel bat influenza subtypes. in summary, bativs are unconventional influenza viruses that resemble to some extent more paramyxoviruses rather than typical orthomyxoviruses. despite the recent findings on the cell entry factors and nas of these viruses, it is clear that we only scratched the surface in terms of characterization of these viruses. the scientific evidence so far indicate a limited spillover risk but data is not conclusive enough to dismiss out of hand the possibility of zoonotic transmission. forecasting viral spillover is a challenging task and additional interdisciplinary and more up-to-date approaches are warranted to fully appreciate the ecology and the implications of these viruses for public health. future studies on bativs hold extra value as they can provide broader mechanistic insights into the molecular biology of influenza viruses and might inform translational studies. the author confirms being the sole contributor of this work and has approved it for publication. eg was supported by funding from a wellcome trust new investigator award to marcus dorner ( /z/ /z). seasonal pulses of marburg virus circulation in juvenile rousettus aegyptiacus bats coincide with periods of increased risk of human infection demographic and environmental drivers of metagenomic viral diversity in vampire bats quantifying global drivers of zoonotic bat viruses: a process-based perspective bats: important reservoir hosts of emerging viruses bat influenza a(hl nl ) virus in fruit bats the bat influenza h n can be neutralized by broadly-neutralizing monoclonal antibodies and its neuraminidase can facilitate viral egress avian and human influenza virus compatible sialic acid receptors in little brown bats human mxa is a potent 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transmission herpesvirus interference with major histocompatibility complex class ii-restricted t-cell activation epithelial mhc class ii expression and its role in antigen presentation in the gastrointestinal and respiratory tracts mutations in the na-like protein of bat influenza h n virus enhance virus replication in mammalian cells, mice, and ferrets characterization of uncultivable bat influenza virus using a replicative synthetic virus crystal structures of two subtype n neuraminidase-like proteins from bat influenza a viruses reveal a diverged putative active site key: cord- -rapinodd authors: vidovic, maria; sparacio, shaun m.; elovitz, michal; benveniste, etty n. title: induction and regulation of class ii major histocompatibility complex mrna expression in astrocytes by interferon-γ and tumor necrosis factor-α date: - - journal: j neuroimmunol doi: . / - ( ) -t sha: doc_id: cord_uid: rapinodd astrocytes can function as antigen-presenting cells (apc) upon expression of class ii major histocompatibility complex (mhc) antigens, which are induced by interferon-γ (ifn-γ). previous data from this laboratory had shown that the cytokine tumor necrosis factor-α (tnf-α) enhances ifn-γ-mediated class ii antigen expression on astrocytes. we have now investigated the effect of ifn-γ and tnf-α on class ii mhc mrna expression in astrocytes using northern blot analysis. astrocytes do not constitutively express mrna for class ii mhc. kinetic analysis of class ii mhc mrna expression in ifn-γ-treated cells demonstrated an h time lag, which was followed by an increase over the next h. optimal expression of class ii mrna was detected after a h incubation with ifn-γ. this level of expression was further enhanced by the simultaneous addition of ifn-γ and tnf-α to the astrocytes, while tnf-α alone had no effect on class ii mrna expression. tnf-α does not act by increasing the stability of ifn-γ-induced class ii mrna, indicating its action is not at that specific level of post-transcriptional control. furthermore, astrocyte class ii mrna expression was inhibited when cycloheximide (chx) was added together with ifn-γ or ifn-γ/tnf-α, and when chx was added up to h after treatment with ifn-γ or ifn-γ/tnf-α. these results indicate that astrocyte class ii mrna expression is mediated by newly synthesized proteins induced by ifn-γ and/or ifn-γ/tnf-α. the expression of class ii antigens on astrocytes, and cytokine modulation of their expression, may be important in the initiation and perpetuation of intracerebral immune responses. the non-neuronal cells of the central nervous system (cns) are made up of the macroglia (astrocytes, oligodendrocytes and ependymal cells) and the microglia. collectively, these glial cells perform a variety of active roles during development of the brain (rakic, ; silver and sapiro, ) and subsequently in the maintenance of normal cns physiology (hertz, ; janzer and raft, ) . recent work has suggested that glial cells such as astrocytes and microglia may be involved in immunological events occurring in the brain. the astrocyte can be stimulated to secrete a number of immunoregulatory molecules, including interleukin- (il-i) (fontana et al., ) , interleukin- (il- ) (frei et al., ) , interleukin- (il- ) (frei et al., ; benveniste eta[., ) , prostaglandins (fontana et al., ) , leukotriene (llartung et al., ) , tumor necrosis factor-c~ (tnf-co (robbins et al., ; lieberman et al., ; chung and benveniste, (i) and ifn-c~/, (tedeschi et al., ) . the microglia can also be stimulated to secrete l- (giulian et al., ) , il- (frei et al., ) and tnf-a (frei et al., ) , thus providing the cns with numerous endogenous sources of cytokines necessary, for immunological response. more importantly, the astrocyte and microglia can function as antigen-presenting cells (apc) in the cns (fierz et al., ; frei et al., ) . these cells are able to internalize, process, express and present antigen to encephalitogenic t cells (fontana et al., ) . however, such a function is only possible upon expression of class i major histocompatibility complex (mhc) molecules. indeed, astrocytes can be induced to express class ii antigens both in the cns and in vitro, following exposure to interferon- (ifn-t) (wong et al.. : fierz et al., or virus (massa et al., ) . mhc-encoded class molecules are heterodimerit glycoproteins which have a central role in the regulation of immune responses (benacerraf, ) . the expression of class ii antigens is primarily restricted to b cells, monocytes/macrophages and dendritic cells (hammerling et al., ) , although certain non-lymphoid cells can be induced to express class ii upon exposure to ifnq,, and function as apc. these include pancreatic beta cells (markmann et al.. ) , keratinocytes (gaspari et al., ) , brain endothelial cells (mc-carron et al., ) , and most pertinent to this study, astrocytes (fontana et al., ) . abnormal control in the level of expression of class i genes, and aberrant expression in cells normally class ii negative have been implicated in autoimmune phenomena. because of the importance of class ii mhc antigens, many studies have been directed toward understanding the regulatory mechanisms involved in class ii mhc gene expression. it is generally accepted that induction of class ii gene expression by ifn-y occurs at the transcriptional level (basta et al., ; blanar et al., : fertsch-ruggio et al., : rosa and fel-ious, : amaldi et al., , and that transacting factors interacting with cis-acting dna regulatory elements are involved in the transcriptional regulation of class ii mhc expression (accolla et al., " salter et al., sherman et al., sherman et al., , blanar et al., , ama[di et al., celada et al., ) . these trans-acting regulatory factors have been postulated to function positively or negatively, and to be expressed ubiquitously, or in a tissue-or stage-specific manner. although ifnq, is considered the primary inducer of class ii antigens, there is evidence for other cytokines contributing to class i expression. we have previously shown that tnf-~ enhances ifnq,-induced class ii antigen expression on astrocytes, and that this is a synergistic interaction as tnf-a alone has no effect on class i expression (benveniste et al., ) . the present study was undertaken to extend these previous findings, and to examine, at the molecular level, the effect of fn-y and tnf-a on astrocyte class ii gene expression. we report that astrocytes express class ii mrna h after treatment with ifn- ¢ or fn-t/tni'-~,, indicating a long lag period between exposure to the cytokines and initiation of class ii gene expression. tnf-~ does not act to stabilize ifnq,-induced class ii mrna, suggesting it may act at other levels of post-transcriptional control or at the transcriptional level. furthermore, the expression of class ii mhc mrna was completely inhibited by cycloheximide (chx), suggesting a role for newly synthesized proteins in astrocyte class ii mhc expression. as astrocytes can be stimulated to secrete tnf-a (robbins et al., ; lieberman et al., ; chung and benveniste, ) , and express high affinity tnf-a receptors (benveniste et al., ) , tnf-a can act in an autocrine fashion to enhance class ii gene expression in astrocytes. by modulating class ii gene expression and thereby stimulating the apc function of astrocytes, ifn-y and tnf-a in concert may play a pivotal role in the regulation of intracerebral immune responses. rat recombinant ifn-y (specific activity: x u/mg) was purchased from amgen biologicais (thousand oaks, ca, u.s.a.), and human recombinant tnf-a (specific activity: . x u/mg) was the generous gift of genentech (south san francisco, ca, u.s.a.). monoclonal antibody to glial fibrillary acidic protein (gfap) was obtained from boeringher mannheim (indianapolis, in, u.s.a.), and monoclonal antibody to rat class i mhc antigens (clone ox- ) was from accurate corporation (westbury, ny, u.s.a.). second antibody was affinity-purified goat anti-mouse lg conjugated to fluorescein-isothiocyanate (fitc) from southern biotechnology (birmingham, al, u.s.a.). cycloheximide and actinomycin-d were purchased from sigma chemical company (st. louis, mo, u.s.a.) . primary glial cell cultures were established from neonatal rat cerebra by a modification of the mccarthy and de vellis technique ( ) as previously described (benveniste and merrill, ) . meninges were removed from rat brains prior to glial cell dissociation and culture. culture medium (cm) was duibecco's modified essential medium (dmem), high glucose formula supplemented with glucose to a final concentration of g/l, mm glutamine, . mm non-essential amino acid mixture, . % gentamicin, and % fetal bovine serum (hyclone, logan, ut, u.s.a.). after days in primary culture, oligodendrocytes were separated from the glial cultures by mechanical dislodging, and the astrocytes were obtained by trypsinization ( . % trypsin/ . % edta) and replated at a density of - x cells/ mm tissue culture plate and allowed to adhere for at least h. the cells were counted using trypan blue; cell viability was - %. the astrocytes were monitored for purity by immunofluorescence, and by non-specific esterase staining for contaminating microglia as previously described (benveniste and merrill, ) . the primary astrocytes were plated ( . x ) on mm glass coverslips, incubated in culture medium for days, washed twice with phosphate-buffered saline (pbs), and fixed for s in cold acetone. the cells were then stained for gfap, an intracellular antigen unique to astrocytes (bignami et al., ) , using a monoclonal antibody to gfap ( : ) for min at room temperature, followed by a min incubation with goat anti-mouse ig/fitc ( : ). the coverslips were then mounted in % glycerol, and visualized by fluorescent microscopy. astrocyte cultures were routinely > % positive for gfap, and less than % of the cells were microglia based on their positive staining for non-specific esterase. total cellular rna was isolated from confluent monolayers of astrocytes that were incubated for various intervals ( - h) without or with ifn-y and/or tnf-a. in some experiments, the protein synthesis inhibitor, chx ( #g/ml) or the rna synthesis inhibitor, actinomycin d ( #g/ml), were added to the cytokine-treated astrocytes for - h. rna isolation followed the procedure of chomczynski ( ) . the cells were collected, washed times with cold pbs, and pelleted. rna was extracted with guanidinium isothiocyanate and phenol, and precipitated with ethanol. samples ( ~g) of total cellular rna were denatured with formaldehyde for rain at °c, and rna was size fractionated by electrophoresis through a . % agarose gel" containing ethidium bromide for visualization of s and s ribosomal rna bands. the visualization of rna bands was useful for assessing the integrity of the rna and for varifying the amount of rna loaded. the rna was then transferred to nitrocellulose paper in x standard saline citrate (ssc) ( m naci and . m sodium citrate) at °c. after the transfer, the nitrocellulose paper was air-dried and the rna cross-linked in a uv stratalinker oven. prehybridization was performed at °( in a solution containing % (v/v) formamide, x ssc, × denhardt's solution, p,g/ml of denatured salmon sperm dna, and . % sodium dodecyl sulfate (sds) for - h. hybridization was carried out at ( ` for h in prehybridization solution containing % dextran sulfate, . mm na phosphate buffer and denatured ? p-labeled murine class i e-e~ cdna probe ( x w' cpm/ml). the blots were then washed in x ssc (twice for min) at room temperature, followed by x ssc containing . % sds (twice for min) at °c and finally in . x ssc for rain at °c. the blots were dried between whatman filter paper and exposed to kodak x-omat ar film plus intensifying screens at - °c. the autoradiographs were quantitated by scanning densitometry with a bio-rad model video densitometer. filters were stripped to remove bound class mhc probe, and rehybridized with a second control probe, cyclophilin. a edna probe (peacll) specific for mouse class ii e-a (mathis et al., ) was the generous gift of dr. jerold woodward, university of kentucky. the . kb ecori insert was isolated, and labeled with [et-s p]deoxyctp using an amersham nick translation kit according to the manufacturer's instructions. a specific activity of . - x () ~ cpm/~g dna was routinely attained. a edna probe for rat cyclophilin (plb ) (danielson et al., ) was the generous gift of dr. jim douglass, the oregon health sciences university. primary rat astrocytes were resuspended in dmem containing % fetal bovine serum (fbs), and plated at - x ~ cells/well into -well ( mm) plates (costar, cambridge, ma, u.s.a.). the plates were incubated overnight to allow recovery of the cells from trypsinization and to assure adherence of the astrocytes. after h the original medium was aspirated off and fresh serum-free medium ( ml) was added to the wells. triplicate wells of primary rat astrocytes were treated with u/ml of recombinant rat ifn-y and/or ng/ml of recombinant human tnf-a for various incubation periods ( days). at each time point, the cells were trypsinized and stained for class it antigens, as previously described (benveniste et al., ) . briefly. astrocytes were incubated with ~ of ox- monoclonal antibody for rain in the cold. washed times with pbs containing . % fbs and . % azide (pbs-fbs-azide), and then incubated with /tl of goat anti-mouse ig-fit(" ( : ) for another rain in the cold. after washing times with pbs-fbs-azide, the cells were fixed in a final volume of ~ of % paraformaldehydc and analyzed on the facstar (becton-dickinson, mountain view, ca, u.s.a.) for class ii antigen expression. negative controls were incubated with /tl of pbs-fbs-azide in place of first antibody, or with an irrelevant monoclonal antibody of the same isotype. the gate window of forward-angle light scatter lay between channels and : the gate window for log of green fifc fluorescence lay between channels and . ten thousand cells were analyzed for each sample. the level of class i mhc mrna was examined in astrocytes following treatment for various times with ifn-y, tnf-a or a combination of the two cytokines. to determine the steady-state level of mrna for class ii, northern blot analysis was performed using a edna probe for murine class ii genes (e-a), with total rna isolated from cultured astrocytes. as seen in fig. , a . kb class ii mhc mrna transcript was present in ifn-~, treated astrocytes (lanes and ) and absent in untreated cells (lanes and ) . class it m}tc mrna expression was more pronounced when the cells were cultured with ifn-y in serum-free medium (sfm) (fig. , lane ) as opposed to serum-containing medium (fig. , lane ) , thus, all the subsequent experiments were con- in primary rat astrocytes. northern blot of rna from astrocytes that were incubated in serum containing media (lanes and ) or serum-free medium (sfm) (lanes and ) without (lanes and ) or with if'n-), ( u/ml) (lanes and ) for h. total rna was extracted and size fractionated by gel electrophoresis. hybridization was performed with a cdna probe (e-a) specific for a murine class i mhc gene. the blot was then exposed at - °c for h to kodak x-omat ar film plus two intensifying screens, kb, kilobases. ducted in sfm. optimal expression of class ii mrna was detected when cells were stimulated with - u/rnl of ifn-'r (data not shown). some variability in the concentration of ifn-t required for induction of class ii mrna was noted, and this variability was dependent on the lot of ifn- used. therefore, it was necessary to do a dose-response study for each lot of ifn-t used. for this study, u/ml of ifn-t was sufficient for maximal expression of class ii mrna. the optimal time required for class ii mrna expression following treatment of astrocytes with ifn-t is illustrated in fig. . astrocytes were incubated in sfm without or with ifn-~, for , or h prior to harvesting. a low level of class ii mhc mrna was detected at h following treatment with ifn-~,, with maximal expression detected after a h incubation with ifn-t. there was a . -fold increase in class ii mhc mrna expression from to h, and a slight reduction at h. we have previously shown that the level of class ii protein expression, based on fluorescenceactivated cell sorting (facs) analysis, was enhanced when the cells were treated with both ifn-t and tnf-a (benveniste et al., ) . similarly, in this present study, the incubation of as- kb fig. . kinetic analysis of ifn-y treatment on astrocyte class ii mhc mrna expression. astrocytes were cultured in sfm without (lanes , , and ) or with ifn-'r (lanes , , and ) for h (lanes and ) , h (lanes and ) or h (lanes and ). total rna was extracted and analyzed for class mrna by northern blot hybridization method. the blot was exposed to kodak x-omat ar film plus two intensifying ~reens at - °c for h. trocytes with both ifn-y and tnf-a resulted in an enhanced expression of class ii mrna compared to ifn- alone (fig. ) . optimal enhancement of class ii mrna was demonstrated using tnf-a at ng/ml (fig. , lane ) , which correlates with the concentration of tnf-a used for synergistic induction of class ii mhc protein (benveniste et al., ) . a . -fold increase in class ii mrna expression in the presence of ng/ml of tnf-a was detected, compared to ifn-y alone. as expected, tnf-a alone did not induce mrna for class ii antigens (data not shown). class ii mhc mrna expression induced by ifn-y/tnf-a was also enhanced when experi- kb , for h. rna was isolated for analysis by northern blot hybridization method. the blot was probed with labeled e-a cdna, and exposed at - °c for h to kodak x-omat ar film plus two intensifying screens. lg ments were performed in sfm (data not shown), indicating that a serum component(s) has a slight inhibitory effect on class i mrna expression. in other cell types, a lag phase of approximately - h precedes the appearance of class ii mrna induced by ifn- (basta et al,, ; blanar et al., , rosa and fellous, ; amaldi et al., ) . we performed a more indepth analysis of the kinetics of induction of class ii mrna by ifn- and ifn- /tnf-a in astrocytes. analysis of mrna was performed at different times after induction with ifn-y (fig. , lanes , , , and ) and ifn-y/tnf-a (fig. , lanes , , , and ). no class ii mrna was detected until h following treatment with ifn- , with maximal exvression detected h after exposure to ifn-y. similarly, class mrna was not detected until h in astrocytes that were stimulated with ifn- /tnf-a; however, the intensity of the rna signal was increased in the presence of both cytokines, as expected. thus, there was an h time lag before class ii mrna was detected in astrocytes. at early time points ( and h), mrna doublets are seen which ultimately merge into a diffuse, and ) . and h (lanes and ). astrocytes in sfm alone (lane ). the blot was exposed to kodak x-omat ar film plus two intensifying screens at - o c for days. more intense . kb band at h. this may be due to multiple transcription initiation sites described for the e-a gene (mathis et al., ) . in addition, a larger mrna species of . kb is seen at h. the significance of this band is unknown at this time. results for the induction of class ii mhc antigen expression and mrna accumulation are summarized in fig. . tnf-a increases ifn-v-induced class ii expression by increasing levels of mrna for the class ii molecule. however, it is not known whether tnf-a acts by increasing transcription or by stabilizing the mrna. experiments were conducted to assess class ii mrna stability in the presence of ifn- or ifn-y/tnf-a. class ii mrna was induced in astrocytes with either ifn-"/or ifn--//tnf-a for h, then actinomycin d (a transcription inhibitor) was added for various times ( , , , , , and h). total cellular rna was isolated and analyzed by northern blotting. preliminary results indicated that a decrease in class ii mrna was not detected until h of actinomycin d treatment (data not shown). subsequent experiments were performed utilizing rna extracted after , and h of actinomycin d treatment. as shown in fig. actinomycin d treatment, tnf-a did not appreciably affect the stability of e-a mrna compared to the stability of ifn-y-induced e-a mrna. in fact, it appears that tnf-a contributes to an accelerated destabilization of class ii mrna. the approximate half-life of e-a mrna in the presence of ifn-v/tnf-a was h, compared to greater than h in the presence of ifn-v alone. these same blots were reprobed for cyclophilin mrna to demonstrate that the integrity and quantity of rna loaded in each lane was similar (data not shown). these data indicate that tnf-a does not act by mrna stabilization to enhance if/q-v-induced class ii expression. the h delay in class ii mrna expression after ifn-v or ifn-v/tnf-a stimulation of as- trocytes suggests that signal transmission initiated by these cytokines involves a number of intermediary steps, possibly the expression of newly synthesized gene products. to test this, we examined whether protein synthesis was required for induction of class i mrna by ifn-v and ifny/tnf-a. chx, an inhibitor of protein synthesis, was added to astrocytes at a concentration ( /.tg/rnl) that inhibited protein synthesis by more than %, while still maintaining cell viability (data not shown). astrocytes were cultured for h in the presence of ifn-y, ifn-y/tnf-a, chx alone, ifn-y plus chx, ifn-y/tnf-a plus chx, rna extracted, and then analyzed. fig. demonstrates the effect of chx on the induction of class ii mrna by ifn-v and tnf-a. no mrna for class ii was detected in cells treated with chx alone, ifn-y plus chx, or ifny/tnf-a plus chx (fig. , lanes , , and ). inhibition of protein synthesis completely abolished the induction of class ii mrna by ifn-y and ifn-y/tnf-ct. however, there was no inhibition of cyclophilin mrna expression (fig. b) , and no alteration in the pattern of ethidium bromide staining of rna in all the samples treated with chx alone or chx plus the cytokines (fig. c) , indicating that chx did not cause a generalized inhibition of mrna expression in astrocytes. cyclophilin was used as a control for these experiments as rna levels do not change upon treatment with ifn-v or ifn-v/tnf-a. that newly synthesized protein(s) is required for the induction of the class ii mhc gene in astrocytes treated with ifn-y or ifn-y/tnf-a was suggested by results in fig. . the duration of protein synthesis required to allow expression of the class ii mhc gene in astrocytes was examined in cells that were pretreated with ifn-y or ifn- /tnf-a for different lengths of time prior to the addition of chx. class ii mhc mrna was measured h after the treatments were started. as shown in fig. a , when chx was added simultaneously with ifn-y/tnf-a or - h after ifn-y/tnf-a treatment, there was no detectable expression of class ii mhc mrna. however, when astrocytes were incubated with ifn- however, in samples that were treated for h with ifn-y/tnf-c~ before ctlx was added, there was still a % reduction in the expression of class ii mhc signal compared to the positive control of fn-y/tnf-a alone (fig. a, lane ) , suggesting that continuous synthesis of protein is required for optimal expression of the class mhc gene. chx treatment had no effect on the expression of cyclophilin rna (fig. b) . similar results were seen when astrocytes were incubated with ifn-t and chx, except that the expression of class ii mhc mrna was detected only after cells were incubated with ifn- for h prior to the addition for h. total rna was extracted, northern blot hybridization performed and the blot was exposed to kodak x-omat ar film plus two intensifying screens at - °c for days. autoradiograph of class ii mrna (a). autoradiograph for cyclophilin mrna was obtained by stripping class i probe and rehybridizing with a second probe to detect cyclophilin mrna expression (b). photograph of the original gel stained with ethidium bronude to show that there was no alteration in the quantity or the quality of rna in all the samples treated with chx (c). ~q /tnf-a for h prior to addition of chx, and class ii rna measured h later, a low level of class ii rna was detected. the increase in the level of class i mhc mrna detected parallels the increase in the amount of time the cells were treated with ifn-t/tnf-a before the addition of chx, i.e., the longer the treatment with ifn- /tnf-a before the addition of chx, the stronger the mrna signal. these results, therefore, suggest that protein synthesis, initiated within h of the cells encountering ifn-t/tnf-a, is critical for subsequent class ii mhc mrna expression. the blot was exposed at - ° c for days to kodak x-omat ar film plus two intensifying screens (a). autoradiograph for cyclophilin mrna obtained by stripping class ii probe and rehybridizing with a second probe to detect cyclophilin mrna (b). photograph of the original gel stained with ethidium bromide (c). of chx (data not shown), indicating that h of protein synthesis was critical for ifn-~-induced class ii mrna expression. in this study we have shown that primary neonatal rat astrocytes, upon stimulation with ifn-~,, express mrna transcripts for class ii mhc genes, and that tnf-a enhances the expression of ifn-~,-induced class ii mrna. these results support previous findings that ifn-~, and tnf-a synergize in the induction of class ii mhc protein expression in rat astrocytes (benveniste et al., ) . kinetic analysis demonstrated that class ii mrna was first detected after h of treatment with ifn-y, followed by an increase in mrna expression over the next h. when astrocytes were treated with ifn-~, and tnf-a simultaneously, the kinetics of class ii mrna expression did not change; however, the overall amount of steady-state class ii mrna was increased. optimal expression of class ii mrna was detected h after incubation with ifn- , alone or ifn-~,/tnf-a. although the predominant forms of gene regulation occur at the transcriptional level, a number of control mechanisms can act on rna once its transcription has been initiated. post-transcriptional regulatory mechanisms include ( ) changes in mrna stability, ( ) alternative rna splicing, ( ) poly a addition, and ( ) control of translational initiation. in our experiments, tnf-a did not increase the stability of ifn- ,-induced class ii mrna, indicating that tnf-a did not act at that level of post-transcriptional control. preliminary results from our laboratory suggest that the increase in class ii mrna occurs primarily by an increase in transcription of the e-a gene since nuclear run-on assays detected no transcription of the class ii genes without induction by ifn-y, and enhanced transcription in the presence of ifn-~, plus tnf-a. further experimentation is necessary to determine conclusively if tnf-a acts solely at the transcriptional level, or whether both transcriptional and post-transcriptional events result in increased class ii mhc mrna and protein. the time required for the appearance of class ii mhc mrna following treatment with ifn-~, or ifn-~,/tnf-a ( h) suggests that cytokine signal transmission is complex and may involve a number of intermediary steps. we examined whether protein synthesis was required for ifn-), or ifn-"t/tnf-a-induced expression of astrocyte class ii genes. the expression of class ii mrna was completely inhibited when chx was included with ifn-~, and ifn-'t/tnf-~ treatment, indicating that newly synthesized protein is required for astrocyte class ii mhc gene expression. a minimum of h of active protein synthesis was required for subsequent ifn-t/tnf-a-induced class ii mrna expression, while h was required for subsequent ifn-), expression. however, in experiments where chx was added h after treatment with ifn-), or ifn-),/tnf-~, there was still a % and % reduction, respectively, in the expression of class ii mrna compared to astrocytes incubated with the cytokines alone. this indicates that the synthesis of novel proteins is required continuously for optimal class ii gene expression in astrocytes. other studies have shown that protein synthesis was required for up to h after ifn-~, was added to murine p d cells to detect an increase in the level of i-aa (boettger et al., ) , while in peritoneal mouse macrophages, a % decrease in i-at~ mrna levels was observed even when chx was added after h of ifn-'rtreatment (fertsch et al., ) . in contrast, celada et al. ( ) demonstrated that protein synthesis was only required for rain after murine macrophages were treated with ifn-), for an increase in i-aft mrna to be detected. thus, different cell types have varying requirements for active protein synthesis to express class ii mrna in response to ifn-),. other reports on ifn-t-induced expression of class i genes have indicated that protein synthesis is not required. induction of dra mrna in the human glioblastoma cell line u -mg (basta et al., ) , dermal fibroblasts (collins et al., ) and i-aa in murine wehi- cells (woodward et al., ) , occurs in the absence of protein synthesis. this suggests that the expression of class ii mrna in these cells is mediated by pre-existing trans-acting factors that are triggered by ifn-'t (woodward et ai., ) . it is also important to note that primary astrocytes (this study) and glioblastoma cells (basta et al., ) differ in their requirements for protein synthesis for class expression, illustrating fundamental differences between normal astrocytes and transformed glial cells. tnf-a may be an important enhancer of class i expression in the cns as it can function in an autocrine fashion on the astrocyte. in addition to responding to tnf-a and expressing specific high affinity receptors for this factor (benveniste et al., ) , astrocytes can also secrete tnf-a (robbins et al., ; sawada et al., ; chung and benveniste, ) . more importantly, ifn-t primes the astrocyte to produce tnf-a (chung and benveniste, ) , thus ifn-t can influence both tnf-a and class ii gene expression in the astrocyte. although class ii expression on astrocytes has been conclusively demonstrated in vitro, in vivo studies have generated conflicting results. direct injection of ifn-t into the brains of mice induced class ii antigens on astrocytes, indicating that astrocytes have the potential to express these antigens in vivo (wong et al., ) . many laboratories have examined whether astrocytes express class i antigens in a variety of immune-mediated disease states to better understand the possible role of the astrocyte as a local apc. traugott et al. ( ) demonstrated class ii expression on astrocytes in active chronic multiple sclerosis (ms) lesions, and then confirmed these studies by performing double-staining for both class ii and gfap (traugott and lebon, ) . a study by hofman et al. ( ) also identified class ll-posirive astrocytes in ms brain by double-staining. rodriguez et al. ( ) have studied class i expression on glial cells in an animal model of cns demyelination induced by theiler's virus. in susceptible strains of mice (bio.s and bio.asr ), the majority of class ii-positive glial cells had morphological characteristics of astrocytes, while uninfected mice or resistant strains (bio.s, ( r)) were class ii negative. in sjl mice with acute or chronic relapsing experimental allergic encephalomyelitis (eae), an animal model for ms, some class ii-positive ceils were identified as astrocytes (sakai et al., ) . however, other studies investigating the eae model in lewis rats failed to detect class ii-positive astrocytes in the brain (hickey et al., : matsumoto et al., vass et al., ) . thesc conflicting results may bc due solely to technical problems involved with antigen fixation and staining methodologies, or may indicate that the ability of astrocytes to function as apc in vivo may only bc relevant in certain diseases or specific stages of disease. another possibility may be the loss of class ii-positive astrocytes by class ii mhc-restricted t cell-mediated cytotoxicity as shown by sun and wekerle ( ) . the disease eae appears to be strain-specific as brown-norway rats and balb/c or c bl/ mice are resistant, whereas lewis rats and sjl mice are susceptible (linthicum and frelinger, ) . recent studies have demonstrated that astrocytes derived from susceptible strains express much higher levels of class ii antigen upon treatment with either ifn-~, or virus compared to astrocytes prepared from eae-resistant strains (massa et al., a, b) . this hyperinduction of class ii in eae-susceptible animals was astrocyte specific as both peritoneal macrophages and microglial cells of susceptible and resistant strains showed identical patterns for class ii induction. this differential expression of class ii on astrocytes in response to ifn-t compared to microglia suggests that regulation of class i expression on astrocytes may correlate with antigen-presenting capacity and ultimately, disease development in the cns. we have begun, at the molecular level, to dissect the regulatory mechanisms utilized by primary rat astrocytes for class i mhc gene expression. future studies will focus on the regulation of gene expression at the transcriptional level, and ifn-t/tnf-a-induced trans-acting regulatory factors required for class ii gene expression. reactivation by a trans-acting factor of human mhc-ia gene expression in interspecies hybrids between an ia-negative human b cell variant and an la-positive mouse b cell lymphoma induction of hla class ii genes by ifn-y is transcriptional and requires a trans-acting protein identification of an interferon-y response region ' of the human histocompatibility leukocyte antigen dret chain gene which is active in human glioblastoma multiform lines detailed delineation of an interferon-y-responsive element important in human hla-dra gene expression in a glioblastoma multiform line role of mhc gene products in immune regulation stimulation of oligodendroglial proliferation and maturation by interleukin- tumor necrosis factor-a enhances interferon-y mediated class i! antigen expression on astrocytes induction and regulation of interleukin- gene expression in rat astrocytes localization of the glial fibrillary acidic protein in astrocytes by immunofluorescence a ( ) transcriptional activation of hla-dra by interferon y requires a trans-acting protein cycloheximide, an inhibitor of protein synthesis, prevents y-interferon-induced expression of class ii mrna in a macrophage cell line lnterferon-y activates multiple pathways to regulate the expression of the genes for major histocompatibility class ii i-a#, tumor necrosis factor and complement component c in mouse macrophages single step method of rna isolation by acid guanidinium thiocyanate-phenolchloroform extraction tumor necrosis fac- tor-alpha production by astrocytes: induction by lipopolysaccharide, interferon-gamma and interleukin- recombinant human tumor necrosis factor increases mrna levels and surface expression of hla-a,b antigens in vascular endothelial cells and dermal fibroblasts in vitro plbl : a cdna clone of the rat mrna encoding cyclophilin induction of macrophage la antigen expression by rlfn-y and down-regulation by ifn-a/fl and dexamethasone are mediated by changes in steady-state levels of la mrna induction of macrophage la antigen expression by rlfngamma and down regulation by ifn-alpha/beta and dexamethasone are regulated transcriptionally astrocytes as antigen presenting cells, t. induction of la antigen expression on astrocytes by t cells via immune interferon and its effect on antigen presentation production of prostaglandin e and an interleukin-l-like factor by cultured astrocytes and c glioma cells astrocytes present myelin basic protein to encephalitogenic t-cell lines astrocytes of the brain synthesize interleukin- - ike factors antigen presentation and tumor cytotoxicity by interferon-y-treated microglial cells on the cellular source and function of interleukin- produced in the central nervous system in viral diseases class ii mhc-bearing keratinocytes induce antigen-specific unresponsiveness in hapten-specific th clones interleukin- of the central nervous system is produced by ameboid microglia tissue distribution of la antigens: la on spermatozoa, macrophages and epidermal cells primary rat astroglial cultures can generate leukotriene b functional interactions between astrocytes and neurons expression of la molecules by astrocytes during acute experimental allergic encephalomyelitis in the lewis rat immunoregulatory molecules and il- receptors identified in multiple sclerosis brain astrocytes induce bloodbrain barrier properties in endothelial cells production of tumor necrosis factor and other cytokines by astrocytes stimulated with lipopolysaccharide or a neurotropic virus acute autoimmune encephalomyelitis in mice. i . susceptibility is controlled by the combination of h- and histamine sensitization genes antigen presenting function of class !i mhc expressing pancreatic beta cells viral particles induce la antigen expression on astrocytes inducibility of la antigen on astrocyte by murine coronavirus jhm is rat strain dependent hypersensitivity of la antigen on astrocytes correlates with strain-specific susceptibility to experimental autoimmune encephalomyelitis the murine e-a immune response gene lmmunohistochemical analysis of the rat central nervous system during experimental allergic encephalomyelitis, with special reference to la-positive cells with dendritic morphology presentation of myelin basic protein by murine cerebral vascular endothelial cells preparation of separate astroglial and oligodendroglial cell cultures from rat cerebral tissues neuron-glial relationship during granule cell migration in developing cerebellar cortex. a golgi and electromicroscopic study in maccacus rhesus production of cytotoxic factor for oligodendroeytcs by stimulated astrocytes immune response gene products (la antigens) on glial and endothelial cells in virus-induced demvelination regulation of hla-dr gene by ifn-~,. transcriptional and post-transcriptional control ) la expression in chronic relapsing experimental allergic encephalomyelitis induced by long-term cultured t cell lines in mice evidence for two trans-acting genes regulating hla class ii antigen expression pr(xluction of tumor necrosis factor-alpha by microgila and astrocytes in culture upstream dna sequences required for tissue-specific expression of the hla-dra gene class ii box consensus sequences in the hla-dra gene: transcriptional function and interaction with nuclear proteins axonal guidance during development of the optic nerve: the role of pigmented epithelia and other intrinsic factors ) la-restricted encephalitogenic t lymphocytes mediating eae lyse autoantigen-presenting astrocytes astrocytes produce interferon that enhances the expression of h- antigens on a subpopulation of brain cells interferon-'), and la antigen are present on astrocytes in active chronic multiple sclerosis lesions on the presence of la-positive endothelial cells and astrocytes in multiple sclerosis lesions and its relevance to antigen presentation the distribution of la antigen in the lesions of rat acute experimental allergic encephalomyelitis inducible expression of h- and la antigens on brain cells mhc class ii transcription in different mouse cell types: differential requirement for protein synthesis between b cells and macrophages this work was funded in part by grants rg -a- and rg -a- from the national multiple sclerosis society (e.n.b) and grant bns- from the national science foundation (e.n.b). we acknowledge the support of the university of alabama at birmingham flow cytometry core facility (am ).we thank mr. keith berry for facs analysis, and ii yup chung, j. gavin norris and john r. bethea for helpful discussions. key: cord- -d yxgv authors: david, ayelet; golani-armon, adi title: polymer-based dna delivery systems for cancer immunotherapy date: - - journal: nanomedicine doi: . / - - - - _ sha: doc_id: cord_uid: d yxgv the use of gene delivery systems for the expression of antigenic proteins is an established means for activating a patient’s own immune system against the cancer they carry. since tumor cells are poor antigen-presenting cells, cross-presentation of tumor antigens by dendritic cells (dcs) is essential for the generation of tumor-specific cytotoxic t-lymphocyte responses. a number of polymer-based nanomedicines have been developed to deliver genes into dcs, primarily by incorporating tumor-specific, antigen-encoding plasmid dna with polycationic molecules to facilitate dna loading and intracellular trafficking. direct in vivo targeting of plasmid dna to dc surface receptors can induce high transfection efficiency and long-term gene expression, essential for antigen loading onto major histocompatibility complex molecules and stimulation of t-cell responses. this chapter summarizes the physicochemical properties and biological information on polymer-based non-viral vectors used for targeting dcs, and discusses the main challenges for successful in vivo gene transfer into dcs. cancer has long represented a major burden on health and longevity [ ] . the high prevalence of the disease has made it the basis of a major research focus, with the disease being investigated in many contexts. intensive research has yielded a large body of information that has served to uncover biological, biochemical and pathophysiological aspects of the disease, as well as their underlying mechanisms. based on the information gathered by the efforts of numerous researchers, novel nanosized medicines (nanomedicines), that can be effectively localized to tumors and actively taken up by cancer cells have been developed, and are expected to replace traditional chemotherapy and possibly irradiation due to increased effi ciencies and attenuated adverse side-effects [ ] . unfortunately, recent years have seen only minor improvements in cancer morbidity and mortality rates , with the most commonly used cancer therapies still being conventional chemotherapy, despite their serious side-effects. this has encouraged researchers to seek answers in the fi eld of immunotherapy [ ] . whereas exogenous anti-cancer agents have failed to generate strong and effective responses against tumors and induce regression, the endogenous activity of the immune system was hoped to elicit specifi c anti-tumor immunity that would result in tumor regression and elimination. in particularly, dendritic cell (dc) vaccinations were expected to induce immunological memory that would enable recognition and destruction of residual tumor cells that evaded earlier treatment, thereby avoiding recurrence of the disease [ ] . previous studies employing mouse models indicated that the establishment of anti-tumor immunity required the presentation of tumorassociated antigens (taa) by dcs [ , ] . two unique properties make dcs the most potent antigen-presenting cells (apcs), namely their ability to present exogenous antigens on major histocompatibility complex (mhc)-i molecules to prime cd + t-cells (i.e., cross-presentation), and their capability to initiate, activate and modulate the various arms of the immune system in a coordinated manner. indeed, in vivo targeting of tumor antigens into dcs was shown to elicit strong taaspecifi c dc + and cd + immune responses [ ] . therefore, dcs loaded with taa or tumor antigen-encoding plasmid dna may facilitate the development of new immunotherapies for cancer treatment. this chapter highlights the repertoire of non-viral, nanosized polymeric dna delivery systems (polyplexes) available to achieve effi cient gene transfer into dcs for immunotherapeutic applications in cancer therapy. the physicochemical characteristics and surface properties of polyplexes required for effi cient gene transfer and gene expression in dcs are discussed. the infl uence of the ligand valency on the targeting of dcs via dendritic cell surface receptors is also described. the fi eld of immunotherapy aims at activating a patient's own immune system against a host disease. immunotherapy includes the investigation of different mechanisms leading to activation of the immune response, and the development of methods to control such responses in a desired manner so as to combat disease, either as a single strategy or in combination with other treatments. in the specifi c case of cancer immunotherapy, it is speculated that the in vivo activity of endogenous immune cells would be far more sensitive and specifi c than would any exogenous treatment [ ] . anti-cancer vaccines have, thus been designed to induce both tumorspecifi c effector t cells and tumor-specifi c memory t-cells [ ] . such anti-cancer vaccination, it is hoped, will induce regression of established tumors, as well as prevent the onset of secondary tumors and metastases, possibly by addressing the issue of minimal residual disease (mrd). in this case, sparse cancerous cells that evaded irradiation or chemotherapy will be detected and destroyed by the immune system cells that, unlike the temporary exogenous treatment, are present in the body at all times. a major requirement for an effi cient anti-cancer vaccination is the generation of a cytotoxic t-lymphocyte (ctl) response, but manipulation of the other arms of the immune response, mainly cd + t helper cells, is critical for a robust and long lasting ctl response [ - ] . ctl response generation depends on antigen (ag) presentation on mhc t o naïve dc + t cells. the poor ag presentation activity demonstrated by tumor cells [ ] highlights the need for professional apcs to generate the desired ctl response. among the several types of apcs in the body, dcs are the most suitable for this purpose. in , ralph steinman was awarded the nobel prize for his discovery of dcs. dcs are a family of professional apcs. as the most powerful apcs, they are equipped with specialized machinery that enables them to regulate the initiation of a primary immune response [ - ] . originally derived from bone marrow, dcs are seeded in all tissues, where they sample their environment in an attempt to detect tissue damage, pathogen entry, infl ammation and malignantly transformed cells by taking up particles and molecules, processing them into short peptides and presenting the resulting peptides on mhc molecules. dcs use different routes to capture ags, including phagocytosis, micro-and macropinocytosis, and receptormediated endocytosis [ , , ] . importantly, receptor-mediated endocytosis can be exploited for the targeting of taas into dcs. immature dcs in peripheral tissues are characterized by high ag capture activity, low surface mhc levels and co-stimulatory molecules, as well as a tendency to induce tolerance to self-antigens. upon recognition of "danger" signals, dcs undergo a maturation process characterized by a reduction of ag capture activity and an increase of mhc and co-stimulatory molecule expression on the cell surface [ ] . mature dcs migrate to adjacent lymph nodes, where they present antigenic peptides to naïve t-cells [ ] . ag presentation, co-stimulatory molecules expression, and the secretion of an appropriate set of cytokines and chemokines by dcs facilitate the differentiation of naïve cd + and cd + t-cells into effector t-helper or t-cytotoxic cells, respectively [ , ] . dcs can, thus, simultaneously activate both a ctl response to directly kill pathogens or affected cells and a t-helper response to recruit and enhance the activity of other immune cells in the right context. this unique ability of dcs to activate the various arms of the immune system to achieve a powerful and effi cient immune response makes them the most promising candidates for immunotherapeutic manipulations . three distinct pathways are used by different cell types for the presentation of antigens, namely mhc class i and class ii antigen presentation, and cross-presentation. this pathway is present in all nucleated cells, and is used to present endogenous antigens derived from the nucleus or the cytoplasm. mhc class i antigen presentation can, therefore, report intracellular bacterial or viral infections, as well as malignant transformation in the presenting cell. the resulting intracellular products are degraded in the cytoplasm by the proteasome, translocated into the endoplasmic reticulum (er) via the transporter associated with antigen presentation (tap), and loaded on newly formed mhc class i molecules. the mhc class i/peptide complexes are then exported along the constitutive secretory pathway to the cell membrane, where they are presented to naïve cd + t-cells. the interaction between mhc class i/peptide complexes on dcs and t-cell receptors (tcr) on cd + t-cells induce the differentiation of naïve cd + t-cells into cytotoxic t-cells that are capable of killing the infected or transformed cells ( fig. . ) [ , , ] . this pathway is restricted to apcs, including macrophages (mΦs), dcs and b lymphocytes, and is used to present exogenous antigens. the exogenous antigens are taken up into these cells by endocytosis, where they are found in early endosomes, a mildly acidic cell compartment containing a small amount of proteases. early endosomes develop into late endosomes that are more acidic and present higher proteolytic activity, which process the captured ag into short peptides suitable for mhc presentation [ ] . the α and β chains of the mhc class ii molecule are assembled in the er and are associated with the invariant chain (ii) that the generation of a ctl response against virally infected or transformed cells is possible through the mhc class i pathway described above. however, other kinds of threats can be addressed by the immune system apcs through the mhc class ii pathway, but this pathway exclusively generates a t-helper response. as it is obvious that not every viral infection or malignant transformation involves apcs, and that many pathogens can impair antigen presentation by their host cells, an alternative mechanism must exist that would enable apcs to generate a cytotoxic t-cell response against the various exogenous threats they encounter [ ] . such a pathway indeed exists and is a unique property of dcs. in addition to the mhc class i and mhc class ii antigen presentation pathways, dcs can present exogenous antigens on mhc class i molecules to cd + t-cells. this pathway is termed cross-presentation, implying that it involves mechanisms from both the mhc class i and the mhc class ii pathways [ ] . although fi rst described more than three decades ago, the details of this pathway remain only poorly understood, with two main theories attempting to explain the mechanisms leading to mhc class i loading of endocytosed exogenous antigens. the cytosolic track suggests that the antigen is translocated from the endosome into the cytosol, where it enters the cytosolic processing pathway. the endocytic track, on the other hand, suggest that mhc i molecules are recycled from the cell membrane to the endosome, where they are loaded with antigenic peptides processed by endosomal proteases [ , ] . as mentioned earlier, anti-cancer immunity requires the generation of a specifi c anti-tumor ctl response that, in turn, requires proper antigen presentation. as part of the many techniques tumor cells use to avoid or weaken the immune system, they can substantially reduce the expression of mhc class i/peptide complexes on their cell surface. since tumor cells are, thus, poor ag-presenting cells, cross-presentation of tumor antigens by dcs is essential for the generation of tumor-specifi c ctl responses [ ] . in addition, given the unique ability of dcs to initiate and orchestrate the various arms of the immune system, including recruitment and activation of macrophages, natural killer (nk) cells, t-helper cells and b-cells, a strong and comprehensive anti-cancer immune response can hopefully be achieved by inducing such cells to present an appropriate tumor antigen [ ] . dc immunization requires that an appropriate antigen be presented to dcs in a proper manner and in the right immune context so as to allow ag uptake, processing and presentation on mhc molecules, parallel to dc maturation and t-cell priming. the fi rst method developed for dc immunization involved the use of whole tumor cells (wtc) or tumor cell lysates, either alone, mixed with an adjuvant, or genetically modifi ed to express an adjuvant. the major advantage of this method is that no ag identifi cation is required, and multiple ags are being delivered simultaneously. nevertheless, all wtc vaccine methods showed limited efficiency in clinical trials, probably due to insuffi cient interaction between the tumor antigens and the dcs [ ] . pulsing dcs with antigenic proteins or peptides corresponds to another approach. protein-pulsed dcs are capable of presenting ags on both mhc class i and mhc class ii molecules, with a long half-life of mhc class i presentation and no hla restriction (i.e., no need for patient selection), but they require an appropriate processing of the protein by dcs, which may be diffi cult to achieve. peptide-pulsed dcs, on the other hand, require no processing. still, they show limited mhc class ii presentation, a short half-life of mhc class i presentation, and a hla restriction. specifi c or total tumor mrna can be processed and presented on both mhc class i and class ii molecules. this, however, requires mrna extraction from a tumor sample and is, thus, patient-specifi c [ , ] . lately, the delivery of tumor ag-encoding plasmid dna has emerged as a promising method for dc immunization [ , ] . dna vaccines are dcs that were genetically modifi ed to express taas. dna vaccines are advantageous for several reasons. they enable the presentation of multiple epitopes of full-length taas on mhc class i and class ii molecules, and since processing occurs within the cell, they are not hla-restricted. in addition, effi cient gene transfer allows for a continuous supply of peptides in the modifi ed dc [ ] . genetic modifi cation of dcs can be achieved either by ex vivo gene delivery methods, including use of a gene gun, electroporation, ultrasound and microinjection, or by in vivo approaches, including naked dna delivery, and viral or synthetic vectors [ , ] . ex vivo delivery offers many advantages. the extracted cells are cultured and undergo maturation under controlled conditions and in the absence of inhibitory signals provided by the tumor cells, such that maturation status can be determined before re-administration. since transfection occurs in culture, high specifi city is achieved as only the selected cells are transfected. nevertheless, ex vivo gene delivery carries substantial limitations. the process is laborious, costly and time-consuming [ ] . patients must undergo cytopheresis, followed by culturing and maturation of the acquired cells, steps that must be performed for each patient separately. reproducibility is very low and different quality control methods are used. thus, only a limited number of patients would be expected to benefi t from this approach. hence, ex vivo gene delivery is not likely to become widely marketable. moreover, transfected mature dcs can show poor distribution from the injection site and ex vivo maturation can impair dc traffi cking to lymph nodes, a critical requirement for cross-priming [ , ] . in contrast, in vivo gene delivery may be expected to become "off the shelf" therapy. as a single product suitable for all patients, in vivo gene delivery can be produced on a large scale with lower costs. simple and uniform manufacturing and product control procedures will enable reproducibility and control over product quality. in addition, dcs can be targeted at different sites and in their natural environment, thereby not impairing their natural course of maturation and activation [ , ] . for these reasons, in vivo gene delivery is considered by many to be the best strategy for ag delivery into dcs [ , , ]. in vivo gene carriers must meet important requirements. first, they should be able to incorporate their plasmid dna cargo into the core of the nanoparticle and be stable enough to carry such cargo in the circulation and protect it from degradation. second, they should be able to selectively target the desired cell type and be properly internalized. third, they must facilitate escape from the endosome, cytoplasm traffi cking, nuclear transport and dna unpacking [ ] . to date, the most effi cient gene delivery systems are viral vectors. using small amounts of dna, viral vectors can induce high transfection effi ciency and stable, long-term gene expression. unfortunately, viral vectors possess some serious safety issues, including toxicity, immunogenicity and oncogenicity, with numerous clinical trials having been terminated because of this [ , , ] . restricted gene size is another major limitation of viral vectors. synthetic vectors correspond to cationic lipids or cationic polymers, respectively termed lipoplexes or polyplexes, which electrostatically bind the negatively charged dna. the main drawback of such vectors is their low transfection effi ciency, as compared to viral vectors. yet, synthetic vectors are simple, safe, easy to manufacture on a large scale and can carry plasmids of unrestricted size. moreover, they can be easily modifi ed to possess desirable properties, including targetability, serum stability, reactivity to external signals and an ability to stimulate an immune response. in fact, cationic polymers themselves may provide immune stimulation and mimic nuclear localization signals (nls), facilitating nuclear transport of their cargo [ , ] . various polymeric carriers for dna and rna delivery have been developed in the last decade as alternatives to viral vectors. cationic polymers with free primary, secondary and/or tetiary amines were the earliest dna carriers investigated as transfection reagents in mammalian cells. such polymers can condense large genes into smaller structures, protect dna from enzymatic degradation, and can mask the negative charges of dna, a prerequisite for successful transfection of most types of cells. some cationic polymers, such as poly-( l -lysine) (pll), l -polyethylenimine ( l -pei) and chitosan, are linear, while others, like b-pei and polyamidoamine (pamam) dendrimers, are highly branched chains [ ] . branched cationic polymers, such as pei and pamam, exhibit high transfection effi ciency in mammalian tissues. micro-and nanoparticulate systems which adsorb or encapsulate oligonucleotides or genes, based on, for example, poly(lactide-co-glycolide) (plga), polycyanoacrylate, polyorthoesters, gelatin, alginate or chitosan, are also under investigation as sustained release matrices for genetic drugs. some of the most important polymer-based systems utilized for dna delivery into apcs are discussed below. poly( l -lysine) (pll) was the fi rst cationic polymer developed for gene delivery. pll is a linear polypeptide presenting l -lysine residues in repeat units ( fig. . c ). this fi rst generation cationic polymer bears ɛ-amino groups that electrostatically bind the negatively charged nucleic acids, and interact with the negatively charged cell membrane. the large number of active functional amine groups allows for easy modifi cation with targeting ligands [ ] . however, the transfection effi ciency of pll is very low because it cannot mediate escape from the endosomal compartment and release into the cytosol. to overcome this barrier, pll is usually used in combination with chloroquine, although use of this agent is limited due to its cytotoxicity [ ] . another disadvantage of pll polyplexes is that transfection effi ciency is signifi cantly infl uenced by serum, probably due to the rapid binding to negativelycharged serum components [ ] . furthermore, in vivo applications of pll polyplexes are complicated by a high level of cytotoxicity [ ] and lack of in vivo stability [ , ] . because of low transfection effi ciency, imidazole groups (pka ~ . ) were introduced into pll to improve buffer capacity, thereby, enhancing transfection effi ciency [ ] . pll with high imidazole content mediated high gene transfection effi ciency, with gene expression levels close to those of pei (discussed below), and exhibited low cytotoxicity. the pegylation of pll can also improve its cytotoxicity profi le [ ] . finally, whereas it has been found that pll/dna complexes can be taken up by dcs, pll/dna did not alter dc phenotype through surface marker expression [ ] . complexation of plasmid dna encoding for chicken egg ovalbumin (ova) with pll-coated polystyrene (ps) particles induced high levels of cd + t-cells as well as ova-specifi c antibodies in c bl/ mice, and further inhibited tumor growth after challenge with ova expressing tumor cells [ ] . pll-based microspheres displaying mannan or mannoside-modifi ed surfaces (for targeting the mannose receptor, discussed below) were readily phagocytosed by both dcs and macrophages, however neither surface-assembled mannan-nor mannoside-modifi ed microspheres could stimulate dc maturation [ ] . thus, despite the promising results shown in early studies, pll is not likely to fi nd clinical applications [ ] . poly(ethylenimine) (pei) is a second generation and one of the most useful polycations for gene delivery [ ] . linear pei has only secondary amino groups that are almost all protonated under physiological conditions. branched pei presents not only primary and secondary amines but also tertiary amines. as such, only about two-thirds of the amino groups in branched pei are protonable under physiological conditions. the transfection effi ciency of peis depends on the molecule weight, the pei nitrogen/dna phosphate charge ratio (n/p) and the cell type. as every third position on the polymer backbone is occupied by a protonable amino group (fig. . a ) , its cationic charge density is very high, allowing for condensation of the negatively charged dna. under physiological conditions, only about % of the pei nitrogen atoms are protonated, leaving the other % to facilitate the important step of endosomal escape by the so-called " proton sponge effect " [ , , ] . thus, pei is the most popular and most effective polymeric transfection reagent cited to date [ ] . however, the positive charge of pei/dna polyplexes cause some serious problems, including adsorption to cells and negatively charged blood components, recognition by the immune system components, resulting in rapid clearance from the circulation, and cytotoxicity to non-target cells [ , ] . to overcome these limitations, pei has been conjugated to hydrophilic polymers (i.e., poly(ethylene glycol) (peg) [ ] , or hyaluronic acid (ha)) of different molecular weights [ ] . peg is wildly used in drug and gene delivery systems to shield charged, immunogenic or toxic segments, resulting in less toxic "stealth" particles that can evade the immune system and, thus, avoid rapid clearance from the circulation [ , ] . the conjugation of peg to pei was previously shown to increase polyplex solubility and serum stability and reduce cytotoxicity by shielding the high positive charge of pei [ ] . pegylated pei, however, showed lower transfection effi ciency, as compared to pei/dna complexes, and also lacked cell-specifi city [ ] . ligation of pei/ dna complexes to molecules targeting dc uptake receptors can signifi cantly increase transfection effi ciency. approaches for targeting dc receptors have generally involved either natural receptor ligands or the use of antibodies raised against specifi c receptors. targeting dc cell surface receptors may also provide cell activation signals [ , ] . different dc receptors have been used to facilitate targeted delivery of gene and drug carriers into dcs, including β-integrins (cd b and cd c) [ ] , cd [ ] and the fc receptor [ ] , but the most studied proteins for this purpose are members of the c-type lectin receptor (clr) family [ ] . clr is a family of receptors sharing structural homology in their carbohydrate recognition domain (crd), where specifi c sugar residues are bound in a calcium-dependent manner. one of the clrs, the mannose receptor (mr, cd ), is the most widely used receptor for targeting dcs, using both mannose and mannan [ ] . while several studies demonstrated that mannosylated-pei conjugates are effective in gene delivery via mr [ - ] , their transfection potential for primary human and mouse dc was found to be rather low [ ] . this might be due to the low affi nity of carbohydrate ligands to their receptors, which is usually in the low millimolar range [ ] , thus, limiting in vivo transfection effi ciency. the eight adjacent crds in mr may help to increase the binding affi nity and specifi city of polyplexes containing mannosylated glycans in a multivalent display [ , ] . we have recently described the design of multivalent mannosylated pei/dna complexes bearing mono-and trivalent mannose as a ligand for targeting mr-positive dcs [ ] . complexes bearing mono-and trivalent mannose (man-peg-b-pei/dna and man -peg-b-pei/dna, respectively) were safe and demonstrated signifi cantly higher in vitro transfection effi ciency in dcs. the mannosylated complexes were injected into the tail base of c /bl mice, and h post-injection, the percentages of gfp positive cells in the entire cell population and in the cd c + cells taken from the inguinal lymph nodes were measured. when examining the entire cells population extracted from the lymph node, only pei/dna complexes showed detectable gfp activity, but no signifi cant change was observed between the treatments (fig. . a ) . however, man -pegb -pei/dna was signifi cantly more effi cient in transfecting cd c+ dcs collected from inguinal lymph nodes, as compared to polyplexes prepared with peg-b-pei/dna or pei/dna (fig. . b ) . one challenge of targeting the mr, as holds true for all of the other abovementioned receptors, is that they are not exclusively expressed on dcs, and are also on several other cell types [ ] . mr, for example, is also expressed on monocytes, subsets of endothelial cells and tumor-associated macrophages [ ] . furthermore, the synthesis of carbohydrate ligands and analogs, especially multivalent or complex carbohydrates, often requires many time-consuming, low yielding steps [ ] . the stereochemistry of such carbohydrate ligands is diffi cult to control, and the products are diffi cult to purify. a frequently used alternative for carbohydrate ligands are antibodies raised against dc receptors (i.e., integrin cd c/cd [ ] , fc receptors [ ] , dec- [ ] , dc-sign [ ] and mr [ ] ), but these, even when humanized, may still elicit adverse immune responses that decrease the effi ciency of treatment and induce auto-immune side-effects [ ] . the use of peptide ligands remains, nonetheless, a promising method to target dc receptors. such ligands are easily synthesized, do not possess immunogenicity, and show high selectivity to dcs (table . ). accordingly, peptide ligands that could serve as dc-targeting moieties have been sought. a dc -nona-arginine fusion peptide (dc - dr) that binds nucleic acids by electrostatic interactions was previously exploited for the specifi c delivery of sirna to dcs in vitro and in vivo [ ] and was also used to silence immunosuppressive molecules in dcs so as to induce strong human t-cell immune responses [ ] . the dc targeting peptide (table . ) was recently studied by our group to mediate the specifi c delivery of pegylated-pei/dna polyplexes into dcs [ ] . polyplexes show signifi cant transfection effi ciency in dcs when decorated with dc peptide but not in endothelial cells. the transfection effi ciency observed was higher than that of pei/dna, signifying the potential use of dc -bearing polyplexes for immunotherapy via dcs . polyamidoamine ( pamam ) dendrimers are hyper-branched, symmetrical, fl exible and monodisperse polymeric molecules (fig. . d ) . szoka et al. fi rstly investigated pamam cascade polymers as non-viral gene delivery vectors [ ] . these polymers have an ammonia initiator core and amido-amine repeat units of different a. david and a. golani-armon generations (g -g ). their stepwise synthesis results in products that are uniform in terms of structure and size [ ] and bear a well-defi ned number of primary and tertiary amines. the surface primary amines are protonated, resulting in an extremely positively charged surface that enables dna binding in stable complexes, and interaction with the cell membrane. the tertiary amines in the interior of the polyplexes are protonable, endowing the dendrimers with ph buffering capacity that enables endosomal escape of the polyplexes following cellular uptake [ ] . all these properties, in addition to their non-immunogenic nature, make pamam dendrimers an alternative to the highly effi cient and highly immunogenic viral vectors. however, a major drawback of pamam dendrimers is that low generation dendrimers (g or lower) show poor transfection effi ciencies, while dendrimers of high generation (g and higher) are effi cient transfection agents, yet possess serious cytotoxicity [ ] . in addition, synthesis of high generation dendrimers is a high cost, labor-consuming process that last several days, making them less likely to be designed for large scale production [ ] . mannosylated g -pamam dendrimers conjugated to ova specifi cally targeted dcs and induced cross-presentation in vivo [ ] . moreover, pre-immunization with mannosylated pamam-ova leads to delayed onset of b -ova melanoma development, slower kinetics of tumor growth and increased survival of ovaimmunized mice. g -pamam dendrimers bearing dc-sign ligand in multivalent presentation also achieved effi cient dc targeting properties, however, did not affect dcs maturation [ ] . it has been postulated that mannosylated dendrimers, as opposed to dc-sign-modifi ed dendrimers, may trigger not only dc-sign, but also other mannose-specifi c clrs that contributes to dc maturation and activation. g -pamam dendrimers conjugated to mhc class ii-targeting peptide (padre , table . ) and surface-loaded dna have been shown to effectively transfect murine and human apcs in vitro [ ] . when applied subcutaneously, this conjugate preferentially transfected dcs in draining lymph nodes, promoted generation of high affi nity t-cells, and elicited rejection of established b tumors. chitosan is a linear polysaccharide (fig. . e ) obtained by deacetylation of its parent polymer chitin, a compound that is widely distributed in nature. chitosan is composed of randomly distributed ß-( - )-linked d -glucosamines (deacetylated units) and n-acetyl-d -glucosamines (acetylated units) and has an apparent pka value of . [ ] . chitosan is a biodegradable and biocompatible polymer, with low or no immunogenicity and antibacterial activity [ , ] . it was previously shown to be non-toxic in both test animals [ ] and humans [ ] . due to these properties, chitosan is attractive for drug and gene delivery. the high density of positive charges along the polymeric chain contributes to the condensation of the negatively charged dna, and more frequently, sirna molecules, into compact structures, thus protecting them from degradation by blood nucleases, promoting their cellular uptake [ ] . chitosan also possess adhesive properties by interacting with glycoproteins in the mucus [ ] , and is thus used in muco-adhesive drug delivery systems as an adsorption enhancer [ ] . dna plasmid incorporated into chitosan nanoparticles was able to induce dcs maturation and increase ifn-γ secretion from t cells after pulmonary mucosal immunization [ ] . however, unless modifi ed, chitosan has low solubility at physiological ph, which signifi cantly limits its use in many applications [ , ] . chemical modifi cations of chitosan have been performed to improve its solubility at physiological ph, including pegylation or quaternization of the amine groups of chitosan. however, the transfection efficiency of chitosan-based derivatives reported so far is generally not superior to that of pei [ ] . various dc-targeted chitosan-based dna delivery formulations has been designed to overcome the major obstacles facing the clinical development of chitosan, namely, the lack of cell-specifi city and low transfection effi ciency. mannosylated chitosan/dna complexes were more effi cient in transfecting dcs, as compared to water-soluble chitosan/dna, and induced better inf-γ production from dcs [ ] . mannosylated-chitosan-entrapping pei/hbv-dna complexes induced signifi cantly enhanced serum antibody production and ctl levels after intramuscular immunization [ ] . biotinylated chitosan nanoparticles were modifi ed with bifunctional fusion protein (bffp) vectors for achieving dc-selective targeting. bffp is a recombinant fusion protein consisting of truncated core-streptavidin fused to an anti-dec- single chain antibody (scfv). intranasal administration of plasmid dna-loaded bffp/chitosan nanoparticles, along with anti-cd dc maturation stimuli, enhanced the amount of mucosal igas, as well as systemic iggs, against nucleocapsid (n) protein of severe acute respiratory syndrome coronavirus (sars-cov) [ ] . finally, to improve low transfection effi ciency, chitosan-linked-pei/dna complexes have been designed, and showed high transfection effi ciency and low cytotoxicity towards dcs [ ] . vaccination with dcs transfected with chitosan-linked-pei/dna encoding gp (melanoma-associated antigen) slightly improved resistance to the b bl melanoma challenge . polymeric particulates have been shown to be effi cient in delivering plasmid dna into apcs (reviewed in [ - ] ). a key advantage of particulate vectors relative to other non-viral gene delivery systems is their superior in vivo stability. the principal types of polymers studied in this context include those made of poly(lactide) (pla; reviewed in [ ] ), poly(lactide-co-glycolide) (plga; reviewed in [ , , ] ), polyorthoesters [ ] , polystyrene (ps) [ , ] and poly(ε-caprolactone) [ ] . of these, plga has been studied most extensively in terms of its capacity to stimulate apcs. poly(lactide-co-glycolide) (fig. . b ) is an fda-and european medicine agencyapproved polymer for drug delivery systems for parenteral administration [ ] . this biocompatible and biodegradable polymer slowly degrades in vivo by hydrolysis, and its byproducts (lactic and glycolic acid) are easily metabolized and excreted. dcs and macrophages appear to have high affi nity for plga particles, as a high level of internalization of antigen-loaded particles has been demonstrated in both in vitro and in vivo settings [ , ] . plga microspheres carrying protein antigens or antigen-encoding plasmid dna are capable of eliciting potent antigenspecifi c immune responses [ , , ] . plga also affected expression maturation markers and cytokine production in dcs [ ] . this capacity appears to be driven by unique physical features of plga particles (i.e., surface charge, shape and the rate of polymer degradation [ ] ). the mechanisms leading to maturation induction in dcs, however, remain unclear. with respect to their stability in plga particles, antigen-encoding plasmid dna offers advantages over protein-based immuno-modulators, which can lose their biological activity in response to small changes in their tertiary and quaternary structures during formulation. dna delivery into dcs can be improved by using ligand-decorated plga particles. mannose-grafted plga nanoparticles lead to a signifi cant enhancement of ova accumulation in the infl amed colon compared to the healthy one, underlining the benefi t of active targeting of macrophages and dendritic cells in diseased tissues [ ] . mannan-decorated ova-loaded plga nanoparticles simultaneously enhanced antigen-specifi c cd + and cd + t-cell responses in vaccinated mice [ ] . modifi cation of plga micro-particles with p-d peptide (table . ) signifi cantly improved dc antigen presentation in vitro, and increased the rate and extent of microsphere translocation by dcs and macrophages in vivo [ ] . plga has been also utilized in combination with other cationic polymers for gene expression in dcs, with [ ] or without targeting ligands [ , ] . plga scaffolds encapsulating pei/dna and granulocyte-macrophage colony-stimulating factor (gm-csf) led to a signifi cant increase in gene expression, and high levels of expression that persisted for a period of time [ ] . yet, since plga nanoparticles are hydrophobic in nature, they tend to form aggregates that reduce the effi ciency of the system [ ] . in addition, plga particles are opsonized by the immune system components, and degraded before reaching their destination. another drawback of plga for delivery of genetic vaccines stems from their sustained release property [ ] . plga particles release their cargo very slowly, over days or even weeks, but in vivo they may be exocytosed from the cell or degraded in the lysosome over much shorter periods of time, before suffi cient amounts of cargo are released [ ] . moreover, most dcs die within days after activation and migration to draining lymph nodes [ ] , hence even fast degrading plga systems, which fully release the encapsulated dna within few weeks, cannot meet with the rapid release kinetic criteria and thus fail to induce high levels of target gene expression [ ] . finally, hydrolysis of plga leads to low ph within the particle and thus to dna degradation [ ] . for these reasons, despite the promising results obtained with animal models [ ] and clinical trials [ , ] , plga has been of limited use in this sense [ ] . the delivery of antigens into dcs carries tremendous potential for immune modulation, and specifi cally, for cancer immunotherapy manipulations. various polymeric nanomedicines for dna delivery, ranging from linear homopolymers to block copolymers, branched polymers, as well as combinations of different types of polymers, selected to reduce cytotoxicity and facilitate endocytosis of particles into dcs, are now been routinely examined for their ability to modulate dcs and macrophages. ultimately, dna delivery systems that can also induce dc maturation and activation would be advantageous. the main challenges for successful in vivo gene transfer into dcs are the cytotoxicity associated with many cationic polymer gene carriers, the lack of cell specifi city and relatively low transfection effi ciency, when compared to viral vectors. together with the fact that dcs compose only - % of cells in peripheral tissues, such gene systems will have limited success unless targeted. given the number of targeting molecules, immune-modulatory agents, chemokines, growth factors and antigens that can be considered for dc-specifi c delivery, large numbers of potentially useful formulations for dc manipulation are available. targeting dcs in vivo with tumor antigen-encoding plasmid dna can elicit effective and long-lasting tumor antigen-specifi c immunity, with minimal inconvenience to the patient. furthermore, dna delivery formulations that are stable for extended periods of time and which can enhance antigen presentation on both mhc-i and mhc-ii molecules are preferable. with respect to clinical translation, effi cacious non-viral gene delivery into dcs will depend on the combination of intelligent material design, the appropriate tumor specifi c antigen-encoding dna and immuno-stimulatory molecules to promote dc maturation and activation. cancer statistics nanoparticle therapeutics: an emerging treatment modality for cancer gene therapy for cancer treatment: past, present and future cancer immunotherapy via dendritic cells type i interferon is selectively required by dendritic cells for immune rejection of tumors host type i ifn signals are required for antitumor cd + t cell responses through cd {alpha}+ dendritic cells in vivo targeting of antigens to maturing dendritic cells via the dec- receptor improves t cell vaccination gene carriers and transfection systems used in the recombination of dendritic cells for effective cancer immunotherapy targeting tumor antigens to dendritic cells using particulate carriers engineering dendritic cells to enhance cancer immunotherapy combining immunotherapy and targeted therapies in cancer treatment dendritic cell based psma immunotherapy for prostate cancer using a cd -targeted adenovirus vector immunobiology of dendritic cells taking dendritic cells into medicine presentation of tumour antigens by dendritic cells and challenges faced intracellular events regulating cross-presentation spinning molecular immunology into successful immunotherapy towards a systems understanding of mhc class i and mhc class ii antigen presentation dendritic cell-based cancer gene therapy multivalent glycopeptide dendrimers for the targeted delivery of antigens to dendritic cells into the intracellular logistics of cross-presentation cross-priming in health and disease a modular and combinatorial view of the antigen crosspresentation pathway in dendritic cells regulation of antigen transport into the cytosol for crosspresentation by ubiquitination of the mannose receptor cellular vaccine approaches whole tumor antigen vaccines improvement of different vaccine delivery systems for cancer therapy third generation dendritic cell vaccines for tumor immunotherapy dendritic-cell immunotherapy: from ex vivo loading to in vivo targeting design and development of polymers for gene delivery rna-based vaccines mesenchymal stem cells: a promising targeteddelivery vehicle in cancer gene therapy cationic polymer based gene delivery systems biodegradable nanoparticles of mpeg-plga-pll triblock copolymers as novel non-viral vectors for improving sirna delivery and gene silencing polymers for intracellular delivery of nucleic acids synthesis of biodegradable multi-block copolymers of poly( l -lysine) and poly(ethylene glycol) as a non-viral gene carrier novel vectors for gene delivery formed by self-assembly of dna with poly( l -lysine) grafted with hydrophilic polymers stabilization of poly-l -lysine/ dna polyplexes for in vivo gene delivery to the liver steric stabilization of poly-l -lysine/dna complexes by the covalent attachment of semitelechelic poly polymer-based gene delivery with low cytotoxicity by a unique balance of side-chain termini role of dna topology in uptake of polyplex molecules by dendritic cells poly-l -lysine-coated nanoparticles: a potent delivery system to enhance dna vaccine effi cacy mannose-based molecular patterns on stealth microspheres for receptor-specifi c targeting of human antigen-presenting cells revisit complexation between dna and polyethylenimine-effect of length of free polycationic chains on gene transfection the proton sponge: a trick to enter cells the viruses did not exploit polycation gene delivery systems: escape from endosomes to cytosol different behavior of branched and linear polyethylenimine for gene delivery in vitro and in vivo polyethylenimine-mediated gene delivery to the lung and therapeutic applications biodegradable poly(ethylenimine) for plasmid dna delivery design of magnetic polyplexes taken up effi ciently by dendritic cell for enhanced dna vaccine delivery polyethylenimine-graft-poly(ethylene glycol) copolymers: infl uence of copolymer block structure on dna complexation and biological activities as gene delivery system the decrease of pamam dendrimerinduced cytotoxicity by pegylation via attenuation of oxidative stress pegylation signifi cantly affects cellular uptake and intracellular traffi cking of non-viral gene delivery particles recent developments in cancer vaccines rapid monoclonal antibody generation via dendritic cell targeting in vivo delivery of antigen to cd induces protective immune responses against tumors targeting antigens to dendritic cells in vivo distinct pathways of antigen uptake and intracellular routing in cd and cd t cell activation the mannose receptor and other macrophage lectins mannose polyethylenimine conjugates for targeted dna delivery into dendritic cells mannosylated biodegradable polyethyleneimine for targeted dna delivery to dendritic cells enhanced immune response against hiv- induced by a heterologous dna prime-adenovirus boost vaccination using mannosylated polyethyleneimine as dna vaccine adjuvant biological roles of oligosaccharides: all of the theories are correct macrophage receptors and immune recognition mannosylated polyion complexes for in vivo gene delivery into cd c(+) dendritic cells peptide ligands for a sugar-binding protein isolated from a random peptide library antigen-antibody immune complexes empower dendritic cells to effi ciently prime specifi c cd + ctl responses in vivo effective induction of naive and recall t-cell responses by targeting antigen to human dendritic cells via a humanized anti-dc-sign antibody antigenic targeting of the human mannose receptor induces tumor immunity strengths and weaknesses of immunotherapy for advanced non-small-cell lung cancer: a meta-analysis of randomized controlled trials interaction of icam- with beta -integrin cd c/cd : characterization of a peptide ligand that mimics a putative binding site on domain d of icam- development of a dendritic cell-targeting lipopeptide as an immunoadjuvant that inhibits tumor growth without inducing local infl ammation peptides identifi ed through phage display direct immunogenic antigen to dendritic cells red-cell icam- is a ligand for the monocyte/macrophage integrin cd c/cd : characterization of the binding sites on icam- targeted delivery of vaccine to dendritic cells by chitosan nanoparticles conjugated with a targeting peptide ligand selected by phage display technique a novel peptide carrier for effi cient targeting of antigens and nucleic acids to dendritic cells prototype alzheimer's disease vaccine using the immunodominant b cell epitope from beta-amyloid and promiscuous t cell epitope pan hla dr-binding peptide enhanced induction of hiv-specifi c cytotoxic t lymphocytes by dendritic cell-targeted delivery of socs- sirna targeted delivery of small interfering rna to human dendritic cells to suppress dengue virus infection and associated proinfl ammatory cytokine production dc -decorated polyplexes for targeted gene delivery into dendritic cells polyamidoamine cascade polymers mediate effi cient transfection of cells in culture cellsurface glycosaminoglycans inhibit intranuclear uptake but promote post-nuclear processes of polyamidoamine dendrimer-pdna transfection disulfi de cross-linked low generation dendrimers with high gene transfection effi cacy, low cytotoxicity, and low cost delivery of antigen using a novel mannosylated dendrimer potentiates immunogenicity in vitro and in vivo peptide-conjugated pamam dendrimer as a universal dna vaccine platform to target antigen-presenting cells chitosan-a versatile semi-synthetic polymer in biomedical applications biocompatibility, cellular uptake and biodistribution of the polymeric amphiphilic nanoparticles as oral drug carriers preparation of chitosan nanoparticles using methacrylic acid chitosan as a nasal delivery system: the effect of chitosan solutions on in vitro and in vivo mucociliary transport rates in human turbinates and volunteers chitosanhyaluronic acid nanoparticles for gene silencing: the role of hyaluronic acid on the nanoparticles' formation and activity preparation and effi cacy of a live newcastle disease virus vaccine encapsulated in chitosan nanoparticles tetraphenylporphyrin tethered chitosan based carriers for photochemical transfection pulmonary delivery of chitosan-dna nanoparticles enhances the immunogenicity of a dna vaccine encoding hla-a* -restricted t-cell epitopes of mycobacterium tuberculosis chitosan and its use as a pharmaceutical excipient receptor-mediated gene delivery into antigen presenting cells using mannosylated chitosan/dna nanoparticles controlled release of pei/dna complexes from mannose-bearing chitosan microspheres as a potent delivery system to enhance immune response to hbv dna vaccine dendritic cell targeted chitosan nanoparticles for nasal dna immunization against sars cov nucleocapsid protein gene-carried chitosan-linked polyethylenimine induced high gene transfection effi ciency on dendritic cells microparticles for the delivery of dna vaccines activation of antigen-presenting cells by dna delivery vectors dna-loaded biodegradable microparticles as vaccine delivery systems and their interaction with dendritic cells microencapsulation of dna using poly( dl -lactide-co-glycolide): stability issues and release characteristics enhancement of poly(orthoester) microspheres for dna vaccine delivery by blending with poly(ethylenimine) gene delivery to dendritic cells facilitated by a tumor necrosis factor alpha-competing peptide polymeric microspheres as stabilizing anchors for oligonucleotide delivery to dendritic cells development of functionalized nanoparticles for vaccine delivery to dendritic cells: a mechanistic approach plga-based nanoparticles: an overview of biomedical applications hydrophilic poly( dl -lactide-co-glycolide) microspheres for the delivery of dna to human-derived macrophages and dendritic cells uptake of poly( d , l -lactic-coglycolic acid) microspheres by antigen-presenting cells in vivo microparticle surface modifi cations targeting dendritic cells for non-activating applications poly-(lacticco-glycolic-acid)-based particulate vaccines: particle uptake by dendritic cells is a key parameter for immune activation composition and surface charge of dnaloaded microparticles determine maturation and cytokine secretion in human dendritic cells drug delivery to infl amed colon by nanoparticles: comparison of different strategies activation of antigen-specifi c t cell-responses by mannan-decorated plga nanoparticles uptake characteristics of ngr-coupled stealth pei/pdna nanoparticles loaded with plga-peg-plga tri-block copolymer for targeted delivery to human monocyte-derived dendritic cells sustained gm-csf and pei condensed pdna presentation increases the level and duration of gene expression in dendritic cells enhancement of gene transfection into human dendritic cells using cationic plga nanospheres with a synthesized nuclear localization signal genetic tagging shows increased frequency and longevity of antigen-presenting, skin-derived dendritic cells in vivo experimental studies and preliminary clinical trial of vinorelbine-loaded polymeric bioresorbable implants for the local treatment of solid tumors an eightmonth clinical study of la- . mg: a new -month, subcutaneous delivery system for leuprolide acetate in the treatment of prostate cancer polymers for gene delivery across length scales acknowledgments this work was supported by a grant from the israeli national nanotechnology initiative (inni), focal technology area (fta) program, nanomedicine for personalized theranostics. key: cord- -gx c mk authors: nan title: cell and tissue reactions date: journal: forensic neuropathology and associated neurology doi: . / - - -x_ sha: doc_id: cord_uid: gx c mk similar types of tissue reaction result as a final common pathway from a wide array of different internal brain pathophysiological states and external insults. since these cellular and tissue reactions are largely independent of the specific type of insults, they are, therefore, non-specific. the tissue reactions are to be differentiated according to their specific pathogenetic mechanisms, though these mechanisms as well as the phenomena are overlapping as demonstrated in fig. . ; brain ischemia as a type of metabolic disturbance, edema, intracranial pressure, necrosis, herniation and inflammation are influencing themselves and are dependent on each other. some will be mentioned again in later chapters as viewed from different forensic aspects; therefore, a certain redundancy is unavoidable. immediately following, we offer a survey of the individual types of reaction and their fundamental pathophysiological principles and morphology. similar types of tissue reaction result as a final common pathway from a wide array of different internal brain pathophysiological states and external insults. since these cellular and tissue reactions are largely independent of the specific type of insults, they are, therefore, non-specific. the tissue reactions are to be differentiated according to their specific pathogenetic mechanisms, though these mechanisms as well as the phenomena are overlapping as demonstrated in fig. . ; brain ischemia as a type of metabolic disturbance, edema, intracranial pressure, necrosis, herniation and inflammation are influencing themselves and are dependent on each other. some will be mentioned again in later chapters as viewed from different forensic aspects; therefore, a certain redundancy is unavoidable. immediately following, we offer a survey of the individual types of reaction and their fundamental pathophysiological principles and morphology. if brain volume increases, both blood and csf are displaced until the intracranial pressure (icp) increases. the consequent pression of the brain against the inelastic dura mater (fig. . ) and the skull can lead to a lethal series of complications in clinical neurology. the following remarks are based largely on ironside and pickard ( ) . brain volume depends on the following factors: . water content (cerebral hydration). the brain has a normal water content of about %. a disturbance of the blood−brain barrier (bbb) can lead to an increase in the fluid content, with a consequent increase in brain volume. . intracranial blood volume (hirai et al. ). this can be driven upward by a number of factors: arterial hypertension (marshall et al. ); enhanced cerebral blood flow secondary to elevated cerebral perfusion pressure (artru et al. ); a decline in the cerebrovascular resistance of arterioles, capillaries, and postcapillary vessels (langfitt et al. ) due to hypercapnia, hypoxemia associated with severe elevation of arterial pressure (marmarou et al. ) , or due to obstruction of the venous outflow of the brain. elevated cerebral blood volume, also known as "brain swelling," is a congestive process. . cerebrospinal fluid (csf) pressure. the central nervous system (cns) of the average adult contains a csf volume of approximately − ml. among the causes of a rise in csf pressure is acute obstructive high pressure hydrocephalus (see pp. f). a number of additional factors may also influence brain volume. the congested brain expands particularly rapidly under high arterial pressure (leech and miller ) . nawashiro et al. ( ) used experimental closed brain injury in rats to demonstrate a rapid and widespread increase in regional cerebral blood flow and impaired cerebral autoregulation. in humans a variety of factors act in concert after the incidence of severe brain injury. cerebral computed tomography (cct) and magnetic resonance imaging (mri) studies have shown that brain edema is the major fluid component of brain swelling (marmarou et al. (marmarou et al. , . a reactive hyperemia is an additional factor and may be the mechanism underlying mechanical/ischemic brain injury (seida et al. ). moreover, regional cerebral ischemia additionally is attributed to a compromised, leaky microvasculature rather than to vasospasm of larger vessels (schröder et al. ). the conclusion that brain swelling is due primarily to edema and not congestion of blood appears to be valid also for children (see chap. , pp. f) . cerebral blood flow in children with severe head injuries is not substantially increased over that in uninjured children (zwienenberg and muizelaar ) . this has also been demonstrated following experimental generation of brain trauma in newborn and juvenile pigs (armstead and kurth ) . the experimental findings of biagas and coworkers ( ) , in contrast, demonstrated a delayed rise in cerebral blood flow following experimental contusion in young and adult, but not in elderly, rats. normal adult icp is less than kpa ( kpa= . mm hg= . torr), mild elevations in pressure range from kpa to kpa, moderate elevations attain kpa, while major intracranial hypertension exceeds kpa (miller and ironside ) . normal csf pressure in adults is − . kpa, with an upper limit of kpa. while a short-term rise in icp pressure of up to kpa may be tolerated so long as it does not cause distortion of the brain (johnston and paterson ) , a mbi-induced rise of more than kpa with distortion of the brain can result in herniation and brain death syndrome. the classic symptoms associated with elevated icp are vomiting, headache, papilledema, and coma. schematic demonstration of overlapping pathogenetic mechanisms that are associated with different types of tissue reactions by a causal link: metabolic disturbance, i.e., edema, increasing cerebral blood volume, perfusion pressure and herniation, i.e., brain swelling and cortical necrosis distortion or pressure on the floor of the fourth ventricle are most likely responsible for the vomiting, while stretching and distortion of the dura mater and major intracranial blood vessels, all sensitive to pain, probably account for the headache. the papilledema is not a direct result of the rise in water content, but the consequence of an accumulation of axoplasm in the optic papilla secondary to the blockade of axoplasmic flow from ganglion cells along the optic nerve. papilledema is a common symptom of chronic intracranial hypertension. it is not, however, a common feature of mbi (selhorst et al. ) , and its absence does not necessarily mean that icp is normal. a further increase of icp leads to a loss of consciousness and coma. elevated icp affects other organ systems as well. it often induces arterial hypertension and systolic blood pressure may climb to kpa or more. the arterial hypertension is caused by an increase in sympathetic activity. cases of raised icp with myocardial involvement often exhibit pathological alterations consisting of subendocardial hemorrhage and widespread focal myocardial necrosis as well as electrocardiographic changes such as t-wave inver- sion and elevation of the st−segment, which point to myocardial ischemia (connor ) . respiratory disturbances associated with elevated icp often precede apnea. central neurogenic hyperventilation is observed in connection with the midbrain lesion. in patients with raised icp, neurogenic pulmonary edema can complicate the clinical course. the mucosa of the digestive and urogenital tracts can become hemorrhagic, eroded, and ulcerated, gastric erosions being particularly common in comatose patients with elevated icp. a fundamental distinction must be made between global and focal cerebral edema. the former follows acute systemic hypoxic events, e.g., transitory cardiac arrest or chronic hypoxia in respiratory diseases. global cerebral edema may also be associated with metabolic diseases, intoxications, and inflammation. focal cerebral edema results from focal tissue destruction or alteration of brain tissue that has undergone membrane failure in cells and vessels due to infarction or traumatic hemorrhage or tumor. the tissue surrounding the central lesion has passed only the upper threshold of electrical silence and thus retains the capacity to recover if perfusion is restored in time (harding and copp ) . this zone resembles the penumbra surrounding the moon in full eclipse (astrup et al. ) . because these tissue changes are partly reversible, they are of considerable therapeutic interest (see pp. f). information on the incidence of intracranial hypertension has been gained mainly from survivors of mbi. miller and his associates ( ) reported that icp levels exceeded . kpa for min or longer in % of patients in one series and in % of another series of patients (miller et al. ) . raised icp was found in more than % of cases in a more detailed prospective study of elevated icp in victims with severe head injury by marmarou and colleagues ( ) . jones and colleagues ( ) found elevated icp in more than % of brain injury patients ( severe, moderate and minor) undergoing artificial ventilation with icp monitoring. these findings indicate that intracranial hypertension is a common event, especially in comatose patients. certain features detected by cct are consistently associated with elevated icp. loss of the images of the third ventricle and perimesencephalic csf cisterns, and dilatation of the lateral ventricle contralateral to a mass lesion, as well as the absence of these features are no guarantee that icp will remain normal (teasdale et al. ; o'sullivan et al. ) . elevated icp is also common in patients who are comatose from causes other than brain injury (chan-dler and kindt ) . among the possible causes are liver failure (hepatic coma), intracranial hemorrhage (subarachnoid and intracerebral), post-hypoxemic states (cardiorespiratory arrest, near drowning), infection (meningitis, abscess, and encephalitis), as well as various other types of inflammation and intoxication. adams and graham ( ) published neuropathological criteria for determining at autopsy whether icp in victims of brain injury was elevated during life. the same team (graham et al. ) compared the nature of the brain damage in patients with and without elevated icp after suffering a non-missile brain injury who had survived long enough to receive treatment in a neurosurgical unit. pressure necrosis of the parahippocampal gyrus, an indicator of high supratentorial icp and tentorial herniation, was present in twothirds of the patients in their most recent study. it was closely associated with skull fracture, brain swelling, diffuse axonal injury, hypoxic brain damage, and extensive supratentorial hematoma. the brain stem was damaged in % of victims with pressure necrosis, the anterior lobe of pituitary was necrotic in %, and there was hemorrhagic infarction in the distribution of the posterior cerebral artery in %. klatzo ( ) distinguishes two types of edema: vasogenic edema and cytotoxic edema (table . ). this distinction continues to be of both theoretical and practical value (cf. kimelberg a, b; mendelow et al. ) . blood vessels in the cns are uniquely restricted in their permeability. many substances are exchanged between the blood and brain parenchyma at slower rates and the concentrations in cns at steady state are lower than in other organs (lee ) . dyes as well as proteins, drugs, and microorganisms introduced into the csf enter the brain freely, while those introduced into the blood stream do not. this limited permeability is attributed to the bbb, a specialized feature of the cns that restricts the entry of viruses and bacteria, emigration of immune cells, and diffusion in the cns of drugs and soluble molecules from the systemic compartment. intravenously applied evans blue will bind to serum albumin and permeate the bbb only under pathological conditions. this phenomenon is demonstrated after experimentally induced ischemia of one hemisphere by means of macroscopic observation ( fig. . a) and by means of fluorescence micros- copy ( fig. . b). the demonstration of albumin in human brain using immunohistochemistry will only succeed during the very early postmortem interval ( fig. . d) − before a general diffusion of blood serum occurs within the brain parenchyma − as a morphological marker of the diffuse postmortem bbb disturbance. under experimental conditions an accumulation of plasma proteins in purkinje cells gencic a, ikegaya et al. ) takes place. anatomically the bbb consists of a capillary endothelium containing intercellular tight junctions and specialized enzymes, such as transpeptidases, dehydrogenases, decarboxylases, and monoamine oxidases (reese and karnowsky ; brightman and reese ; lee ) . the intercellular junctions are most conspicuous near the luminal surface where the cell membranes fuse. a basement membrane in contact with astrocytic foot processes surrounds the endothelial cells. pericytes are enclosed by an envelope of the perivascular basement membrane, which splits to enclose the pericyte. brain capillaries are almost totally invested by astrocytic processes. astrocytes exert inductive actions during development and are thereby largely responsible for the special attributes exhibited by endothelial cells, such as the presence of tight junctions between the cells (abbott et al. ) . astrocytes and microglia both contribute to the formation of the bbb (prat et al. ) . there is an inverse hemodynamic relation between icp and cerebral blood flow (cbf): the higher the icp, the lower the cbf. if cerebral circulation and circulatory autoregulation are normal, a drop in icp will induce only a slight increase in cbf until a threshold level of about kpa is attained. cbf is regulated by mechanisms such as compensatory dilatation of small arteries and arterioles. patients suffering from acute mbi, intracranial hemorrhage, or hypoxic brain injury need a mean arterial blood pressure above kpa to maintain perfusion. the brain damage in such circumstances is associated with a rise in cerebrovascular resistance due to the vessels' spastic reactivity. baseline icp levels may even need to be higher in order to drive sufficient blood through the brain tissue (chan et al. ) . should cbf drop below (ml/min)/ g, potassium levels in the intercellular spaces rise, while intracellular sodium and calcium increase. cellular edema causes the cells to swell and a calcium influx triggers a series of autodestructive processes. the bbb can be compromised by the following three mechanisms (see miller and ironside ) : . enhanced vesicular transport and creation of transendothelial channels by perturbation of endothelial plasmalemma, increased pinocytic activity, the activity of free oxygen radicals, or an increase in superoxides. subarachnoid application of hemoglobin and hemoglobin degradation are known to cause brain edema (huang et al. ) . . disconnection of the interendothelial tight junctions, e.g., by substances of very high osmolarity. . structural or biochemical modification of the endothelial membrane that intensifies its permeability. it has also been known for a long time that the ability of certain substances to pass through the bbb depends on their specific properties: ▬ their nature regarding the capacity of the bbb for active transport (broman and steinwall ) ▬ their affinity for carrier molecules (lajtha ). ▬ their molecular radius (thompson ) . ▬ their lipid solubility (oldendorf ) . as shown in detail later, the permeability of the bbb also depends on age. a good example of this is bilirubin encephalopathy, which is caused by bilirubin crossing the bbb of certain nuclei during the perinatal period − a feat it is incapable of in later life − and inflicting damage on nerve cells and, to a lesser vasogenic perifocal edema in a case of a traffic accident associated with liver failure and elevated bilirubin level. a massive intracerebral hemorrhages and green-colored edema; b focal hemorrhage; perifocal as well as contralateral greencolored edema degree, on astrocytes (for further information see pp. ff). thus for bilirubin at least the bbb appears to be less efficient at birth than in children or adults (haymaker et al. ) . in mbi with intracerebral hemorrhages and associated elevation of the bilirubin level the perifocal edema can be marked by a green color demonstrating extravascular bilirubin as demonstrated in fig. . . in senile and mentally disturbed patients, the bbb has been found to have a lower rate of transport, a reduced uptake of glucose and other nutrients, plus a diminished outflow of metabolic wastes (quadbeck (quadbeck , . the movement of water from the vascular compartment to intracellular or interstitial spaces is not regulated biochemically by the bbb since hydrostatic and more powerful osmotic gradients enable free water to diffuse passively across capillary membranes. the passage of ions and molecules of various sizes is controlled by lipid-soluble substances in the endothelial wall and by ionic channels and active pumps. the white matter of the brain is % water, the gray matter % (adachi and feigin ) . a rise in brain water content entails an increase in brain volume, i.e., brain edema. as a result of energy failure (deprivation of oxygen and glucose), which disables the sodium-potassium membrane atpase pump system, water accumulation within the cells follows an osmotically obliged response to an increase in intracellular sodium and loss of potassium ( fig. . c) . the influx of osmotically drawn water causes swelling of the cell. the energy failure is accompanied by an influx of sodium and chloride and an efflux of hydrogen ions, potassium, and bicarbonate. there is a parallel disturbance of the voltage-and ligand-gated mechanisms that regulate the entry of calcium into the cell that initiates a calcium-mediated destructive sequence. cytotoxic edema is the result of the action of various cytotoxic agents, e.g., of cyanide or triethyl tin (also see chaps. , ). brain cells can also swell without a concomitant increase in brain volume if fluid shifts from an extracellular to an intracellular space. although this does not produce an immediate rise in icp, cellular edema ultimately draws water from the vasculature into the brain, increasing brain volume and precipitating a rise in icp. ischemic edema is a cytotoxic edema whose clinical effects depend on how much and for how long cerebral blood flow is reduced (bell et al. ) . klatzo ( ) showed that the initial cytotoxic edema following permanent occlusion of a major blood vessel causes irreversible ischemic cell damage, resulting in a secondary vasogenic edema when endothelial cells are damaged. even temporary ischemia with subsequent reperfusion will induce reactive hyperemia and secondary endothelial damage that produces a secondary vasogenic edema (greenwood ) . as a whole, the brain is resistant to pure hypoxia (diminished oxygen supply) (pp. ff, see also miyamoto and auer ) , which causes no or only partial breakdown of the bbb and which may be reversible after recovery (bakay and lee ; auer ) . anoxia (complete lack of oxygen), by contrast, results in a rapid rise in bbb permeability that becomes irreversible after just a few minutes (pp. ff). if anoxia acts in combination with complete ischemia secondary to ligation of both common carotid and subclavian arteries, the bbb can retain its impermeability for as long as h (broman ; blank and kirshner ) . incomplete ischemia, however, will rapidly and completely break down the bbb (bakay and lee ) . kimelberg and colleagues ( ) could demonstrate the potential toxic mechanisms of this type of edema on neurons. they describe a primary cytotoxic effect on astrocytes that induces astrocytic swelling. this swelling in turn leads to the release of excitatory amino acids such as glutamate, whose levels increase in extracellular spaces following the incidence of mbi (kanthan and shuaib ) . a rise in extracellular glutamate levels causes cell death due to an influx of ca + via the neurons' activated ionotropic glutamate receptors (choi and rothman ) . other authors have offered a somewhat different explanation for the irreversible injury: it may be induced by simultaneously generated free radicals or extravasated plasma components that stimulate the activation of nitric oxide synthase (nos) in reactive cells. nitric oxide thus generated may contribute to diffuse degeneration of the white matter (gotoh et al. ). the accumulation of plasma proteins within neurons and microglia in combination with cytochrome-c release by astrocytes can lead to dna fragmentation and cell death (matz et al. ). klatzo's ( ) classification of brain edema has proved to be a useful aid in distinguishing between various pathogenetic mechanisms and their sequelae. in experimental and clinical practice however it must be assumed that brain swelling is caused by a combination and/or a temporal overlapping of a number of processes, as described by kimelberg et al. ( ) . non-invasive diffusion-weighted (dw) mri is able of calculating changes in the apparent diffusion coefficient (adc) of water protons in the brain (garcia et al. ; chu et al. ) . a decline in adc has been attributed mainly to a reduction of the extracellular space and a rise in intracellular volume, although other contributing factors are possible (pierpaoli et al. ) . in this manner cellular (cytotoxic) edema can be differentiated from extracellular (vasogenic) edema and a correlation made with the severity of injury and consequent deficit. because dw-mri (see also mendelow et al. ) enables edema types to be determined intra vitam under clinical conditions, current classifications of edema types could be revised in light of new findings. however, we should remember that all edema ultimately arises from the blood. it is the size of the leak in the brain vasculature that gives rise to the artificial distinction between cytotoxic and vasogenic edema, cytotoxic edema being mainly water and vasogenic edema including proteins also derived from the blood. brain swelling caused by edema, congestion or a rise in csf pressure can obliterate the subarachnoid space, flatten the gyri, reduce ventricular size (squier , fig. . ), and cause herniation (see pp. ff). at autopsy the white matter seems softer in consistency and paler than normal. the normal, sharp demarcation between gray and white matter is lost, often with thinning of the cortex overlying the zone of white matter edema. the arcuate fibers are spared. in rare cases of vasogenic edema associated with liver insufficiency combined with elevated bilirubin levels in serum the spread of edema may be characterized by a greenish-yellow color ( fig. . ) . under normal conditions bilirubin is not able to permeate the bbb, with one exception: the newborn (see pp. ff). cytotoxic edema, which predominates in gray matter, is characterized by astrocytic swelling and the enlargement of perineuronal and perivascular spaces indicative of the swelling of astrocytic foot processes around neurons, capillaries, and arterioles ( fig. . ). the hallmarks of vasogenic edema include swelling of pericapillary astrocytic processes and of oligodendrocyte cytoplasm, plus the spread of exudate in the extracellular space of white matter ( fig. . ). macroscopically vasogenic edema induces a slight green discoloration of the white matter. histologically, edema, vasogenic edema in particular, features extensive cytoplasmic vacuolation in the white matter with status spongiosus where a clear space surrounds small vessels and nuclei ( fig. . ) . ultrastructurally, few visible changes are evident in the cerebral capillaries. the brain parenchyma, in contrast, exhibits swelling of glial processes or dendrites, splits in the myelin laminae and, less often, enlargement of the extracellular space. vacuolation may be especially prominent in myelinated fiber bundles and constitute the earliest and most consistent elementary edema-induced change. following immersion fixation, however, these phenomena can often be difficult to distinguish from (postmortem) artifacts. these phenomena may be associated in the beginning with a leukocyte emigration ( fig. . a) and in the last stage with astrocytic hyperplasia and hypertrophy ( fig. . c, d). astrocytes and macrophages also ingest extravasated plasma proteins. myelin sheaths undergo increasing fragmentation and macrophages phagocytose lipid breakdown (figs. . f, . b, . ) . oligodendrocytes are much less likely to partake in the alterations of edematous brain tissue. most cases of brain edema exhibit a combination of cytotoxic and vasogenic edema. inhibition of ion pumping or secondary retrograde reaction can cause swelling of neurons. the usual reaction is neuronal shrinkage, commonly combined with swelling of neighboring glial cells, especially of astrocytic processes. irreversible changes in the myelin sheath are unequivocally manifested by the apposition of mac-rophage reaction in the form of compound granular cells. involvement of the white matter by edema of this severe degree coincides with a so-called edematous necrosis (jacob ) . the final phase of terminal edema can be marked by cystic alteration or glial scaring. brain swelling is one of a wide variety of neurological conditions, among them tumors, hemorrhages, and ischemia/hypoxia, that can induce an increase in icp. a rise in icp leads not only to compression of the brain, but to diminished csf volume, shifting, and herniation, as well as to secondary complications such as ischemia and hemorrhage. if not treated, icp can rapidly progress to death due to brain stem compression secondary to cerebellar or uncal herniation (meyer ) . focal expanding mass lesions must be distinguished from diffuse space-occupying processes. ▬ diffuse brain lesions such as inflammation, bilateral intracranial hemorrhage, total brain ischemia (cardiac arrest) or intoxication are macroscopically characterized ( fig. . ) by a tension of the dura, gyral flattening, and by narrow ventricles that are symmetrically compressed. no lateral shift of midline structures is seen, but rather central herniation of the diencephalon (centrencephalic herniation) and by cerebellar tonsillar herniation and compression of the medulla oblongata. bilateral herniation may occur and various types of herniation result from caudal displacement of the brain parenchyma (for types of herniation, see below). ▬ focal intracranial processes such as abscess, tumor, infarction or subdural hemorrhage ( fig. . a, b) are also capable of inducing a life-threatening homolateral rise in icp. because they allow time for intrinsic compensatory mechanisms to operate, particularly reduced csf volume, slowly expanding focal lesions are less likely to cause an early increase in icp and brain shift. however, the distortion and herniation of the brain in such cases can be considerable. rapidly expanding focal lesions, by contrast, are more likely to produce an immediately elevated icp. brain death often supervenes in such cases before much distortion or herniation can occur. distortion of the brain results from compressive forces exerted by adjacent structures, which leads to overall expansion of the hemispheres. the dura mater may become so tense as to compress the terminal branches of the cerebral arteries, with consequent ischemic or hemorrhagic necrosis of cortical structures (lindenberg ) or impairment of perfusion (janzer and friede ) accompanied by perisulcal infarcts. continued expansion of the mass may provoke contralateral displacement of the midline structures (see chap. and fig. . a). if the contralateral foramen of monro is obliterated, the contralateral lateral ventricle may become enlarged, triggering a further rise in icp. a lesion that expands in the frontal lobe may displace the free margin of the anterior part of the falx cerebri ( fig. . a, d). if a lesion expands in the temporal lobe, a disproportionately pronounced shift of the third ventricle will occur ( fig. . a), with upward displacement of the sylvian fissure and neighboring branches of the middle cerebral artery. the ultimate result of the space-occupying process is development of lateral and then downward herniation, visible at several loci: . falx cerebri (cingulate, or subfalcine herniation). . tentorium cerebelli (lateral, or uncal herniation). . thalamus/hypothalamus (central, or diencephalic herniation) which may result in downward displacement and hemorrhage in the midbrain and pontine tegmentum. . foramen magnum (tonsillar herniation). a bilateral expanding supratentorial mass can cause herniation-induced notches as well as hemorrhages of the uncal area. this in turn exerts downward pressure on the medial part of the parahippocampal gyrus toward and through the tentorial incisura (figs. . b, . ). the clinical and morphological se- d a hemorrhage in the upper frontal lobe may lead to a notch, a hemorrhage or a softening of the corpus callosum; e a rare tentorial displacement caused by an infratentorial space-occupying process is demonstrated by notches on the upper cerebellar surface (arrows) quelae of uncal herniation depend on the magnitude of the supratentorial pressure and on anatomical variations in the size of the tentorial notch, position of the brain stem within the notch, position of the oculomotor or third nerve, and the inter-oculomotor nerve angle. they also depend on the structure and course of the posterior cerebral artery, known to play a role in herniation syndromes (adler and milhorat ) . herniation of the parahippocampal gyrus ( fig. . ) creates narrowing of the midbrain along its transverse axis and compression of the aqueduct. this pushes − in the case of a unilateral expanding mass − the contralateral cerebral peduncle against the opposite free tentorial edge ( fig. . b), pinning the ipsilateral oculomotor nerve between the petroclinoid ligament or free edge of the tentorium and the posterior cerebral artery. the lesion of the ipsilateral oculomotor nerve is associated clinically with ptosis and dilatation of the ipsilateral pupil, which becomes unresponsive to light. the elevated icp produces a wedge of hemorrhagic necrosis along the parahippocampal gyrus groove (so-called pressure necrosis, to be differentiated from "herniation contusion" − see figs. . b, . b) . pressure necrosis can result from an icp exceeding . kpa (see adams and graham ) . it is analogous to necrosis due to brain retractor pressure in neurosurgery. clinically, uncal herniation is accompanied by an abrupt worsening of the patient's neurological status, with loss of consciousness and onset of decerebrate rigidity, both due to midbrain impairment caused by pressure coming from above. compression of arteries can also cause secondary necrosis: if the anterior choroidal artery is occluded, the result can be infarction of the medial part of the globus pallidus; posterior cerebral artery occlusion can cause hemorrhagic infarction of the thalamus, of the medial and inferior surfaces of the cortex of the occipital lobe (figs. . , . ) , and of the temporal lobe including the hippocampus. herniation of the ipsilateral cingulate gyrus under the free edge of the falx results from the unilateral growth of a mass in the frontal or parietal lobe and causes selective displacement of the pericallosal arteries away from the midline (fig. . a, d) . should this compromise circulation through the pericallosal arteries, the parietal parasagittal cortex can become infarcted, which is expressed clinically as weakness or sensory loss in one or both legs. a frontal or parietal mass lesion can induce downward axial displacement of the diencephalon (fig. . a) and rostral brain stem (figs. . c, . a) . the consequent symmetrical herniation of both parahippocampal gyri (fig. . a , b) may be manifested clinically by bilateral ptosis and failure of upward gaze. the final clinical result is loss of consciousness, decerebrate rigidity, and bilateral dilatation of the pupils with loss of the pupillary light reflex. the blood pressure rises due to increased sympathetic activity. hemorrhage or necrosis of the midbrain and/ or pons are the possible sequelae of supratentorial space-occupying processes located adjacent to the midline (figs. . a, . a, . ). these lesions are caused by caudal displacement and anterior-posterior elongation of the rostral brain stem and by sideto-side compression by the tentorial hernia, coupled with relative immobility of the basilar artery. progressive displacement stretches and narrows the central perforating branches of the basilar artery which supply the rostral brain stem, causing spasm, infarction or hemorrhage. an early complication of expanding masses in the posterior cranial fossa is displacement of the cerebellar tonsils through the foramen magnum (figs. . d, . b, c) . this may also be caused, however, by lesions occupying the supratentorial space. morphologically, the tips of the tonsils display hemorrhagic necrosis and grooving of the ventral surface of the medulla where it impinges on the anterior border of the foramen magnum. clinically these changes give rise to apnea, which can occur while the victim is still conscious. among the other common neurological deficits are decerebrate rigidity and impairment of brain stem reflexes. upward tentorial displacement (fig. . e) is produced by enlargement of an infratentorial mass in the posterior cranial fossa. both the fourth ventricle and aqueduct become compressed and displaced contralaterally. there is upward herniation of the superior surface of the cerebellum, which is distinguished clinically by the abrupt manifestation of bilateral extensor rigidity and loss of pupillary light response. a number of clinical complications associated with brain swelling, brain edema, and bbb can arise during diagnostic and therapeutic interventions in the cns carried out by physicians or nursing personnel. since the sequelae are foreseeable − and in most cases avoidable − these complications will be dealt with briefly in the following. ▬ in patients with elevated icp, a lumbar puncture of the csf can give rise to herniation. papilledema, though not always associated with icp, must be excluded before every csf puncture. should other clinical signs of high icp be present, a ct examination must be carried out prior to puncture. ▬ pharmacotherapy must not be performed without knowing whether the agent can permeate the bbb and affect the cns. this is especially true of substances such as antibiotics or cytostatics that are intended to reach and act upon the cns. other substances are not intended to reach the cns because they are toxic there; thus, the contraindication for intrathecal application of vincristine (see p. ). ▬ if cns edema already exists, the bbb may be (pathologically) permeable to substances not intended to reach the cns, some of which can then have a toxic effect. a mbi-induced perifocal edema, for example that arises in the context of polytrauma-induced shock, can produce greenish discoloration of the perifocal edema as a consequence of an accompanying hepatic insufficiency, which can have an additional neurotoxicologic effect on the neurons. ▬ the status of the bbb may well be age dependent, its postnatal status differing from that of adults. blood group incompatibility between mother and child during pregnancy or after birth can cause bilirubin encephalopathy (see pp. ff). the bbb appears to be less permeable in the elderly. ▬ a cytotoxic effect can be mediated by alcohol in mbis with consequent loss of neurons. alcohol lowers cerebral perfusion pressure (cbf) and depresses ventilation. it diminishes respiratory drive in response to elevated paco levels. ethanol-induced respiratory depression and hypotension can increase the morbidity and mortality associated with brain injury. the theoretical considerations of kimelberg et al. ( ) appear to contradict these empirical findings, arguing rather that alcohol inhibits both the excitotoxin receptor function of neurons (simson et al. ) and the influx of ca + via nmda receptor ion channels. hydrocephalus is characterized by abnormal accumulation of fluid within csf spaces, i.e., within the cerebral ventricles and subarachnoid space. by this time, there is atrophy of the brain parenchyma and additional ventricular enlargement. csf is formed by the choroid plexus at a rate that remains unchanged within a wide range of icp values: − ml/h will avert a long-lasting imbalance between its formation and absorption. elevated csf pressure is associated only with acute or obstructive hydrocephalus (see below). the subarachnoid space and ventricular system are connected via the foramina of luschka and magendie in the basal cisterns. csf is absorbed by the arachnoid villi, which do not fully develop until adolescence or young adulthood (grassman and potts ) . in fetuses and infants, csf is absorbed mainly through nerve roots and periventricular and arachnoid veins. external hydrocephalus ex vacuo involves diffuse loss of gray matter that gives rise to external atrophy, with dilatation of the subarachnoid space ( fig. . a , b). diffuse loss of white matter can cause expansion of the ventricular system, the so-called internal hydrocephalus ex vacuo (fig. . c ). the causes of the gray and white tissue damage may vary, but csf kinetics and anatomic pathways are important considerations (see also pp. f). a distinction is made between normal pressure hydrocephalus of still unknown etiology (adams et al. ; hurley et al. ) , low pressure hydrocephalus, and high pressure hydrocephalus. the latter is caused by accumulation of fluid secondary to elevated csf production (hypersecretory hydrocephalus) or insufficient resorption (malabsorptive hydrocephalus or communicating hydrocephalus). disten-sion of the ventricles results from pressure-induced fluid build up in the cerebral ventricles (reversible) or from pressure-induced (irreversible) atrophy as a result of parenchymal loss. fluid may also enter the periventricular tissue (trans-ependymal resorption of csf seen on neuroimaging). normal pressure hydrocephalus can feature repeated brief episodes of elevated icp, possibly in the form of an intermittent pressure or occult hydrocephalus. normal pressure hydrocephalus can also result from a hydrocephalus ex vacuo, which is associated with primary ventricular system enlargement and white matter destruction. it is often found in victims of severe brain injury, in alcoholics, and vascular disease patients with multi-infarct dementia or other types of progressive degenerative brain disorders, especially age-dependent dementia (miller and ironside ) . hydrocephalus ex vacuo can also be caused by long-lasting generalized brain edema with high icp. the causes of obstructive hydrocephalus are mbi, subarachnoid hemorrhage, meningitis and arachnoid fibrosis, arnold-chiari malformation, aqueductal stenosis (for complete list see table . , for review see garton and piatt ) . the latter can be either congenital due to atresia or acquired due to inflammation, compression or reactive gliosis, hemorrhages or tumor. the responsible congenital abnormalities consist in most cases of replacement of the aqueduct lumen by numerous random, narrow channels or ependymal nests. external hydrocephalus, whatever the causes are, exhibits dilation of the subarachnoid space, with no increase in collagenous fibers or cellular elements, but an increase in csf. the causative encephaloclastic disease may be diagnosed on the basis of other phenomena, such as liver cirrhosis in alcoholics, or loss of neurons in the presence of plaques and tangles in senile dementia or alzheimer's disease. typical macroscopic features of internal hydrocephalus include an enlarged ventricular system ( fig. . a, b) (weller and shulman ) , interstitial edema, disruption of the ependymal cells lining the ventricle, and axonal and myelin destruction in the periventricular white matter (del bigio ) . secondary changes in neurons reflect compensation to the stress or ultimately the disconnection. proliferating astrocytes and/or gliosis (fig. . c) replace in part the interrupted ependymal cell line. these glial nodules appear granular or like small tumors (fig. . c ) upon macroscopic inspection of the inner surface of the ventricles (fig. . b) . there is also a partial reestablishment of flattened ependymal cells, and a decline in the number of axons with parallel proliferation of glial fibers in the periventricular white matter. in chronic hydrocephalus with high pressure hydrocephalus, a flattening of the gyral crests is seen (fig. . a ); in addition a small reactive glial zone around the ventricular system (fig. . b ) may develop which can be separated from the intact white matter at autopsy (fig. . c) . in adults, the clinical symptoms of hydrocephalus are non-specific; in infants and children they may be specific (see chap. − pp. f) and depend on the causes and the time course of the hydrocephalus. the salient symptoms comprise psychopathological alterations such as dementia, memory disturbances, and loss of orientation, culminating in the most severe cases in loss of consciousness. the first step in diagnosing hydrocephalus involves the use of imaging techniques, mri and cct, to establish its presence. the second step seeks to determine the underlying cause, and again employs as its methods of choice mri and cct, in combination with clinico-chemical (fishman ) and cytological analysis of the csf itself (oehmichen a ). "necrosis" (for review see lindenberg ) is commonly used to designate the death of tissue components, including that of cells and their processes in a defined area of blood and oxygen supply. after a severe episode of ischemia, mbi or epilepsy, it is typical to find necrotic cell death within the injury core. in addition, a substantial number of neurons in regions surrounding the injury core have been observed to die via the programmed cell death pathways due to secondary effects derived from the various types of insults (liou et al. ) . "apoptosis" (for review see vermes et al. ) is applied to the selective (programmed) death of one or more individual cells. apoptosis is the more active and physiological form of cell death. in necrotic cell death, a stimulus such as ischemia, hemorrhage, mechanical or chemical damage alters cell homeostasis thus causing cell death, whereas in apoptosis an internal death stimulus triggers the innate cellular suicide program, the latter (not the stimulus itself) mediating the cellular demise (beal ) . in the following, the various pathogeneses and mechanisms of these types of cell death will be described, along with their different morphologies and underlying molecular factors. the most common cause of necrosis is ischemia. other causes include mechanical injury (contusion fig. . a−c. internal hydrocephalus. a expanding ventricular system associated with an atrophy of white matter; b the ventricular surface is commonly marked by a granular surface structure, which (c) microscopically is characterized by multiple glial nodules which replace lost segments of the ependymal layer (h&e, magnification × ) necrosis), toxic agents (formic acid in methyl alcohol), heat (thermocoagulation), freezing (cryosurgery), infections (poliovirus), and overexposure to ultrasound. each case, however, involves the action of additional factors independent of the type of primary traumatic event. chief among these factors are free radicals and nitric oxide (no). reaction products of no and o , including potent oxidizing molecules such as peroxynitrite and nitrogen dioxide, can be more toxic than no itself. the type of brain necrosis depends in large part on the duration of local circulatory arrest: . transient ischemia only destroys neurons and oligodendrocytes, inducing incomplete necrosis or selective neuronal necrosis (scholz ). . prolonged ischemia, termed "infarction," gives rise to complete necrosis of all tissue components. morphologically cell necrosis, especially neuronal necrosis, features irreversible changes of the cytoplasm (condensation, hydropic swelling, intense eosinophilia, loss of structure, homogenization) ( fig. . ) and of the nucleus (pyknosis, karyolysis, karyorrhexis) (majno and joris ) . time course. the data vary on how soon after the onset of ischemia the first microscopic neuronal changes become evident. some authors report an interval of min (jacob and pyrkosch ) , others / h (müller ) . the data may differ in part due to prolongation of the necrotic process for as long after death as brain temperature remains favorable (lindenberg ). prolonged ischemia-induced tissue necrosis is termed "infarction" or liquefactive necrosis. the infarcted area displays macroscopically evident pallor on h&e, nissl, and myelin preparations within − h as an indication of acidosis. a narrow halo of even greater pallor surrounds the necrotic area ( fig. . a) . around the necrotic area, vessels are distended and release fluid into the infarcted tissue and surrounding tissue by way of a vascular network (perifocal edema). neurons become thorny and severely shrunken within − h, with darkly staining incrustation of their pericellular structures. a survival time of h leads to homogenization of the cytoplasm and nuclear and cytoplasmic pallor. between h and h, the neurons disappear except for the nuclei (see fig. . c) . within − h the necrotic tissue is characterized by an emigration of neutrophil leukocytes (fig. . a, b) . within h the necrotic area exhibits proliferation and extensive activation of microglial cells (fig. . c−f). along the infarct margin, macrophage numbers increase. hypertrophic astrocytes appear along the border zone within the brain parenchyma after − days ( fig. . ) . the infarct liquefies at its center and macrophages phagocytose the debris. the final stage of cortical liquefactive necrosis is termed "laminar necrosis" (fig. . a−c) associated with intense gliosis during the final phase (fig. . d) . the final stage of ischemic involvement of the basal ganglia and the thalamic nuclei is a cystic necrosis (fig. . ) . ischemic damage of the hippocampal area is characterized by a segmental loss of neurons in the hippocampal cortex ( fig. . a, b) associated with compensatory (early) microglial activation (fig. . c ) and secondary gliosis (fig. . d) . another type of brain tissue necrosis is the rare phenomenon "persistent coagulative necrosis" first described by spielmeyer ( ) (fig. . ) , which affects cell tissue elements. within gray matter the outlines of dead neurons are recognizable, their cytoplasm is intensely eosinophilic and usually contains numerous, often large, vacuoles, and the nucleus stains poorly with hematoxylin. this picture, which is recognizable within the first − h, is followed by decreasing stainability of nucleus and cytoplasm until a barely recognizable ghost cell is all that re-mains. acute and enduring deprivation of blood supply causes necrosis of cells and tissue components, the lesion remaining in a state of "coagulation" for a prolonged period. the cells appear only as shadows and the necrotic tissue persists within the brain as a foreign body and sometimes becomes encapsulated in mesenchymal tissue. the pathogenesis of this rare type of necrosis is still unknown (escolá and hager ; cervos-navarro and ferszt ) . apoptosis can be differentiated from necrosis based on differences in their pathogenesis, cell reactions, and morphologic features. apoptosis is the programmed death of a cell as regulated by specific death genes (for review see clarke ). it initiates a delayed secondary death of neurons in response to environmental changes, deficient metabolic and trophic supply, and changed gene transcription. during apoptosis, the integrity of mitochondria is compromised and various pro-apoptotic proteins are released into the cytoplasm. this results in activation of caspases, proteases that orchestrate the death of the cell (waterhouse ). apoptosis requires active protein synthesis (mcintosh et al. ; raghupathi et al. ) . a single cell can undergo a switch between the two types of cell death based on several pathways (mcconkey ; fiskum et al. ; see also leist et al. ) (for further informations, see p. ). the characteristic morphology of apoptosis exhibits cleavage of the internucleosomal chromatin that can be identified in situ using the terminal deoxynucleotidyl transferase-mediated dutp nick end-labeling (tunel) method (gavrieli et al. ) . apoptosis causes pyknosis of the nucleus and condensation and shrinkage of the cell body. as it progresses, budding and karyorrhexis occur, and ultimately a breakup into clusters of apoptotic bodies (majno and joris ) . the time course of apoptosis following a traumatic event is as follows: about h after a traumatic event, apoptosis begins, and remains demonstrable for about days (yakovlev and faden ) . three major factors are known to participate in the apoptotic cascade of "delayed" neuronal death (for review kermer et al. ; see also huppertz et al. ): . immediate early gene transcription factors (cjun, jun-b, jun-d, c-fos, ap- , atf, nf-κb) . proteases (calpains, caspases) . glutamate-mediated toxicity (free radicals, protein-kinases, ca + homeostasis, second messenger systems) the death-inducing activity of the bax, bad, bid, bcl-x s family members is thought by raghupathi et al. ( ) to be in dynamic equilibrium with their survival-promoting cognates bcl- , bcl-x l . shifts in the protective intracellular bcl- -family-protein levels can tilt the balance toward cell death by activating the death-inducing cysteine proteases, caspases (thornberry and lazebnik ) . the death of single cells releases insufficient quantities of chemoattractants to allow effective concentrations of molecular species to reach the vascular endothelium. for this reason a genuine cell reaction does not occur. neighboring cells that are not professional phagocytes cannibalize the cell debris in a process specific to apoptosis (majno and joris ) . in inflammatory diseases, an essential factor in the resolution of the inflammatory attack is the clearance of apoptotic leukocytes by tissue-specific phagocytes (platt et al. ). this process has been termed the "safe, phagocytic clearance of dying, yet intact leukocytes undergoing apoptosis" involving rapid recognition, uptake, and degradation. microglial cells were recently shown to be capable of protecting neurons, cerebellar granule neurons in particular, from apoptosis (polazzi et al. ): molecules are released by apoptotic neurons that enable the anti-apoptotic activity of microglia. in vitro, normal microglia release molecules capable of rescuing neurons from apoptotic death. microglia, astrocytes, and oligodendroglia may participate in apoptotic or necrotic processes. the reaction of neurons highly sensitive to injuries such as ischemia, hypoglycemia, infection, and mechanical trauma are described above and classified systematically in table . . a review by rosenblum ( ) includes a comparison of various hypotheses regarding the underlying causes of "delayed" neuronal death, among them excitotoxicity, calcium, and apoptosis. rubin ( ) and abe ( ) review the phenomenon of neuronal apoptosis as it appears in various neurological diseases. astrup et al. ( ) first defined the ischemic penumbra as brain tissue perfused at a level within the thresholds of functional impairment and morphologic integrity, which has the capacity to recover if perfusion is improved. because tolerance of tissue to ischemic damage is dependent on residual flow and duration of flow disturbance (heiss and rosner ) , the ischemic penumbra is a dynamic process; it exists for a short period even in the center of ischemia, from which the conversion into irreversible necrosis propagates over time to the neighboring tissue. focal ischemia results in necrosis at the infarct core and activation of complex signal pathways for cell death and cell survival in the penumbra. increased expression of caspase- , - , - , and - , and of cleaved caspase- has been observed in the penumbra (for details, see ferrer and planas ) . but for a limited time interval, penumbral tissue has the potential for recovery and, therefore, is the target for interventional therapy in acute ischemic stroke (time window, heiss ). relatively little is known about the effects of injury of the dendritic processes of neurons. axotomy of a motor neuron induces loss of some presynaptic terminals and the retraction of processes (blinzinger and kreutzberg ; summer and watson ) . abnormalities in dendritic spines have been report- fig. . a−f. dendritic and axonal injury. a dendritic processes and neuronal perikarya are reactive with a map antibody which (b) lose their reactivity within a short time after an ischemic period as well as in cases of (c) mbi-induced hemorrhage; d axonal injury is demonstrable by axonal bulbs or balls using h&e stain as well as (e, f) by their reactivity to β-app antibody (magnification a × ; b × , c × ; d−f × , ) ed during the perinatal period in cases of developmental retardation (purpura (purpura , . li et al. ( ) showed that expression of microtubule-associated protein (map ) in perikaryons and dendrites is a sensitive marker of dendritic lesions in spinal cord trauma (fig. . b ; see also chap. − pp. f). as early as h after moderate or severe compression of the spinal cord, loss of map immunoreactivity in dendrites and nerve cell bodies became evident in the injured segment. this phenomenon continued for the duration of the -day experimental period. how much map immunoreactivity is lost depends on how hard the cord is impacted (li et al. ) . the loss of immunoreactivity may result from an (impact-induced) influx of calcium, activating calcium-dependent proteolytic enzymes capable of degrading map (inuzuka et al. ). the same phenomena may be observed as a result of hypoxic lesions in the hippocampal area ( fig. . a, b) . axonal injury is characterized by an interruption of axonal fibers (fig. . b ) as demonstrable − for example − as a result of a gunshot: the axons will be fragmented (figs. . b, . ) . moreover, axonal injury induces an anterograde (wallerian) and retrograde degeneration of the injured axons. the terms "anterograde" and "retrograde" refer to the directions of conduction of the nerve impulse along the axon, i.e., the degeneration following focal damage proceeds in a centrifugal or centripetal direction (for review see brodal ). an interrupted anterograde flow of proteins along the axon can cause the phenomenon of axonal injury. this phenomenon was once demonstrated by h&e stain and by silver staining techniques within − h after a traumatic event (strich ; adams et al. ) . but since the injured axon is selectively characterized by expression of β-amyloid precursor protein (β-app) (fig. . d−f; cf. gentleman et al. ; sherriff et al. ) , it is now routinely confirmed in − min (blumbergs et al. ; oehmichen et al. ) , in adult brains as well as in infant brains (reichard et al. ) . β-app expression is seen even if the axonal injury is moderate or the axotomy delayed or incomplete (povlishock ) . although axonal injury was long thought to be a morphological correlate of mbi, it is now known to be a non-specific phenomenon also associated with acute intoxication (nies et al. ) or hypoxia/ischemia (oehmichen et al. ) . in a recent study graham et al. ( ) evaluated the pattern of β-app immunoreactivity. the authors identified three different types: . a diffuse − multifocal type − in mbi, co poisoning and hypoglycemia; . a type corresponding to the outlines of an infarct or hematoma with evidence of raised intracranical pressure; . a mixture of ( ) and ( ) which was seen in serve mbi. it is still unclear which events trigger axonal degeneration within the cns and pns. kapoor et al. ( ) suggest a link between nitric oxide (no) and subsequent molecular events that previously have been indicated as contributors to reversible and irrevers- ible axonal injury. waxman ( ) demonstrates the axonal death cascade induced by hypoxia, ischemia, mechanical trauma, inflammation or no. the molecular events involving sodium channels and the na + /ca + exchanger lead to an increase in intracellular calcium, which can provoke axonal degeneration. axonal injury of myelinated axons is always accompanied by a demyelinating process, i.e., a loss of myelin, which will be eliminated by mononuclear phagocytes (see: fig. . b) . the sequelae will be a glial scar lacking myelin as demonstrable by luxol fast blue stain (fig. . a) or by immunohistochemistry using an antibody against myelin basic protein ( fig. . b, c) . interrupted axons or injured neurons exhibit disintegration of the distal stump. breakdown of the distal axonal stump is known as "wallerian degeneration" and begins several days after a traumatic event. its salient morphological features are lysis of axons and myelin, schwann cell proliferation, and phagocytosis by invading macrophages. axonal and myelin degeneration follow a time course described in detail by brodal ( ) (see also p. ). axotomy induces a characteristic centripetally directed morphological change of the neuron termed retrograde axonal degeneration. this retrograde alteration is characterized by rounding of the neuronal contours, swelling of the neuronal cytoplasm, and a decline in the number of nissl bodies at the center of the cytoplasm (central chromatolysis − see fig. . c). the nucleus becomes slightly deformed and is displaced to the periphery of the perikaryon. these changes correspond to the changed or increased neuronal gene expression after axotomy (graeber et al. ) . axotomy is followed in hours by rapid upregulation of the immediate early genes, such as cfos, c-jun, jun-b (haas et al. ) , in association with the upregulation of heat shock proteins (kalmar et al. ) . the one constant change associated with acute retrograde changes may be chromatolysis. these changes are sometimes accompanied by local proliferation of satellite glial cells whose time course has also been described by brodal ( ) (see also pp. and f). it is accepted as common knowledge that plasticityassociated molecular and structural events occur in the injured brain. these are at least partly responsible for functional recovery. increases in dendritic arborization, spine density, and synaptogenesis in both peri-injury and intact cortical areas are the potential morphological strategies that enable the brain to reorganize its neuronal circuits (keyvani and schallert ) . on the other hand we have to consider that the scarring process is an ineffective regenerative process which is associated with cell proliferation. the cell proliferation will be − especially within the first h after the traumatic event − non cell-specific, as immunostaining with markers for immature and mature astrocytes, activated microglia, neural precursors, and mature neurons will be negative (chirumamilla et al. ) . nevertheless, it is also accepted that both retrograde degeneration and the axonal injury-induced bulbs and swelling of the proximal axonal stump are markers of a regeneration process. although the cns has little innate capacity for repair, this capacity does exist for the peripheral nervous system. it is not known why axons in the adult cns are not capable of better regeneration; it is known that they do in fact regenerate, as was recently reconfirmed by von euler et al. ( ) . the observations of schwab and caroni ( ; schwab ) are of major importance. they show that proteins released by oligodendrocytes inhibit axonal elongation. this inhibitory protein has since been identified and cloned (chen et al. ; grand-pré et al. ; prinjha et al. ) . these authors also identified the protein (nogo) of a previously unknown gene that encodes the inhibitory myelin protein in rats and humans. the myelin-derived axon outgrowth inhibitor nogo protein binds to an axonal nogo- receptor and at least accounts for the lack of cns repair (li and strittmatter ; mcgee and strittmatter ) . it remains unclear, however, what effect other factors may have, whether negative (e.g., the release by neurons of large quantities of glutamate following the incidence of spinal cord injury), or positive [e.g., release of tissue growth factors (ramer et al. ) and cytokines, or induction of the macrophage scavenger function (see also schwab ) ]. it is now thought that neurons are renewed throughout life from endogenous stem cells and added to the dentate gyrus. adult neurogenesis could be demonstrated in the subgranular and subventricular zones of the hippocampus (kempermann et al. ; cameron and mckay ) and the olfactory bulb (craig et al. ). these are the only zones where differentiation of neural stem cells into neurons is known to take place in the intact adult cns (reilly ) . because this process does not occur in the adult spinal cord, svendsen ( ) postulates that the growth of neurites can only be stimulated by newly regenerated astrocytes. additional findings suggest that adult neurogenesis is actively regulated in large part by astrocytes (reilly ; song et al. ) . gage ( ) and colleagues (kempermann et al. ) were able to show that exposure to an environmental challenge evokes much greater neurogenesis in old than in young animals. more recent data suggest that the old brain reacts quickly with a neurogenic response to functional challenges, a type of cellular plasticity associated with the continued sensory stimulation and activity of the aging brain (kempermann et al. ; mckhan ) . the cns was once described as an "immunologically privileged site" (medawar ) . this hypothesis was based mainly on the existence of the bbb (pachter et al. ) , which restricts diffusion of soluble molecules and the migration of immune cells out of the systemic circulation into the cns (oehmichen ) . pericytes, endothelial cells, microglia and astrocytes, by contributing to bbb function (see above), participate in cns immune regulation (prat et al. ) . meanwhile a number of basic processes are known (bauer et al. ) that explain how the cns responds to inflammatory attack by regulating local antigen presentation, by the different cell types, and by its special cytokine environment. it also eliminates inflammation via emigrating macrophages (oehmichen et al. − for review oehmichen cserr and knopf ) and destruction of apoptotic t-cells. in vitro and in vivo studies have elucidated the mechanisms underlying immune-mediated (via cytokines, macrophage/microglial toxins, and antibodies) tissue damage, featuring many different potential pathways. the normal cns has limited expression of major histocompatibility complex (mhc) class i antigens, primarily on the endothelium and glial cells, and no expression of mhc-ii and the various immunoactive adhesion molecules. fontana et al. ( ) were the first to point out that cytokines such as interferonγ (ifn-γ) are able to stimulate astrocytes to express mhc class ii to secrete cytokines (il- , il- , tnf-α), and to present antigens such as myelin basic protein (mbp) to specific t-cell lines (fontana et al. (fontana et al. , massa et al. ) . frei et al. ( ) focused on microglia and found that they too could respond to ifn-γ by upregulating mhc-ii. if ifn-γ or ifn-γ and tnf-α are introduced directly into the cns, there is independent, progressive upregulation of both mhc classes and of adhesion molecules, such as the intercellular adhesion molecule- (icam- ), which are expressed first by perivascular macrophages (hickey and kimura ) , subsequently by microglia and macrophages throughout the cns, and finally by astrocytes (massa et al. ). experimental induction of autoimmune encephalitis (eae) revealed that perivascular macrophages are the chief presenters of cns antigens to circulating t-cells (hickey and kimura ) . the role of the endothelium in antigen presentation within the cns is ambiguous. astrocytes too are thought (waksman ) to play an ambiguous role. when presenting antigen to specific t-cells in vitro, they are lysed. it is not known whether t-cells are induced to proliferate or to release inflammatory cytokines, or possibly both, or whether they shut down for lack of suitable co-stimulatory signals (so-called clonal anergy). the duality of the inflammatory response is crucial to host repair and defense. it may also however cause loss or impairment of function, i.e., although otherwise beneficial, inflammation may impair neuronal function (perry et al. ). instead of the bbb being a limiting factor of the cns immune response as once believed, today it is thought that the brain itself is an immune system organ (fabry et al. ; chao et al. ). this conclusion is based on the numerous cytological and immunological findings of the past two decades showing that the targets to be protected are neurons, axons, and myelin. in the absence of protection these can become necrotic or succumb to apoptosis, be phagocytosed and disappear. ultimately all signs of degenerative alteration can be demonstrated. there is no direct correlation however between leukocyte emigration and parenchymal cell death in vivo (schmid-schönbein et al. ) . glial cells outnumber neurons in the cortex, where there are eight glial cells for every neuron. among glial cells in the cortex, astrocytes comprise %, microglia %, and oligodendroglia % (chao et al. ). these cells possess many immunological features marking them as important immunoregulatory cells. hallmarks of cns inflammation in particular are activated microglia and astrocytes. inflammation undoubtedly serves primarily as a host defense mechanism in peripheral tissue, facilitating essential repair processes by altering local blood flow in the injured tissue, with accumulation of fluid and specialized cells. the brain possesses cellular host defense mechanisms since activated t-cells are capable of crossing the bbb (hickey et al. ) . cellular mediators of cns inflammation in the brain have been shown to differ with regard to type and number from those of the periphery (see below). these differences are mainly due to the brain's tight regulatory environment and the balance between inflammation-induced tissue damage and tissue repair (parsons and hunter ) . the specificity of the immune response appears to be controlled largely by cns antigen presentation (sedgwick and hickey ) , though the precise nature of the control remains unresolved. cns antigen presentation according to hart and fabry ( ) occurs outside and inside the cns, with the bbb playing a major role in the regulation of cns immune function. parsons and hunter ( ) showed that the early events leading to t-cell activation by antigenpresenting cells result from mhc binding. co-stimulatory molecules then bind to the antigen, presenting cell−t-cell complexes for further development of the cascade. mhc is expressed not only on astrocytes and microglia, but under certain conditions also on neurons and oligodendrocytes (sedgwick and hickey ) . mhc ii antigens are also expressed under normal conditions in a population of macrophages inhibiting the perivascular space, subarachnoid space, and choroid plexus. this expression may indicate that these cells perform a modulatory function at the blood/csf interface (matyszak et al. ) . under highly specific circumstances, physiological response mechanisms resembling those at the periphery also appear to take place in the brain. under pathological conditions, hematogenous cells that are absent or extremely rare under normal circumstances appear to accumulate in the brain, i.e., platelets (fig. . a) , neutrophilic leukocytes ( fig. . b,c) , lymphocytes (fig. . d) , and macrophages. the number and type of inflammatory cells in the cns vary widely depending on the attracting stimulus or on their inherent ability to attack a cns antigen. the intravascular cells, especially the leukocytes, interact with vessel walls as determined by integrins (hynes ) , selectins (bevilaqua ) , and immunoglobulins of the supergene family (springer ). among the heterogeneous supergene family are mhc molecules, t-cell receptors, and icam- . during emigration, i.e., extravasation, leukocytes initially come into loose contact with the walls of vessels of the microcirculation via selectin molecules or lectins, producing a rolling motion along the vessel wall (mcever ) . they are then expressed on the endothelium. the selectin molecules are e-selectin (elam- ), l-selectin (lam- , leccam- ), and p-selectin (cd ). p-selectin plays a role in recruitment of neutrophils to the brain parenchyma (bernardes-silva et al. ) , while e-selectin is thought to participate in both neutrophil and cd + t-cell adhesion (harlan and liu ) . endothelial selectins can be rapidly upregulated following wounding, p-selectin within minutes (< h − zoppo ) , and e-selectin within a few hours (granger and kubes ; mcever ) . among the stimuli for expression of endothelial cell adhesion receptors are tnf-α and il- . adhesion of leukocytes to endothelial cells is mediated by adhesion molecules such as mac- , the intracellular adhesion molecules (icams), lymphocyte function-associated antigen- (lfa- ), and vascular cell adhesion molecule- (vcam- ), all of which, as already mentioned, are upregulated in endothelial cells. in the cns, icam- and vcam- ( baron et al. ) are constitutively expressed on perivascular cells and some astrocytes. in areas of cns inflammation they are readily upregulated on endothelial cells and astrocytes (sobel et al. ) , which stimulates recruitment of neutrophils to the site of injury. such neutrophil emigration can be experimentally induced by extravasal injection of cytokines such as il- β (bernardes-silva et al. ) or tnf-α (schmid-schönbein et al. . neutrophils are the first circulating leukocytes to reach the site of injury. increase in vascular permeability is caused by the release of free radicals and lysosomal enzymes, giving rise to edema (weiss ) . despite mechanisms evolved to restrict entry of neutrophils into the brain parenchyma, neutrophil recruitment is clearly a feature of acute brain injury, such as that caused by stroke or mechanical trauma. numerous studies employing a transient or permanent model of focal ischemia in rats and mice have demonstrated that cerebral tissue injury is lessened by neutrophil depletion (jean et al. ) . a correlation between the development of cerebral edema and neutrophil recruitment has also been shown in models of mbi (schoettle et al. ). this deleterious effect of neutrophil recruitment contrasts with their beneficial function and phagocytic ability as scavenger cells in cases of bacterial inflammation and of particular importance in cases of sterile inflammation. the aforementioned recruitment of t-lymphocytes to the site of injury clearly depends on an accumulation of adhesion molecules, especially of vcam- , in the endothelial wall. when activated, t-cells express lfa- and can bind icam- on endothelium (van kooyk et al. ) , thus facilitating entry of t-cells into the cns (baron et al. ) . few data have been published on the migratory requirements of b-cells. it is thought, however, that b-cells in their fully mature form as plasma cells have no or only limited migratory potential. immunization of rats with a foreign, non-pathogenic antigen behind the bbb was found to result in the presence of b-cells and plasma cells specific for that antigen in the cns (knopf et al. ) . this finding appears to indicate that, after entering, b-cells remain in the cns at least in part because they have found their specific antigen . after entering the cns, the function of t-and bcells is to recognize their antigen. among the potential antigen-presenting cells of the cns are endothelial cells and astrocytes. under inflammatory conditions, microglial and perivascular cells (members of the mononuclear phagocyte family residing in the cns) constitute the chief antigen-presenting cells. as already mentioned (pp. f), a distinction must be made between activated and resident microglia, and leptomeningeal, perivascular, and choroidal macrophages. all of these cells represent different functional stages of blood monocytes (oehmichen and huber ; oehmichen a oehmichen , b, oehmichen , cf. also perry and gordon ) . microglia residing in the white matter do not express the mhc i antigen, and only a few express mhc class ii (hart and fabre ) . leptomeningeal, perivascular, and choroidal macrophages, in contrast, do express mhc class ii. the resident microglia also feature a downregulation of other antigens, the leukocyte common antigen (lca), and ed- or cd . monocytes emigrate under pathological conditions into the brain parenchyma, where their morphology and antigenic characterization both change. they now participate in the immunological process as macrophages and express mhc class ii antigens. they also scavenge cell debris and myelin fragments left over from damaged tissue. astrocytes are among the first local cells to respond to cns injury. the main responses are reactive gliosis and swelling of reactive (hypertrophic) astrocytes upregulating gfap. reactive astrocytes express acute phase reactive protein (koo et al. ) , mhc class ia (frank et al. ) and mhc class ii antigens (fierz et al. ) , il- (griffin et al. ) , plus multiple other factors (for review see norenberg ) . astrocytes are known to act in conjunction with cells of the immune system and to be involved in immune/ inflammatory processes. they are immunocompetent cells capable of augmenting, amplifying, and sometimes even of regulating an immune response. they produce many immune mediators and can in return be affected by them. by helping to eliminate infectious or foreign agents, astrocytes may contribute to a beneficial response. given their direct contact with leukocytes in the blood, endothelial cells constitute the ideal site for antigen recognition in the cns (sedgwick and hickey ) . the scarcity of t-cells in the cns may be an indication that endothelial cells are the sole site capable of adequate t-cell antigen−mhc interaction. the finding that activated t-cells can enter the cns through an intact bbb (hickey et al. ) , however, is a clear sign that antigen recognition at the endothelial cell surface need not occur. endothelial cells are thus regarded as major players in the inflammatory and immune response (sternberger et al. ) and simultaneously guarantee the bbb. they also enable alterations in the form of receptor-mediated events (dietrich ) , i.e., an inflammatory response (for details, especially on the expression of adhesion receptors, see above). endothelial cells proliferate at the site of brain wounds (fig. . a) . therefore, the number of capillaries increases and − in a final phase − decreases near hemorrhages or infarcts (fig. . b, c) in association with an increase in collagen fibers, especially collagen type iv (fig. . c ). this process leads to a network of collagen fibers and glial fibers, which is the last stage in the formation of a brain scar. among the inflammatory mediators are cytokines and their subgroups, chemokines, in the sense of adhesion molecules. other mediators of inflammation include effector molecules such as no, nos, reactive oxygen species (ros), and free radicals. cytokines mediate the initiation, propagation, regulation, and suppression of immune and inflammatory responses (benveniste ) . they are proteins with low molecular weight and are synthesized during effector phase immunity. they are secreted by cells and are also expressed on their surfaces. many different cell types are capable of producing the individual cytokines, which for their part can have a variety of effects on different cell types. usually acting locally, cytokines begin to act on target cells by binding to specific cell-surface receptors, which generally have a high affinity for their ligands. it takes only minute amounts of a cytokine to evoke a biological response. in multiple sclerosis or experimental allergic encephalitis, ifn-γ and il- are known to be products of activated t-cells. tnf-α and il- derive from activated astrocytes and macrophages. astrocytes can be activated by ifn-γ and/or tnf-α, produce il- and il- , tnf-α, transforming growth factor β (tgf-β), granulocyte-macrophage colony-stimulating factor (gm-csf), and other types of molecules such as prostaglandin e (pge ) (for review see waksman ) . in human brains, tnf-α, il- , and il- in particular can be induced by mbi or cerebral ischemia. brain ischemia triggers rapid production of tnf-α mrna, which peaks − h after ischemia and subsides − days later. it remains above baseline, however, for up to days (barone ) . neuronal cells in and around the ischemic tissue acutely express tnf-α in a so-called penumbra, but it also turns up several days later in macrophages in the infarcted tissue. tnf-α triggers adhesion molecule expression on activated glial cells and the endothelium, in this manner regulating gliosis, tissue remolding, and scar formation. il- levels rise before and during glial activation and neuronal damage (rothwell et al. ). chemokines are chemoattractant cytokines. during inflammation they mediate leukocyte entry into the cns. among their known functions is the interaction of leukocytes with the endothelial surface, a multistep and sequential process mediated by selectin molecules by which the leukocyte rolls on the endothelium. the end result is firm adhesion. the entire process is mediated by interaction of icam- and vcam- expressed by endothelial cells and their leukocyte-associated ligands. moderate levels of icam- and very low levels of vcam- , two molecules responsible for the adhesive properties of granulocytes and of t-cells, are expressed by brain endothelial cells. chemokines constitute a subgroup of small cytokines ( − kda) that attract certain inflammatory cell populations, among them lymphocytes, neutrophils and monocytes, to the target tissue (meeusen et al. ; bonecchi et al. ). the number of known chemokines and chemokine receptors continues to expand rapidly (for review see prat et al. ) . three classes of chemokines are known, as defined by the arrangement in the mature protein of conserved cysteine (c) residues, cxc or α-chemokines, cc or βchemokines, and of cc or γ-chemokines. astrocytes, endothelial cells (zach et al. ; weiss et al. ), perivascular cells, and macrophages (simpson et al. ) produce and release chemokines. ros and no are generated in astrocytes and activated macrophages (hartung et al. ). ros, no, and other free radicals are effector molecules that contribute to the inflammatory cascade and tissue damage. what role complement plays in cns damage is not clear (morgan ) . the enzyme no synthase (nos) synthesizes no. inducible nos (inos) is an isoform of nos that is induced transcriptionally by immunological stimuli. inos, which synthesizes large quantities of no, participates in inflammation-induced cytotoxicity. in the brain, inos message, proteins, and enzymatic activity are induced de novo by cerebral ischemia (iadecola ) . inos is expressed by neutrophils in permanent ischemia and in vascular cells in transient ischemia. high levels of no are synthesized by human astrocytes upon stimulation with ifn-γ, tnf-α, il- and potentiate il- . dalkara et al. ( ) showed that no plays a detrimental role in experimentally induced cerebral infarction in neuronal nos knockout mice. the nos knockout infarcts h after permanent vessel occlusion were % smaller than those of wild type. these finding seem to indicate that expression of inos is a factor contributing to ischemic brain damage. an apoptotic pathway mediates no-induced neuronal cell death and an nmda receptor antagonist blocks no-mediated neurotoxicity. neuronal cell death was shown by chao et al. ( ) to be initiated by the release of il- by the microglial cell, this in turn inducing the generation of astrocytes. neurons are destroyed by no via nmda receptor-linked apoptosis. cerebral trauma, ischemia, and reperfusion are known to generate hydrogen peroxide and superoxide radicals, which then produce ros and hydroxyl radicals (chan ) . under normal conditions and following reperfusion injury, mitochondrial respiration creates ros. microglia and astrocytes that have been activated by cytokines produce vast quantities of neurotoxic free radicals. an intramitochondrial antioxidant enzyme, manganese superoxide dismutase (mnsod), scavenges superoxide radicals and thus constitutes the first line of antioxidant defense. as already pointed out, inflammatory responses are based on an inflammatory cascade, whose details have become increasingly clear in recent years. three basic types of inflammatory response are known: sterile inflammation, cell-mediated inflammation, and antibody-mediated inflammation. these types of response are mutually exclusive, but characterized by occasional overlapping. in cases of mechanical violence, spontaneous intracerebral hemorrhage or stroke, sterile inflammation features an initial phase of infiltrating neutrophils and a second phase of infiltrating mononuclear phagocytes (bone marrow-derived monocytes, i.e., activated microglia and macrophages). macrophages and neutrophils produce cytotoxic cytokines such as tnf-α, proteolytic enzymes (anthony et al. ) , reactive oxygen intermediates (cross et al. ) , cell death-inducing surface molecules such as fasligands (d'souza et al. ) , or even excitotoxins (lipton ) . the macrophages in particular take up the scavenger function and eliminate tissue and cellular debris. they also release mediators that promote the scarring process, i.e., induce fibroblasts to produce collagenous fibers and stimulate astrocytes to proliferation and produce fibrils, aiding healing by the production of a fibrillary glial-collagenous scar. multiple sclerosis (ms) is the classic model of t-cellmediated inflammation whose inflammatory infil-trates are chiefly comprised of t-lymphocytes, fewer b-lymphocytes, as well as activated microglial cells and macrophages (brück et al. ; gay et al. ) . ms features local expression and/or upregulation of markers of t-lymphocyte and macrophage activation (brück et al. ) , of class i and class ii mhc antigens (traugott ) , of chemokines and adhesion molecules in addition to their receptors (lassmann ) , and of co-stimulatory molecules (windhagen et al. ) . diseases with an autoimmune background such as ms or virus-induced inflammatory diseases exhibit a uniform cellular and mediator profile. unlike ms, lesions associated with viral inflammation of the brain display considerable differences in topography and in their patterns of structural damage. they also vary with regard to the nature of the immune response and its associated cellular tropism. in lesions of virus encephalitis cd + -lymphocytes abound; in ms lesions both active and inactive cd + -cells usually outnumber cd + -cells (gay et al. ) . virus-infected cells generally evoke a cell-mediated immune response, although humoral mechanisms play a role as well (for review see esiri and kennedy ) . phagocytosis of infected cells by macrophages can also be promoted by antibodies. antibody-dependent cell-mediated cytotoxicity (adcc) is a process in which lymphocytes bearing fc receptors for igg lyse virus-infected cells bearing relatively small amounts of surface-bound antibody. the immunological specificity of the reaction derives from the antibody not the lymphocytes, which are not specific and have been designated killer cells. an important cell-mediated specific mechanism for killing infected target cells is provided by virus-specific cytotoxic t-cells. the cytotoxic effect is seen even if an antibody is lacking and is restricted by mhc class i antigens. viral antigens on the surface of infected host t-cells are recognized by virus-induced cytotoxic t-cells in association with class i mhc antigens. the infected target t-cells are then killed by these t-cells only if they share the same mhc antigens, i.e., the t-cell killing is restricted by mhc (zinkernagel and doherty ) . viral infection induces secretion of cytokines within the cns, either by lymphomononuclear cell infiltrates or infected brain cells. cytokines play an important role in the induction of mhc molecules. they stimulate humoral and cell-mediated immune responses by acting on immune system cells and neighboring brain cells, evoking the expression of surface recognition molecules such as mhc antigens and antiviral proteins such as mx (campbell ) . viral infections can provoke or amplify mhc class ii expression on the surface of astrocytes and microglial cells, which is important in light of the significance of these antigen-presenting cells in the cns. this process can occur as a direct effect of the infection even in the absence of ifn-γ, as shown for measles virus-infected astrocytes and the murine coronavirus j. howard mueller virus (massa et al. ) . in viral and bacterial inflammation, both the cell-mediated immune response and the humoral response are highly important, the latter also usually dominating. blood-borne dissemination of virus from the primary infection site to other organs is restricted by circulating antibodies, igg or igm. antibodies in tissue spaces can stop the spread of infection from one cell to another by neutralizing extracellular viruses. however, viruses able to fuse cell membranes, such as measles or herpes viruses, can elude this mechanism without ever being exposed to antibody. viruses can be inactivated by antibodies in a variety of ways. antibodies can assist phagocytosis by coating the surface of the virion, or they may thwart attachment of the virus to specific receptors on vulnerable cells, or in the case of enveloped viruses they may promote viral lysis via attachment and activation of complement. in the absence of antibody, direct viral lysis can also be produced by complement alone. edema can produce an increase in tissue pressure that disturbs the microcirculation of portions of the cns that are anatomically prevented from swelling by bone or tight meningeal constraints. it is thought that this mechanism contributes to the formation of necrotic lesions in transverse myelitis. the inflammatory process often has a vasculitic component (gray ) that causes occlusion of small veins and venules and is commonly associated with massive tissue damage. ischemia can contribute to the development of structural damage and functional deficits in inflammatory cns lesions. involvement of arteries and veins is rare in bacterial inflammation but the exudate is frequently accompanied by strands of fibrin. the edematous cortex exhibits large artificial perineuronal spaces and a spongiform neuropil. the cytoplasm of neurons often reveals ischemic cell necrosis and is acidophilic. if the course is subacute, fibrinoid necrosis and thrombosis may appear in a few blood vessels in the exudate, resulting in small foci of cortical necrosis. in the field of neuropathology both gross and microscopic changes can be misinterpreted. only a few aspects will be discussed here, each involving routine immersion fixation with % buffered formalin, dehydration and embedding in paraffin (see above). a detailed overview of the problems associated with postmortem changes, artifacts, and misinterpretation has been provided by lindenberg ( ) . autolysis, a process involving self-digestion of tissue, can cause slides of the adult brain to be discolored or poorly staining. the morphology of the changes associated with autolysis are identical with those of respirator brain (brain death). they arise if fixation is done too late, i.e., if the interval between intracranial circulatory arrest and autopsy or brain fixation is too long. depending on the ambient conditions, the brain will liquefy after a certain postmortem interval without fixation. the brain emits a foul odor if it has undergone microorganism-induced putrefaction, and the central parts of brain slides display a faint pink coloration. variably large bullous cavities ( fig. . a, b) give an appearance of swiss cheese to large sections of the brain. such cavities are created by the activation of gas-producing microorganisms due to poor quality formalin. macroscopic necrosis of the cerebellar granular layer was interpreted by ikuta et al. ( ) as a postmortem phenomenon, whereas lindenberg ( ) thought that the necrotic process precedes or accompanies the onset of sublethal hypoxemia, i.e., shortly before death. neurons in the substantia nigra and locus coeruleus of the brains of infants and children up to years of age normally possess no melanin pigment. this finding is neither an artifact nor pathological. the spinal cord is especially sensitive to postmortem mechanical injury in an unfixed state. in this manner the so-called toothpaste-artifact can occur (hughes ) . if the tissue in a segment of the spinal cord is artificially constricted, proximal portions of the cord are squeezed upward, distal portions downward, thus appearing in histological sections at the wrong level. any tissue processing produces artifacts that have to be interpreted: fixation procedures as well as staining techniques. the artifacts are dependent on the time periods between death and fixation, the duration of the fixation process, the temperatures, etc. for example the freezing process of (brain) tissue leads to crystalline vacuoles within the tissue sections ( fig. . a, b) . additionally, the forensic neuropathologist fundamentally needs to be able to reliably distinguish between vital and postmortem changes (cf. oehmichen ) . this requires among other things the testing and use of novel histochemical and immunohistochemical staining techniques that can help to establish the length of the postmortem interval. so-called dark neurons (fig. . c) can appear among otherwise normal neurons. dark neurons are irregularly contoured, shrunken neurons that are created by excessive pressure on unfixed postmortem tissue (scharrer ; cammermeyer cammermeyer , . apical dendrites also have a dark coloration, sometimes in combination with a corkscrew-like appearance. there is a danger of confusing dark neu-rons with ischemic cell necrosis. in our own investigations (oehmichen and gencic a) we could observe that most, but not all dark neurons have a potent albumin uptake, an indication that they represent lesions of neuronal metabolism (and/or membrane) . animal experiments demonstrate that the following three types of altered neurons appear at different postmortem intervals in rats (oehmichen and gencic b) : . shrunken, hyperchromatic neurons, whose number declines as the postmortem interval proceeds. . swollen and autolytic neurons, with a pale perikaryon and nucleoplasm, and an absence of nissl bodies. the nucleus can no longer be differentiated from the vacuolized and autolysing cytoplasm. the cells themselves have lost their contours and appear swollen and spherical. the swelling in particular represents the most fundamental postmortem change (whose differential diagnosis is retrograde degeneration). . pericellular spaces surrounding neurons may be caused by postmortem autolytic processes that mimic edema, especially in the gray matter. children two years old and younger almost invariably exhibit periventricular and perivenous accumulation of cytoplasm-poor, lymphocyte-like mononuclear cells which suggest an encephalitis (so-called pseudoencephalitis). those cell aggregations consist of neuroblasts as an indication of development, not of an inflammatory process. this age group also regularly shows a superficial granule layer of the cerebellar cortex composed of germinal cells (matrix cells). these usually disappear some time between the second and fourth years of life. in cases of sudden death with brief agony or if tissues have been poorly fixed (hirano ) , extensive acute swelling of oligodendroglia is common. in addition, a generalized swelling and clasmatodendrosis of astrocytes is often seen. these changes are rather slight compared to the neuronal changes. they must also be regarded as non-specific and do not constitute markers of edema. lindenberg ( ) points out that the state of the brain before circulatory arrest also plays a role. if the agony was brief (healthy person who died within minutes), neurons and glial cells may show marked postmortem ischemic cell injury (vacuolation, homogenization, acute shrinkage associated with nuclear changes). agony of long duration results in a largely unchanged cytology. not only does the architecture of the cells change, but their functional state and/or stainability with various reagents changes as well. postmortem function and staining are influenced by many factors in addition to fixation procedures (time, temperature, fig. . a, b . putrefactive bullous cavities in cerebrum and cerebellum and type of procedure). it is further known that the enzyme activity of cells and tissue depends in large part on the length of the postmortem interval (for a survey of findings prior to see oehmichen ) . numerous variables are known to affect the immunoreactivity of cells and tissue in the antigen−antibody reaction for immunohistological demonstration of epitopes (grizzle et al. ). among these variables are the 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ced-related genes modulating neuronal apoptosis expression of a chemotactic cytokine (mcp- ) in cerebral capillary endothelial cells in vitro immunological surveillance against altered self components by sensitized t-lymphocytes in lymphocytic choriomeningitis selectins, icams, and integrins in cns injury severe pediatric head injury: the role of hyperemia revisited key: cord- - lybzcz authors: stockwin, luke h; mcgonagle, dennis; martin, iain g; blair, g eric title: dendritic cells: immunological sentinels with a central role in health and disease date: - - journal: immunol cell biol doi: . /j. - . . .x sha: doc_id: cord_uid: lybzcz immunological effector cells must be sensitive to the antigens or environmental signals that indicate that a pathogen is present. to this end, a group of cells known as the professional antigen‐presenting cells have the ability to educate t, b and nk cells as to the fingerprints of specific infections. the most adept of these cells are a closely related family termed dendritic cells (dc). a subset of these act as peripheral sentinels, specializing in the uptake, processing and presentation of antigenic material combined with an ability to detect a wide variety of ‘danger’ signals. these ‘danger’ or activation signals induce profound changes in dendritic cell physiology, facilitating the efficient stimulation of both adaptive and innate immunity. in the present review, a number of recent advances in the understanding of dc biology are discussed. these advances offer insights into the pathogenesis of a wide variety of diseases and point towards future strategies for immunotherapy. dendritic cells (dc) are now recognized as essential regulators of both innate and acquired arms of the immune system. dendritic cells, in addition to their distinctive morphology, have a number of phenotypic and functional characteristics that make them formidable apc (table ) . dendritic cells bear sole responsibility for the stimulation of virgin t lymphocytes, a property that distinguishes them from all other apc (e.g. b cells). , the dc are also essential accessory cells in the generation of primary antibody responses and are powerful enhancers of nk cell cytotoxicity. conversely, dc are also involved in the maintenance of tolerance to antigens, with dc in the thymus contributing towards shaping of the t cell repertoire by deleting autoreactive lymphocytes. , as a consequence of this heterogeneity in vivo, it is now acknowledged that dc are a family of cells, with each subset exerting control over a different area of immunity. in terms of origin, dc are bone marrow derived cells originating from both myeloid and lymphoid precursors. , examples of dc in vivo include the epidermal langerhans cell, the interstitial dc (found in the heart, lungs, liver and other organs), the veiled dc found in afferent lymph, interdigitating dc (found in t cell-rich areas of lymphoid tissue) and thymic dc. follicular dc (fdc) are an exception to this group because they are believed to be involved in the long-term maintenance of b cell memory by retaining immune complexes; these cells differ markedly from the aforementioned group and will not be discussed here (reviewed by tew et al. ). the main function of langerhans and interstitial type dc is as immunological sentinels, being strategically placed to detect invading microbes. sentinel dc remain dormant until signals in the extracellular milieu (derived either from microbes or distressed bystander cells) induce a rapid change in function, also known as 'activation'. activation induces a number of important changes in dc (fig. ) , not least of which is migration into local lymphoid tissue where they communicate antigenic information to lymphocytes. recent clarification of the biochemistry of activation signals and the effects of dc activation have prompted research into the part played by activated dc in the pathogenesis of disease and whether dc-modulating immunotherapies can be used in the treatment of a wide range of diseases, including autoimmunity, allergy, immunodeficiency, transplant rejection, persistent viral infection and cancer. dendritic cells were initially characterized on the basis of their distinctive morphology, with numerous cytoplasmic processes giving rise to a stellate appearance. as a consequence, the dc has a high surface area permitting intimate contact with a large number of surrounding cells. as proof of this point, in vitro dc form large spherical aggregates with lymphocytes, and experimentally, only one mature dc is required to stimulate - t cells. dendritic cells also possess an array of mechanisms for sampling antigen. these include macropinocytosis, where fluid from the extracellular milieu is taken up into pinocytic vesicles and antigen is concentrated by expelling excess water via channels called aquaporins. the dc also expresses a repertoire of receptors for efficient receptor-mediated antigen uptake. additional receptors expressed by dc include fcγrii (cd ), fcγri (cd ), , fcεri and the c bi complement receptors (cd b), which increase the efficiency of immune complex endocytosis. dendritic cells express c-type lectin receptors, including the macrophage mannose receptor and dec- , which bind bacterial carboydrates. , dendritic cells also express markedly higher levels of antigen presentation molecules than any other cell (e.g. cd a, mhc-i and mhc-ii). in addition to this, dc have a high surface density of accessory/costimulatory molecules, including intercellular adhesion molecule (icam)- /cd , icam- /cd , leucocyte functional antigen (lfa)- /cd , b - /cd and b - /cd (fig. ) , which facilitate both the interaction with, and stimulation of, lymphocytes. , an additional noteworthy property of dc is that exogenous antigen (e.g. immune complexes) can be 'cross primed' into the mhc class i presentation pathway. this pathway would normally only present antigen from the endogenous compartment. the dc are thought to possess a specific mechanism that allows fluid from an endocytic vesicle to gain direct access to the cytoplasm. in essence, this allows the dc to present new peptides to cd + t lymphocytes without themselves being infected or damaged. , generated dc. ultrastructural analysis reveals the presence of many fluid-filled endosomes. antigen sampling is also by receptormediated uptake, for example fc receptors (fcγrii/cd , fcγri/cd ), complement receptors (c bi/cd b) and c-type lectin receptors (mmr, dec- ). . dendritic cells are distinct from macrophages in that they do not express cd or macrophage characteristic antimicrobial enzymes (e.g. lysozyme and myeloperoxidase). . high surface density of antigen presentation molecules, for example mhc-i and mhc-ii (expression - -fold greater than on other apc, e.g. b cells). . mature dc have a high surface density of accessory/costimulatory molecules (cd , icam- /cd , icam- /cd , lfa- /cd , b - /cd and b - /cd ). . production of large quantities of il- after treatment with activation signals, including cd l or lps. . an ability to cross prime antigen into the mhc-i presentation pathway: protein from the exogenous milieu can be presented to cd + t cells. this allows specific class-i mediated immunity to be generated without the dc becoming infected. cross priming is therefore of prime importance in the generation of ctl responses against pathogens that infect non-haemopoietic cells. dc, dendritic cell; icam, intercellular adhesion molecule- ; lfa, leucocyte functional antigen. dendritic cell life history can be subdivided into a number of phases, all with discrete cellular functionality. transition between phases is mediated by diverse signals and is accompanied by changes in expression patterns of many surface markers and secreted factors. mdc, myeloid dendritic cell; scf, stem cell factor; gm-csf, granulocyte-macrophage colony stimulating factor; tgf, transforming growth factor; hsp, heat shock protein. throughout evolution, the presence of a pathogen has been accompanied by a series of unique markers. for example, this may be dsrna for influenza virus or lps in the case of gram-negative bacteria. the dc are sensitive to a wide range of these stimuli that serve not only to activate innate immunity via the release of chemokines and proinflammatory mediators, but also trigger dc migration towards local lymphoid tissue in order to generate antigen-specific (adaptive) immunity. it is hardly surprising that intact, viable microbes, such as influenza virus or mycobacteria, can be observed to activate dc. , however, the question remains as to which individual molecules mediate dc activation and via which receptors. factors specific to bacteria, such as lps and, more surprisingly, cpg motifs in dna, have been shown to activate dc. , lipopolysaccharide-induced activation is mediated by cd acting in concert with the recently described toll-like receptors (tlr) and . , however, the receptor that mediates dna-induced activation still remains to be identified. the ligation of fc receptors during the uptake of immune complexes has also been shown to induce the maturation of dendritic cells. a further interesting observation is that several dc-activating factors (including lps, tnf-α and cd l) are able to rescue immature dc from apoptosis. the second group of dc-activating signals is derived from distressed or dying cells. the phenomenon of langerhans cell migration into tissue culture media after in vitro culture of skin explants for h is thought to reflect inflammatory stimuli resulting from skin damage promulgating dc migration. cells, as a consequence of infection, will release pools of pro-activating factors, which will indirectly activate dc. the action of pro-inflammatory cytokines (tnf-α) and prostaglandins (pge ) on immature dc produces a maturation event characterized by an increase in the levels of membrane hla-dr (mhc-ii) and a reduction in the propensity of the cells to take up soluble antigen. in addition, dc that encounter apoptotic cells undergo maturation in vitro; this maturation involves autocrine/paracrine secretion of il- β and tnf-α and is thought to be mediated by α v β integrin and cd . , one of the primary events after activation is the release of chemokines, such as rantes, monocyte chemotactic protein (mcp)- , macrophage inflammatory protein (mip)- α, and mip- , which not only attract new mononuclear cells (including dc precursors) but also serve to activate nk cells, supporting the inference that the dc is an important bridge between the acquired and innate immune systems. other chemokine-related phenomena that accompany infection include a reduction in the chemotactic response of dc towards mip- α, mip- β, mip- β, rantes, mcp- and fmlp and an increase in the chemotactic response towards chemokines stromal cell-derived factor (sdf)- (cxcr ) and monocyte-derived chemokine (mdc), ccr . chemokines play an important role in targeting dc to lymphoid tissue. the chemokine receptor ccr is gradually up-regulated after activation. the ligands for this receptor, ckine and mip- β, are chemoattractants for dc and are produced primarily in the t cell-rich parafollicular areas of lymphoid tissue. , for the langerhans cell, activation is also accompanied by the loss of specific markers, such as cutaneous lymphocyte antigen (cla) and birbeck granules, along with altered surface expression of cell adhesion molecules that facilitate movement into the afferent lymph. for example, langerhans cells express e-cadherin, which mediates homotypic interactions with keratinocytes. the production of this molecule is down-regulated during maturation. , mhc class ii molecules, stored within vesicles termed miic, are transported to the cell surface and there is a concomitant increase in surface half-life of both mhc class i and mhc class ii molecules. activation-induced changes serve to change the immature dc, which are adept at antigen sampling, into dc dedicated to the presentation of antigen. immediately after exposure to an activation signal, such as tnf-α, there is a short-term increase in antigen uptake followed by an almost complete cessation in uptake preceding migration into afferent lymphatics. , interaction of dendritic cells with t lymphocytes. antigen is presented as peptide mhc class-i/ii complexes (signal ). t lymphocytes are activated by the presence of costimulatory molecules, which communicate that the presented antigen is associated with a 'threat' (signal ). absence of these secondary signals induces tolerance towards the presented peptide. lfa, leucocyte functional antigen; icam, intercellular adhesion molecule; vla, very late antigen. in summary, dc activation is accompanied by a number of changes, including increased expression of costimulatory and antigen presentation molecules, a decrease in the propensity to capture soluble antigen, the increased stability of mhc class i and ii molecules, changes in chemokine ligand and receptor expression and translocation into lymphoid tissue. once inside the lymphoid tissue, the dc interacts with t, b and nk cells. the unique ability of dc to attract and stimulate naïve t cells at this point is still poorly understood; however, it appears that dc secrete a c-c chemokine (dc-ck ) that selectively attracts these cells, while both dc and activated b cells produce a c-c chemokine abcd- that attracts mature t cells. dendritic cells, by virtue of their high levels of surface mhc-ii and costimulator expression, readily cluster cd + t helper cells. two different subsets of dendritic cells have recently been characterized that control the fate of naïve t helper cells. one subset, designated dc , secretes large quantities of il- that promotes the development of a th -type phenotype, which is important in the generation of immunity to intracellular parasites. another subset, the dc , promotes a th pattern of cytokine production via the release of an as yet uncharacterized factor. the th responses are important in the generation of immunity to extracellular infections and also in allergic responses. the interaction of the cd /tcr complex with dc mhc-ii has significant effects on the cytokine production of dc, one of the most important being enhancement of il- production. expression of cd l by t helper cells further stimulates the production of il- and il- from dc. , the secretion of these th -type cytokines by dc is important in enhancing the generation of cytotoxic t cells from naïve cd + t cells. the cd / l pathway has many other important functions in the context of dc. the interaction of dc-expressed cd with cd l on nk cells has also been shown to enhance nk cell cytotoxicity and ifn-γ production. dendritic cells also interact both directly and indirectly with b cells. as alluded to previously, one of the first observations made regarding dc was that they are essential accessory cells for the generation of primary antibody responses. the addition of dc to a culture of b cells promotes an lfa- -dependent clustering, proving that dc/b cell communication is not mediated solely via th cell intermediates. , the dc stimulate the proliferation of cd activated b cells and also enhance maturation into igm-producing plasma cells. , this differentiation of naïve b cells into igm-producing plasma cells is dependent on dc secretion of il- . in the presence of il- and tgf-β, dc have also been observed to promote immunoglobulin class switching towards iga. the final stage in the dc life cycle is apoptosis, mediated either by t lymphocytes or nk cells. this process makes way for the next wave of dc migrating from local tissues into the afferent lymph , and provides an explanation as to why dc are never found in the efferent lymph. in summary, immature dc are activated by a number of environmental signals that are associated with cellular stress or the presence of a microbe. these signals are transduced by the dc into costimulators, cytokines and chemokines, which activate innate and acquired immunity. dendritic cells are commonly the first immunological cells to encounter foreign organisms. not surprisingly, dc play an important role in the generation of protective immunity towards intracellular parasites. , however, as a consequence, dc function may also be subverted as part of the life cycle of a pathogen. a number of viruses use molecules expressed by dc as receptors; examples include cd , ccr and cxcr (hiv), cd (coronavirus and cytomegalovirus) , and cd (measles virus; mv). the most extensively studied example of dc involvement in infection is hiv. this lentivirus can remain latent in dc and exploits the trafficking of dc towards lymphoid tissue as a strategy to enhance the infection of permissive cd + lymphocytes. , the hiv appears to be activated in dc by cd ligation or the presence of th cells. the activation status of the dc themselves is thought to have an impact on viral replication, with immature and cutaneous dc supporting productive infection of macrophage tropic virus, while mature dc are able to transport hiv but appear unable to replicate both t cell and macrophage tropic strains of virus. a further interesting observation is that viruses derived from infected dc carry t cell-specific factors that make them highly infectious. the mechanisms used by hiv to subvert antigen presentation by dc undoubtedly have direct parallels in other viral infections. for example, activation of mv-infected dc with cd l or with cd + t cells results in a profound viral replicative event, which ultimately leads to cell death. the aforementioned activation of dc by cd l occurs primarily in the lymph node and it appears that this mechanism will prevent both mv-and hiv-infected dc from successfully presenting antigen to t cells. mechanisms for interfering directly with antigen presentation, such as those used by adenoviruses, ebv and cmv, may also prevent dc from effectively modulating ctl responses. for example, many human herpesviruses, including ebv (hhv ), encode a viral homologue of il- , which will suppress the ability of professional apc such as the dc to produce effective ctl responses. human cytomegalovirus (hcmv) causes severe morbidity in immunocompromised patients and again subverts immunosurveillance using a latent 'protein-free' replicative stage. human cytomegalovirus remains latent in the cd + myeloid progenitors of monocytes and dendritic cells. reactivation has been observed when these cells are treated with cytokines that promote differentiation of dc (tnf-α, il- and granulocyte-macrophage colony stimulating factor (gm-csf)). reactivation under the influence of these signals would be an ideal mechanism for interfering with antigen presentation. the inadequate antigen-presenting ability of langerhans cells that express hepatitis c virus genes is thought to reflect active interference with antigen presentation by an indeterminate mechanism, perhaps involving perturbed il- production. the picture for other intracellular parasites is somewhat similar. effective th -type immune responses are essential for the generation of immunity towards parasites such as histoplasma, leishmania and mycobacterium spp. [ ] [ ] [ ] it appears that dc secretion of th -promoting cytokines, such as il- , is inhibited by infection with these parasites. other microbes, such as the sexually transmitted intracellular pathogen chlamydia spp., have been detected in dc, although the mechanisms that afford this organism relative freedom from immunosurveillance still remain to be clarified. a salmonella sp. was recently discovered to both infect and survive within dc and, as with hiv, this may be an important mechanism for disseminating the disease away from mucosal sites. in summary, many organisms use dc as bases for the evasion of immunosurveillance. subversion of dc antigen presentation and activation is a newly recognized phenomenon, which helps to explain the persistent nature of some infections. dendritic cells have been studied in a number of common autoimmune conditions. despite the diverse nature and tissue distribution of autoimmune diseases, dc share certain common characteristics irrespective of disease and sites from which they are recovered. these include increased numbers of tissue dc, particularly in a perivascular distribution, dc infiltration at the earliest stage of disease and an altered dc phenotype. early dc infiltration contributes to the recruitment of other immune cells. rheumatoid arthritis (ra) is a chronic destructive autoimmune disease of poorly defined pathogenesis with a prevalence approaching %. for almost two decades, the helper t cell has been considered to be the mediator of the immune response leading to joint destruction. this is based on the strong hla dr association with ra, on histological features of the synovium and on the trend towards a response in ra with t cell-modulating therapies. however, the primacy of t cells in ra has been challenged and several authors have investigated dc expression and function in ra. increased numbers of dc have been shown in both the peripheral blood, synovial fluid exudates , and synovial tissues in patients with ra. it has been argued that ra is due to dc presentation of endogenous self peptides. this is a feasible hypothesis, but a more important question is: what factors result in synovial dc activation? both tnf-α and gm-csf are abundant in the synovium, which could contribute to activation, as could mechanical factors such as trauma. pro-inflammatory mediators, such as tnf-α, could be up-regulated in the synovium by rheumatoid-factor auto-antibodies directed against the fc moiety of immunoglobulins, which could lead to macrophage activation by complement. the plethora of cellular and molecular abnormalities reported in ra could therefore be secondary to the ravages of chronic inflammation rather than being of primary pathogenic importance. anti-tnf-α therapy is an exciting development in the treatment of ra. the sustained immunomodulatory effect of anti-tnf therapy in early ra could be due to modulation of dc function downregulating both costimulatory signals and dc trafficking. seronegative polyarthritis includes the related conditions ankylosing spondylitis (as), reactive arthritis (rea), psoriatic arthritis and undifferentiated arthritis. these diverse clinical entities are interrelated clinically by spinal inflammation and at an immunological level by the presence of the mhc class i molecule hla b . in the hla b transgenic rat model of as there is evidence that dc play a critical function, because disease can be transferred by bone marrow cells presumed to be dc. putative activation signals for seronegative arthritis have been identified. bacteria may preferentially home to the joint tissues and activate an immune response. , another signal could be tnf-α, which can induce similar disease phenotypes in experimental models to human as. the synovial inflammation seen in these conditions could be related to proinflammatory cytokines released from the adjacent joint capsule. direct mechanical trauma to the tissues could also serve to initiate cell-mediated immunity in these conditions. dendritic cell infiltration is an early feature of islet cell autoimmunity in diabetes mellitus and contributes to local lymphoid tissue formation in the islets cells. what could the signal for dc activation be in autoimmune diabetes? the destructive processes are directed against the pancreatic β cell. a viral tropism for the β cell is the one mechanism that has been postulated to initiate autoimmunity. clues to dc activation signals for endocrine disease in general come from studies in thyroid disease, which show that dc infiltration is preceded by metabolic abnormalities in the thyroid gland itself, suggesting that some types of autoimmunity are due to primary abnormalities in the tissue targeted. psoriasis is a common skin condition with a prevalence of about %. the skin has a rich source of langerhans cells, which are thought to be important in the pathogenesis of inflammation at that site. streptococcal bacteria and ifn-γ have both been implicated in the pathogenesis of psoriasis and will also influence the activation status of dc. a recently identified cutaneous danger signal may be il- . this is based on experiments that show that mechanical stressing of keratinocytes results in liberation of high concentrations of il- , which is noteworthy because the most common sites of psoriasis are the elbows and knees, where the skin is subject to considerable trauma and stretching. knowledge of the central role of dc in autoimmune disease is important for both determining the mechanism of action of currently available therapies and for the development of future therapeutic strategies. not all studies on dc support an activated dc phenotype in autoimmune disease, with some studies showing that expression of costimulatory molecules is down-regulated at the sites of disease. [ ] [ ] [ ] this could be due to an immunomodulatory response at these sites to reduce the severity of inflammation. however, these changes have been interpreted as representing a primary defect in dc function that could theoretically allow auto-aggressive t cell clones to emerge during thymic selection. for such a contention to be supported, dc abnormalities would need to be demonstrated prior to the chronic phases of the respective diseases. the cell-mediated arm of the immune system plays an important role in the detection and elimination of malignant cells. t lymphocyte responses to tumour cells will require initial antigen presentation by professional apc such as the dc. dendritic cell infiltration into a tumour has for many years been linked to increased patient survival and reduction in the number of metastases in a variety of malignancies (including endometrial, gastric and lung cancers). this positive prognostic indicator is tempered by the observation that dc isolated from cancer patients show an impaired ability to present antigen as a product of decreased surface expression of the costimulators cd and cd . , it also appears that many sporadic tumours actively exploit immunosuppressive pathways to interfere with antigen presentation. tumour-derived il- acts directly on tissue dc to prevent maturation and therefore subsequent immunogenic presentation to t lymphocytes. increased expression of fasl on tumour cells may also induce apoptosis in dc and t cell effectors. , vascular endothelial growth factor (vegf) is produced by a majority of carcinomas and has been shown to inhibit maturation of dc within the tumour and to impair differentiation of haemopoietic progenitors into dc. an in vitro study using a renal cell carcinoma cell line has shown that factors are released that inhibit the differentiation of dendritic cells from cd + progenitors. interleukin- and macrophage (m)-csf have been subsequently identified as the factors mediating this inhibition via the down-regulation of the gm-csf receptor. , these mechanisms represent parodies of the normal processes intended to establish tolerance between a tissue and the immune system. the role of the dc in the pathogenesis of sporadic cancers is still poorly understood. there is a great deal of interplay between the acquired and innate arms of the immune system during allotransplantation. however, it is the action of cytotoxic t cells that determine whether a graft survives or is rejected. the dc, with its ability to control primary t lymphocyte responses, will therefore initiate attack on an allograft by the presentation of alloantigen. tissue damage during transplantation will 'stress' the host and graft tissues to thresholds that induce dc maturation via the release of, for example, the pro-inflammatory cytokines tnf-α and il- β. activated alloantigen-presenting dc will then migrate away from the graft to present antigen primarily in host lymphoid tissue. the presentation of peptide by donor dc in the context of foreign mhc molecules will activate both helper (cd + ) and cytotoxic (cd + ) t cells. cytotoxic t cells generated against alloantigen will then move into the circulation and eventually initiate an attack on graft cells expressing the specific peptides that were presented by the dc. the role of the dc in graft rejection is exemplified by the observation that graft survival can be dramatically increased by dc depletion. in addition, infusions of costimulatornegative dc are able to prolong the life of similar allografts by inducing t cell hyporesponsiveness. indeed, the paucity of corneal allotransplant rejection is thought to represent the low level of dc within this tissue. modulation of cytokines that directly affect the maturation of dc will also influence graft survival; for example, blockade of cd signalling in dc has been shown to prolong cardiac graft survival. in a recent study on the biology of the cytokine il- , it was shown that not only does this factor induce the functional maturation of dc, but a novel antagonist to il- has been shown to promote the survival of cardiac grafts. , therapeutic applications the observation that dc play a role in the progression and severity of many diseases has led many researchers to investigate how modulation of dc function could be used therapeutically. the power of the activated dc could be used in cancer or intracellular parasite infection to redirect an immune response towards defective (malignant) or infected cells that have evaded immunosurveillance (fig. ) . alternatively, dc maturation could be suppressed to alleviate the symptoms of autoimmunity and allotransplant rejection. advances in the extraction and in vitro culture of dc have been a major driving force behind the recent increased interest in these cells and have facilitated the inclusion of these powerful adjuvants in therapeutic trials. refined laboratory protocols are now available for either the generation of dc from a number of readily available sources or for the direct isolation of dc from mixed cell populations. cells that have been found to yield dc, after culture in lineagerestricting cocktails of cytokines, include cd + stem cells [ ] [ ] [ ] [ ] and cd + monocytes. [ ] [ ] [ ] cd + monocytes are perhaps the most readily available precursors used to generate human dc, because they constitute some - % of human pbmc. the simplest method of obtaining dc is by allowing pbmc to adhere to tissue culture dishes for h. after several washes, the adherent fraction will be enriched for cd + monocytes. immature dc can then be produced using gm-csf + il- /il- . the gm-csf has the effect of restricting differentiation towards a myelomonocytic lineage, while il- or il- inhibits the development of macrophages. the overall result is a forced differentiation towards a dc lineage. cells obtained from in vitro culture with high doses of gm-csf and il- /il- resemble immature dc, in that they have a high affinity for soluble antigen and express lower levels of costimulatory molecules than the activated form. subsequent addition of tnf-α, lps or cd l to a culture of these dc promotes maturation and activation, as would be expected from the discussion of dc life history presented earlier. high-purity dc cultures can also be obtained by immunomagnetic selection. contaminating leucocyte subsets can be depleted with a cocktail of antibodies against t cells (cd ), b cells (cd ), nk cells (cd ), monocytes (cd ) and macrophages (cd ), leaving a fraction enriched for dc. alternatively, dc can be positively selected from mixed cultures using antibodies against antigens that have dc-restricted expression, for example cd or cmrf- . dendritic cell-based immunotherapy of cancer has been demonstrated in a number of studies using murine tumours. established malignancies ranging from melanoma to mammary carcinoma have all been successfully regressed using infusions of antigen-loaded, activated dc. the generation of specific tumour-specific ctl requires the introduction of antigen into the dc, thus providing a target for immunity. the dc are able to present antigen that has been introduced by a variety of methods, including plasmid, mrna, peptide (eluted and synthetic), cell lysate and recombinant viral vectors, including adenovirus and vaccinia virus. data relating to human dc clinical trials are scarce, but certain studies have reported encouraging results. one study has used dc pulsed with tumour lysate or peptide for the treatment of patients with metastatic melanoma. of the patients immunized, five had objective responses ( %) with two subjects experiencing considerable disease regression. malignant melanoma, due to its intrinsic antigenicity, is an ideal target for a dc-based immunotherapy. of the published studies on the use of dc for the treatment of prostate cancer, one of most promising cites that nine of patients ( %) responded to immunization. hsu et al. treated patients suffering from follicular b cell lymphoma with dc and tumourspecific idiotype protein. this resulted in a marked antitumour immune responses in all four patients. for b cell lymphoma, the presence of a truly tumour-specific antigen is the ideal in terms of a dc-based immunotherapy, because it will reduce autoimmune complications arising from coexpression of antigens on normal tissue. for example, patients immunized against melanoma-derived antigens (e.g. mart- ) sometimes experienced an autoimmune vitelligo resulting from destruction of normal melanocytes by autoreactive ctl. it is suspected that dc immunotherapy of solid tumours will be considerably less efficacious than for melanoma or leukaemia, perhaps due in part to the higher incidence of hla antigen dysregulation in these tumours and the paucity of good tumour-restricted antigens. further increases in our understanding of how dc are activated will allow us to associate more 'danger' with the tumour antigens under investigation. culturing dc ex vivo for tumour immunotherapy represents a considerable challenge. one alternative may be to use infusions of factors that directly promote dc differentiation, in an effort to increase the level of antitumour immunosurveillance. a case in point is the haemopoietic growth factor flt- . this growth factor has been shown to increase the numbers of dc and monocytes in peripheral blood without producing the serious side effects that are a consequence of using other cytokines/growth factors as adjuvants (e.g. il- , ifn-γ). flt- is able to induce protective antitumour immunity in some animal models, , which is thought to be a consequence of increased presentation of tumour antigens combined with increased nk cell activity. the synergy of a dc growth-promoting factor, such as flt- ligand, with a dc activation signal, such as cd l or tumour necrosis factor-related activation-induced cytokine (trance), may provide additional benefits by promoting ctl generation from an already expanded pool of dc. with respect to persistent viral disease, dc function could be modulated to reactivate silent t cells and lessen the effects of relapse. diseases that may be amenable to this form of intervention include viral hepatitis (hbv and hcv), papillomavirus infection and chronic hcmv infection. optimizing dc therapies for persistent viral disease is hampered by a lack of appropriate animal models. however, it is encouraging to note that in a murine model of chronic hepatitis, where mice are transgenic for hepatitis b surface antigen (hbsag), tolerance to hbsag can be broken by immunization with cytokine-activated dc. it is inferred from the earlier discussion of dc life history that, for immunotherapy of cancer and viral disease, dc activated by members of the tumour necrosis family of ligands (e.g. tnfα or cd l) will be the best candidates for breaking immunological tolerance. , dendritic cells in health and disease in situations where intracellular parasites interfere with dc function, there may be some opportunity to correct the defects. for example, dc secretion of il- is inhibited by leishmania sp. and prevents an effective th -type immune response, which is crucial for parasite clearance. infusions of parasite-pulsed dc, in combination with il- or other th promoting cytokines, may help to reduce parasite burden by repolarizing immunity. it has been suggested that a possible therapy for hiv could involve infusions of il- -transfected dc in an attempt to counter the suppression of th immunity that is a characteristic of this infection. conversely, t lymphocytes could be anergized by direct suppression of dc activation for the treatment of autoimmune diseases and for reducing the severity of allotransplant rejection. enhancement of the ability of dc to tolerize t lymphocytes could be accomplished in a number of ways, for example by pretreatment of dc with maturation-suppressing factors such as il- and tgf-β. one recent study has shown that infusions of flt- ligand can increase the survival time of skin allografts when administered in combination with a factor such as anti-cd l, which will prevent dc activation. in a further landmark study, it has been found that dc transfected with cd l could induce antigen-specific tolerance after being pulsed with a peptide to which they had previously been sensitized. this observation provides proof that it may also be possible to delete autoreactive t cells from the repertoire using modified dc. in the present review, we have described how an in-depth understanding of dc biology has provided insights into the coordination of both innate and adaptive immunity. the importance of these cells in a number of disease states is also being gradually revealed. it is therefore realistic that in the near future, once the lineage, danger and tolerance paradigms have been fully resolved, it will be possible to modulate dc function for considerable therapeutic benefit. dendritic cells and the control of immunity response of naive antigenspecific cd + t cells in vitro: characteristics and antigen presenting requirements b lymphocytes in vivo fail to prime naive t cells but can stimulate antigen-experienced t lymphocytes dendritic cells are critical accessory cells for thymus dependent antibody responses in mouse and man the natural killer t (nkt) cell ligand alpha-galactosylceramide demonstrates its immunopotentiating effect by inducing interleukin (il)- production by dendritic cells and il- receptor expression on nkt cells mhc class ii expression on dendritic cells is necessary and sufficient for survival of cd + t cells targeted expression of mhc class ii molecules demonstrates that dendritic cells can induce negative but no positive selection of thymocytes in vivo human peripheral blood dendritic cell subsets. isolation and characterisation of precursor and mature antigen-presenting cells thymic dendritic cells and t cells develop simultaneously in the thymus from a common precursor population follicular dendritic cells and presentation of antigen and costimulatory signals to b cells constitutive macropinocytosis allows tap-dependent major histocompatibility complex class i presentation of exogenous soluble antigen by bone marrow-derived dendritic cells type i (cd ) and type ii (cd ) fc gamma receptor-mediated phagocytosis by human blood dendritic cells characterisation of expression, cytokine regulation, and effector function of the high affinity igg receptor fc gamma ri (cd ) expressed on human blood dendritic cells fc epsilon receptor i on dendritic cells delivers ige bound multivalent antigens into a cathepsin s-dependent pathway of mhc class ii presentation antigen presentation and allogeneic stimulation by langerhans cells dendritic cells use macropinocytosis and the mannose receptor to concentrate macromolecules in the major histocompatibility complex class ii compartment: downregulation by cytokines and bacterial products the receptor dec- expressed by dendritic cells and thymic epithelial cells is involved in antigen processing differential gene expression in cultured human langerhans cells in response to phagocytic stimulation new cd from the b cell section of the fifth international workshop on human leukocyte differentiation antigens cloned dendritic cells can present exogenous antigens on both mhc class i and class ii molecules presentation of exogenous protein antigens on major histocompatibility complex class i molecules by dendritic cells: pathway of presentation and regulation by cytokines activation of human dendritic cells following infection with mycobacterium tuberculosis lanzavecchia a. maturation activation, and protection of dendritic cells induced by double stranded rna regulation of dendritic cell numbers and maturation by lipopolysaccharide in vivo bacterial dna and immunostimulatory cpg oligonucleotides trigger maturation and activation of murine dendritic cells toll-like receptor- mediates lipopolysaccharide-induced cellular signalling defective lps signaling in c h/hej and c bl/ sccr mice: mutations in tlr gene fcγr-mediated induction of dendritic cell maturation and mhc class i-restricted antigen presentation after immune complex internalisation spontaneous apoptosis of dendritic cells is efficiently inhibited by trap (cd -ligand) and tnf-alpha, but strongly enhanced by interleukin- human cutaneous dendritic cells migrate through dermal lymphatic vessels in a skin organ culture model pro-inflammatory cytokines and prostaglandins induce maturation of potent immunostimulatory dendritic cells under fetal calf serum free conditions bystander apoptosis triggers dendritic cell maturation and antigen-presenting function immature dendritic cells phagocytose apoptotic cells via alpha beta and cd , and cross-present antigens to cytotoxic t lymphocytes distinct patterns and kinetics of chemokine production regulate dendritic cell function differential regulation of chemokine receptors during dendritic cell maturation: a model for their trafficking properties mature dendritic cells respond to sdf- , but not to several beta-chemokines dendritic cell chemotaxis and transendothelial migration are induced by distinct chemokines and are regulated on maturation the cc chemokine receptor- ligands ckine and macrophage inflammatory protein- beta are potent chemoattractants for in vitro and in vivo derived dendritic cells adhesion of epidermal langerhans cells to keratinocytes mediated by e-cadherin langerhans cells require signals from both tumour necrosis factor alpha and interleukin beta for migration developmental regulation of mhc class ii transport in mouse dendritic cells efficient presentation of soluble antigen by cultured dendritic cells is maintained by granulocyte/macrophage colony-stimulating factor plus interleukin and down regulated by tumour necrosis factor alpha a comparison of murine epidermal langerhans cells with spleen dendritic cells a dendritic-cellderived c-c chemokine that preferentially attracts naive t cells activated murine b lymphocytes and dendritic cells produce a novel cc chemokine which acts selectively on activated t cells reciprocal control of t helper cell and dendritic cell differentiation ligation of cd on dendritic cells triggers production of high levels of interleukin- and enhances t cell stimulatory capacity: t-t help via apc activation dendritic cell secretion of il- is induced by recombinant hucd lt and augments the stimulation of antigen-specific cytolytic t cells dendritic cells and resting b cells form clusters in vitro and in vivo: t cell independence, partial lfa- dependence, and regulation by cross-linking surface molecules human interdigitating dendritic cells directly stimulate cd -activated naive b cells dendritic cells enhance the differentiation of naive b cells into plasma cells in vitro towards a role of dendritic cells in the germinal center reaction: triggering of b cell proliferation and isotype switching critical role of il- in dendritic cell-induced differentiation of naive b lymphocytes human dendritic cells skew isotype switching of cd -activated naive b cells towards iga and iga maturation stages of mouse dendritic cells in growth factor-dependent long term cultures cytolysis of human dendritic cells by autologous lymphokine-activated killer cells: participation of both t cells and nk cells in the killing dendritic cells efficiently induce protective antiviral immunity antigen-pulsed epidermal langerhans cells protect susceptible mice from infection with the intracellular parasite leishmania major the role of skin dendritic cells in the initiation of human immunodeficiency virus infection human aminopeptidase n is a receptor for human coronavirus e cd (human aminopeptidase n) mediates human cytomegalovirus infection induction of maturation of human blood dendritic cell precursor by measles virus is associated with immunosuppression dendritic cells route human immunodeficiency virus to lymph nodes after vaginal or intravenous administration to mice virus replication begins in dendritic cells during the transmission of hiv- from mature dendritic cells to t cells immature dendritic cells selectively replicate macrophage tropic (m-tropic) human immunodeficiency virus type , while mature cells efficiently transmit both m-and t-tropic virus to t cells human immunodeficiency virus type derived from cocultures of immature dendritic cells with autologous t cells carries t-cell-specific molecules on its surface and is highly infectious measles suppresses cell-mediated immunity by interfering with the survival and functions of dendritic and t-cells measles virus infects human dendritic cells and blocks their allostimulatory properties for cd + t-cells viruses use stealth technology to escape from host immune system viral interleukin (il- ), the human herpes virus cellular il- homologue, induces local anergy to allogenic and syngeneic tumours low stimulatory capacity of lymphoid dendritic cells expressing hepatitis c virus genes cd ligand is not essential for induction of type cytokine responses or protective immunity after primary or secondary infection with histoplasma capsulatum interleukin- is capable of generating an antigen-specific th -type response in the presence of an ongoing infectiondriven th -type response acquired resistance but not innate resistance to mycobacterium bovis bacillus calmette-guerin is compromised by interleukin- ablation human dendritic cell (dc)-based anti-infective therapy: engineering dcs to secrete functional ifn-gamma and il- internalisation of chlamydia by dendritic cells and stimulation of chlamydia-specific t cells salmonella efficiently enter and survive within cultured cd c+ dendritic cells initiating cytokine expression identification of immunostimulatory dendritic cells in the synovial effusions of patients with rheumatoid arthritis the distribution of dendritic cells in the synovial fluids of patients with arthritis rheumatoid synovium is enriched in mature antigen-presenting dendritic cells presentation of self peptides by dendritic cells: possible implications for the pathogenesis of rheumatoid arthritis the future role of anti-tumour necrosis factor (tnf) products in the treatment of rheumatoid arthritis classification of inflammatory arthritis by enthesitis transfer of the inflammatory disease of hla-b transgenic rats by bone marrow engraftment borderlinetissues as sites of antigen deposition and persistence -a unifying concept of rheumatoid inflammation? bacterial dna as an evolutionary conserved ligand signalling danger of infection to immune cells transgenic mice expressing a truncated peromyscus leucopus tnf-alpha gene manifest an arthritis resembling ankylosing spondylitis dendritic cells and macrophages are the first and major producers of tnf-alpha in pancreatic islets in the nonobese diabetic mouse dendritic cells induce autoimmune diabetes and maintain disease via de novo formation of local lymphoid tissue preautoimmune thyroid abnormalities in the biobreeding diabetesprone (bb-dp) rat: a possible relation with the intrathyroid accumulation of dendritic cells and the initiation of the thyroid autoimmune response psoriasis induced at the injection site of recombinant interferon gamma. results of immunohistologic investigations mechanical deformation promotes secretion of il- alpha and il- receptor antagonist defective maturation and function of antigen-presenting cells in type diabetes impaired yield, phenotype, and function of monocyte-derived dendritic cells in humans at risk for insulin-dependent diabetes defective dendritic cell (dc) function in a hla-b transgenic rat model of spondyloarthropathy (spa) prognostic significance of p , bcl- , vimentin, and s protein-positive langerhans cells in endometrial carcinoma dendritic cells prevent lymph node metastasis in patients with gastric cancer s positive dendritic cells in human lung tumours associated with cell differentiation and enhanced survival decreased antigen presentation by dendritic cells in patients with breast cancer inflammatory cells infiltrating human colorectal carcinoma express hla-ii but not b - and b - costimulatory molecules of the t-cell activation il- ): an immunosuppressive factor and independent predictor in patients with metastatic renal cell carcinoma lymphocyte apoptosis induced by cd (apo- /fas) ligand-expressing tumor cells -a mechanism of immune evasion? upregulation of fas (apo- /cd ) ligand and down-regulation of fas expression in human esophageal cancer production of vascular endothelial growth factor by human tumors inhibits the functional maturation of dendritic cells defective function of langerhans cells in tumour-bearing animals is the result of defective maturation from hemopoietic progenitors inhibition of the differentiation of dendritic cells from cd (+) progenitors by tumor cells: role of interleukin- and macrophage colonystimulating factor migration of dendritic cells to the draining lymph node after allogenic or congenic rat skin transplantation enhanced survival of skin grafts depleted of langerhans' cells by treatment with dimethylbenzanthracene costimulatory molecule deficient dendritic cell progenitors induce t cell hyporesponsiveness in vitro and prolong the survival of vascularised cardiac allografts blockade of the cd -cd ligand pathway potentiates the capacity of donor-derived dendritic cell progenitors to induce long-term cardiac allograft survival evidence for a role of il- in organ allograft rejection: il- promotes the functional differentiation of dendritic cell progenitors evidence for a role of il- in alloimmunity: a novel il- antagonist promotes heart graft survival in vitro generation of human dendritic cells and cell therapy isolation and function of human dendritic cells generation of human dendritic cell/langerhans cells from circulating cd + hematopoietic progenitor cells expansion of immunostimulatory dendritic cells among the myeloid progeny of human cd + bone marrow precursors cultured with c-kit ligand, granulocyte-macrophage colony stimulating factor, and tnf-alpha tnf in combination with gm-csf enhances the differentiation of neonatal cord blood stem cells into dendritic cells and macrophages improved methods for the generation of dendritic cells from nonproliferating progenitors in human blood cd + blood monocytes can differentiate into functionally mature cd + dendritic cells molecular and functional characteristics of dendritic cells generated from highly purified cd + peripheral blood monocytes differentiation of human dendritic cells from monocytes in vitro dendritic cells and macrophages can mature independently from a human bone marrow-derived, post colony-forming unit intermediate generation of dendritic cells from bone marrow progenitors using gm-csf, tnf-alpha and additional cytokines: antagonistic effects of il- and ifn-gamma and selective involvement of tnf-alpha receptor- rapid purification of human langerhans cells using paramagnetic microbeads human blood dendritic cells selectively express cd , a member of the immunoglobin superfamily isolation of human blood dendritic cells using the cmrf- monoclonal antibody: implications for studies on antigenpresenting cell function and immunotherapy eliciting t cell immunity against poorly immunogenic tumors by immunisation with dendritic cell-tumor fusion vaccines pulsing of dendritic cells with cell lysates from either b melanoma or mca- fibrosarcoma yields equally effective vaccines against b tumours in mice transgene expression in dendritic cells to induce antigen-specific cytotoxic t cells in healthy donors induction of primary carcinoembryonic antigen (cea)-specific cytotoxic t lymphocytes in vitro using human dendritic cells transfected with rna dendritic cells in anti tumor immune responses. ii. dendritic cells grown from bone marrow precursors, but not mature dc from tumor-bearing mice, are effective antigen carriers in the therapy of established tumors vaccination with dendritic cells inhibits the growth of hepatic metastases in b mice generation of melanoma-specific cytotoxic t lymphocytes by dendritic cells transduced with a mart- adenovirus antigen expression by dendritic cells correlates with the therapeutic effectiveness of a model recombinant poxvirus tumor vaccine vaccination of melanoma patients with peptide or tumor lysate-pulsed dendritic cells evaluation of phase i/ii clinical trials in prostate cancer with dendritic cells and psma peptides vaccination of patients with b-cell lymphoma using autologous antigen-pulsed dendritic cells dramatic increase in the numbers of functionally mature dendritic cells in flt ligand-treated mice: multiple dendritic cell subpopulations identified flt ligand induces the generation of functionally active dendritic cells in mice flt ligand induces tumour regression and antitumor immune responses in vivo dendritic cells directly trigger nk cell function: cross-talk relevant in innate anti-tumour immune responses in vivo dendritic cell immunisation breaks cytotoxic t lymphocyte tolerance in hepatitis b virus transgenic mice dendritic cells require maturation via cd to generate protective antitumour immunity generation of tumor immunity by bone marrow-derived dendritic cells correlates with dendritic cell maturation stage phenotype, function, and in vivo migration and survival of allogenic dendritic cell progenitors genetically engineered to express tgf-β prolonged skin allograft survival in mice treated with flt -ligand-induced dendritic cells and anti-cd monoclonal antibody. transplant induction of antigen-specific immunosuppression by cd l cdna-transfected 'killer' dendritic cells we would like to offer our sincere thanks to stephen richards, richard jones and peter cotterrell for their constructive comments regarding the manuscript. laboratory work is supported by grants from yorkshire cancer research, the arnold tunstall fellowship and the medical research council. key: cord- -ez yq xi authors: suzumura, akio title: immune response in the brain: glial response and cytokine production date: - - journal: neuroimmune biology doi: . /s - ( ) - sha: doc_id: cord_uid: ez yq xi abstract although the brain has been considered as an immunologically privileged site, the evidence to date suggests that this is no longer the case. cytokines such as interferon (ifn)-γ, tumor necrosis factor (tnf)-α, and interleukin (il)- induce class i major histocompatibility complex (mhc) antigen expression on neural cells. ifn-γ, the most potent inducer of mhc antigen, also induces class ii mhc antigen expression on microglia and astrocytes, which enable them to function as antigen-presenting cells. thus, in some pathological conditions, invading t cells can interact with neural cells to induce central nervous system (cns) damage. glial cells have also been shown to produce various cytokines and chemokines. almost all cytokines and chemokines known to occur in the immune system are also produced in the cns. in this chapter, the glial responses contributing to neuroimmune interactions are reviewed, with a focus on production and functions of cytokines in the cns. the brain has long been considered as an immunologically privileged site based on a large body of evidence: the lack of major histocompatibility complex (mhc) antigen expression on neural cells; the lack of lymphoid drainage in the central nervous system (cns); and the presence of the blood-brain barrier (bbb), which blocks the invasion of immune cells or high molecular substances including antibodies into the brain. however, as has been shown by research published in the s, some cytokines or viral infections induce mhc antigen expression on both neuronal and glial cells. interferon-g (ifn-g), the most potent inducer of mhc antigen, also induces class ii mhc antigen expression on microglia and some populations of astrocytes, which enable them to function as antigen-presenting cells (apcs). in order to effectively present antigens to t cells, apcs have to express other costimulatory molecules. microglia and astrocytes have been shown to express these costimulatory molecules, and this expression is also enhanced by exposure to ifn-g. thus, if activated t cells enter the cns, either microglia or some populations of astrocytes are able to present cns antigens to expand a t-cell clone specific for a particular cns antigen. in fact, it has also been shown that activated t cells can enter the brain through an intact bbb. consequently, in certain pathological conditions, glial cells may alter their functions to actively interact with immune cells. in most cases these glial changes are mediated by cytokines. another remarkable glial response in pathological conditions is the production of cytokines. in the late s, many laboratories, including ours, have demonstrated the production of cytokines by glial cells. almost all cytokines known to occur in the immune system were produced in the cns. thus, the brain should no longer be considered as an immunologically privileged site. in this chapter, i will review the glial responses in neuroimmune interactions in the cns with a focus on the production and functions of cytokines. in normal or unstimulated conditions in vivo and in vitro, neuronal and glial cells do not usually express class i or class ii mhc antigens on their surface, whereas microglia only weakly express class i mhc antigens in vitro. consequently, in the normal brain, neural cells cannot interact with their own immune cells in a specific manner. however, it has been shown that both neuronal and glial cells can be induced to express class i mhc antigens in response to lymphokines [ , ] . as a result of this induction, the cytotoxic t cells acquire the capacity to lyse the cns cells in a mhc-restricted manner. although ifn-g is a principal factor for the induction of mhc antigen expression, tumor necrosis factor-a (tnf-a) can also induce class i mhc antigen expression on astrocytes, but not on oligodendrocytes [ ] . lymphokines, especially ifn-g, also induce the expression of class ii mhc antigens on astrocytes [ ] and microglia in vitro [ ] . this expression is associated with the induction of mrna for class ii mhc antigens. induction of class ii mhc antigens is also observed in vivo in certain pathological conditions. in the brains of experimental allergic encephalomyelitis (eae), microglia near the infiltrating t cells are reported to be class ii mhc antigen-positive [ ] [ ] [ ] , suggesting that a t cell-derived cytokine, most probably ifn-g, can induce class ii mhc antigen expression in vivo as well. after axotomy there are increased numbers, relative to controls, of microglia in and around facial nerve nuclei. moreover, these cells are reportedly class ii mhc antigen-positive [ ] . since the bbb is not damaged in this experimental condition and since there is no definitive evidence that neural cells produce inf-g in the cns, it is unlikely that ifn-g is responsible for the induction of class ii mhc antigen expression in this model. another candidate for the induction of class ii mhc antigens in microglia is interleukin (il)- . we have shown that il- induces, in a dose-dependent manner, surface expression and mrna for class ii mhc antigens in microglia, which is completely inhibited by anti-il- antibody [ ] . although we do not detect il- or il- mrna in either microglia or astrocytes in the mouse cellular system under study, it has been reported that rat microglia produce il- in vitro [ ] , and that il- mrna is detected in some populations of astrocytes and neurons by in situ hybridization [ ] . therefore, it is possible that il- derived from degenerating neurons, reactive astrocytes, or microglia in vivo may themselves induce class ii mhc antigens on microglia in certain pathological conditions. in contrast to il- , granulocyte-macrophage colony-stimulating factor (gm-csf) downregulates ifn-g-induced class ii mhc antigen expression in microglia. the suppression occurs in a dose-dependent manner and is neutralized by anti-gm-csf antibody [ ] . as we have shown previously, gm-csf is produced by astrocytes [ ] and induces the proliferation of microglia in vitro [ , ] . it is possible that astrocytes downregulate immunoregulatory functions of microglia. however, so far, there is no evidence that gm-csf contributes to the modulation of microglia proliferation and suppression of their ia antigen expression in either physiological or pathological conditions in vivo. all the macrophage deactivating cytokines, or inhibitory cytokines, such as il- , il- , and transforming growth factor-b (tgf-b) downregulate the inf-g-induced class ii mhc antigen expression in microglia [ ] [ ] [ ] . as we and other groups have shown, astrocytes and microglia produce il- [ ] and tgf-b [ , ] , but neither cells produce il- , although both cell types express il- receptors [ , ] . thus, microglia may downregulate their own immunoregulatory functions in an autocrine fashion, or the astrocyte may suppress the functions of microglia in a paracrine manner. it is also possible that invading t helper cells, especially t helper (th ), may downregulate class ii mhc antigen expression in microglia by these inhibitory cytokines. induction of mhc antigen expression on glial cells occurs without breakdown of bbb, without invasion of immune cells. we, and another group, have shown that infection with neurotropic corona virus induces class i mhc antigen expression on oligodendrocytes and astrocytes [ , ] and class ii mhc antigen on astrocytes [ ] . these inductions permit glia to interact with invading immune cells to produce cns pathology. table , and chemokines [ , , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . rat microglia reportedly produce il- in culture [ ] . only a trace amount of il- , but not the other cytokines, is detectable in the supernatant of unstimulated microglial culture. however, lipopolysaccharides (lps), and/or ifn-g in some cases, induce cytokine production. since microglia express receptors for most of the cytokines produced (see table ), these components may function as an autocrine regulator. they also express receptors for cytokines, which are produced by other cells, but not by themselves, such as il- or gm-csf. thus, the latter may function as paracrine mediators (for functions of these cytokines on microglia, refer to our previous review [ ] ). unstimulated microglia do not express the il- receptors (il- r); however, lps treatment will induce il- r expression on these cells. moreover, il- can also induce the proliferation of lps-stimulated microglia [ ] . although il- treatment has been shown to induce the proliferation of oligodendrocytes as well [ ] , we could not confirm these effects [ ] . microglia also express the receptor for il- , the cytokine produced by t cells, but not in the cns. thus, il- may be a paracrine mediator exerting its effects only in cases of an inflammatory process occurring in the cns, but not in the normal brain. we have shown the production of il- and the upregulation by ifn-g in murine microglia by means of rt-pcr for mrna expression and the bioassay to assess il- activity [ ] . however, since we have not detected il- receptors on neural cells, the functions of il- in the cns remain to be elucidated. in contrast to murine microglia, lee et al. [ ] failed to detect il- mrna expression in human microglia as assessed by rt-pcr, while they detected mrna for the il- receptor. astrocytes produce cytokines very similar to those produced by microglia (table ) . however, microglia, rather than astrocytes, seem to be a principal source of most critical cytokines, immune response in the brain such as tnf-a, il- , and il- as discussed later [ , ] , in both pathological conditions and in culture systems. astrocytes sometimes have suppressive effects on microglia or microgliaderived cytokines. for, example, in contrast to microglia after stimulation with lps and ifn-g, astrocytes produce il- p , but not p [ ] . when the astrocyte-derived p forms a homodimer, it may suppress the functional heterodimer il- p produced by microglia. in addition, the production of il- p by activated microglia was inhibited by coculture with astrocytes [ ] . thus, it is possible that astrocytes suppress microglial cytokine production and/or the effects of produced cytokines. the suppression of ifn-g-induced mhc class ii expression on microglia by astrocyte-derived gm-csf is another example of this type of interaction [ ] . as discussed above, ifn-g activates various functions of glial cells including the induction of cytokines. the production of ifn-g was thought to be restricted to lymphoid cells. however, it has recently been shown that human fetal forebrain cells can be induced to express ifn-g mrna and produce ifn-g protein when stimulated with trypanosome lymphocyte-triggering factor (tltf) [ ] . the authors claimed that astrocytes were the major producer of ifn-g in response to tltf. we have shown recently that microglia produce ifn-g in response to il- and/or il- [ ] . thus, microglia may be another source of inf-g production in the cns. it has been shown that apcs such as macrophages and dendritic cells also produce ifn-g in response to il- [ ] . candidates for nonfunctional apcs in the cns are microglia, astrocytes, and endothelial cells [ , [ ] [ ] [ ] [ ] [ ] . they usually do not express class ii mhc antigen constitutively, although some populations of microglia reportedly may express class ii mhc antigens constitutively [ ] . these cells induced the expression of class ii mhc molecules after treatment with certain inflammatory cytokines, especially ifn-g [ , , , ] , in addition to expressing some of the costimulatory molecules as well [ ] [ ] [ ] . there is published evidence that endothelial cells [ ] , astrocytes [ ] , and pericytes [ ] can process and present protein antigens to primed cd -positive t cells in vitro, but the specific role of these cells as apcs in vivo is still unclear. astrocytes do not usually express class ii mhc antigens in vivo, even in the presence of inflammatory cells [ ] . since microglia have functional characteristics very similar to macrophages and can be induced to express class ii mhc antigens as discussed above, microglia are the most possible candidates for apcs in the cns. the expression of costimulatory molecules, such as b , icam, lfa , in microglia, but only a few in astrocytes, further supports this hypothesis. menendez iglesias et al. [ ] detected b - , but not b - , in murine microglia only after stimulation with lps and ifn-g. satoh et al. [ ] have shown that human microglia, but not astrocytes, express both b - and b - , suggesting that microglia is a much more suitable candidate for local apcs in the cns. in fact, microglia when stimulated with ifn-g reportedly presented antigen to ovalbumin-specific or myelin basic protein (mbp)-specific t cells in vitro [ , ] . in a carefully executed study, hickey and kimura [ ] have shown that microglia function as apcs in pathological conditions in vivo. they used bone marrow chimeras of eaesusceptible and -resistant animals, and found that eae lesions developed only when the perivascular microglia were replaced with those of an eae-susceptible strain, suggesting that antigen presentation by perivascular microglia is critical for the development of eae lesions. professional apcs such as dendritic cells and macrophages produce il- and il- . both cytokines have been shown to be key cytokines in the development of autoimmune processes, regulating differentiation of naïve t cells into th . in order to exert its activity, il- has to form a heterodimer of p and p ; the homodimer of p suppresses the functional heterodimer. immature il- is cleaved by caspase- to become a functionally mature il- that induces the differentiation of th and the cytotoxic activity of nk and t cells. it has been reported that both microglia and astrocytes produce il- upon stimulation with lps [ ] , while we detected functional il- p production only in microglia, but not in astrocytes, after stimulation with lps and ifn-g [ ] . since soluble tnf receptors reportedly suppress il- production by human microglia [ ] , tnf signal may also be involved in il- production. microglia and astrocytes also express il- mrna after stimulation with lps [ , ] . lpsstimulated microglia have enough il- bioactivity to induce inf-g production by thymocytes and splenocytes in synergism with il- . this suggests that microglia express caspase- as well. in fact, caspase- mrna expression is elevated in microglia in multiple sclerosis (ms) plaques [ ] where il- is also reported to be elevated [ ] . interestingly, there is a group of microglia that produce only il- p , but not il- p , resulting in the failure to produce functional il- p heterodimers [ ] . the population did not produce il- even after lps stimulation (unpublished observation). therefore, microglia may have subpopulations, which regulate the differentiation of t cells in a different manner. both microglia and astrocytes produce the same cytokines, such as il- , il- , tnf-a, and tgf-b. however, there are several differences in the response to stimulation in these two cell types. for example, microglia produce tnf-a in response to lower doses of lps than are required for astrocytes and more rapidly than astrocytes as well. il- production is induced by tnf-a in astrocytes, but not in microglia [ ] . similarly, gm-csf produced by astrocytes induces il- production in microglia, but not in astrocytes [ ] . these observations indicate that microglia and astrocytes may mutually regulate their individual cytokine production. since microglia are activated in the earlier phase than are astrocytes under various pathological conditions, microglia may initiate the cascade of cytokine actions in the cns cytokine network. inhibitory signals are also included in the network (table ) . tgf-b, produced by astrocytes and microglia, suppresses all the functions of microglia. it suppresses m-and gm-csf-induced proliferation of microglia, lps-induced activation of enzymatic activity in microglia, ifn-ginduced class ii mhc antigen expression and cytokine production by microglia. tgf-b along with il- and il- is known to be a macrophage-deactivating factor. therefore, these cytokines may function as negative regulators in the cns cytokine network by suppressing cytokine production and activation of microglia. in fact, it has been found that these inhibitory cytokines exert their influence on microglia differently. tgf-b functions as if it is a total inhibitory factor [ ] . il- suppresses cytokine production and ifn-g-induced class ii mhc antigen expression in microglia, but does not suppress the proliferation or the activation of lysosomal enzymes in microglia [ ] . il- also suppresses ifn-g-induced class ii mhc antigen expression in microglia [ ] . however, unlike other inhibitory cytokines, il- induces the proliferation of microglia in either unstimulated or m-, or gm-csf-stimulated conditions. il- does not suppress lps-induced cytokine production, though it suppresses gm-csf-induced il- production by microglia [ ] . we also found that il- , but neither tgf-b nor il- , suppressed the expression of cytokine receptors [ ] . thus, it would appear that all these three inhibitory cytokines regulate the functions of microglia in a distinct manner, and that il- may be the most potent inhibitor for the functions of cytokines on microglia because it suppresses both cytokine production and receptor expression. several lines of evidences suggest that tnf-a plays a critical role in the pathogenesis of inflammatory demyelination, either directly or indirectly via induction of other cytokines, nitric oxide (no), or free radicals (fig. ) . increased cerebrospinal fluid levels of tnf-a have been demonstrated in patients with ms [ ] . tnf-a-positive microglia and astrocytes have been identified, especially in new active plaques. in vitro studies have demonstrated that tnf-a kills oligodendrocytes, myelin-forming cells in the cns [ , ] , and that microglia are the principal table . ", upregulate; !, no effect; #, downregulate. a il- upregulates il- receptor, but does not affect the expression of other receptors on microglia. immune response in the brain effectors for oligodendrocyte killing [ ] . it has also been shown that anti-tnf-a antibody suppresses the development of eae, an animal model of ms [ , ] . demyelination has been demonstrated to be much more severe in transgenic mice producing tnf-a in the cns [ ] . in addition, tnf-a induces inflammatory cytokines or chemokines in endothelial cells and impairs the tight junctions of the bbb [ ] . up until now, several substances that suppress tnf-a production have been used for the treatment of eae and ms. most of them, such as phosphodiesterase inhibitors, n-acetyl-l-cysteine, have been shown to effectively suppress the development of eae and ms [ ] [ ] [ ] , further supporting the hypothesis that tnf-a is critical for the development of inflammatory demyelination. however, experimental demyelination could also be induced in tnf-a knockout mice, though eae was delayed in the onset and inflammatory leukocytes failed to move normally into the cns parenchyma [ ] . more recently, tnf-a has been identified as a factor that promotes remyelination [ ] . thus, although tnf-a is an important cytokine, it may not be the sufficient effector molecule for inflammation and demyelination. it is also possible that tnf-a may exert different effects on inflammatory demyelination, depending on whether the tnf signaling through type tnf receptor (tnfr ) or tnfr is dominant. gliosis is a rather common pathological finding observed as a glial scar following inflammation, demyelination, ischemia, and neuronal degeneration. it consists of astrocyte proliferation, hypertrophy, and increased synthesis of glial fibrillary acidic protein (gfap), a phenotypic marker for astrocytes. evidence to date suggests critical roles for cytokines in the development of astrocytic gliosis. fontana et al. [ ] first demonstrated that factors from activated lymphocytes stimulated astrocyte proliferation and designated the factor(s) as glial cell-stimulating factor (gsf). merrill et al. [ ] also demonstrated increased proliferation of astrocytes after treatment with lymphokines. using enriched cultures of astrocytes and recombinant cytokines, selmaj et al. [ ] showed that tnf-a is a primary factor to bring about the proliferation of rat astrocytes. however, giulian et al. [ , ] have shown that il- derived from microglia [ ] is the principle factor to induce astrocyte proliferation in gliosis. in contrast, yong et al. [ ] claimed that the primary factor that induced the proliferation of human astrocytes was ifn-g and not il- or tnf-a. these differences in experimental results may be attributed to either species differences or redundancy of functions for these cytokines. alternatively, it is possible that other factors induced by either il- , tnf-a, or ifn-g may also play a role in the proliferation of astrocytes. in view of these diverse results, it can be concluded that cytokines contribute to the pathogenesis of gliosis. however, precise identification of individual cytokine contributions to the overall process will require additional experimental inquiries. tnf-a has also been implicated as an effector for neuronal degeneration [ ] [ ] [ ] . tnf-a exerts its cytotoxicity directly via tnfr . alternatively, it also induces no or free radicals to form the toxic peroxinitrite. it has been shown that b-amyloid stimulates microglia to produce factors toxic to neurons. it is possible that neuronal apoptosis induced by b-amyloid is also mediated by glia-derived tnf-a [ ] . combs et al. [ ] concluded that the most critical factor in b-amyloid-induced, microglia-mediated neuronal apoptosis might be no, because neurotoxicity was decreased by the selective inhibitors against inducible nitric oxide synthase. apoptosis of motor neurons and dorsal root ganglion neurons by peripherin aggregates is also reportedly mediated by tnf-a [ ] . tnf-a also exerts its neurotoxicity by activating astrocytes to release glutamate [ ] . recently, we have shown that the most neurotoxic factor from activated microglia is glutamate [ ] . tnf-a dose not exert direct neurotoxicity, but induces neurotoxicity via glutamate production by microglia. glutamate disturbs the mitochondrial respiratory chain to cause energy depletion in neurons, which results in neuronal damage toward cell death [ ] . in contrast, il- , but not tnf-a, may be involved in neurotoxicity during some variants of viral encephalitis [ ] . the protein fas associated with death domain (fadd) is an adaptor protein of the tnf receptor family death pathway. a number of fadd-positive dopaminergic neurons in the substantia nigra pars compacta have been shown to be significantly decreased in patients with parkinson's disease (pd), as compared to levels in normal subjects [ ] . this decrease correlated with the known selective vulnerability of nigral dopaminergic neurons in pd. on the basis of the latter, the authors concluded that the tnf-fadd pathway contributed to the susceptibility of dopaminergic neurons in pd to the effects of tnf-mediated apoptosis [ ] . interestingly, cytokines described above as toxic also have protective roles for neurons against oxidative stress. tnf-a and il- have been shown to increase the level of manganese superoxide dismutase (mn-sod) in astrocytes, in a dose-and time-dependent manner [ ] . since sod functions as protective against oxidative stress, and since the increased mn-sod activity has been demonstrated in the substantia nigra of parkinsonian patients [ ] , these cytokines may function to protect degenerating neurons, via induction of sod. il- reportedly increases the production of nerve growth factors by astrocytes [ ] . therefore, a balance between toxic and protective factors induced by cytokines may determine neuronal damage (see fig. ). microglia undergo various morphological changes to become either ramified, amoeboid, or rodshaped. we have shown that all of these morphological changes could be reproduced in vitro with various cytokines [ , ] . microglia also form a unique phenotype of multinucleated giant immune response in the brain cells (mngc), which are observed in aids encephalopathy, tuberculosis, etc. lee et al. [ ] have demonstrated that treatment with il- , il- , ifn-g, and gm-csf induces mngc in rat microglia, while addition of il- , il- , or tnf-a failed to form mngc. in mouse experiments using microglia, there was no single cytokine that induced mngc in culture. however, when stimulated with il- or il- in the presence of gm-csf or m-csf, mngc formation occurred in the cultures of mouse microglia [ ] . the different results between these studies may be attributable to species differences. nevertheless, the results of these studies indicate that introduction of cytokines, most probably those that are t cell-derived, can induce mngc formation without infectious agents. inducible expression of h- and ia antigens on brain cells expression of h- antigen on oligodendrocytes is induced by soluble factors from concanavalin a activated t cells tumor necrosis factor induces expression of mhc antigens on mouse astrocytes astrocytes as antigen-presenting cells. i. induction of ia antigen expression on astrocytes by t cells via immune interferon and its effect on antigen presentation mhc antigen expression on bulk isolated macrophage-microglia from newborn mouse brain: induction of ia antigen expression by gamma-interferon immunohistochemical analysis of the rat central nervous system during experimental allergic encephalomyelitis, with special reference to ia positive cells with dendritic morphology an immunoelectron microscopical study of class ii major hsitocompatibility complex during chronic relapsing experimental allergic encephalomyelitis in biozzi ab/h mice microglial involvement in autoimmune inflammation of the central and peripheral nervous system expression of ia antigen on perivascular and microglial cells after sublethal and lethal motoneuron injury induction of mhc class ii antigen expression on murine microglia by interleukin- rat microglial interleukin- in situ hybridization histochemistry localization of interleukin- mrna in mouse brain production of granulocyte/macrophage colony stimulating factor by cultured astrocytes effects of colony stimulating factors on isolated microglia morphological transformation of microglia in vitro transforming growth factor beta suppresses activation and proliferation of microglia in vitro il- induces proliferation and activation of microglia but suppressed their induction of class ii major histocompatibility complex antigen expression production of interleukin- by mouse glial cells in culture macrophage-, and astrocyte-derived transforming growth factor beta as a mediator of central nervous system dysfunction in acquired immunodeficiency syndrome differential expression of transforming growth factor beta , , and by glioblastoma cells, astrocytes, and microglia expression of cytokine receptors in cultured neuronal and glial cells corona virus infection induces h- antigen expression on oligodendrocytes and astrocytes viral particles induce ia antigen expression on astrocytes interleukin- of the central nervous system is produced by ameboid microglia on the cellular source and function of interleukin- produced in the central nervous system in viral diseases production of tumor necrosis factor alpha by microglia and astrocytes in culture tnfa induces il- production by astrocytes but not by microglia production of interleukin- by mouse astrocytes and microglia in culture production of interleukin- and the expression of its receptors by murine microglia ifns are critical regulators of il- receptor antagonist and il- expression in human microglia murine microglial cells produce and respond to interleukin- production of interferon-g by microglia production of il- and il- family cytokines by microglia and their subpopulations cytokine network in the central nervous system and its roles in growth and differentiation of glial and neuronal cells induction of functional il- receptor in mouse microglia proliferation of astroglia and oligodendroglia in response to human t cell-derived factors cytokines, chemokines, and cytokine receptors in human microglia il- production by central nervous system microglia is inhibited by astrocytes african trypanosomes activate human fetal brain cells to proliferation and ifn-g production ifn-a production by antigen presenting cells: mechanisms emerge astrocytes present myelin basic protein to encephalitogenic t-cell lines expression of ia molecules by astrocytes during acute experimental allergic encephalomyelitis in the lewis rat perivascular microglial cells of the cns are bone marrow-derived and present antigen in vivo antigen presentation and tumor cytotoxicity by interferon-gamma-treated microglial cells immune regulation by brain cells in the central nervous system; microglia but not astrocytes present myelin basic protein to encephalitogenic t-cells under in vivo-mimicking conditions normal adult ramified microglia separated from other central nervous system macrophages by flow cytometric sorting the costimulatory molecule b is expressed in human microglia in culture and multiple sclerosis acute lesions t-cell costimulatory molecules b (cd ) and b - (cd ) are expressed in human microglia but not in astrocytes in culture analysis of b - and b - costimulatory ligands in cultured mouse microglia: upregulation by interferon-gamma and lipopolysaccharide and downregulation by interleukin- , prostaglandin e and cyclic amp-elevating agents antigen-specific damage to brain vascular endothelial cells mediated by encephalitogenic and nonencephalitogenic cd t cell lines in vitro antigen presentation by brain microvessel smooth muscle and endothelium soluble tumor necrosis factor receptor inhibits interleukin production by stimulated human adult microglial cells in vitro cultures of astrocytes and microglia express interleukin caspase- expression in multiple sclerosis plaques and cultured glial cells ccr (+) and cxcr (+) t cells are increased in multiple sclerosis and their ligands mip- alpha and ip- are expressed in demyelinating brain lesions selective induction of interleukin- in mouse microglia by granulocyte-macrophage colony stimulating factor il- inhibits both production of cytokine and expression of cytokine receptors in microglia cytokine levels in the cerebrospinal fluid and serum of patients with multiple sclerosis tumor necrosis factor mediates myelin and oligodendrocyte damage in vitro cytokine cytotoxicity against oligodendrocytes. apoptosis induced by lymphotoxin microglial cell cytotoxicity of oligodendrocytes is mediated through nitric oxide an antibody to lymphotoxin and tumor necrosis factor prevents transfer of experimental allergic encephalomyelitis anti-tumor necrosis factor therapy abrogates autoimmune demyelination increased severity of experimental autoimmune encephalomyelitis, chronic macrophage/microglial reactivity, and demyelination in transgenic mice producing tumor necrosis factor-alpha in the central nervous system exposure of tumor necrosis factor-alpha to luminal membrane of bovine brain capillary endothelial cells cocultured with astrocytes induces a delayed increase of permeability and cytoplasmic stress fiber formation of actin phosphodiesterase inhibitor pentoxifylline, a selective suppressor of t helper type -but not type -associated lymphokine production, prevents induction of experimental autoimmune encephalomyelitis in lewis rats oral administration of the oxidant-scavenger n-acetyl-l-cysteine inhibits acute experimental autoimmune encephalomyelitis drop in relapse rate of multiple sclerosis patients using combination therapy of three different phosphodiesterase inhibitors challenging cytokine redundancy: inflammatory cell movement and clinical course of experimental allergic encephalomyelitis are normal in lymphotoxin-deficient, but not in tumor necrosis factor-deficient, mice tnfa promotes proliferation of oligodendrocyte progenitors and remyelination glia cell stimulating factor (gsf): a new lymphokine. part . cellular sources and partial purification of murine gsf, role of cytoskeleton and protein synthesis in its production proliferation of astrocytes in vitro in response to cytokines. a primary role for tumor necrosis factor interleukin- stimulation of astroglial proliferation after brain injury interleukin- is an astroglial growth factor in the developing brain g-interferon promotes proliferation of adult human astrocytes in vitro and reactive gliosis in the adult mouse brain in vivo tumor necrosis factor alpha-induced apoptosis in human neuronal cells: protection by the antioxidant n-acetylcysteine and the genes bcl- and crma inhibition of p tumor necrosis factor receptor by antisense oligonucleotides increases hypoxic injury and beta-amyloid toxicity in human neuronal cell line neuronal death in cytokine-activated primary human brain cell culture: role of tumor necrosis factor-alpha the inflammatory response system of brain: implications for therapy of alzheimer and other neurodegenerative diseases b-amyloid stimulation of microglia and monocytes results in tnf-alpha-dependent expression of inducible nitric oxide synthase and neuronal apoptosis apoptotic death of neurons exhibiting peripherin aggregates is mediated by the proinflammatory cytokine tumor necrosis factor-a cxcr -activated astrocyte glutamate release via tnf-a: amplification by microglia triggers neurotoxicity neuritic beading induced by activated microglia is an early feature of neuronal dysfunction toward neuronal death by inhibition of mitochondrial respiration and axonal transport tumor necrosis factor-a induces neurotoxicity via glutamate release from hemichannels of activated microglia in an autocrine manner neuronal apoptosis mediated by il- b expression in viral encephalitis caused by a neuroadapted strain of the mumps virus (kilham strain) in hamsters fadd: a link between tnf family receptors and caspases in parkinson's disease induction of manganese superoxide dismutase by cytokines and lipopolysaccharide in cultured mouse astrocytes a selective increase in particulate superoxide dismutase activity in parkinsonian substantia nigra regulation of nerve growth factor mrna by interleukin- in rat hippocampal astrocytes is mediated by nfkb lymphokine induction of rat microglia multinucleated giant cell formation multinucleated giant cell formation by microglia: induction by interleukin(il)- and il- key: cord- -hypqpqs authors: sigal, luis j. title: activation of cd t lymphocytes during viral infections date: - - journal: encyclopedia of immunobiology doi: . /b - - - - . - sha: doc_id: cord_uid: hypqpqs cd t lymphocytes are a major cell population of the adaptive immune system. a fundamental characteristic of the cd t lymphocyte pool is that it is composed of millions of clones; each with a unique t cell receptor capable of recognizing a limited number of peptides displayed at the cell surface bound to the grooves of major histocompatibility complex class i (mhc i) molecules. naïve cd t lymphocytes are normally resting and circulate between the blood and secondary lymphoid organs in search of their cognate peptide–mhc complexes. during viral infections, bone marrow–derived professional antigen-presenting cells (papcs) in secondary lymphoid organs display viral peptides on their mhc i molecules. specific cd t lymphocytes that recognize these peptide–mhc adducts become activated (primed), proliferate extensively, and develop into effectors capable of killing infected cells, identified by the presence at their surface of the pertinent viral peptide–mhc complexes. this article describes how the process of priming naïve cd t lymphocytes occurs. during viral infections, cells of the adaptive immune system become activated when they recognize antigens through their antigen-specific receptors. the b cell receptor (bcr) is a membrane immunoglobulin that binds to the antigen directly. for t cells, however, the process is more complex because the t cell receptor (tcr) only recognizes antigens as small peptides bound to major histocompatibility complex (mhc) molecules at the surface of antigen-presenting cells. while the basic principles of antigen recognition by cd and cd t cells are similar, there are also major differences. this article focuses on the activation of cd t cells. effector cd t lymphocytes kill virus-infected cells and produce antiviral cytokines such as interferon gamma. in this way, cd t lymphocytes contribute to resisting primary and secondary viral infections. for example, cd t lymphocytes have been shown to be essential for the efficient control of several viral infections of mice and humans including influenza virus, poxviruses, coronavirus, and herpes viruses (lau et al., ; karupiah et al., ; welsh et al., ; fang and sigal, ; xu et al., ; doom and hill, ; channappanavar et al., ; terrazzini and kern, ) . once an infection is cleared, most effector cd t cells die but many of them remain in the circulation and tissues as resting memory cells. these memory cells can help control secondary infections more rapidly (welsh et al., ) . the activation of cd t lymphocytes requires an antigenspecific signal through the tcr (yanagi, ; matis, ) , which recognizes viral peptides (townsend et al., (townsend et al., , a townsend and bodmer, ) bound to a groove on mhc class i (mhc i) molecules (bjorkman et al., a,b) . the interaction of the tcr with peptide-mhc i is enhanced by the binding of the cd coreceptor on the t cell to a region of the mhc i molecule away from the peptide-binding groove (gao et al., ) . in addition, optimal cd t lymphocyte priming may require other signals such as costimulation (signal ) (duttagupta et al., ; welten et al., ) . direct signals to cd t cells by proinflammatory soluble cytokines such as type i interferons and interleukin- , known as signal , are also important for efficient cd t cell responses to viruses but their effect seems to be during their early proliferation rather than priming (haring et al., ; curtsinger and mescher, ; kim and harty, ) . mhc i molecules are heterodimers composed of a polymorphic alpha (a) chain with three extracellular domains, one transmembrane domain, one cytosolic domain, and a monomorphic beta- microglobulin chain with a single extracellular domain whose role is to stabilize the a chain. the membrane-proximal a domain of the a chain interacts with the cd coreceptor, while the membrane-distal a and a domains form a polymorphic groove that binds a diverse array of peptides, - amino acid long (bjorkman et al., a,b) , with a sequence motif that is characteristic for each a chain allele and determined by its polymorphic amino acids (rammensee, ; rammensee et al., a,b; grey et al., ) . the tcrs of cd t lymphocytes bind very loosely to most peptide-mhc i-peptide combinations but with much higher affinity to a small subset. this specificity is acquired during the development of the cd t lymphocyte in the thymus. in this process, the variable (v), diversity (d), and joining (j) segments of the tcr b and the v and j segments of the tcr a genes recombine randomly to generate unique complementarity-determining regions that bind with higher strength to particular peptide-mhc i adducts. this gives rise to millions of cd t lymphocyte clones, each with a distinctive peptide-mhc i specificity, that circulate between the blood and secondary lymphoid tissues in search of their cognate peptide-mhc i combinations (gras et al., ; godfrey et al., ) . importantly, most cd t lymphocytes that randomly recognize self peptides are eliminated or rendered tolerant during t cell development. on the other hand, those that recognize nonself peptides, such as those derived from the degradation of viral proteins, become part of the activatable cd t lymphocyte pool (klein et al., ; parello and huseby, ; morris and allen, ). during normal cellular housekeeping, the proteasome and other peptidases in the cytosol progressively degrade mature proteins and defective ribosomal products to their constitutive amino acids. the intermediate peptides generated in this process are very short-lived, if free in the cytosol. however, some of them are rapidly shuttled into the lumen of the endoplasmic reticulum (er) by the transporter associated with antigen presentation. in the er, the peptides can be further trimmed by er aminopeptidase and those with the appropriate motif bind to mhc i molecules that protect them from further degradation. after acquiring their peptide cargo, the mhc i molecules transit to the plasma membrane, where they display the peptides at the cell surface for cd t lymphocyte recognition. in this process, known as 'direct presentation' (dp), mhc i molecules present to cd t lymphocytes a sampling of the cellular proteome, which, in virus-infected cells, includes viral proteins (york and rock, ; cresswell et al., ; rock and shen, ; rock et al., ) (figure a ). of note, dp is the only mechanism of mhc i antigen presentation available to most cells. hence, for most cells, only direct viral infection triggers recognition and killing by antiviral cd t lymphocyte effectors (rock et al., ) . therefore, the quasiuniversal expression of mhc i allows effector cd t lymphocytes to kill almost every type of infected cells. cross-presentation (cp) : viral proteins are phagocytosed (here in cell debris such as apoptotic cells, but could also be in other forms such as viral particles). in the cytosolic pathway ( ), the protein is transferred to the cytosol and then follows a pathway similar to dp ( a). alternatively, tap acquired from er membranes can transport peptides back to the phagosome, where it is loaded to mhc i and transported to the cell surface (( b), never demonstrated for viral infections). in the vacuolar pathway ( ), the phagosome fuses with lysosomes. lysosomal peptidases, mainly cathepsins, degrade the antigen and some of the resulting peptides are loaded into recycling mhc i molecules for transport to the cell surface. cp is active only in bone marrow-derived professional antigen-presenting cells and, in particular, in cd a dendritic cells. cytosolic cp is known to be important for the priming of cd t cells against herpes simplex virus and to be operative for poliovirus, vacv, influenza a virus (iav), and lymphocytic choriomeningitis virus. vacuolar cp has been shown to play a role in cd responses to some peptides of iav. although effector cd þ t cells can recognize and kill every cell displaying their cognate peptide-mhc i combination, the initial activation (priming) of cd t lymphocytes generally requires antigen presentation by bone marrow-derived cells (bmdcs) (lenz et al., ; sigal et al., ; sigal and rock, ) . the importance of bmdcs in the priming of antiviral cd t lymphocyte responses was initially shown in the mouse for vaccinia virus (vacv), poliovirus (pv), influenza a virus (iav), and most antigenic peptides of lymphocytic choriomeningitis virus (lcmv). yet, for one peptide from lcmv, bmdc dependency could not be demonstrated (sigal et al., ; sigal and rock, ; lenz et al., ) . this may indicate that there might be exceptions to the need of professional antigen-presenting cells (papcs) for cd t lymphocyte activation. the bmdc responsible for the priming of cd t cells are called papcs. the ability of papcs to initiate cd t lymphocyte responses relies in their expression of high levels of mhc, costimulatory ligands and cytokines, and the capacity to migrate to the areas of secondary lymphoid organs where cd t lymphocyte responses are primed. traditionally, dendritic cells (dcs), monocytes/macrophages, and b lymphocytes have been considered papcs. however, it has been shown that when recovered from secondary lymphoid tissues of mice infected with herpes simplex (hsv- ), vacv, and iav, the only papc that can prime cd t lymphocytes ex vivo are those dcs that express the cd a molecule (allan et al., ; smith et al., ; belz et al., ) . moreover, mice deficient in the transcription factor batf , which lack cd a dcs, do not generate cd t lymphocyte responses to hsv- or mcmv (nopora et al., ) . thus, there is current consensus that cd a dcs are key papcs during viral infections. however, it remains to be formally demonstrated that these are the only papcs that prime cd t lymphocytes in vivo. in addition to dp, some papcs have the capacity to phagocytose dead or dying cells, processing some of their proteins, and presenting their peptides on their own mhc i molecules. this process is known as cross-presentation (cp) and allows papcs to cross-present viral antigens inside virus-infected cells (shen and rock, ; gutierrez-martinez et al., ; cresswell et al., ) . two major pathways of cp have been defined. in the cytosolic pathway, the exogenous proteins in vacuoles, such as endosomes and phagosomes, gain access to the cytosol, are processed by the proteasome, and are transferred to the er or back to phagosomes to be loaded onto newly synthesized mhc i molecules. alternatively, in the vacuolar pathway, access to the cytosol is not necessary and the peptides are generated in endosomes and phagolysosomes by proteases in the vacuoles (shen and rock, ) (figure b) . the initial demonstration that cytosolic cp can be sufficient to prime an antiviral cd t lymphocyte response in vivo was done with pv in a situation where dp was not possible (sigal et al., ) . however, the physiological role of cp in the normal priming of antiviral cd t lymphocytes remained hotly debated with some arguing that cp is essential and others considering it irrelevant (melief, ; heath et al., ; wilson et al., ; zinkernagel, ; amigorena, ; lizee et al., ) . in the middle ground, it appears that cp is important for some viral infections or viral antigens and not for others. for example, cp by cd a dcs is necessary for an efficient cd t cell response to hsv- (nopora et al., ; wilson et al., ) . also, vacuolar cp is partly responsible for the cd t cell response to iav (shen et al., ) . on the other hand, dp is sufficient for efficient priming of cd t cell responses to wt vacv, even though at least some of its antigens can be crosspresented (larsson et al., ; ramirez and sigal, ; serna et al., ; xu et al., ) . conversely, it has been reported that cp dominates the cd lymphocyte response to the replication-deficient strain of vacv-modified vaccinia ankara (gasteiger et al., ) . however, this finding has been challenged (wong et al., ) . in addition to tcr ligation, optimal cd t lymphocyte responses require costimulation, which is also known as 'signal .' most costimulatory molecules expressed by t cells belong to either the cd or the tumor necrosis factor receptor (tnfr) families. the cd family includes cd , which binds cd and cd on papcs and icos which binds b h on most cells. the tnfr family includes cd , ox , and - bb, which respectively bind cd , ox -l, and - bbl (duttagupta et al., ; welten et al., ) . for the priming of cd t cells, the most important costimulatory molecule is cd , while the others play roles at other stages of the response such as expansion or maintenance (duttagupta et al., ) . in contrast to the cd t cell responses to inert antigens, where absence of cd costimulation results in anergy, the cd t lymphocyte responses to virus in the absence of cd still occur, but their timing and strength may vary according to the specific pathogen. for example, absence of cd and cd results in reduced responses to vesicular stomatitis virus, murine gammaherpesvirus , and iav, but the response to lcmv is normal (duttagupta et al., ) . of note, during vacv infection, the strength of the cd t lymphocyte response in cd -deficient mice is normal, but the kinetics is delayed by one or two days. yet, because vacv is only mildly pathogenic to mice, this delay is inconsequential to the health of the mice. on the other hand, when cd deficient mice are infected with the related ectromelia virus, a poxvirus that is very pathogenic to mice, this delay in the cd response results in the death of the majority of cd deficient mice (fang and sigal, ) . in contrast to cd t cells, cd t cells recognize antigens bound to mhc ii molecules. for cd t cells, the major papcs are also dcs, but b cells that acquire antigen through the bcr also play a very important role. the process of antigen presentation on mhc ii is quite different because the peptide is loaded in vesicular compartments and not in the er. this requires a special mechanism to prevent the binding of peptides to mhc ii in the er (blum et al., ) . in general, it is thought that cd t cells recognize viral antigens acquired by the papcs from the outside. yet, endogenous viral proteins can also be presented on mhc ii (eisenlohr, ) . nkt cells are another type of t cells that recognize antigen as exogenous and endogenous glycolipids bound to the nonclassical mhc i molecule cd d. the mechanisms of antigen loading on cd d and the role of nkt cells in antiviral immunity are less well understood. naïve cd t cells become activated when they recognize peptide antigen bound to mhc i at the surface of bone marrow-derived papcs. in contrast to other cells, papcs produce cytokines and express costimulatory molecules that are important for optimal cd t cell activation. for several viral infections, it has been shown that the key papcs are cd a þ dcs. as most other cells, papcs can directly present antigen synthesized within the cell. yet, papcs, and in particular cd a þ dcs, can also acquire viral antigens from other infected cells and cross-present them with their own mhc i. while dp is more efficient, it is thought that cp is important for cd t cell responses to viruses that do not infect papcs or those that produce immune evasion molecules that target dp. once activated by papcs, effector cd t cells can recognize any infected cell expressing mhc i loaded with its cognate peptide. this results in the killing of the infected cell and/or the production of antiviral cytokines both being important to control or clear viral infections. after the infection subsides, some antiviral cd t cells remain as memory cells, with their numbers being proportional to the strength of the initial response. these memory cd t cells can help control secondary infections, and their induction is a main goal of vaccination. thus, understanding the process of cd t cell activation can help develop better vaccines. see also: cytokines and their receptors: epidermal viral immunity induced by cd alpha þ dendritic cells but not by langerhans cells virus-specific t cells as correlate of (cross-)protective immunity against influenza y in x priming cutting edge: conventional cd alpha þ dendritic cells are generally involved in priming ctl immunity to viruses the foreign antigen binding site and t cell recognition regions of class i histocompatibility antigens structure of the human class i histocompatibility antigen, hla-a pathways of antigen processing t cell-mediated immune response to respiratory coronaviruses mechanisms of mhc class i-restricted antigen processing and cross-presentation inflammatory cytokines as a third signal for t cell activation mhc class i immune evasion in mcmv infection costimulation signals for memory cd þ t cells during viral infections alternative generation of mhc class ii-restricted epitopes: not so exceptional? antibodies and cd þ t cells are complementary and essential for natural resistance to a highly lethal cytopathic virus direct cd costimulation is required for cd þ t cellmediated resistance to an acute viral disease in a natural host molecular coordination of alphabeta t-cell receptors and coreceptors cd and cd in their recognition of peptide-mhc ligands crosspriming of cytotoxic t cells dictates antigen requisites for modified vaccinia virus ankara vector vaccines the fidelity, occasional promiscuity, and versatility of t cell receptor recognition a structural voyage toward an understanding of the mhc-i-restricted immune response: lessons learned and much to be learned class i mhc-peptide interactions: structural requirements and functional implications cross-presentation of cellassociated antigens by mhc class i in dendritic cell subsets inflaming the cd þ t cell response crosspresentation, dendritic cell subsets, and the generation of immunity to cellular antigens different roles for cd þ and cd þ t lymphocytes and macrophage subsets in the control of a generalized virus infection impact of inflammatory cytokines on effector and memory cd þ t cells positive and negative selection of the t cell repertoire: what thymocytes see (and don't see) efficiency of cross presentation of vaccinia virusderived antigens by human dendritic cells cytotoxic t-cell memory without antigen requirements for bone marrow-derived antigen-presenting cells in priming cytotoxic t cell responses to intracellular pathogens control of dendritic cell cross-presentation by the major histocompatibility complex class i cytoplasmic domain the molecular basis of t-cell specificity mini-review: regulation of cytotoxic t lymphocyte responses by dendritic cells: peaceful coexistence of cross-priming and direct priming? how the tcr balances sensitivity and specificity for the recognition of self and pathogens mhc class i cross-presentation by dendritic cells counteracts viral immune evasion indoctrinating t cells to attack pathogens through homeschooling macrophages and dendritic cells use the cytosolic pathway to rapidly cross-present antigen from live, vaccinia-infected cells chemistry of peptides associated with mhc class i and class ii molecules mhc molecules as peptide receptors peptides naturally presented by mhc class i molecules proteases in mhc class i presentation and cross-presentation cross-presentation: underlying mechanisms and role in immune surveillance cutting edge: efficient mhc class i cross-presentation during early vaccinia infection requires the transfer of proteasomal intermediates between antigen donor and presenting cells priming of t cells by exogenous antigen cross-presented on mhc class i molecules important role of cathepsin s in generating peptides for tap-independent mhc class i crosspresentation in vivo cytotoxic t-cell immunity to virusinfected non-haematopoietic cells requires presentation of exogenous antigen bone marrow-derived antigen-presenting cells are required for the generation of cytotoxic t lymphocyte responses to viruses and use transporter associated with antigen presentation (tap)-dependent and -independent pathways of antigen presentation cutting edge: conventional cd alpha þ dendritic cells are preferentially involved in ctl priming after footpad infection with herpes simplex virus- cell-mediated immunity to human cmv infection: a brief overview antigen recognition by class i-restricted t lymphocytes cytotoxic t lymphocytes recognize influenza haemagglutinin that lacks a signal sequence the epitopes of influenza nucleoprotein recognized by cytotoxic t lymphocytes can be defined with short synthetic peptides cytotoxic t cells recognize fragments of the influenza nucleoprotein immunological memory to viral infections the distinct role of t cell costimulation in antiviral immunity systemic activation of dendritic cells by toll-like receptor ligands or malaria infection impairs cross-presentation and antiviral immunity systemic toll-like receptor ligation and selective killing of dendritic cell subsets fail to dissect priming pathways for antivaccinia virus cd (þ) t cells memory cd þ t cells are gatekeepers of the lymph node draining the site of viral infection direct presentation is sufficient for an efficient anti-viral cd þ t cell response t-cell receptor and t-cell-resistant virus variants antigen processing and presentation by the class i major histocompatibility complex on cross-priming of mhc class i-specific ctl: rule or exception? acknowledgments i thank ms holly gillin for assistance in the preparation of the manuscript. the work in the author's laboratory was supported by nih grants r ai , r ai , and r ag . key: cord- -s l yxc authors: ranga, vipin; niemelä, erik; tamirat, mahlet z.; eriksson, john e.; airenne, tomi t.; johnson, mark s. title: immunogenic sars-cov- epitopes: in silico study towards better understanding of covid- disease—paving the way for vaccine development date: - - journal: vaccines (basel) doi: . /vaccines sha: doc_id: cord_uid: s l yxc the emergence of the covid- outbreak at the end of , caused by the novel coronavirus sars-cov- , has, to date, led to over . million infections and nearly , deaths. consequently, there is an urgent need to better understand the molecular factors triggering immune defense against the virus and to develop countermeasures to hinder its spread. using in silico analyses, we showed that human major histocompatibility complex (mhc) class i cell-surface molecules vary in their capacity for binding different sars-cov- -derived epitopes, i.e., short sequences of - amino acids, and pinpointed five specific sars-cov- epitopes that are likely to be presented to cytotoxic t-cells and hence activate immune responses. the identified epitopes, each one of nine amino acids, have high sequence similarity to the equivalent epitopes of sars-cov virus, which are known to elicit an effective t cell response in vitro. moreover, we give a structural explanation for the binding of sars-cov- -epitopes to mhc molecules. our data can help us to better understand the differences in outcomes of covid- patients and may aid the development of vaccines against sars-cov- and possible future outbreaks of novel coronaviruses. the ongoing pandemic outbreak of covid- has resulted in the declaration of a global health emergency around the world on january by the world health organization (who) [ ] . the first reported case was on december from the chinese city of wuhan, from which the virus quickly spread to other countries and territories and, as of july , resulted in at least . million infections and over , deaths [ ] . based on the early epidemiology of the covid- disease statistics, the who estimated that the fatality rate of the novel coronavirus is around %, significantly higher than the mortality rate caused by common human coronaviruses [ ] . there are currently numerous scientific, clinical, and socio-economical efforts aimed at combating the covid- disease and its ramifications around the world [ , ] . peptides are loaded on mhc receptors using a peptide loading complex consisting of tap and several other proteins [ ] . in order to narrow down the specific epitopes that could elicit an effective mhc class-i-mediated t cell response, we predicted linear -mer immunogenic sars-cov- peptides and their prominent interacting hla allotypes using the immune epitope database and analysis resource (iedb) and netctl . web servers. the identified peptides were then analyzed in conjunction with the available experimental data for sars-cov-derived linear t-cell epitopes. the three-dimensional structural models of selected ternary complexes of sars-cov- epitope-hla allotype-t cell receptor were created to assess interactions at the structural level. our results can at least partially explain individual differences in the covid- severity and could potentially be used for vaccine development against sars-cov- . all protein sequences encoded by the most up-to-date sars-cov- genomic sequence (refseq: nc_ . ) were retrieved from ncbi refseq database [ ] on march (full accession identifiers in table ). the iedb (http://tools.iedb.org/mhci/) [ ] and netctl . (http://www.cbs.dtu.dk/services/ netctl/) [ ] web servers were used with default parameters to predict sars-cov- epitopes and their binding affinities (expressed as ic ) to different hla allotypes. the iedb server sorts the predicted mhc-i-binding viral epitopes based on the percentile score, which is calculated by comparing the predicted binding affinities of sars-cov- peptides and affinities calculated for a large set of peptides, randomly selected from the swissprot database [ ] ; the iedb server integrates an artificial neural network (ann), stabilized matrix method (smm) and combinatorial library (comblib). the iedb method is highly accurate in classifying mhc class i epitopes, having an auc for the roc curve greater than . [ ] . the netctl . server integrates prediction of proteasomal cleavage, tap transport and peptide-binding to mhc-i supertypes (see table s a for a list of hla-a and hla-b allotypes that belong to these supertypes, with data extracted from the published scientific literature [ , ] ). the netctl . method allows for the identification of class i mhc epitopes with a sensitivity of . and specificity of . based on the default filtering threshold score of . [ ] . since the netctl . and iedb servers use different multistep approaches to predict the binding of sars-cov- peptides to hlas, we used both servers. mhc-i allotypes are known to bind epitopes with lengths of to amino acids. the optimal epitope length was determined by potting the ic values of the predicted (iedb) top percentile epitopes against -to -mers. moreover, the immunogenicity of the top percentile epitopes were predicted using the mhc-i immunogenicity server of iedb (http://tools.iedb.org/immunogenicity/) [ ] . the epitopes with an immunogenicity score greater than . were considered for comparison with epitopes predicted using the netctl . server. in order to identify experimental epitopes matching the predicted sars-cov- epitopes, the data of experimentally known sars-cov epitopes and their interacting mhc allotypes (validated using t-cell assays and mhc ligand assays) were downloaded from the iedb database on april (https://www.iedb.org/database_export_v .php) [ ] . the characteristics of each "match" (protein name, sequence, mapped start-end, mhc-i allotype, etc.) were tabulated and are presented in table s . the grand average of hydropathicity index (gravy) was calculated using the kyte-doolittle hydropathy index scale [ ] . tmhmm . was used to search potential transmembrane helices [ ] . the jpred web server was used to predict secondary structures in non-transmembrane proteins [ ] . the half-lives of the predicted emhc-i complexes was predicted using the netmhcstabpan . web server [ ] . data were plotted using graphpad prism (graphpad software, version . . ; https://www.graphpad.com). the crystal structures of an influenza a virus epitope in complex with the hla-a* : allotype (pdb id: tez, chain a and c; . Å) [ ] and hepatitis b core antigen with hla-a* : (pdb id: oxr, chain a and c; . Å) [ ] were downloaded from the protein data bank (pdb) [ ] (see table s b for the pdb codes of mhc-i allotype structures indicated in table s a ). for docking to hla allotypes, the rosetta flexpepdock web server [ ] was used, after the -mer influenza a epitope was mutated to match selected sars-cov- epitopes using pymol (the pymol molecular graphics system, schrödinger, llc). for ternary complex analysis, the t-cell receptor (tcr)-hla-influenza a epitope complex (pdb id: tez) was used as a template. coordinates of the tcr α and β chains (pdb id: tez; chain i and j, respectively) and the docked epitope-hla-a* : complex were saved using pymol, and interacting residues visually inspected at the interface. molecular dynamics (md) simulations followed the general protocol detailed in [ ] . briefly, the ternary composite structure of hla-a* : in complex with the sars-cov- s protein epitope fiagliaiv and tcr was used as a starting structure to perform molecular dynamics simulation. the structure was initially prepared using maestro (schrödinger release - : maestro, version, schrödinger, llc) by adding hydrogen atoms, determining the protonation states of ionizable side-chains and energy minimizing the structure to remove bad contacts. three independent simulations were carried out using the amber program (version ) [ ] and the ff sb protein forcefield [ ] . the ternary structure was solvated with tip p water molecules [ ] in an octahedral box, keeping a Å distance between solute atoms and the surface of the box. the simulation system was neutralized by adding na + ions, with additional na + /cl − ions incorporated to bring the system salt concentration to . m. the system was then energy-minimized for cycles using the steepest descent and conjugate gradient methods. the minimization was carried out in six-stages, where a restraint on solute atoms was gradually lowered from to kcal mol − Å − . subsequently, the system was heated to k during ps with a kcal mol − Å − restraint on solute atoms. next, equilibration was performed for ns in four steps by systematically reducing the restraint force to kcal mol − Å − . finally, a restraint-free ns production simulation was carried out at constant temperature ( k) and pressure ( bar). coordinates were saved every ps and the sampled conformations were analyzed using vmd [ ] , cpptraj [ ] and chimera [ ] programs. root-mean-square fluctuation (rmsf) calculation was computed using the cα atoms of the initial structure as a reference. hydrogen bonds were defined with a bond length ≤ . Å and a bond angle ≥ • . in order to estimate the potential antiviral cytotoxic t-cell response linked to specific hla allotypes, we predicted the binding affinity of all possible linear -to -mer peptides derived from the proteins (table ) of the sars-cov- proteome (n = , n = , n = and n = ) to hla-a and hla-b supertypes using the iedb web server [ ] . the hla-c supertype-an extremely good ligand for killer-cell immunoglobulin-like receptor (kir) on natural killer (nk) cells-was not selected for this analysis because it is known to be less effective in presenting antigens to cytotoxic t-cells than either hla-a or hla-b [ ] . the class i mhc-epitope (emhc-i) complexes were classified into three different groups based on the predicted epitope-to-mhc binding affinity scores [ ] : strong binders (ic ≤ nm), weak binders ( nm < ic ≤ nm) and non-binders (ic > nm). out of the highest scoring sars-cov- epitopes (top percentile), the -and -mers had, on average, a higher binding affinity to mhc class i supertypes than either the -or -mer epitopes ( figure a ). moreover, there were % more -mer peptides ( ) predicted to bind to class i mhc receptors (ic ≤ nm) in comparison with -mer peptides ( ) ( figure b) . consequently, the top one percentile -mer peptides were selected for further analysis. the predicted (iedb) sars-cov- -mer epitopes (top one percentile) for different hla allotypes (table s a) with an immunogenicity score ≥ . were compared to those predicted using the netctl . server [ ] . the mhc class i epitopes identified from this consensus/combined approach were classified using the binding affinity values from iedb prediction method as strong mhc binders (table s ) , weak binders (table s ) or non-binders (table s ). based on this analysis, many peptides derived from nonstructural proteins (nsp), surface glycoproteins (s) and membrane proteins (m) of sars-cov- are likely to be presented by mhc class i receptors ( figure c , table s ), and hence have a high potential to activate an immune response or the destruction of infected host cells, with many epitopes being derived from the s, e, c-like proteinase ( clpro) and -to- exonuclease ( exo) proteins. in order to predict the most potent epitopes, including those without experimental data, we screened in silico the proteins of sars-cov- . we ranked the epitopes based on predicted binding to mhc-i molecules (affinity values; iedb) and "combined score" (netctl . ), predicting the potency of an epitope to be presented by mhc-i (tables s and s ). the top-ranked, common epitopes from both prediction methods (table ) were selected for further analysis; all five of these predicted epitopes are unique to sars-cov- and not identical in sars-cov. this comparison, we identified common epitopes of sars-cov/sars-cov- (table s ) and hla allotypes hla-a* : and hla-a* : molecules as the top antigen presenters; both allotypes having strong binding affinities (ic ≤ nm) to six peptide epitopes (table ) : residues fiagliaiv from the s protein, vllflafvv and flafvvfll to the e protein, vlawlyaav to clpro, llsagifga to nsp and vlwahgfel to exo of sars-cov- . in order to obtain experimental proof that the mhc-i-binding sars-cov- epitopes predicted in this study are presented by the mhc class i antigen processing pathway in vivo, we compared the in-silico-identified -mer epitopes (n = , table s ) to the equivalent, experimentally identified epitopes of sars-cov strains (n = ; mhc ligand assays data from the iedb database) [ ] . in this comparison, we identified common epitopes of sars-cov/sars-cov- (table s ) and hla allotypes hla-a* : and hla-a* : molecules as the top antigen presenters; both allotypes having strong binding affinities (ic ≤ nm) to six peptide epitopes (table ) : residues fiagliaiv from the s protein, vllflafvv and flafvvfll to the e protein, vlawlyaav to clpro, llsagifga to nsp and vlwahgfel to exo of sars-cov- . table . sars-cov- -derived hla-a* supertype-binding epitopes that are identical to the epitopes of sars-cov strains experimentally known to activate cytotoxic t-cells. to examine the evolutionary conservation or "sequence stability" of the six common epitopes of sars-cov/sars-cov- binding strongly to hla-a* : and hla-a* : , we performed a blastp (ncbi) search against the non-redundant database of sars-cov- [ ] . only one epitope- llsagifga from nsp -was found to have heterogeneity in its sequence (table ) , whereas the other five epitopes were fully conserved, i.e., not yet having changed during the evolution of sars-cov- , suggesting an important role for these peptide sequences for the virus and, at the same time, making these peptides top-candidate antigens for activating the cytotoxic t-cell response against sars-cov- itself. the five conserved and experimentally proven epitopes we selected for further analyses. since mhc class i molecules must retain bound epitopes long enough at the cell surface to successfully induce t-cell-specific immune responses [ ] , we estimated the half-life in hours for each of the experimentally identified epitope-hla complexes (table ) using the netmhcstabpan . web server [ ] . this analysis revealed that the hla-a* : and hla-a* : allotypes have longer predicted half-lives than hla-a* : when in complex with the immunogenic epitopes (table ): all five hla-a* : -epitope complexes and three out of five hla-a* : -epitope complexes had a predicted half-life longer than h, whereas the single hla-a* : -epitope complex had the lowest half-life of less than one hour. table . predicted half-lives of complexes of the conserved sars-cov- -derived most immunogenic experimentally identified epitopes and hla-a* allotypes shown in table . secondary structures, localization within sars-cov- and gravy (grand average of hydropathicity index) scores of the epitopes. allotypes in order to examine the distribution of the hla-a* -antigen complex half-lives in general, we analyzed more than experimentally known complexes of hla-a* supertypes (see table s a for a list of allotypes) bound to bacterial-and viral-pathogen-derived epitopes, which were extracted from the iedb database [ ] . this revealed that immunogenic (ic ≤ nm) hla-a* : -epitope complexes with predicted half-lives of more than three hours were almost double in number in comparison to the hla-a* : -epitope complexes, whereas no immunogenic hla-a* : -epitope complexes were found that would have a similar half-life ( figure s a ). interestingly, the amino acid sequences of the α chains of hla-a* : and hla-a* : are . % identical; the only difference in the sequences-a phenylalanine to tyrosine substitution-is located at the epitope-binding site and is the likely reason for the difference in the epitope binding affinities and half-lives. out of the five most stable epitopes (table ) , three- fiagliaiv (s protein), vllflafvv (e protein) and flafvvfll (e protein)-were derived from transmembrane proteins and were, as expected, more hydrophobic (gravy score > ) than the vlawlyaav ( clpro) and vlwahgfel ( exo) epitopes originating from intravirion proteins ( table ). the epitopes derived from the s and e proteins respectively map to transmembrane helical segments wyiwlgfiagliaivmvtimlcc and livnsvllflafvvfllvtlail that, based on analysis using the tmhmm . web server [ ] , are bitopic in nature, i.e., the predicted transmembrane helices span the lipid bilayer only once. in fact, our predicted epitopes share features, such as being membrane associated, with the well-studied and clinically important epitopes in hiv [ ] and tuberculosis [ ] . this supports the idea that the transmembrane helical epitopes of sars-cov- could potentially stimulate cytotoxic t-cell-mediated immune responses. in order to assess whether hydrophobic residues are enriched in the immunogenic epitopes, we compared the gravy score distribution of the immunogenic (n = , ic ≤ nm) and non-immunogenic (n = ; ic > nm) hla-a* -bound epitopes of bacterial and viral pathogens (retrieved from the iedb database) ( figure s b ). we found that, for epitopes with a gravy score greater than one (having at least seven non-charged residues in a -mer epitope), the immunogenic epitopes were more enriched in hydrophobic residues ( %) in comparison to the non-immunogenic epitopes ( %). thus, our analysis suggests that hla-a* supertype molecules prefer binding to hydrophobic epitopes (ic ≤ nm), and this agrees with published results [ ] . moreover, we obtained similar results from our in-silico-identified sars-cov- -derived novel epitope-mhc-i complexes ( table ) : the most potent identified epitopes had long half-lives and were derived from either hydrophobic, transmembrane regions of sars-cov- or from intravirion proteins that were also found to be mutated among sars-cov- sequences (table ) . table . predicted half-lives of the novel sars-cov- -derived, most immunogenic in-silico-identified epitopes in complex with the allotypes shown in table . secondary structures, localization within sars-cov- , gravy scores and known mutations in the epitopes. in order to compare the interaction patterns adopted by the predicted top five immunogenic epitopes (table ) of mhc-i molecules, we docked the epitopes to the cleft between the α and α helices of hla-a* : (pdb id: tez, chain a) and hla-a* : (pdb id: oxr, chain a). this docking analysis agrees with our other prediction data and suggests that the immunogenic epitopes bind to both the hla-a* : and hla-a* : allotypes by adopting a similar backbone conformation, as has been observed for the canonical epitope gilgfvftl of the influenza a virus (pdb id: tez, chain c) ( figure s c,d) . in more detail, we observed that the residues at position (pos) , , and are fully buried within the antigen-binding cleft of hlas and act as anchoring residues, providing steric constraints to the nand c-terminus of the epitopes (figure a,b) . comparison of the root-mean-square deviation (rmsd) of the superposed cα atoms of the epitopes revealed a maximum deviation for the vlwahgfel epitope ( exo), possibly due to the bulky, aromatic side chain of w buried at pos and two charged residues: h at pos and e at pos (table ) . interactions between the partially solvent-exposed hydrophobic residues at pos -pos of the docked epitope fiagliaiv (s protein) and the solvent-exposed residues a , k , v , t , k , v and q of the α and α helices of hla-a* : , complement what is seen in the x-ray crystal structure of the influenza a virus epitope-hla-a* : complex (pdb id: tez, chain a and c), and suggest that the sars-cov- epitope-hla complex interacts with tcr ( figure c ). and cdr β of the tcr-β chain, recognize the hla-a* : -sars-cov- fiagliaiv epitope ( figure d ); the cooperative interacting nature of residues in these loops could provide specificity towards class i mhc molecules. moreover, the residues l and w within the cdr β loop and i within the cdr α loop could be important for antigen-hla complex recognition due to their likely direct contacts with residues l , i and i of the fiagliaiv epitope and residues a , v and q of hla-a* : ( figure e ). to understand the molecular basis of tcr binding to the sars-cov- antigen-loaded mhc-i cell surface molecules, hla-a* : , in complex with the s protein epitope fiagliaiv , was superimposed with the hla-a* : allotype of the influenza a virus ternary complex structure (pdb id: tez, chain a), and the atomic coordinates of tcr (tcr-α and tcr-β chain; pdb id: tez, chain i and j, respectively) were then utilized for visual analysis of binding. this visualization suggests that the loops cdr α, cdr α and cdr α of the tcr-α chain, and loops cdr β, cdr β and cdr β of the tcr-β chain, recognize the hla-a* : -sars-cov- fiagliaiv epitope ( figure d ); the cooperative interacting nature of residues in these loops could provide specificity towards class i mhc molecules. moreover, the residues l and w within the cdr β loop and i within the cdr α loop could be important for antigen-hla complex recognition due to their likely direct contacts with residues l , i and i of the fiagliaiv epitope and residues a , v and q of hla-a* : ( figure e) . to assess the conformational and intermolecular interaction dynamics of the hla-a* : - fiagliaiv s protein epitope-tcr complex, a ns simulation was carried out on the ternary structure in triplicate. the global conformational dynamics was assessed by computing the rmsf over the cα atoms ( figure a ), which shows a stable tcr and epitope, with an average rmsf of . ± . Å and . ± . Å, respectively. the α and α domains of hla-a* : were also stable ( . ± . Å, . ± . Å), unlike the α domain, which exhibits a higher fluctuation ( . ± . Å) likely arising from the flexibility of the loop between domains α and α and the lack of stabilizing β -microglobulin. figure b compares the complex at , and ns during the simulation. these observations were consistent among the three independent simulations ( figure s ). the intermolecular interactions taking place in the complex structure were also examined by visually inspecting the trajectory and calculating the number of hydrogen bonds formed during the simulation (table s ) . for example, in simulation , the highest number of hydrogen bond interactions recorded were between hla-a* : and backbone atoms of the epitope peptide, with t -v and w -i interactions topping the list, as they were respectively formed during % and % of the simulation time. on the other hand, hydrogen bond interactions between the tcr and the epitope were almost exclusively from interactions between w (β chain)-i ( %) and q (α chain)-i ( %). the hla-a* : -tcr hydrogen-bonding interactions were mostly between residues from the cdr α, cdr α, cdr α, cdr β and cdr β loops of the tcr and the α and α helices of hla-a* : . visual analysis also revealed that hydrophobic interactions are integral to the interaction the epitope is making, both with hla-a* : and the tcr, as the hydrophobic epitope peptide was tightly enclosed by hydrophobic clusters from the proteins throughout the simulation, reflecting observations made on the basis of the original docked complex. thus, our structural analysis suggests that the s protein epitope fiagliaiv of sars-cov- (and sars-cov), and the other epitopes listed in table , could form strong complexes with hla-a* : and hla-a* : allotypes, and that the epitope-hla complexes can also be recognized by tcrs to initiate cytotoxic t-cell-mediated immune responses. the intermolecular interactions taking place in the complex structure were also examined by visually inspecting the trajectory and calculating the number of hydrogen bonds formed during the simulation (table s ) . for example, in simulation , the highest number of hydrogen bond interactions recorded were between hla-a* : and backbone atoms of the epitope peptide, with t -v and w -i interactions topping the list, as they were respectively formed during % and % of the simulation time. on the other hand, hydrogen bond interactions between the tcr and the epitope were almost exclusively from interactions between w (β chain)-i ( %) and q (α chain)-i ( %). the hla-a* : -tcr hydrogen-bonding interactions were mostly between residues from the cdr α, cdr α, cdr α, cdr β and cdr β loops of the tcr and the α similar to the docking results obtained from the experimentally known epitopes with the hla-a* supertype, we observed that residues at pos - and pos (a , e , w and l ) of the novel epitope aewflayil -derived from a transmembrane segment of the nsp protein of sars-cov- -are fully buried within the antigen-binding cleft of hla-b* : (pdb id: iex, chain a). moreover, the location of the partially solvent-exposed residues at pos -pos (l , a , y and i ) of the docked epitope along with solvent-exposed residues r , t , t , e , q , y , e and w of the α and α helices suggest an interaction of the aewflayil -hla-b* : complex with tcr ( figure f ). in order to tackle the current covid- pandemic, it is critically important to better understand the underlying mechanism that gives rise to the individual differences in disease severity as well as to aid the vaccine development against the causative virus, sars-cov- . effective vaccinations are needed to eradicate the virus from populations all over the world and knowledge regarding the immunological response should have a significant impact on understanding disease progression. however, due to the limited experimental and clinical data currently available on the specific immune responses against sars-cov- , the development of an effective vaccine against covid- will be a challenge. this study sought to better understand the individual differences in the viral antigen presentation pathway and to aid the development of vaccines against covid- by predicting in silico sars-cov- immunogenic epitopes. based solely on in silico predictions, the most potent sars-cov- -derived mhc class i binding epitopes are aewflayil and lvaewflay in terms of binding affinity, hydrophobicity and stability. however, for these "in silico epitopes", only limited experimental data are available to correlate with. therefore, in this study we mainly focused on potential sars-cov- epitopes that were conserved with sars-cov epitopes experimentally known to activate cytotoxic t-cells, and hence could be used in vaccine development. the s glycoprotein-derived epitope fiagliaiv binds to the hla-a* : and hla-a* : allotypes with experimental ic values lower than nm (table ). our docking analysis supports these predictions, i.e., that epitope fiagliaiv could bind tightly to these allotypes and that ternary complexes with tcrs could form. moreover, recent data demonstrate that patients with a severe form of covid- have a stronger t-cell response after stimulation with the sars-cov- s-protein peptide pool compared to those with a mild manifestation of the disease [ , ] . the disease progression of covid- is also associated with a higher magnitude of inflammatory cytokine-producing cd + t cells [ ] . whether these immune responses are due to strong binding of sars-cov- epitopes, including fiagliaiv , to certain hla allotypes, such as hla-a* : and hla-a* : , or whether tight virus epitope-hla interaction in general can actually be harmful for covid- patients by causing, e.g., an immunological over-reaction, is not yet fully understood [ , ] . furthermore, both cd + and cd + t-cells have been shown to be stimulated by overlapping peptides ( -mers overlapping by amino acids) of the entire s glycoprotein sequence [ ] . does this mean that the s protein might function as a double-edged sword-that is, being crucial for viral entry into the host cell, but also important for overstimulating the immune responses, causing severe inflammation that aids the spread of the virus to surrounding cells? this still needs to be answered. the latter "sword" is known to be avoided at least by hiv, which has a sophisticated mechanism to limit the infection rate in order to better avoid immune surveillance [ , ] . a recent in vivo study shows that epitopes derived from the c-terminus of the s protein had a significantly stronger cd + t helper cell response in healthy donors in comparison to those infected with sars-cov- [ ] . the cd + t cells' cross-reactivity to the s protein might represent the key for understanding the different disease manifestations of covid- , particularly in the asymptomatic infections in children and adolescents. our predicted epitope fiagliaiv overlaps with the c-terminal sequence of the s protein containing the s subunit, which is internalized after tmprss cleavage. therefore, this particular amino acid sequence may also be important for inducing a protective immunological response towards immunity in covid- . however, canonically t helper cells recognize mhc class ii molecules, whereas our prediction is based on the mhc class i molecules' ability to present viral antigens for a possible cytotoxic t-cell response [ ] . indeed, we found four -mer epitopes that include the intact -mer fiagliaiv epitope sequence and showed binding affinities < nm with drb * : allotype of mhc class ii (http://tools.iedb.org/mhcii/) [ ] , an allotype that is common in caucasoid and oriental ethnic backgrounds (https://www.ebi.ac.uk/ipd/imgt/hla/ethnicity.html) [ ] . nevertheless, there are a few reports regarding mhc class i-reactive cd + t helper cells, including the study where co-cultures of highly purified cd + t cells, together with a stimulatory mhc class ii-negative cell line transfected with mhc class i molecules, were used to show the direct interaction of t helper cells with mhc class i molecules [ ] . however, whether or not the fiagliaiv epitope is presented to both cytotoxic and helper t cells needs to be experimentally verified; a recent study that appeared while the current work was under review suggested that cross-reactivity could affect disease progression in covid- [ ] . furthermore, the latest experimental reports suggest that the s glycoprotein of sars-cov- is both o-and n-glycosylated, especially on the rdb domain, which could mask immunogenic epitopes and may play an important role in sars-cov- immune evasion [ ] [ ] [ ] . fortunately, the predicted epitope fiagliaiv is part of a transmembrane helix, and consequently is neither o-nor n-glycosylated; the closest glycosylation site is at pos , rendering this particular amino acid sequence potentially suitable for vaccine development. intriguingly, the sars-cov- -derived, membrane glycoprotein epitope hlriaghhl , which has low binding affinity (ic = . nm; table s ) towards hla-b* : , is % identical in sequence with the intravirion sars-cov epitope hlrmaghsl also from a membrane glycoprotein and shown to elicit a strong t-cell response in patients with the hla-b* : allotype [ ] . furthermore, hla-b* : has been shown to have a protective role against the severe forms of sars-cov [ ] . this inspired us to do a separate in silico binding affinity analysis of the sars-cov hlrmaghsl epitope to the hla-b* : allotype: a high binding affinity (ic = . nm) of the hlrmaghsl epitope with the hla-b* : was predicted and agrees with the reported [ ] protective immune response against sars-cov. thus, there seems to be a difference between the highly conserved sars-cov-derived hlrmaghsl and sars-cov- -derived hlriaghhl epitopes in their potency towards the hla-b* : allotype. moreover, the predicted low binding affinity of the sars-cov- epitopes with the hla-b* : allotype (table s ) might not be sufficient to induce immune responses, thus rendering the potential of this specific epitope unfavorable for vaccination. based on our predictions, hla-b* : is one of the worst allotypes for presenting sars-cov- -derived epitopes with an average binding score (ic ) of nm for the four epitopes predicted to bind (table s ). this is in line with similar results from the predictions for sars-cov- and previous clinical data from sars-cov patients, demonstrating that this particular hla allotype gives susceptibility to a more severe form of the viral disease [ ] . furthermore, our prediction shows that hla-b* : is not binding to either the s or m protein-derived peptides, strengthening the reported general view that hla-b* : is not optimal for eliciting an immune response in covid- patients [ ] . taken together, identification of the predicted most immunogenic epitopes of sars-cov- could aid vaccine development. since the sequences of the "top" epitopes of sars-cov- and sars-cov are highly conserved and the sars epitopes are known to elicit an immunological response based on a previous study [ ] , a common vaccine protecting against both viruses and potential future strains is possible. moreover, the selected experimentally known epitopes have been shown not to evoke an unwanted t cell cross-reactivity in vitro, further validating the potential use of the conserved sars-cov/cov- peptides in vaccine development without disrupting self-tolerance [ ] . however, there are many hurdles that need to be addressed; for example, due to the hydrophobic nature of these epitopes, they probably would need to be loaded in a liposome or nanocarrier for efficient vaccine delivery [ ] . moreover, as these conserved epitopes are presented by only a few hla allotypes, i.e., a* : , a* : , a* : , which are common in american indian, caucasoid, hispanic and oriental ethnic backgrounds [ ] , the estimated world population coverage using these epitopes would only be around . % (http://tools.iedb.org/population/) [ ] . therefore, developing a globally effective sars-cov- vaccine will probably require a pool of both novel and conserved epitopes, making a globally effective sars-cov- vaccine development a challenging task. in the present study, we identified sars-cov- epitopes that were predicted to be presented by the mhc class i antigen processing pathway to the cytotoxic t cells. we report five purely in-silico-predicted most potent epitopes unique to sars-cov- and five potent sars-cov- epitope peptides identical to and experimentally determined for sars-cov. the novel sars-cov- epitopes were analyzed for their interaction with hla allotypes using the iedb and netctl . web servers and three-dimensional structural models of selected, molecular dynamics simulation proven ternary complexes of sars-cov- phla-tcrs were created to assess interactions at structural level. hla-a* : and hla-a* : were found to have the greatest potential to present the selected epitopes, which are hydrophobic in nature and originated mainly from the transmembrane region of sars-cov- proteins. our results could assist in the understanding of the individual and varying disease progression of covid- , as well as paving the way towards vaccine development against sars-cov- . supplementary materials: the following material are available online at http://www.mdpi.com/ - x/ / / / s , figure s : (a): distribution of the predicted half-lives (log scale) of predicted (iedb) -mer epitope-hla-a* supertype complexes. the complexes are classified as immunogenic (ic ≤ nm) and non-immunogenic (ic > nm) based on the epitope binding affinity with the mhc molecule, (b): distribution of the gravy scores of immunogenic (ic ≤ nm) and non-immunogenic (ic > nm) epitopes, figure s c and s d: comparison of the backbone conformation of the five epitopes docked into the cleft of hla-a* : (c) and hla-a* : (d) molecules against the canonical epitope gilgfvftl of the influenza a virus (pdb id: tez, chain c), figure s : structural dynamics of the hla-a* : - fiagliaiv s protein epitope-tcr complex during three replicate ns simulations. (a) cα atom rmsf of the ternary complex. (b) average cα atom rmsf. table s a : mhc class i allotypes association with supertypes described in published scientific literature [ , ] . superscript "x" indicates availability of x-ray crystal structure in protein data bank (pdb) [ ] , table s b : pdb codes for the x-ray crystal structures of mhc class i allotypes, table s : sars-cov- -derived mhc class i binding epitopes identified with iedb and netctl . prediction methods as having strong binding affinity (ic ≤ nm) with mhc molecules, table s : sars-cov- -derived mhc class i binding epitopes identified with iedb and netctl . prediction methods as having weak binding affinity ( nm < ic ≤ nm) with mhc molecules, table s : sars-cov- -derived mhc class i binding epitopes identified with iedb and netctl . prediction methods as having non-binding affinity (ic > nm) with mhc molecules, table s : sars-cov- -derived epitopes (immunogenicity score ≥ . ) and their prominent interacting hla allotypes identified with iedb and netctl . prediction methods, table s : most potent sars-cov- -derived epitopes having immunogenicity score ≥ . and predicted binding affinity (ic ) ≤ nm with their prominent interacting hla allotypes identified with iedb prediction method, table s : most potent sars-cov- -derived mhc class i binding epitopes identified with netctl . (combined score ≥ ) prediction method, table s : most potent sars-cov- -derived mhc class i binding epitopes that are identical to the experimentally known epitopes (mhc ligand assays data from the iedb database) of sars-cov strains to activate cytotoxic t-cells. predicted half-lives of the epitope-mhc-i complexes, gravy scores and mutations in the epitopes are shown. table s world health organization declares global emergency: a review of the novel coronavirus (covid- an interactive web-based dashboard to track covid- in real time preliminary identification of potential vaccine targets for the covid- coronavirus (sars-cov- ) based on sars-cov immunological studies data sharing and outbreaks: best practice exemplified potential inhibitors against -ncov coronavirus m protease from clinically approved medicines a review of coronavirus disease- (covid- ) acute respiratory distress syndrome advances in diagnosis and treatment an outbreak of severe kawasaki-like disease at the italian epicentre of the sars-cov- epidemic: an observational cohort study middle east respiratory syndrome coronavirus (mers-cov) in oman: current situation and going forward emerging coronaviruses: genome structure, replication, and pathogenesis structural genomics of sars-cov- indicates evolutionary conserved functional regions of viral proteins universal three-dimensional construction of eleven amino acids near the catalytic nucleophile and base in the superfamily of (chymo)trypsin-like serine fold proteases sars-cov- cell entry depends on ace and tmprss and is blocked by a clinically proven protease inhibitor a pneumonia outbreak associated with a new coronavirus of probable bat origin structural basis of receptor recognition by sars-cov- functional assessment of cell entry and receptor usage for sars-cov- and other lineage b betacoronaviruses human leukocyte antigen (hla) and immune regulation: how do classical and non-classical hla alleles modulate immune response to human immunodeficiency virus and hepatitis c virus infections? front association of hla class i with severe acute respiratory syndrome coronavirus infection memory t cell responses targeting the sars coronavirus persist up to years post-infection hla-a* t-cell epitopes in severe acute respiratory syndrome (sars) coronavirus nucleocapsid and spike proteins efficient induction of cytotoxic t lymphocytes specific for severe acute respiratory syndrome (sars)-associated coronavirus by immunization with surface-linked liposomal peptides derived from a non-structural polyprotein a presence of sars-cov- -reactive t cells in covid- patients and healthy donors pathways of antigen processing ncbi viral genomes resource the immune epitope database (iedb): update large-scale validation of methods for cytotoxic t-lymphocyte epitope prediction the immune epitope database and analysis resource in epitope discovery and synthetic vaccine design definition of supertypes for hla molecules using clustering of specificity matrices hla class i supertypes: a revised and updated classification a simple method for displaying the hydropathic character of a protein predicting transmembrane protein topology with a hidden markov model: application to complete genomes jpred : a protein secondary structure prediction server pan-specific prediction of peptide-mhc class i complex stability, a correlate of t cell immunogenicity structural basis for clonal diversity of the human t-cell response to a dominant influenza virus epitope structural insights into the binding of hepatitis b virus core peptide to hla-a alleles: towards designing better vaccines the rcsb protein data bank: new resources for research and education rosetta flexpepdock web server-high resolution modeling of peptide-protein interactions deciphering the structural effects of activating egfr somatic mutations with molecular dynamics simulation improving the accuracy of protein side chain and backbone parameters from ff sb comparison of simple potential functions for simulating liquid water visual molecular dynamics software for processing and analysis of molecular dynamics trajectory data ucsf chimera-a visualization system for exploratory research and analysis friends or foes? retrovirology systematically benchmarking peptide-mhc binding predictors: from synthetic to naturally processed epitopes ncbi blast: a better web interface peptide-mhc class i stability is a better predictor than peptide affinity of ctl immunogenicity primary human immunodeficiency virus type -specific cd + t-cell responses induced by myeloid dendritic cells identification of antigens presented by mhc for vaccines against tuberculosis tcr contact residue hydrophobicity is a hallmark of immunogenic cd + t cell epitopes a sequence homology and bioinformatic approach can predict candidate targets for immune responses to sars-cov- targets of t cell responses to sars-cov- coronavirus in humans with covid- disease and unexposed individuals phenotype of sars-cov- -specific t-cells in covid- patients with acute respiratory distress syndrome key words natural killer cells in hiv- infection: a double-edged sword structural basis of transmembrane coupling of the hiv- envelope glycoprotein human leukocyte antigen susceptibility map for sars-cov- major histocompatibility complex class i-restricted alloreactive cd + t cells deducing the n-and o-glycosylation profile of the spike protein of novel coronavirus sars-cov- site-specific analysis of the sars-cov- glycan shield site-specific n-glycosylation characterization of recombinant immunogenetics in sars: a casecontrol study hla studies in the context of coronavirus outbreaks a epitopes described in-immune epitope database (iedb) quantitating t cell cross-reactivity for unrelated peptide antigens harnessing self-assembled peptide nanoparticles in epitope vaccine design conflicts of interest: e.n. has filed a patent application regarding a method for preventing the spreading and lowering the infection rate of pathogens, finnish patent application number , filing date . . . all other authors declare that they have no competing interests. the funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.vaccines , , key: cord- -jdp kgg authors: deschler, barbara; waller, cornelius; engelhardt, monika; müller, antonia; luebbert, michael; finke, jürgen; bertz, hartmut; illerhaus, gerald; kaskel, anna-katharina; mackensen, a.; veelken, hendrik; rosenthal, f. m.; müller, claudia i.; scheele, jürgen; martens, uwe title: particular treatment procedures date: journal: concise manual of hematology and oncology doi: . / - - - - _ sha: doc_id: cord_uid: jdp kgg nan in preclinical and clinical studies attempts have been made to grow ("expand") cultured stem cells (ex vivo) in order to provide new treatment options. while it is possible to increase cell numbers, relevant stem cell expansion has not been achieved yet. in addition, clinical studies did not demonstrate a significant cost / benefit advantage despite the option of tumor cell depletion. the following potential areas of use are being evaluated in clinical studies: • removal of contaminated tumor cells from autologous stem cell grafts • gene transfer into repopulating stem cells to correct hereditary enzyme deficiencies or for gene marking analyses • expansion of stem cells and partially differentiated progenitor cells from a single stem cell harvest for repeated clinical use in sequential therapy or tandem transplantation • expansion of lineage-determined progenitor cells (e.g., myeloid or megakaryopoietic postprogenitor cells) to accelerate hematopoietic regeneration or for differentiation into antigen-presenting dendritic cells • expansion of bone marrow repopulating stem and progenitor cells from umbilical cord blood samples for transplantation in adult patients • cell support in allogeneic transplantation • de-/redifferentiation into other organ-type cells ("stem cell plasticity") • preparation laboratory (equipped according to national and international guidelines) which allows production of cultivated and gene-transfected cells according to standards • central filing of all protocols of studies concerned with somatic cell and gene therapy • suspension culture using unseparated or cd + -separated stem cells (advantage of reduced culture volume). • addition of recombinant growth factors. optimal cytokine combinations are currently being tested. stem cell factor (scf), flt ligand, thrombopoietin, and il- seem to preserve part hematological treatment aimed to accelerate bone marrow and blood reconstitution by use of autologous peripheral blood hematopoetic stem cells (pbsc) after intense (myeloablative) chemotherapy and / or radiotherapy (total body irradiation, tbi). in , , autologous transplantations in europe (ebmt) the intensity of conventional chemotherapy is limited, in particular due to hematotoxcity (myelosupression) with neutropenia and thrombocytopenia. dose-intensive myeloablative therapies require transplantation of hematopoietic stem cells from the patient (autologous transplantation) or a donor (allogeneic transplantation, chap. . ). • in early studies, hematopoietic stem cells (hsc) were obtained from the bone marrow by aspiration. • meanwhile, hsc are mainly harvested via blood cell separation (leukapheresis) after stimulation with colony-stimulating factors (csf) and mobilization into the peripheral blood. cells are cryopreserved and retransfused after high-dose therapy (peripheral blood stem cells transplantation, pbsct). in healthy donors (no stimulation with hematopoietic growth factors, i.e., steady-state conditions), hscs are a rare population primarily located within the bone marrow. they do not circulate within the peripheral blood without mobilization. • the number of circulating pbsc increases during the phase of hematopoietic reconstitution after conventional chemotherapy. • the use of hematopoietic growth factors (e.g., granulocyte colony-stimulating factor, g-csf) after conventional chemotherapy leads to a further increase and is used as a standard method for mobilizing and collecting pbsc. • a sufficient pbsc yield is the prerequisite for high-dose chemotherapy with stem cell transplantation (pbsct). • identification of hematopoietic progenitor cells by detection of cd antigen expression ( - % of mononuclear cells in the bone marrow or mobilized blood are cd -positive; however, recent data have shown the existence of cd -negative hematopoietic stem cells, characterized by cd , thy- , oct- , c-kit/cd , sp phenotype). ep: ep: phys: phys: • over % of these cells are "committed progenitor cells" which have lost the ability to renew themselves. only pluripotent stem cells hold this feature and have the potential for complete hematopoietic reconstitution ( chap. . ). • in mice, sufficient numbers of pluripotent stem cells for complete hematopoietic reconstitution can be attained with highly purified cells. in humans after myeloablative chemotherapy (conditioning), - × cd -positive cells/kg body weight (bw) are regarded as sufficient. optimal reconstitution of all cell lineages is achieved by transplantation of ≥ × cd -positive cells/kg bw. patients who are candidates for high-dose chemotherapy with peripheral blood stem cell transplantation (pbsct) should initially be treated with conventional chemotherapy in order to combine maximum clinical response with early pbsc mobilization and harvesting. myelosuppression generated by pbsc mobilizing chemotherapy should ideally be brief (without affecting the stem cell compartment) and have a maximum effect on the underlying disease: • so far, there is no optimal mobilization protocol which could be used for the entire range of cancer patients. most commonly, cyclophosphamide is used to facilitate pbsc mobilization, e.g., in the vcp-e protocol (etoposide (vp ), cyclophosphamide, cisplatin, and epirubicin protocol . . ) followed by administration of recombinant human g-csf. • administration of rh g-csf increases the number of circulating multipotent progenitor cells by factor . maximum pbsc mobilization occurs concomitantly with an increase in neutrophil granulocytes after the leukocyte nadir has been passed. pbsc are harvested as an outpatient procedure when the leukocyte count (wbc) is > , - , /µl and cd + cells are > - /µl blood, using a standard cell separator ("leukapheresis"). the procedure is well tolerated. possible electrolyte imbalances can be compensated. • in patients without prior chemo-or radiotherapy, sufficient numbers of pbsc can usually be harvested with - leukaphereses. • for reasons of quality control, volume, differential leukocyte count, percentage of cd -positive cells, viability, and sterility of every stem cell apheresis sample must be determined. • leukapheresis pbsc may be further processed (e.g., cd -positive or -negative selection chap. . ) or directly preserved in liquid nitrogen (at − °c) . cryopreserved cells can be stored for many years prior to transplantation. • both processing and storage of the cell samples are carried out under gmp conditions (good manufacturing practice, eu gmp guidelines) in accordance with national laws. randomized studies have shown high-dose chemotherapy to be advantageous in the treatment of high-grade (and indolent) non-hodgkin's and hodgkin's lymphoma compared with standarddose chemotherapy. ebmt (european group for blood and marrow transplantation) has published guidelines for autologous stem cell transplantation as follows: • in certain patient groups with high-grade non-hodgkin's lymphomas (nhl), hodgkin's disease, multiple myeloma, and chemosensitive relapses of germ cell tumors, high-dose chemotherapy with autologous transplantation is effective and regarded as standard of care. • in solid tumors (e.g., sarcoma, breast cancer, or ovarian carcinoma), high-dose chemotherapy should only be carried out within clinical studies. • high-dose chemotherapy with stem cell support has been proven to be a potentially effective treatment for various other malignancies. however, the clinical benefit as compared to standard treatment has to be established in randomized clinical trials for each individual disease entity. ind: ind: . • bone marrow aplasia: high-dose chemotherapy causes bone marrow aplasia, which is overcome within approximately (granulocytes) to days (thrombocytes) after autologous pbsc transplantation. infections and hemorrhage may occur within this period, and most co: co: patients require antibiotics and blood products for - days. fungal infections are rare. viral infections may occur due to reactivation (hsv, vzv, rarely cmv). • gastrointestinal toxicity: oropharyngeal mucositis, gastroenteritis • pulmonary toxicity: with use of certain cytostatics (e.g., busulfan, cyclophosphamide, thiotepa, bcnu), inflammatory changes (fibrosis, alveolar hemorrhage, infection), pulmonary edema, pulmonary damage, and "acute respiratory distress syndrome" (ards) • cardiotoxicity: cardiac damage due to cytostatics, e.g., cyclophosphamide (cardiac insufficiency, transmural hemorrhagic myocardial necrosis), anthracyclines (acute and chronic cardiotoxicity); cardiac complications may be intensified in cases of preceding radiotherapy or anthracycline treatment • renal dysfunction: renal insufficiency or acute renal failure due to cytostatic drugs, antibiotic treatment with aminoglycosides, insufficient hydration during treatment, tumor lysis, and blood pressure fluctuations; renal insufficiency is usually reversible • hepatic dysfunction: besides fully reversible short-term increases in hepatic enzymes, rare occurrences of veno-occlusive disease (vod) progress in pbsct is to be expected in the following areas: • sequential transplantation (e.g., multiple myeloma) • use of new hematopoietic growth factors for stimulation of recovery of platelets and neutrophils to further shorten the cytopenic phase after chemotherapy • improved management of side effects of induction therapy • generation of immunocompetent cells for the treatment of minimal residual disease . . web: web: . autologous transplantation transfer of pluripotent hemato-/ lymphopoietic stem cells from healthy donors to recipients. • bone marrow transplantation (bmt) • peripheral blood stem cell transplantation (pbsct) • umbilical cord blood transplantation (ucbt) in , allogeneic transplantations were performed in europe (ebmt). the success of allogeneic transplantation is based on two therapeutic principles which distinguish this method from conventional chemotherapy and autologous transplantation: • conditioning: immuno-and myeloablative high-dose chemotherapy and/or total body irradiation (tbi) • graft versus leukemia effect (gvl effect): immunological reaction of donor lymphocytes from the graft against malignancy in the recipient. while cytostatic treatment largely reduces the malignant clone, the gvl effect seems to ensure long-term reduction of the relapse rate. complete t-cell depletion of the graft leads to increased relapse rates. donor search strategy . hla-typing of the patient and close family (siblings, parents) . in urgent cases, parallel search for donors among members of the extended family and unrelated donors of particular relevance are hla alleles of the classes i a, i b and ii dr. • in patients with related donors, one allele difference in graft versus host direction (gvh direction) and three allele differences in host versus graft direction (hvg direction) are acceptable. this configuration provides for equally successful transplantation results as complete hla identity. • class ii dr alleles should be identified using genetic typing methods (dna typing). • the relevance of the class ii loci dq and dp is not yet known. when several hla-a-, b-, and dr-identical donors are available, further selection criteria are considered (sex, age, cmv status, blood group). in the typing of unrelated donors, hla antigens (two alleles each of a, b, drb *, dqb *) of the patient and the prospective unrelated donor are important. recently, the relevance of hla-c has been emphasized, especially for graft rejection and nk cell-induced gvl effects. dp seems to be of little importance in relation to alloreactions. • dr typing should be based on high-resolution dna typing ( digits), just as serological hla-a, -b, and -c typing should be replaced by dna methods ( digits; recently digits). • particularly in younger patients, minor differences may be acceptable. • additional hla-dqb * and/or hla-dpb * differences are at present no reason for exclusion of a donor. allele differences in the a or b locus as well as minor drb * differences may also be acceptable, especially in patients with an aggressive underlying disease. • due to its low predictive value, mixed lymphocyte culturing (mlc) is no longer a routine procedure. • the donor search should be coordinated by recognized immunogenetics laboratories together with a transplantation center. options in case of unsuccessful donor search: • transplantation from donor after t-cell depletion • transplantation of umbilical cord blood (ucb): of particular importance in pediatric patients and young adults. cord blood banks are currently being established. so far, over , ucb transplants have been carried out worldwide. if necessary, several hla differences may be accepted, especially in pediatric patients. the main limiting factor is the cell content of the graft in relation to the patient's body weight. hematopoietic stem cells are characterized by their expression of specific surface markers (cd , cd ). the following hematopoetic stem cell compartments are used for transplantation: • bone marrow (bm) → bone marrow transplantation (bmt) • peripheral blood stem cells (pbsc, after stimulation with g-csf) → peripheral blood stem cell transplantation ((pbsct) • cryopreserved umbilical cord blood stem cells (ucb) → ucb transplantation (ucbt) normally, the freshly collected grafts are transplanted without being manipulated or cryopreserved. however, in specific situations, the graft needs to be modified: treatment procedures • t-cell depletion: prevention of graft versus host reactions • selection of cd -positive cells ("stem cell selection"): reduction of immune reactions and elimination of malignant cells (autologous transplantation) (→ chap. . ) bone marrow is collected under general anesthesia (approximately min) by bilateral puncture and aspiration from multiple sites of the iliac crest. • the bone marrow is anticoagulated and can be stored without cryopreservation for up to day without significant stem cell loss (duration of transport with unrelated donor transplants). • potential disadvantages for the donor include blood loss, local pain and hematomas and side effects related to anesthesia. recently, peripheral blood stem cells have increasingly been used for allogeneic transplantation. • pretreatment of the donor with - µg/kg/day g-csf s.c. for - days. • stem cell harvest by one or more leukaphereses. • depending on the rate of yield, the graft may contain more cd + stem cells than comparable bone marrow products as well as times more cd + t-lymphocytes. • in randomized studies, allogeneic transplantation of peripheral blood hematopoetic cells conferred an increased risk of gvhd compared with allogeneic bmt. • hematopoietic engraftment is faster with pbsc than with bone marrow. • pbsc grafts allow manipulation (stem cell enrichment, selective t-cell depletion, "graft engineering"), facilitating gvhd prophylaxis and transplantation despite hla barriers. successful allogeneic transplantation is ultimately "myeloablative, " which is a result of both the conditioning therapy and the donor t-lymphocytes. side effects: • toxic side effects of the conditioning therapy (chemotherapy, radiotherapy) depending on the therapy protocol • infections during the phase of bone marrow aplasia (until bone marrow reconstitution): bacterial infections, fungal infections (candida, aspergillus), viral infections (cmv, hsv) • long-term consequences: pulmonary fibrosis, bronchiolitis obliterans, gonadal insufficiency, hormonal deficiencies, cardiomyopathy, cataract, secondary neoplasia • lymphocytes in the graft play a major role in "alloreactions, " i.e., immunological reaction of transplanted immunologically active donor cells versus recipient organism → inflammatory immunological reactions in immunogenic structures, such as skin (dermatitis), intrahepatic bile ducts (cholestasis), and intestinal epithelium (enteritis); in extreme cases: destruction. • the period of immunological adaptation and development of tolerance is divided into an acute phase ( days after transplantation, acute gvhd) and a chronic phase ( - months after transplantation, chronic gvhd). • acute gvhd especially affects the skin, liver, and intestine. • chronic gvhd can potentially affect any organ. particularly skin and mucosal dryness, generalized sicca syndrome with conjunctivitis, malabsorption syndrome, chronic cholestasis, weight loss, and increasing pulmonary obstruction may occur. in extreme cases, sclerodermatype skin symptoms and other autoimmune disorders may develop. • gvhd prophylaxis with immunosuppressive drugs (e.g., cyclosporin a + methotrexate) is usually required in the initial weeks and months following transplantation. unlike transplanted solid organs, an immune system developing from transplanted stem cells gradually becomes tolerant of the recipient. • especially in the first few months of this process, the patient is at risk of immune reactions and opportunistic infections (cmv, vzv, pcp, pneumococcal pneumonia) → close monitoring by the transplantation center in collaboration with the referring physician. a high degree of patient compliance is required. since different transplantation units may use center-specific protocols, consultation with experienced centers is always advisable. variations may exist in relation to the following: • conditioning: based on high-dose chemotherapy with or without total body irradiation (ibi) • management of immunosuppression • stem cell graft engineering / manipulation furthermore, new protocols with less intensive conditioning regimens are currently being developed, for different indications and entry criteria (age). in protocols with reduced conditioning regimens, the age limit for allogeneic transplantation from related or unrelated donors has been raised from years to approximately years. see table ( p. ) remission-inducing therapy (transplantation in complete remission generally leads to improved long-term outcome and cure rates) • chemotherapy ± radiotherapy, high-dose or intensity-reduced (see below) • transplantation: transplantation of bm, pbsc, or cord blood from healthy donors via intravenous infusion; engraftment of hematopoietic stem cells in the bone marrow • supportive administration of antibiotics, erythrocytes and platelets during the neutropenic ( - days) and thrombocytopenic phase ( - days) • parenteral nutrition and pain therapy in cases of severe mucositis ( - days). mucositis prevention (palifermin) • prophylaxis and, if necessary, treatment of graft versus host disease (gvhd), (cyclosporine levels). monitoring for clinical signs of gvhd, exanthema, diarrhea, icterus, dryness of the mouth, conjunctivitis, mucositis • rehabilitation, outpatient follow-up, return to work after approximately - months terms such as "mini-transplantation, " "micro-transplantation, " or "non-myeloablative transplantation" have been used to describe reduced-intensity conditioning protocols which are based on dose modifications of traditional regimens (containing tbi - . gy or busulfan mg/kg). however, these protocols represent a variation of allogeneic sct with otherwise similar procedures and comparable immunological and infection-related problems (gvhd, opportunistic infections). due to reduced acute toxicity, these regimens may preferably be used in patients with additional comorbidities or in older patients. • the immunotherapeutic benefit of the gvl effect was demonstrated in patients with relapse of cml ( chap. . . ) after allogeneic bone marrow transplantation: the transfer of immunologically active donor lymphocytes resulted in complete remission in > % of these patients. • so-called myeloablative therapy (e.g., with - gy tbi or busulfan mg/kg in combination with cyclophosphamide) is an aggressive conditioning regimen with multiple side effects limiting use to younger patients without significant comorbidity. older patients therefore do not equally benefit from allogeneic transplantation with standard conditioning regimens. • dose escalation studies with radiotherapy or chemotherapy in patients with aggressive, therapy-refractory leukemias have shown that despite excessive toxicity and mortality, the risk of relapse after allogeneic transplantation was not significantly reduced. • recent animal experiments have shown that stable lympho-hematopoietic engraftment of donor cells can be achieved with significantly lower irradiation doses of gy, causing less side effects (so-called immunoconditioning). parallel to the gvl effect, the treatment induces "donor chimerism, " i.e., simultaneous existence of lymphatic and hematopoietic cells of the donor and the recipient. • "allogeneic transplantation with reduced conditioning" is aimed at utilizing the gvl effect without the disadvantages of maximum tolerable conditioning treatment. this therapeutic approach eventually constitutes a form of immunotherapy based on t-cell-mediated cytotoxicity. • initial clinical studies, including patients over years of age, have demonstrated the feasibility of this therapeutic approach. in individual cases, post-therapeutic complete and partial remission has been described. its applicability in patients of > years of age allows curative therapeutic attempts, particularly in patients with aml, mds, and low grade nhl. • donor chimerism of lymphatic and hematopoietic cells without significant myelosuppression (leuko-or thrombocytopenia) was achieved. • other approaches use fludarabine in combination with alkylating agents to increase tolerance prior to allogeneic transplantation. fludarabine has a particularly toxic effect on t-cells and has added benefit in the treatment of lymphomas. • the long-term outcome (overall survival) after reduced-intensity conditioning has not been established yet. randomized clinical trials for individual disease entities are necessary. ( chap. . ), all acute lymphatic leukemia ( chap. . . ) , cll chronic lymphatic leukemia, mps myeloproliferative syndrome ( chap. . ), nhl non-hodgkin's lymphomas ( chap. . ) , omf osteomyelofibrosis, cr complete remission, pr partial remission experimental treatment procedure. transfusion of donor granulocytes with the aim of correcting neutropenia (e.g., after chemotherapy). the use of rhg-csf for mobilization of donor granulocytes has been a prerequisite for successful development of granulocyte transfer. several phase i/ii studies have produced promising results in neutropenic patients with severe infections. prospective phase iii studies with comparison of granulocyte transfusion vs. standard of care are still pending. • donors and recipients must be abo and rh compatible (granulocyte concentrates contain erythrocytes). cmv status has to be considered • stimulation and mobilization of granulocytes with rhg-csf ( µg/kg) s.c. • after h, leukapheresis → with haes % (to accelerate sedimentation) and sodium citrate (anticoagulant) • granulocyte yield depends on donor wbc count, leukapheresis efficiency, and volume of processed blood • irradiation of granulocyte concentrates with gy to avoid graft versus host reaction (large numbers of lymphocytes and stem cells in the leukapheresis sample) • store concentrates at room temperature, without agitation for up to hrs. • carry out transfusion as soon after collection as possible • therapeutic success depends primarily on the number of transfused cells → transfusion of ≥ . × granulocytes per kg body weight of recipient clinical studies in patients with severe infections not responding to anti-infective treatment, with concomitant neutropenia (< neutrophils/µl) and without foreseeable bone marrow recovery. . granulocyte transfusion • direct pulmonary toxicity, dyspnea, hypoxemia → monitor o saturation • transfusion-induced acute pulmonary insufficiency (trali: transfusion-related acute lung injury) ( chap. . ) • alloimmunization against hla class i antigens and granulocyte-specific antigens → inefficiency of further transfusions, fever, respiratory symptoms, anaphylactic reactions • inclusion in clinical study, information and signed consent of donor and recipient • abo and rh compatible donor, check cmv serology of donor and recipient (no transfusion from cmv-positive donor to cmv-negative recipient) • clinical diagnosis: case history, physical examination • laboratory tests: full blood count with differential, blood group, hepatic and renal function parameters, coagulation parameters, serology (hav, hbv, hcv, cmv, hiv, treponema pallidum); female patients: pregnancy test where appropriate • cross-match (blood group testing) before each granulocyte transfusion • ecg, optional chest x-ray, abdominal sonography (splenic enlargement) • g-csf µg/kg s.c., h before each leukapheresis • prior to each new leukapheresis as well as and days after the final donation: blood count with differential, urea and electrolytes, serum creatinine, bilirubin, alt • leukapheresis sample irradiated with gy • administer as soon as possible (within h) • serological (erythrocytic) and leukocytic (lymphocyte toxicity test) compatibility must be assessed prior to each granulocyte transfusion. after administration of amphotericin b, wait at least h before giving a granulocyte transfusion (pulmonary toxicity). • ten minutes prior to transfusion, premedication with antihistamines and antipyretics (e.g., paracetamol). • recommended transfusion rate: × cells per hour. use standard filters (pore size - µm). • monitor blood pressure, pulse, respiratory rate, and o saturation from the beginning until h after transfusion. • evaluate transfusion success by measuring the post-transfusion granulocyte increase. specific or non-specific modulation of the immune system with the objective of immunologically mediated destruction of malignant cells. immunotherapy approaches to cancer treatment have been studied since the nineteenth century. four different approaches can be distinguished: • active specific immunotherapy • active non-specific immunotherapy • passive immunotherapy • adoptive immunotherapy the t-cellular immune response is crucial to recognize and eliminate tumor cells. t-cell subtypes include: a more recent classification is based on the cytokines produced by t-lymphocytes. cd -positive t-lymphocytes are subclassified as: t-lymphocytes do not recognize intact proteins, but peptides bound to mhc (major histocompatibility complex) molecules. different types of proteins can be processed by intracellular proteases, the resulting peptides are bound to mhc molecules and expressed on the cell surface of antigen-presenting cells. consequently, t-cell recognition is not limited to surface markers, but may include intracellular antigens, thus multiplying the diversity of t-cell target epitopes. prerequisite for the recognition of peptides is the binding to mhc molecules: • peptides binding to mhc i: - amino acids • peptides binding to mhc ii: - amino acids the specifics of antigen recognition by t-lymphocytes are dependent on the interaction of the variable region of the t-cell receptor (tcr) molecule with the mhc-peptide complex. this binding site of the tcr is coded by a unique gene segment, which is formed by the recombination of a v-d-j segment for the β locus and a v-j segment for the α locus during t-cell differentiation. this combinatorial diversity is amplified by the random addition of nucleotides and is the basis for the diversity of the t-cell repertoire. def: def: meth: meth: . immunotherapy proteins (e.g. melanoma antigens tyrosinase, mage - , melan-a) are processed by intracellular proteases. resulting peptides are bound by mhc molecules and presented at the cell surface. t-cell receptor (tcr) binds to mhc-peptide-complex. er endoplasmatic reticulum the binding of a tcr to a specific peptide-mhc complex alone is not sufficient for activation and proliferation of naive t-cells. additional signals are required: • adhesion molecules which facilitate contact with the targeted cell • costimulatory signal: costimulatory molecules are the antigens of the b -family (b - , b - ), which are expressed by antigen-presenting cells (apc). "professional apcs" play a central role for the initiation of the immune response. the antigens interact with suitable ligands on t-cells (cd , ctla- ). if a naive t-cell meets a non-professional apc (e.g., tumor cell presenting a peptide matching a specific tcr), a second costimulatory activation signal is missing. the result is the induction of anergy, i.e., the t-cell is refractory to further stimulatory signals. the immune system of a tumor patient is rarely capable of inducing regression of manifest tumors and metastases. this observation supports the hypothesis, that tumor specific antigens lead to incomplete activation of the immune system, or tumor cells "escape" immunologic toxicity through induction of immunosuppression. various "tumor escape" mechanisms of neoplastic cells have been described: • lack of expression of costimulatory molecules on tumor cells • loss or downregulation of mhc molecules (β -microglobulin, hla-a or -b) or receptors for apoptosis • loss of transport proteins (tap) → reduced presentation of tumor peptides with mhc molecules • selection of so-called antigen-loss variants, without expression of tumor-associated antigens (mage, tyrosinase, gp ) • induction of angiogenetic or antiapoptotic factors • secretion of immunoinhibitory cytokines, such as tgf-β and il- , by tumor cells dendritic cells or monocytes (professional antigen-presenting cells, apc) ingest antigenic material and disintegrate and process it to peptides. peptides are attached to mhc-i or mhc-ii molecules and transported to the cell membrane where they are presented. the tcr binds to peptide-mhc complexes, and cellular inter-action is supported by adhesion molecules and costimulatory signals such as b (on apc) and cd (t-cells). depending on the antigen and the cytokine environment, cell types preferably induced are th or th helper cells. th cells produce cytokines (such as , which are particularly important for the stimulation and differentiation of b-cells to antibody-producing plasma cells. th cells also interact with b-cells, which can ingest and process the same antigen complex (e.g., bacterium, tumor cells) with the help of membrane antibody molecules or the b-cell receptor -similar to dendritic cells. the interaction of b-and t-cells leads to a coordinated antigen-specific b-and t-cell immune response. th cells produce interferon-γ and il- , which particularly promote the maturation of mhc-i-peptide complex specific cytotoxic t-cells the term "active non-specific immunostimulation" relates to the administration of modifiers, which can directly modulate the immune system. "non-specific" indicates the lack of antigen specificity. non-specific immunity is mainly based on activated macrophages, but also nk cells and neutrophils. the following are possible biological response modifiers: . immunotherapy treatment of a disease by expression of one / multiple specific genes in a cell or group of cells. the production of the desired gene product corrects the genetic defect or alters cellular function. • somatic gene therapy: expression of genes in differentiated somatic cells • germline therapy: expression of genes in fertilized human oocytes or embryonic stem cells adequate methods of gene transfer are a basic requirement for gene therapeutic approaches. in most cases, so-called vectors are used to transport the therapeutic gene construct into the target cells. due to directed genetic deletions, viral vectors are usually unable to replicate after transfection of the target cell. viral vectors are produced with the help of "packaging cells, " which provide the necessary structural proteins for replication. after transfection of the genetic construct, these cells produce the required vector. the following are important criteria for the evaluation of gene transfer methods: • efficacy: transfection quality (transient or stable), transfection efficiency, tropism (potential of organ-specific gene transfers), biological efficacy (expression of gene products) • safety: tolerance, adverse effects, immunogenicity • production effort, cost and compliance with gmp / glp / gcp criteria the first somatic gene therapy was carried out in september at the nih, usa, on a -year-old girl with adenosine deaminase (ada) deficiency. since then, more than clinical gene therapy studies have been licensed worldwide and , patients have been treated. besides monogenic hereditary diseases, gene therapy is used particularly in patients with advanced malignancy, aids, or multifactorial diseases such as coronary heart disease or rheumatoid arthritis. meth: meth: . gene therapy gene therapy • induction of specific immune responses by transfer of immunostimulating genes (e.g., interleukin or interferon genes) into tumor cells, bystander cells, or immunoeffector cells • transfer of tumor suppressor genes → correction of regulation defects in tumor proliferation • blockade of oncogenic effects via antisense strategies • transfer of "suicide genes" into tumor cells: suicide genes usually encode enzymes (e.g., hsv thymidine kinase), which by means of phosphorylation convert non-toxic prodrugs (e.g., ganciclovir) into toxic substances, thus selectively killing hsv-tk-expressing cells • transfer of cytostatic drug resistant genes (mdr- , aldehyde dehydrogenase, o -alkylguanine-dna alkyltransferase, cytidine deaminase) into hematopoietic stem cells to increase the in vivo resistance of the hematopoietic system to cytostatic drugs and alleviate the hematotoxicity of subsequent chemotherapy until now, mainly critically ill patients with short life expectancy were included in gene therapy studies. therefore, the results of these studies focus primarily on safety and side effects, rather than on curative aspects. initial data on the safety of gene therapy methods showed that close surveillance is required: • after administration of high doses of adenoviral vectors, immune reactions to adenoviral proteins and pulmonary toxicity were detected in patients with cystic fibrosis. • severe adverse reactions also occurred during a clinical trial in patients with the x-chromosomal form of severe combined immunodeficiency (x-scid), carried out in paris. newborn children suffering from this so far incurable disease were cured by transfer of the normal gene. however, approximately years after treatment, out of children developed t-cell leukemia, the cause of which seemingly involved the insertion of the retroviral vector. the exact causes of this severe adverse effect are under intense medical and molecular biological investigation. • a -year-old patient with a severe congenital metabolic disease died in september in the usa after infusion of high doses of genetically modified adenovirus into the liver artery. the importance of strict adherence to the highest standards for production and quality control of gene therapy drugs as well as the conduct of controlled clinical trials was underlined by these cases. at present, gene therapy is used in clinical studies only ( chap. . ) . besides legal aspects mentioned above, national and international guidelines and regulation for gene therapy products and studies have to be followed. target entities • infections / parasitic diseases % • cardiovascular diseases % therapeutic approaches (number of trials) • immunotherapy • tumor suppressor regulation • proof-of-concept has been established for the biological or clinical efficacy of gene transfer in studies: • induction or amplification of tumor-specific immune responses in tumor vaccination studies • occasional tumor regression or stable disease after transfer of tumor suppressor genes (p ) • decreased incidence of gvhd after allogeneic hematopoietic transplantation due to transfer of hsv-tk suicide genes into allogeneic donor lymphocytes after administration of ganciclovir • correction of the immunodeficiency in x-chromosomal severe combined immunodeficiency (x-scid) • in patients suffering from hemophilia, factor viii use was reduced by - % after intramuscular injection of aav vectors carrying the wildtype gene furthermore, genetic marking of hematopoietic stem cells showed that while contributing to long-term hematopoietic reconstitution, transplanted stem cell products can potentially contain malignant cells which act as a starting point for relapse. although this method is a diagnostic procedure, the results of these studies are seminal for future advancement of transplantation strategies and trends in gene therapy with respect to the hematopoietic system. . inhibition of angiogenesis in , folkman described the transition of solid tumors from an avascular resting state to a vascularized phase with optimal tumor oxygenation and nutrition. only in the vascularized state, i.e., after the "angiogenic switch", accelerated tumor proliferation, metastasis, and generalization can occur, as for example in prostate carcinoma, breast cancer, and renal cell carcinoma. similarly, increased bone marrow microvessel density has been described in proliferating hematological neoplasia, particularly in patients with leukemia (aml, all, cml) and myelodysplasia. inhibition of tumor-induced angiogenesis and the angiogenic switch of human tumors (transition from avascular state to vascularized proliferating tumor) was first demonstrated as an effective means of treatment with the vegf-receptor antibody bevacizumab ( chap. . compound has been approved for treatment of metastatic colorectal cancer and non-small cell lung cancer (nsclc). similar approaches are followed with small molecule inhibitors of angiogenesis, e.g. sorafenib and sunitinib ( chap. . ) . increasing understanding of the molecular mechanisms of cancer and hematologic malignancies led to new strategies in the development of therapeutic agents, and this has resulted in the introduction of new drugs such as tyrosine kinase inhibitors in the treatment of malignant diseases. promising new approaches include: • rna-targeted therapy • aurora kinase inhibition • hsp (heat shock proteins) as targets for cancer therapeutics • telomerase therapeutics in recent years the members of the rna family have grown rapidly. in addition to the coding messenger rnas (mrnas) and transcriptional rnas [ribosomal rnas (rrnas) and transfer rnas (trnas)], another subfamily, called small rnas, has been discovered, each member of which has its own particular function. small rnas do not code for proteins, but instead control the transcription and translation of protein-coding rnas. the small rna subfamily contains small interfering rnas (sirnas), micrornas (mirnas), small nucleolar rnas (snornas), and small nuclear rnas (snrnas). sirnas and mirnas have attracted much attention due to their potential diagnostic and therapeutic applications in different diseases. sirnas and mirnas are generated using the same pathway by processing long doublestranded rna (dsrna) or microrna precursors with an endonuclease known as "dicer". subsequently, the rnas attach to an rna-induced silencing complex (risc) and are directed to the messenger rna (mrna) of interest which is marked for cleavage or inhibition of translation. developmental therapeutics these rnas are - nucleotides in length, double-stranded, and have ' overhangs of nucleotides. sirnas mediate the phenomenon of rna interference (rnai) which is a pathway for silencing the transcript of an active gene. rnai, discovered in , is now a standard laboratory tool for knocking down gene expression. exogenous synthetic sirnas or endogenously expressed sirnas attach to an rna-induced silencing complex (risc) and are directed to the messenger rna (mrna) of interest which is marked for destruction. the highly specific silencing effect of rnai has emerged as an attractive way to allow specific inhibition of the function of any chosen target genes, including those involved in diseases such as cancer, aids, and hepatitis. • the biggest obstacle to the development of rnai-based therapeutics is the delivery. trigger rnas (dsrnas from which sirnas are derived by the action of dicer) can be expressed from vectors or delivered as artificial sirnas. inserting foreign vector sequences (gene therapy) into chromosomal dna includes the problem of insertional activation and inactivation of cellular genes. direct administration of sirnas would require sirnas that are stable and modified to be resistant to nucleases. • drug therapies using sirnas are now in clinical trials for treating age-related macular degeneration and respiratory syncytial virus infection (rsv). micrornas are short - nucleotide rna molecules that are negative regulators of gene expression in a variety of eukaryotic organisms. mirnas are involved in numerous cellular processes including development, differentiation, proliferation, apoptosis, and stress response. about mirnas have been identified in humans, with the total predicted to eventually reach , or more. mirna mutations or altered expression correlate with various human cancers and indicate that mirnas can function as tumor suppressors or oncogenes ("oncomirs"). like sirnas, mirnas are generated from long primary precursor rnas before being processed by the dicer protein in the cytoplasm and incorporated as mature mirna into the rna-induced silencing complex (risc). whereas sirnas perfectly match with the target mrna, most mirnas do not match the target sequence exactly, enabling them to bind to multiple mrnas. instead of destruction of mrna as in rnai interference, imprecise matching results in inhibition of translation. about half of the annotated human mirnas map within fragile regions of chromosomes in cancer genomes. expression profiling of about mirnas has been shown to be a more accurate method of classifying cancer subtypes than using the expression of protein-coding genes. gene therapies using mirnas might be an effective approach to restore tumor suppressor function or to block oncogene activation. • administration of synthetic antisense oligonucleotides that encode sequences that are complementary to mature oncogenic mirnas-termed anti-mirna oligonucleotides (amos)might effectively inactivate mirnas in tumors and slow their growth. . developmental therapeutics part treatment procedures • antagomirs, a novel class of chemically engineered oligonucleotides appear to be specific and effective silencers of mirna expression in mice when conjugated with cholesterol. the serine-threonine kinases aurora a, b, and c represent a family of mitotic regulators which are essential for mitotic progression, spindle formation, centrosome maturation, chromosomal segregation, and cytokinesis. selectively inhibiting aurora kinase activity by rnai or small molecules leads to chromosome segregation errors and deregulation of the spindle checkpoint associated with cell death. aurora a localizes to centrosomes / spindle poles and is required for spindle assembly, whereas aurora b is a chromosome passenger protein required for phosphorylation of histone h , chromosome segregation, and cytokinesis. elevated expression of aurora a and b has been detected in many human cancers and overexpression of aurora a has been shown to induce oncogenic transformation in vitro. tumor cells treated with aurora kinase inhibitors show normal timing of expression of core cell cycle regulators, such as cyclins, and do not undergo arrest or delay transit through mitosis as classic antimitotic agents do. the antiproliferative effect is unique, in that tumor cells, especially those lacking functional p damage response, are catastrophically driven forward and out of an aberrant mitosis, which finally leads to cell death due to massive chromosomal instability. although aurora a has received most of the attention so far in terms of a link with human cancer, aurora b might be the more suitable anticancer drug target, because inhibition of aurora b rapidly results in a catastrophic mitosis. • small molecule inhibitors in development: hesperadin, azd (phase i), mk (= vx- , inhibits also flt- , phase i), mln (aurora a, phase i), jnj- (inhibits also cyclin-dependent kinases, cdks) • aurora kinases are only expressed and active as kinases during mitosis, therefore, it is assumed that non-proliferating cells would not be adversely affected by aurora kinase inhibitors • reduction or abrogation of histone h phosphorylation could serve as biomarker • mk (= vx- ) is effective against imatinib-and dasatinib-resistant bcr-abl(t i) kinase in vitro the heat shock protein (hsp) family of proteins has emerged as a target for cancer drug discovery because it is important for mediating the action of oncogenically relevant growth factor receptors and their downstreaming signaling elements. many hsps form multimolecular complexes that act as chaperones binding other proteins, denoted as client proteins. hsp consists of two isoforms and is one of the most abundant cellular chaperone proteins. it is a cellular chaperone required for refolding of denatured proteins, cellular survival under stress conditions, and the maturation of a subset of proteins that play key roles in oncogenesis. therefore, hsp does not catalyze a single reaction, but mediates the stability and function of multiple client proteins such as: natural products, including the ansamycin antibiotic geldanamycin and radiciol, that bind selectively to hsp and inhibit its chaperone function have been identified. however, geldanamycin is limited by its hepatotoxicity for clinical use, but a less toxic derivative -allylamino- -demethoxygeldanamycin ( -aag) has been identified. • due to poor chemical stability and bioavailability subsequent geldanamycin-and non-geldanamycin-based compounds are in development: kos- (tanespimycin, cremaphor-based formulation of -aag, phase i/ii), kos- ( -dmag, alvespimycin hydrochloride, orally active, phase i), cnf (oil-in-water nanoemulsion of -aag, phase i), cnf (orally, phase i), ipi- (water soluble, phase i), snx- (orally active, preclinical). • tumors which are dependent on a given client protein are particularly sensitive to degradation of hsp inhibitors. the proteins observed to be most sensitive to hsp inhibitor-induced degradation are the her and met receptor tyrosine kinases, raf- kinase, and the estrogen and androgen receptors. • hsp inhibitors might be particularly effective in cancer cells in which rb is mutationally inactivated (e.g., small cell lung cancer). typically, hsp inhibition induces cell cycle arrest in g phase. however, in rb-defective cells, tumor cells fail to arrest in g and enter a mitotic block with disordered prometaphase and unstable kinetochore assembly which is followed by apoptotic cell death. • hsp inhibitors enhance the activity of cytotoxics including taxanes, anthracyclines, hormonal agents, bortezomib, trastuzumab, and radiation. there is a schedule dependence in context with an intact retinoblastoma (rb) function due to its growth arrest in g phase of the cell cycle. • inhibitors of angiogenesis may also sensitize tumor cells to hsp inhibitors, because hypoxic tumor cells are under greater stress and hif alpha is also a client protein required for survival under these conditions. maintenance of telomeres at the ends of chromosomes is essential for unlimited cellular proliferation and confers immortality in cancer cells. since most cancer cells are reliant on telomerase for their survival, this enzyme represents an attractive mechanism-based target for the development of new cancer therapeutics. telomeres consist of repetitive double-stranded repeats of the sequence ttaggg associated with telomere-binding proteins. their major function is to cap the ends of chromosomes and to provide genetic stability. telomerase is a ribonucleoprotein enzyme, which synthesizes telomere repeats de novo. in human cells, the telomerase holoenzyme consists of a high-molecular weight complex with a template-containing rna subunit, htr, and protein components including the catalytic subunit human telomerase reverse transcriptase, htert. in addition, several additional molecules might play a role in regulating in vivo activity of telomerase such as the chaperone hsp /p . in most normal somatic cells telomerase activity is absent and telomere repeats are lost with cell division and with ageing. telomere attrition beyond a certain threshold is assumed to uncap chromosome ends which subsequently induces dna damage and onset of replicative senescence. in contrast, about - % of cancer cells have detectable telomerase activity, which leads to stabilization of telomeres and unlimited growth potential. strategies targeting telomeres / telomerase in cancer cells: • oligonucleotide antagonists against htr or htert (e.g., grn l, a thio-phosphoramidate oligonucleotide targeting the template region of htr as a "template antagonist"; phase i/ii). like sirna therapeutics (see above) there remains the issue of delivery and stability of antisense oligonucleotides. • small molecule inhibitors of the catalytic component htert (e.g., bibr ; preclinical); the antiproliferative effect is not induced by inhibition of the enzyme itself but through consecutive telomere dysfunction. the lag period of telomere shortening limits the widespread use of this approach. • heat shock protein (hsp ) inhibitors which compromise telomerase assembly by targeting hsp /p (phase ii) dose intensity and the toxicity and efficacy of allogeneic hematopoietic cell transplantation allogeneic stem-cell transplantation from related and unrelated donors in older patients with myeloid leukemia the graft-versus-lymphoma-effect: clinical review and future opportunities hematopoietic stem-cell transplantation how i treat refractory acute gvhd results of the ebmt activity survey on haematopoietic stem cell transplantation: focus on increasing use of unrelated donors adult umbilical cord blood transplantation ref: web: web: . allogeneic hematopoetic stem cell transplantation side effects of retroviral gene transfer into hematopoietic stem cells gene therapy strategies in prostate cancer good manufacturing practice production of adenoviral vectors for clinical trials regulatory considerations for novel gene therapy products: a review of the process leading to the first clinical lentiviral vector a review of gene therapy for haematological disorders survival of the fittest: in vivo selection and stem cell gene therapy angiogenesis: formation of new blood vessels; mostly formation of new capillaries from pre-existing blood vessels. inhibition of angiogenesis: through inhibition of endogenous angiogenic factors or administration of physiological / pharmacological angiogenesis inhibitors physiological angiogenesis is essential for the development of embryonic organs as well as the regulation of the adult vascular system: • embryogenesis: vasculogenesis, i.e., formation of new angioblast-derived blood vessels • proliferation of uterine epithelia, menstruation • proliferation and vascularization of muscle tissue • wound healing, bone growth, nerve regeneration, hair growth • regulation of vascular permeability → homeostasis in adult organisms, angiogenesis is typically strictly regulated and of limited duration ve-cadherin • fibroblast growth factors (afgf, bfgf), hepatocyte growth factor (hgf) • platelet-derived growth factor • transforming growth factors (tgfα, tgfβ), tumor necrosis factor alpha (tnfα) • interleukin • integrins α v β , α v β , α β • prostaglandins e (pge ) and e (pge ) • matrix metalloproteinases (mmps) angiogenesis in health and disease use of angiogenesis inhibitors in tumour treatment the biology of vegf and its receptors role of angiogenesis in tumor growth and metastasis angiogenesis and leukemia the pathophysiologic role of vegf in hematologic malignancies: therapeutic implications angiogenesis-dependent diseases and angiogenesis therapy disrupting tumour blood vessels ref: web: web: . inhibition of angiogenesis such folding is incompatible with telomerase function and may induce rapid telomere uncapping. the toxicity of such molecules is not yet clarified • immunotherapy with vaccines targeting htert-specific epitopes on cancer cells (gv ; primovax and telovax trial for pancreatic cancer, phase ii) telomerase-directed gene therapy: − suicide gene therapy: the htert promoter is linked to a proapoptotic gene or cytotoxic prodrug oncolytic viral therapy: viral genes which are critical for replication are placed under control of the htert gene promoter. this results in virus vectors that are replicated only in telomerase-positive cells, and then spread to adjacent cells on cell lysis heat shock protein modulators in hematologic neoplasms the major world of micrornas. horizon symposia inhibition of drug resistant mutants of abl, kit, and egf receptor kinases sirnas: applications in functional genomics and potential as therapeutics oncomirs-micrornas with a role in cancer aurora-kinase inhibitors as anticancer agents overcoming the immortality of tumour cells by telomere and telomerase based cancer therapeutics: current status and future prospects silencing of micrornas in vivo with "antagomirs" telomerase therapeutics for cancer: challenges and new directions heat shock protein as therapeutic target in solid tumors therapeutic potential of rna interference treatment with monoclonal antibodies directed against tumor antigens. mechanisms of tumor cell lysis by monoclonal antibodies: • antibody-dependent cellular cytotoxicity; adcc • complement-dependent cytotoxicity; cdc • intrinsic cytotoxic activity / induction of apoptosis • carrier of a cytotoxic substance (toxins, radionuclides, cytostatics) • antibody variants: murine antibodies, chimeric / humanized antibodies, bispecific antibodies, immunotoxins / radioconjugates indications ( • web: web: key: cord- - m nzi authors: lundegaard, claus; lund, ole; keşmir, can; brunak, søren; nielsen, morten title: modeling the adaptive immune system: predictions and simulations date: - - journal: bioinformatics doi: . /bioinformatics/btm sha: doc_id: cord_uid: m nzi motivation: immunological bioinformatics methods are applicable to a broad range of scientific areas. the specifics of how and where they might be implemented have recently been reviewed in the literature. however, the background and concerns for selecting between the different available methods have so far not been adequately covered. summary: before using predictions systems, it is necessary to not only understand how the methods are constructed but also their strength and limitations. the prediction systems in humoral epitope discovery are still in their infancy, but have reached a reasonable level of predictive strength. in cellular immunology, mhc class i binding predictions are now very strong and cover most of the known hla specificities. these systems work well for epitope discovery, and predictions of the mhc class i pathway have been further improved by integration with state-of-the-art prediction tools for proteasomal cleavage and tap binding. by comparison, class ii mhc binding predictions have not developed to a comparable accuracy level, but new tools have emerged that deliver significantly improved predictions not only in terms of accuracy, but also in mhc specificity coverage. simulation systems and mathematical modeling are also now beginning to reach a level where these methods will be able to answer more complex immunological questions. contact: lunde@cbs.dtu.dk supplementary information: supplementary data are available at bioinformatics online. the adaptive immune system of vertebrates is thought to be only million years old and exists in most fish, amphibians, reptiles, birds and mammals (thompson, ) . adaptive immunity is induced by lymphocytes and can be classified into two types: humoral immunity, mediated by antibodies, which are secreted by b lymphocytes and can neutralize pathogens outside the cells; and cellular immunity, mediated by t lymphocytes that eliminate infected or malfunctioning cells, and provide help to other immune responses. diversity is the hallmark of the adaptive immune systems. both the b and t lymphocyte-specific receptors for antigen recognition are assembled from variable (v), diversity (d), and joining (j) gene segments early in the lymphocyte development. there are multiple copies of v, d and j segments, and a huge repertoire of t and b cells is generated by the recombination of these segments, reviewed by li et al. ( ) . another task faced by the immune system is the tolerance to self, which is handled by continuously removing receptors that react to self-epitopes. special immunoglobulin molecules (antibodies) mediate the humoral response. as mentioned above, the antibodies are produced by b lymphocytes that bind to antigens by their immunoglobulin receptors, which is a membrane bound form of the antibodies. when the b lymphocytes become activated, they start to secrete the soluble form of this receptor in large amounts. the antibody is y-shaped, and each of the two branches functions independently and can be recombinantly produced and is then known as fabs. the highly variable tip of the fab, which can bind to epitopes is called the paratope and is made up of the so-called complementary determining regions (cdrs) . antibodies can coat the surface of an antigen such as a virus, so that it cannot function or infect cells, reviewed by burton ( ) . antibody-covered viruses or bacteria are easily phagocytosed and destroyed by scavenger cells of the immune system, e.g. the macrophages. antigenic proteins can be recognized by the antibodies in their native form without any cleavage or interactions with other molecules. thus the humoral immune response reacts to extracellular pathogens, and the response is crucial in the defense against most pathogens. b-cell epitopes are normally classified into two groups: continuous and discontinuous epitopes. a continuous epitope, (also called a sequential or linear epitope) is a short peptide fragment in a protein that is recognized by antibodies specific for that protein. a discontinuous epitope is composed of residues that are not adjacent in the primary structure (amino acid sequence), but are brought into proximity by the folding of the polypeptide. the classification is not clear-cut as discontinuous epitopes may contain linear stretches of amino acids, and continuous epitopes may show conformational preferences. the cellular arm of the immune system consists of two parts; cytotoxic t lymphocytes (ctl), and helper t lymphocytes (htls). ctls destroy cells that present non-self peptides (epitopes). htls are needed for b cells activation and proliferation to produce antibodies against a given antigen. ctls on the other hand perform surveillance of the host cells, and recognize and kill infected cells, generally explained in janeway et al. ( ) . both ctl and htl are raised against peptides that are presented to the immune cells by major histocompatibility complex (mhc) molecules, which are the most polymorphic of mammalian proteins. the human versions of mhcs are referred to as the human leucocyte antigen (hla). the cells of an individual are constantly screened for such peptides by the cellular arm of the immune system. in the mhc class i pathway, class i mhcs presents endogenous antigens to t cells carrying the cd receptor (cd þ t cells). to be presented, a precursor peptide is normally first generated by the large cytosomal protease complex called the proteasome (loureiroa and ploegha, ) . generally, it then binds to the transporter associated with antigen processing (tap) for translocation into the endoplasmic reticulum (er), reviewed by abele and tampe´( ) , but some peptides can enter the er independently of tap. this should be considered when dealing with virus-infected cells or tumors cells that might have reduced or absent tap function. there are several ways that the peptide can enter the er without tap function depending on the origin and properties of the peptide. the most well-established model, however, is for proteins containing a signal peptide. such proteins are translated directly into the er through the sec transporter complex and sometimes the cleaved-off signal peptide will end up in er. this model is especially relevant for peptides binding to hlas belonging to the abundant a hla serotype where tap-independent presentation is responsible for up to % of the a restricted epitopes, reviewed in . during or after the transport into the er the peptide must bind to the mhc class i molecule (stoltze et al., ; zhang and williams, ) before it can be transported to the cell surface through the golgi system. the most selective step in this pathway is binding of a peptide to the mhc class i molecule. in an older review, yewdell and bennink ( ) states that only in binds with an affinity strong enough to generate an immune response. this has been challenged, and it might be that up to % of the possible peptides bind strong enough to generate a subsequent immune response (assarsson et al., ) . in another recent work of moutaftsi et al. ( ) , however, it is found that of the epitopes that are responsible for % of the total cd þ t-cell response against a vaccinia challenge in mouse % binds mhc with an affinity stronger than nm. in any case a peptide must go through the processes in a greater number than competing peptides to be immunodominant. the mhc is the most polymorphic gene system known. this polymorphism is a huge challenge for t-cell epitope discoveries, enhancing the need for bioinformatical analysis and resources. however, it also highly complicates immunological bioinformatics, as predictive methods for peptide mhc binding have to deal with the diverse genetic background of different populations and individuals. on a population basis, hundreds of alleles have been found for most of the hla encoding loci ( in release . . of the imgt/hla database, http://www.ebi.ac.uk/imgt/hla/). in a given individual either one or two different alleles are expressed per locus depending on whether the same (in homozygous individuals) or two different (in heterozygous individuals) alleles are coded for on the two different chromosomes. the number of mhc expressing loci, however, differs highly among species. while a fully heterozygous human has six different mhc class i genes, a rhesus macaque may host up to active mhc class i genes (daza-vamenta et al., ) . each mhc allele binds a very restricted set of peptides and the polymorphism affects the peptide binding specificity of the mhc; one mhc will recognize one part of the peptide space, whereas another mhc will recognize a different part of this space. the very large number of different mhc alleles makes reliable identification of potential epitope candidates an immense task if all alleles are to be included in the search. however, many mhc alleles share a large fraction of their peptide-binding repertoire, and it is often possible to find promiscuous peptides, which bind to a number of hla alleles. a way of reducing the problem is to group all the different alleles into supertypes in a manner so that all the alleles within a given supertype have roughly the same peptide specificity (hertz and yanover, ; reche and reinherz, ; sette and sidney, , ) . this allows the search to be limited to a manageable representative set. representing a supertype by a well-studied allele might lead to selection of epitopes that is very restricted to this allele, but not to any other alleles within the supertype. thus another, and potentially more rational approach, would be to select a limited set of peptides restricted to as many alleles as possible. this should be within reach with new methods that directly predict epitopes that can bind to different alleles (promiscuous epitopes) (brusic et al., ) , or pan-specific approaches that can make predictions for all alleles where the sequence is known (jojic et al., ; nielsen et al., a) . when the peptide-mhc complex is presented on the surface of the cell, it might bind to a cd þ t cell with a fitting t-cell receptor (tcr). if such a tcr clone exists depends on, among other factors, if the tcr-peptide complex is too similar to mhc-peptide complexes generated with peptides from the host proteome (selfpeptides). this effect is called tolerance and might be broken by so-called self-epitopes, reviewed by andersen et al. ( ) . b cells must be activated to produce antibodies against a given antigen, and helper t cells specific for peptides from the antigen must be activated to get a strong b-cell response. the epitope recognized by the helper t cell is usually somehow connected to the epitope that is recognized by the b cell, but the two cells do not necessarily recognize overlapping epitopes. t cells can recognize internal peptides that do not need to be a part of the surface-surface interactions with the b-cell receptor. actually, the t-cell and the b-cell epitopes might not even come from the same protein (janeway et al., ) . the peptides recognized by the cd þ t cells are presented by the mhc class ii molecule, and peptide presentation on mhc class ii molecules follow a different path than the mhc class i presentation pathway (castellino et al., ) : mhc class ii molecules associate with the invariant chain (ii) in the er and the mhc-ii complex accumulates in endosomal compartments. here, ii is degraded, while another mhc-like molecule, called hla-dm in humans, loads the mhc class ii molecules with the best available ligands originating from endocytosed antigens. the peptide-mhc class ii complexes are subsequently transported to the cell surface for presentation to t helper cells. immunological predictions and simulations have been demonstrated highly useful in applied immunology in general, and in vaccinology in particular. it can be used as an efficient tool to lower the experimental workload in epitope discovery for use in rational vaccine design, immunotherapeutics and development of diagnogstic tools. a number of recent publications describe in great detail the values and benefits obtained by the use of immunoinformatics and predictions in applied immunology and vaccinology (davies and flower, ; de groot, ; de groot and moise, ; korber et al., ; lund et al., ; petrovsky and brusic, ; tong et al., ) . here, we will not engage in this discussion, but rather limit ourselves to describing the available methods for making such predictions, and deliver some of the background information needed to be able to choose the appropriate method for a given task. a large variety of machine-learning techniques are commonly used in the field of immunological bioinformatics ranging from the conventional techniques of position-specific scoring matrices (pssms) (altschul et al., ) , gibbs sampling (lawrence et al., ; nielsen et al., ) , artificial neural networks (anns) described in baldi and brunak ( ) , hidden markov models (hmms) explained in hughey and krogh ( ) , and support vector machines (svms) described in cortes and vapnik ( ) , to more exotic methods like ant colonies (karpenko et al., ) and other motif search algorithms (bui et al., ; chang et al., ; murugan and dai, ) . anns and svms and are ideally suited to recognize non-linear patterns, which are believed to contribute to, for instance, peptide-hla-i interactions (adams and koziol, ; brusic et al., ; buus et al., ; gulukota et al., ; nielsen et al., ) . in an ann, information is trained and distributed into a computer network with an input layer, hidden layers and an output layer all connected in a given structure through weighted connections (baldi and brunak, ) . in a pssm on the other hand, all positions in the motif are assumed to contribute in an independent manner, and the likelihood for matching a motif is calculated as a sum of individual matrix scores. the gibbs sampler method is a particular implementation of the pssm search algorithm, where the optimal pssm is determined by a search for a sequence alignment that provides maximal information content for a given motif length. conventionally pssms are log-odds matrices (altschul et al., ) , where the weight matrix elements are estimated from the logarithm of the ratio of the observed frequency of a given amino acid to the background frequency of that amino acid. however, many other techniques including the stabilization matrix method (smm) (peters and sette, ) , and evolutionary algorithm (brusic et al., ) exist to construct a pssm. the pssms might also be coupled with other information available to compensate for lack of data (lundegaard et al., ) . finally, hmms have been used in the field of immunological bioinformatics. these are well suited to characterized biological motifs with an inherent structural composition, and have been used in the field of immunology to predict for instance peptide binding to mhc class i (mamitsuka, ) and class ii (noguchi et al., ) molecules. beside machine-learning techniques, also (empirical) molecular force field modeling techniques (logean et al., ) and d quantitative structure-activity relationship ( d-qsar) (doytchinova and flower, ; zhihua et al., ) analysis have been used to predict features of the immune system. as an evaluation of the general quality of a prediction method a measure describing this quality is needed. however, no single measure can capture all qualities of a prediction, and not all types of data and predictions can be reasonably described by the same measure. so to be able to compare different systems, it is often needed to present several measures of quality. most measures need the data to be classified into two groups, i.e. positives and negatives. the number of classified (experimentally measured) positives is often designated as actual positives (ap), and the number of negatives, actual negatives (an), the number of predicted positives (pp), predicted negatives (pn), truly predicted positives (tp), falsely predicted positives (fp), truly predicted negatives (tn), and falsely predicted negatives (fn). some of the most often used measures are briefly described here. the equations for the mentioned measures are given at the end of the section. the fraction correct predicted (fcp) is the fraction of the total predictions that falls into the correct group. this measure is intuitively easily captured, but has the weakness that if a large fraction of the total evaluation data falls into a single group one will get high performance by just blindly predicting most or even everything to belong to this category. the positive predicted value (ppv) is the fraction of the positive predictions that actually falls into the positive class. the sensitivity is the fraction of the ap that is predicted as positives using a given threshold. the specificity is the fraction of the an that is predicted as negatives. the three latter measures are also easily grasped, however they are all dependent on the chosen prediction cutoff classifying the data into positive and negative predictions. a high sensitivity can be obtained by setting your prediction cutoff so that most of your evaluation data will fall into the positive group, but this will then be at the expense of the specificity and the ppv. which cutoff to use is determined by the purpose of the prediction, i.e. how many verified epitopes is needed versus the resources available for experimental validation. a plot of the sensitivity against the false positive rate ( -specificity) is called a receiver operating characteristic (roc) curve (swets, ) . such a plot can be a help to set the best prediction cutoff. one of the best ways of measuring the predictive power of a method is to calculate the area under the roc curve (auc) since this is a threshold-independent measure. another robust measure is the pearson correlation coefficient (pcc), which is a measure of how well the prediction scores correlate with the actual value on a linear scale. in situations where the correlation is not necessarily linear, the spearman's rank correlation coefficient (src) is more appropriate. in this measure each prediction is ranked on the basis of the prediction score and the pcc is calculated on the basis of this rank rather than the prediction score. the src, like the auc, is a threshold-independent measure of how well the predictor ranks the data when compared with the actual ranking. when comparing different methods, the thresholdindependent measures are to be preferred. otherwise a threshold has to be set under the same assumptions for all predictors. as an example one can estimate the specificity for each predictor by setting the threshold for the given predictor to a value where the sensitivity will be . (i.e. half of the total available positives is over the threshold), or estimate the sensitivity at a threshold where the specificity will be . (i.e. % of the an are predicted as negatives). the choice of an evaluation set is also absolutely crucial and several considerations must be taken. a large and diverse dataset is to be preferred to avoid any biases in prediction space. extreme care should also be taken to ensure that none of the predictors have been trained on the data used for evaluation even though that might not always be possible. to make the evaluation as broad as possible cross-validation is often used, i.e. the method is trained on a large part of the available data and a smaller part is left out for evaluation. this is done until all data has been included in the evaluation set and in this way it is possible to estimate the performance on the complete dataset. caution has to be taken, however, that the part used for training is not too similar to the evaluation part, as this will lead to an overestimation of the performance due to overtraining. this is especially true when using the leave-one-out version of cross-validation where everything except one data point is used for training, and the evaluation is then performed on the ensemble of the left out data points. equations are as follows: the state-of-the-art class i t-cell epitope prediction methods are today of a quality that makes it highly useful as an initial filtering technique in epitope discovery. studies have demonstrated how it is possible to rapidly identify and verify mhc binders from upcoming possible threats such as the sars virus (sylvester-hvid et al., ) with high reliability, and take such predictions a step further and validate the immunogenecity of peptides with limited efforts, as has been shown with the influenza a virus (wang et al., ) . it is also possible to identify the vast majority of the relevant epitopes in a rather complex organism as the vaccinia virus using class i mhc binding predictions and only have to test a very minor fraction of the possible peptides in the virus proteome (moutaftsi et al., ) . mhc class ii predictions can be made fairly reliable for certain alleles, and a number of helper epitopes have been identified by the help of bioinformatical approaches (consogno et al., ) . b-cell epitopes are still the most complicated task. however, some consistency between predicted and verified epitopes is starting to emerge using the newest prediction methods (dahlback et al., ) . in the following, we describe some of the best-performing prediction methods within each area. b-cell epitope prediction is a highly challenging field due to the fact that the vast majority of antibodies raised against a specific protein interact with discontinuous fragments (van regenmortel, ) . the prediction of continuous, or linear, epitopes, however, is a somewhat simpler problem, and may be still useful for synthetic vaccines or as diagnostic tools (regenmortel and muller, ) . moreover, the determination of continuous epitopes can be integrated into determination of discontinuous epitopes, as these often contain linear stretches (hopp, ) . in the early s, hopp and woods (hopp and woods, , ) developed the first linear epitope prediction method. this method takes the assumption that the regions of proteins that have a high degree of exposure to solvent contain the antigenic determinants. according to the hydrophilicity scale generated by levitt ( ) , hopp and woods ( ) assigned the hydrophilicity propensity to each amino acid in a sequence and looked at groups of six residues. this gave promising results and a number of methods have since been developed with the aim of predicting linear epitopes using a combination of different amino acid propensities (alix, ; debelle et al., ; jameson and wolf, ; maksyutov and zagrebelnaya, ; odorico and pellequer, ; parker et al., ) . , pellequer et al. ( proposed an evaluation set containing continuous epitopes in proteins and found that the method based on turn propensity (i.e. the propensity of an amino acid to occur within a turn structure) had the highest sensitivity using this set. seventy percent of the residues predicted to be in epitopes by this method were actually part of epitopes. the sensitivity for methods based on other propensities was in the range of - % (pellequer et al., ) . analyzing the epitope regions in the pellequer dataset reveals that almost all the hydrophobic amino acids are underrepresented, supporting the assumption that linear b-cell epitopes will occur in hydrophilic regions of the proteins. an extensive study of linear b-cell epitope prediction methods was published by blythe and flower ( ) . to test how well peaks in single amino acid scale propensity profiles are (significantly) associated with known linear epitope locations, amino acid propensities from the aaindex database (http://www.genome.ad.jp) (kawashima and kanehisa, ) were used. as test set they used epitope-mapped proteins defined by polyclonal antibodies, which were the best non-redundant test set available. blythe and flower ( ) found, however, that even the predictions based on the most accurate amino acid scales were only marginally better than random, suggesting that more sophisticated approaches is needed to predict the linear epitopes. bepipred , an algorithm that combines scores from the parker hydrophilicity scale (parker et al., ) and a pssm trained on linear epitopes, shows a small, but significant, increase in auc over earlier scale-based methods. the sequence parametrizer algorithm (sollner, ; sollner and mayer, ) , along with its associated machine-learning methods uses the common single amino acid propensity scales, but also incorporates neighborhood parameters reflecting the probability that a given stretch of amino acids exists within a predefined proximity of a specific amino acid residue. training and testing on epitope sequences pulled from a high-quality proprietary database, as well as several publicly accessible databases, yields a degree of accuracy that is greatly increased over single-parameter methods. different experimental techniques can be used to define conformational epitopes. probably the most accurate, and easily defined is using the solved structures of antibody-antigen complexes (fleury et al., ; mirza et al., ) . the amount of this kind of data is unfortunately still scarce, compared to linear epitopes. furthermore, very few antigens have been studied in a way where all possible epitopes on a given antigen has been identified. unidentified epitopes within the dataset will lower the apparent performance of an accurate prediction method by increasing the apparent false positive rate. the simplest way to predict the possible epitopes in a protein of known d structure is to use the knowledge of surface accessibility (novotny et al., ; thornton et al., ) . two newer methods using protein structure and surface exposure for prediction of b-cell epitopes have been developed. the cep method (kulkarni-kale et al., ) calculates the relative accessible surface area for each residue in the structure. then it is determined which parts of the protein that are exposed enough to be antigenic determinants. regions that are distant in the primary sequence, but close in three-dimensional space are considered as one epitope. the tool was tested on a dataset of antigen-antibody complexes and the algorithm correctly identified % of the epitope residues. discotope (haste et al., ) uses a combination of amino acid statistics, spatial information and surface exposure. it is trained on a compiled dataset of discontinuous epitopes from x-ray structures of antibody-antigen protein complexes. this method outperforms methods that predict linear epitopes. recently a workshop was held on the subject of b-cell epitope predictions attended by a broad range of the current method developers. the workshop resulted in a published review containing conclusions on the present common ground, and suggestions for the future especially concerning coordination and evaluation (greenbaum et al., ) . different ways of measuring the accuracy of b-cell epitope predictions have been suggested (hopp, ; van regenmortel and pellequer, ) . pellequer suggested using the specificity as a measure of accuracy, while hopp suggested using the ppv, but, as described earlier, neither measure will alone give a good description of the performance. in accordance to this the recent workshop concluded that the auc measure is to be preferred (greenbaum et al., ) . another issue is whether to make the statistics on a per-residue or on a per-epitope basis. however, as the latter have the additional complications of defining how much of an epitope that must be included in a prediction to be considered correct, and how much extra included residues is allowed, the per residue measure is to be preferred. epitope mapping can be performed experimentally by other methods than structure determination, e.g. by phage display (jesaitis et al., ; smith and petrenko, ) . the low sequence similarity between the mimotope [i.e. a macromolecule, often a peptide, which mimics the structure of an epitope, (meloen et al., ) ] identified through phage display and the antigen complicates the mapping back onto the native structure of the antigen. a number of methods have been developed to facilitate this (batori et al., ; enshell-seijffers et al., ; halperin et al., ; huang et al., ; moreau et al., ; mumey et al., ; schreiber et al., ; tarnovitski et al., ) . however, these are to be considered as interpreters of experimental data rather than predictors, which are the main focus of this review. a number of methods for predicting the binding of peptides to mhc molecules have been developed (schirle et al., ) since the first motif methods were presented (rothbard and taylor, ; sette et al., ) . the majority of peptides binding to mhc class i molecules have a length of - amino acids. position and the c-terminal position have turned out generally to be very important for the binding to most class i mhcs and these positions are referred to as anchor positions (rammensee et al., ) . for some alleles, the binding motifs further have auxiliary anchor positions. peptides binding to the human hla-a* allele thus have positions , and as anchors (kondo et al., ; kubo et al., ; rammensee et al., ) . the importance of anchor positions for peptide binding and the allele-specific amino acid preference at the anchor positions was first described by falk et al., . the discovery of such allele-specific motifs led to the development of the first reasonable accurate algorithms (pamer et al., ; rotzschke et al., ) . in these prediction tools, it is assumed that the amino acids at each position along the peptide sequence contribute a given binding energy, which can independently be added up to yield the overall binding energy of the peptide (meister et al., ; parker et al., ; stryhn et al., ) . similar types of approaches are used by the epimatrix method (schafer et al., ) , the bimas method (parker et al., ) , the syfpeithi method (rammensee et al., ) , the rankpep method (reche et al., ) and the gibbs sampler method (nielsen et al., ) . several of these matrix methods use an approach in the development where the method is build using exclusively positive examples defined after certain criteria, like eluted peptides and interferon gamma response data. this data can be used in training as well as affinity binding data defining binding stronger than a certain threshold (usually nm). other matrix methods, like the smm method, aim at predicting an actual affinity and thus use exclusively affinity data. as described earlier, matrix-based methods cannot take correlated effects into account, i.e. where the contribution to the binding affinity by a given amino acid at one position is influenced by amino acids at other positions in the peptide. higher order methods like anns and svms, on the other hand, are ideally suited to take such correlations into account. these methods can be trained with data either in the format of binder/non-binder classification, or as real affinity data. some of the recent methods combine the two types of data and prediction methods, either by averaging over predictions made by either (bhasin and raghava, ) , or by feeding the predictions from the positive data-trained pssms to anns together with sequence/affinity data (nielsen et al., ) . a study by yu et al. ( ) clearly shows the influence of having a large dataset on the performance of the resulting method. however, including knowledge of important positions reduce the need for data significantly (lundegaard et al., ) . several prediction methods have been made publicly available, and when selecting between these several cautions should be taken. the published performance, and how it is evaluated should be examined, but it is also very important that the method is able to generate predictions for the actual allele of interest. a major study comparing the predictive performance of a large part of the available methods was recently performed by peters et al. ( ) showing that in general the smm and the ann methods (table ) perform the best, even when taken into account the number of training data for each method. the cross-validated performance of these methods for several human and mouse mhc class i alleles was compared with the best performing other method available as web tool. the full results of this work are listed in supplementary table . the tools and urls are listed in table . it should be mentioned, however, that tools known to be trained on a significant part of the test set were excluded from this comparison. to achieve binding predictions for an allele with uncharacterized specificity, the supertype concept (sette and sidney, ) can be used for the limited number of alleles with well-defined supertype relationships (lund et al., ) . note, however, that predictions with methods predicting the specific allele is most often to be preferred, as the accuracy of these will be better (nielsen et al., a) . in general, hla-i binding predictions depend on sufficient experimental data being available for the exact hla-i molecule in question. unfortunately, % of the registered hla-i proteins (lefranc, ) have been examined experimentally, and % have been characterized with more than examples of peptide binders (rammensee et al., ; sette et al., ) . several groups have suggested prediction strategies to span these 'uncharacterized' regions of the hla diversity (brusic et al., ; jojic et al., ; nielsen et al., ; zhu et al., ) . in different forms, all these methods exploit both peptide and primary hla sequence as input information for training, aiming at simultaneously incorporating all hla specificities. in a recent paper (nielsen et al., a) , it is successfully demonstrated that such an approach can, to a very high degree, accurately characterize the binding motif for previously untested hla-i molecules. unlike the mhc class i molecules, the binding cleft of mhc class ii molecules is open-ended, which allows for the bound peptide to have significant overhangs in both ends. as a result mhc class ii binding peptides have a broader length distribution even though the part of the binding peptide that interacts with the mhc (the binding core) still includes only amino acid residues. this complicate binding predictions as identification of the correct alignment of the binding core is a crucial part of identifying the mhc class ii binding motif (nielsen et al., ) . the mhc class ii binding motifs have relatively weak and often degenerate sequence signals. while some alleles like hla-drb * show a strong preference for certain amino acids at the anchor positions, other alleles like hla-drb * allow basically all amino acids at all positions (rammensee et al., ) . however, there are other issues affecting the predictive performance of most mhc class ii binding prediction methods. the majority of these methods take as a fundamental assumption that the peptide-mhc binding affinity is determined solely from the nine amino acids in binding core motif. this is clearly a large oversimplification since it is known that peptide flanking residues (pfr) on both sides of the binding core may contribute to the binding affinity and stability (godkin et al., ) . some methods for mhc class ii binding have attempted to include pfrs indirectly, in terms of the peptide length, in the prediction of binding affinities (chang et al., ) . recently, nielsen et al. ( b) published a method for mhc class ii prediction that directly include pfrs and demonstrated that these pfrs improves the prediction accuracy. most of the methods for mhc class ii binding predictions have been trained and evaluated on very limited datasets covering only a single or a few different mhc class ii alleles, making it very difficult to compare the different performance values and generality of the methods. nielsen et al. ( b) have made available a large-scale benchmark set-up for evaluating mhc class ii peptide binding affinity prediction algorithms. the benchmark covers hla-dr (human mhc) and three mouse h -ia alleles, and consists of peptide/ic affinity data downloaded from the publicly available iedb database , and could set the start for large-scale unbiased evaluations of novel methods for mhc class ii prediction. successful prediction of the proteasome cleavage site specificity should provide valuable additional information useful in the design of treatments based on ctl responses. however, the complexity of proteasomal enzymatic specificity complicates such predictions. the proteasome have a highly stochastic element, exemplified by the observation that only $ % of the cleavage sites observed in one in vitro experiment can be verified in a second identical experiment (hansjo¨rg schild, personal communication). it is thus expected that the accuracy for prediction of proteasomal activity will be relatively low when compared to that of methods for mhc peptide binding. fragpredict, which is publicly available as a part of mappp service (http://www.mpiibberlin.mpg.de/mappp/), combines proteasomal cleavage predictions with mhc-and tap-binding predictions. fragpredict consists of two algorithms. the first algorithm uses a statistical analysis of cleavage-enhancing and -inhibiting amino acid motifs to predict potential proteasomal cleavage sites (holzhutter et al., ) . the second algorithm, which uses the results of the first algorithm as an input, predicts which fragments are most likely to be generated. this model takes the time-dependent degradation into account based on a kinetic model of the s proteasome (holzhutter and kloetzel, ) . at the moment, fragpredict is the only method that can predict fragments, instead of only possible cleavage sites. paproc (http://www.paproc.de) is a prediction method for cleavages by human as well as wild type and mutant yeast proteasomes. the influences of different amino acids at different positions are determined by using a stochastic hillclimbing algorithm (kuttler et al., ) based on the experimentally in vitro verified cleavage and non-cleavage sites (nussbaum et al., ) . both the fragpredict and paproc methods make use of the limited in vitro proteasomal digest data available. fragpredict is a linear method, and it may not capture the non-linear features of the specificity of the proteasome. the netchop (kesmir et al., ) method tries to address these two issues. the prediction system is a multilayered ann and uses naturally processed mhc class i ligands to predict proteasomal cleavage. since some of these ligands are generated by the immunoproteasome, and some by the constitutive proteasome, such a method should predict the combined specificity of both forms of proteasomes. in , netchop- . were evaluated to be the best-performing predictor on an independent evaluation set (saxova´et al., ) . pcleavage is another web accessible proteasomal cleavage predictor, which is svm based and have a published performance comparable to netchop- . (bhasin and raghava, ) . an update of the netchop method [netchop- . , nielsen et al. ( ) ] consists of a combination of several anns, each trained using a different sequence-encoding scheme of the data. netchop . has an increase in the prediction sensitivity as compared to netchop . , without lowering the specificity, and is thus probably the current best predictor of proteasomal cleavage. tenzer et al. ( ) have published a weight matrix based method for prediction of both constitutive-and immunoproteasomal cleavage specificity. both matrices are trained on in vitro digest data. relatively few methods have been developed to predict the specificity of tap. daniel et al. ( ) have developed anns using peptide mers for which tap affinity was determined experimentally. surprisingly, they found that some mhc alleles have ligands with very low tap affinities, e.g. hla-a . however, it has been shown that tap ligands can be trimmed in er before binding to mhc molecules (fruci et al., ) , i.e. a tap ligand might be an epitope precursor and thus does not need to be amino acids long. hla-a might easily have precursors of its optimal ligands, which are also good tap binders. peters et al. ( ) used an smm to predict tap affinity of peptides. this method has the advantage of not being bound to only mers but can also be used for longer peptides. the method assumes that only the first three positions in the n-terminal and the last position at the c-terminal influences the tap binding. the method is very well evaluated and the accuracy is high. the significance of tap binding in the epitope presentation pathway is much lower than the mhc binding (see later) and the auc value when this method is used alone as an epitope predictor of . is thus significantly lower than most mhc-binding prediction methods. two methods were published in . bhasin and raghava ( ) published a method for which they do only compare to the method of daniel et al. ( ) and it is not determined how it performs compared to the peters' method. the method of doytchinova et al. ( ) is evaluated by comparing the resulting method (matrix) with other matrices. from such a comparison it can only be concluded that this method is closer to peters' model than to the model of bhasin and raghava ( ) but not how it actually performs. recently a new tap predictor, predtap, have been published . this method does not have an auc value for the methods performance in epitope prediction making a direct comparison to other models impossible. with increasing numbers of tap ligands available on the internet (e.g. jen-pep database, http://www.jenner.ac.uk) (blythe et al., ) , it will likely soon be possible to obtain more accurate tap predictions. with respect to tap-independent transport and cleavage of peptides, the most established model is especially connected to the most abundant hla supertype (a ) and is related to the signal peptides and the processing of such . prediction of potential signal peptides that can be transported by sec can be made with tools for prediction of signal peptides, and some of these will also predict the signal peptidase cleavage site (bendtsen et al., ; kall et al., ; zhang and henzel, ) , but the value in the context of cd þ t-cell epitope predictions remains to be elucidated. the tcrs are generated by highly stochastic processes that secures that the tcrs in general will be able to recognize the entire probable space of mhc-peptide complexes. however, tcrs that recognize self-peptides will be eliminated so peptides that form complex with mhc are indistinguishable from self-peptides will not be recognized. it is still not clear how close peptides must be to the self to be able to escape recognition in this way (louzoun et al., ) . reliable predictions of immunogenic peptides can reduce the experimental effort needed to identify new epitopes, and though reliable predictions of the mhc binding alone can indeed be used to rank the possible epitopes very accurately, even better predictions should be possible if the other steps in the pathway were integrated in the predictions. accordingly, many attempts have been made to predict the outcome of the steps involved in antigen presentation, mapp (hakenberg et al., ) , netctl (larsen et al., ) , mhcpathway (tenzer et al., ) , epijen (doytchinova et al., ) and wapp (donnes and kohlbacher, ) . all these methods attempt to predict antigen presentation by integrating peptide-mhc binding predictions with one or more of the other events involved in the antigen presentation pathway. to benchmark these, a set of verified epitopes can be used as the positive dataset. negative examples (peptides that cannot induce an immunologic response) are hard to identify, as it is very hard to determine that a peptide will never be an epitope in any persons with a given hla haplotype. instead, epitopes from well-studied pathogens (e. g. hiv) are often used as the positive set, and all other peptides from the genome of the same pathogen that have never been shown to be an epitope are assumed negative as they have a very low probability of being an epitope. running a large-scale benchmark calculation comparing the predictive performance of several publicly available mhc-i presentation prediction methods evaluated on a large set of known hiv epitopes (http://www.cbs.dtu.dk/suppl/immunology/ctl- . / hiv_dataset) reveals that the updated netctl and mhcpathway methods have the highest predictive performance with % if the epitopes being within the top % peptides with the highest prediction scores (mette volby larsen, personal communication). improved understanding of the immune systems, and its population-wide variation, is one of the major challenges in the next decade within biology and medicine. many of the steps by which the immune system deal with infectious agents and disease can now successfully be modeled by computational techniques, and it is clear that the theoretical approaches will be a major player in this area, adding a systems view to the massive experimental effort being carried out at the moment. in this review, we have summarized how a number of bioinformatics tools that use genomic sequences as input to predict epitopes, have been developed over the past decade. at the same time, theoretical models have been developed that describe the dynamics of different immune-cell populations and their interactions with microbes (borghans and de boer, ; carneiro et al., ; davenport et al., ) . these models have been used to interpret experimental findings where timing is of importance, such as the interval between administration of a vaccine and infection with the microbe that the vaccine is intended to protect against. moreover, these dynamic models allowed for generating a quantitative picture of immune system kinetics and diversity during health and disease. the quantitative approach is necessary to understand the functioning of the immune system, which consists of many different cell types and molecules interacting in complicated regulatory pathways involving positive and negative feedback loops. surprisingly little is known about the population dynamics, i.e. the production rates, division rates and distribution of life spans of mouse or human lymphocyte populations. as a consequence, fundamental questions like the maintenance of memory, the maintenance of a diverse naive repertoire and the role of homeostatic mechanisms, remain largely unresolved. having so little insight in the normal lymphocyte population dynamics also hampers our understanding of immune responses during disease and immune reconstitution after therapeutic interventions such as chemotherapy, irradiation and/or bone marrow transplantation. several areas in immunology call for a better interpretation of data by means of theoretical models. a simple pubmed search reveals that at least % of the recent papers in the immunological literature involve labeling experiments in which lymphocytes are labeled radioactively, with deuterium, or with dyes. however, the interpretation of such labeling data is controversial and is notoriously difficult (boer et al., a, b; deenick et al., ; gett and hodgkin, ; hellerstein, ; mohri et al., ; mohri et al., ; revy et al., ; ribeiro et al., ) , which emphasizes the enormous demand to develop a quantitative mathematical approach to immunology. similar examples of how difficult it is to properly interpret kinetic data come from the attempts to characterize the division history of cells from the length of the telomeres, or from the presence of autosomal dna circles (trecs) that are formed in the thymus (boer and noest, ; douek et al., ; dutilh and de boer, ; hazenberg et al., ; hazenberg et al., ) . integrating the dynamic (using mathematical models and computer simulations) and bioinformatics approaches clearly could lead to a better understanding of the immune responses and their role during normal, disease and reconstitution states, where both timing and sequence specificity are highly significant. diseases that are characterized by complex interactions between the host cellular immune system and evolving pathogens such as hiv infection, or diseases where molecular similarities between self and non-self are important such as in autoimmune diseases could be investigated in such integrated models. complex generalized cellular automata have been proposed as models of the immune system (kohler et al., ; seiden and celada, ) . these methods have now developed to a stage where it is possible successfully to simulate the outcome of cancer vaccine protocols using a mouse simulation model (castiglione and piccoli, ; lollini et al., ; motta et al., ; pappalardo et al., ) . in a recent paper, rapin et al. ( ) outline a framework for integration of these bioinformatics and simulation approaches by developing a simple model in which hiv dynamics are correlated with genomics data. this model is the first one where, the fitness of wild-type and mutated virus is assessed by means of a sequence-dependent scoring matrix that links protein sequences to growth rates of the virus. further refinements of these approaches may involve increasing the spatial resolution by including different tissues and their geometry. the abcs of immunology: structure and function of tap, the transporter associated with antigen processing prediction of binding to mhc class i molecules predictive estimation of protein linear epitopes by using the program people gapped blast and psi-blast: a new generation of protein database search programs cytotoxic t cells a quantitative analysis of the variables affecting the repertoire of t cell specificities recognized after vaccinia virus infection bioinformatics: the machine learning approach an in silico method using an epitope motif database for predicting the location of antigenic determinants on proteins in a structural context improved prediction of signal peptides: signalp . analysis and prediction of affinity of tap binding peptides using cascade svm a hybrid approach for predicting promiscuous mhc class i restricted t cell epitopes pcleavage: an svm based method for prediction of constitutive proteasome and immunoproteasome cleavage sites in antigenic sequences benchmarking b cell epitope prediction: underperformance of existing methods jenpep: a database of quantitative functional peptide data for immunology t cell renewal rates, telomerase, and telomere length shortening different dynamics of cd þ and cd þ t cell responses during and after acute lymphocytic choriomeningitis virus infection estimating average cellular turnover from -bromo- -deoxyuridine (brdu) measurements quantification of t-cell dynamics: from telomeres to dna labeling 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tumor antigen mage- support-vector networks epitope mapping and topographic analysis of var csa dbl x involved in p. falciparum placental sequestration relationship between peptide selectivities of human transporters associated with antigen processing and hla class i molecules understanding the mechanisms and limitations of immune control of hiv harnessing bioinformatics to discover new vaccines genetic divergence of the rhesus macaque major histocompatibility complex immunomics: discovering new targets for vaccines and therapeutics prediction of immunogenicity for therapeutic proteins: state of the art predictions of the secondary structure and antigenicity of human and bovine tropoelastins stochastic model of t cell proliferation: a calculus revealing il- regulation of precursor frequencies, cell cycle time, and survival integrated modeling of the major events in the mhc class i antigen processing pathway changes in thymic function with age and during the treatment of hiv infection 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cleavage quantitative, pan-specific predictions of peptide binding to hla-a and-b locus molecules prediction of mhc class ii binding affinity using smm-align, a novel stabilization matrix alignment method hidden markov model-based prediction of antigenic peptides that interact with mhc class ii molecules antigenic determinants in proteins coincide with surface regions accessible to large probes (antibody domains) {paproc}: a prediction algorithm for proteasomal cleavages available on the {www} bepitope: predicting the location of continuous epitopes and patterns in proteins expression and deletion analysis of the trypanosoma brucei rhodesiense cysteine protease in escherichia coli analysis of vaccine's schedules using models new hydrophilicity scale derived from high-performance liquid chromatography peptide retention data: correlation of predicted surface residues with antigenicity and x-ray-derived accessible sites scheme for ranking potential hla-a binding peptides based on independent binding of individual peptide side-chains predicting location of continuous epitopes in proteins from their primary structures correlation between the location of antigenic sites and the prediction of turns in proteins generating quantitative models describing the sequence specificity of biological processes with the stabilized matrix method identifying mhc class i epitopes by predicting the tap transport efficiency of epitope precursors the immune epitope database and analysis resource: from vision to blueprint a community resource benchmarking predictions of peptide binding to mhc-i molecules bioinformatics for study of autoimmunity syfpeithi: database for mhc ligands and peptide motifs modelling the human immune system by combining bioinformatics and systems biology approaches definition of mhc supertypes through clustering of mhc peptide binding repertoires prediction of mhc class i binding peptides using profile motifs synthetic peptides as antigens functional antigen-independent synapses formed between t cells and dendritic cells modeling deuterated glucose labeling of t-lymphocytes a sequence pattern common to t cell epitopes exact prediction of a natural t cell epitope predicting proteasomal cleavage sites: a comparison of available methods prediction of well-conserved {hiv}- ligands using a matrix-based algorithm combining computer algorithms with experimental approaches permits the rapid and accurate identification of t cell epitopes from defined antigens d-epitope-explorer ( dex): localization of conformational epitopes within three-dimensional structures of proteins a model for simulating cognate recognition and response in the immune system hla supertypes and supermotifs: a functional perspective on hla polymorphism nine major hla class i supertypes account for the vast preponderance of hla-a and-b polymorphism prediction of major histocompatibility complex binding regions of protein antigens by sequence pattern analysis phage 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susceptibility new insights into {v}({d}){j} recombination and its role in the evolution of the immune system location of 'continuous' antigenic determinants in the protruding regions of proteins methods and protocols for prediction of immunogenic epitopes predicting antigenic determinants in proteins: looking for unidimensional solutions to a three-dimensional problem? mapping epitope structure and activity: from one-dimensional prediction to four-dimensional description of antigenic specificity ctl epitopes for influenza a including the h n bird flu hla-wide screening immunodominance in major histocompatibility complex class i-restricted t lymphocyte responses methods for prediction of peptide binding to mhc molecules: a comparative study pred(tap): a system for prediction of peptide binding to the human transporter associated with antigen processing assembly of mhc class i molecules within the endoplasmic reticulum signal peptide prediction based on analysis of experimentally verified cleavage sites toward the quantitative prediction of t-cell epitopes: qsar studies on peptides having affinity with the class i mhc molecular hla-a* improving mhc binding peptide prediction by incorporating binding data of auxiliary mhc molecules conflict of interest: none declared. key: cord- -rhji io authors: popko, brian; corbin, joshua g.; baerwald, kristine d.; dupree, jeffrey; garcia, annie m. title: the effects of interferon-γ on the central nervous system date: journal: mol neurobiol doi: . /bf sha: doc_id: cord_uid: rhji io interferon-gamma (ifn-γ) is a pleotropic cytokine released by t-lymphocytes and natural killer cells. normally, these cells do not traverse the blood-brain barrier at appreciable levels and, as such, ifn-γ is generally undetectable within the central nervous system (cns). nevertheless, in response to cns infections, as well as during certain disorders in which the cns is affected, t-cell traffic across the blood-brain barrier increases considerably, thereby exposing neuronal and glial cells to the potent effects of ifn-γ. a large portion of this article is devoted to the substantial circumstantial and experimental evidence that suggests that ifn-γ plays an important role in the pathogenesis of the demyelinating disorder multiple sclerosis (ms) and its animal model experimental allergic encephalomyelitis (eae). moreover, the biochemical and physiological effects of ifn-γ are discussed in the context of the potential consequences of such activities on the developing and mature nervous systems. interferon-gamma (ifn-?) was originally discovered by wheelock ( ) as an activity that interfered with viral replication. tlymphocytes and natural killer cells are the only cells known to be capable of expressing this cytokine (reviewed in trinchieri and perussia, ) . because under normal condi-under these conditions, neuronal and glial cells that normally do not encounter ifn-',v are exposed to the potent, pleotropic effects of this cytokine. there is considerable evidence that suggests that the effects of ifn-?~ are important in a variety of cns disorders (reviewed in ransohoff and benveniste, ) . in this article, we discuss various molecular, biochemical, cellular, and physiological properties of ifn-~, and its evoked response. moreover, the potential role this cytokine plays in cns disorders is discussed. a considerable amount of information has been learned concerning the molecular biology and biochemistry of ifn-~, since the human and mouse cdnas were cloned over a decade ago (gray et al., ; goeddel, , ) . the human and mouse proteins are and amino acids in length, respectively, and are encoded for by four exons that reside on kb of dna. interestingly; the human and mouse proteins share only limited homology, approx % at the amino acid level, and do not bind to the other's receptor to an appreciable extent (reviewed by farrar and schreiber, ) . both proteins contain two glycosylation sites (gray and goeddel, ; ealick et al., ) , although glycosylation is not essential for ifn-'i activity (kelker et al., ) . one factor that may play a role in regulating the biological activity of ifn-y is message stability, since the ifn-y mrna contains an au rich sequence in the '-untranslated region that has been shown to reduce [he mr_na halfqife of other cytokines dramatically (shaw and kamen, ) . biologically active ifn-~/from both human and mouse is a noncovalently bound homodimer that contains two receptor binding sites . the ifn- receptor is a multimeric receptor complex composed of a ligand binding subunit (a-chain) (aguet et al., ) and a transmembrane, accessory factor (~-chain) (soh et al., ; hemmi et al., ) . the c~-chain is a ubiq-uitously expressed, glycosylated, cell-surface protein that binds ifn-~ , with high affinity (reviewed by farrar and schreiber, ) . the extent of glycosylation is variable depending on cell type and accounts for the range of sizes of the a-chain, between and kda. on binding, ifn- induces (r-chain dimerization, which is believed to be critical to the initiation of the signal transduction cascade (greenlund et al., ; fountoulakis et al., ) . this feature of the ifn- receptor has been exploited to generate mutant forms that confer ifn- , unresponsiveness to cells in a dominant manner (dighe et al., (dighe et al., , . the crystal structure of the complex between ifn-y and the a-chain of its receptor has recently been reported (walter et al., ) . the ~-chain of the receptor, which associates with the or-chain in an ifn-,f-dependent manner, is essential for the induction of the ifn- signaling cascade (bach et al., ) . recently, considerable progress has been made in the elucidation of the ifn-,~-induced signal transduction pathway (sadowski et al., ; darnell et al., ; shuai, ; vilcek and oliveira, ; david, ; schindler, ) . the janus kinases jak and jak become phosphorylated following binding of ifn- to its receptor, with which the jak kinases associate (muller et al., ; wafting et al., ) . the jak kinase appears to associate with the a-chain, and the jak kinase associates with the [ -chain (sakatsume et al., ) . following activation, the jak kinases phosphorytate the transcriptional factor known as signal transducer and activator of transcription (stat-i~z), w~hich binds to a specific dna element referred to as ~,-activation site (gas) . stat-la binding results in the rapid transcriptional induction of genes containing the gas element. for example, the genes encoding the gaunylate binding protein and the igg fc receptor are induced in this way: other genes that are stimulated by ifn-~/, such as the class i and class ii molecules of the major histocompatibility complex (mhc), require a longer period (several hours) before transcriptional induction is detected. these genes lack the gas element and are likely activated through ifn-?-induced intermediates (benveniste and benos, ) . (summarized in table ) ifn-? plays a critical role in the regulation of the immune response (young and hardy, ) . for this reason, it is often referred to as immune interferon. ifn-y facilitates the stimulation of the ~ subpopulation of t-lymphocytes, which control cell-mediated immunity and which, in fact, express ifn-y; whereas it impedes the stimulation and activity of the th subpopulation of t-cells, :which express il- and are involved in regulating humoral immu-ni~ (gajewski et al., ; swain et al., ; paul and seder, ; seder and paul, ; reiner and seder, ) . cytotoxic t-cells and natural killer cells are also activated by ifn- (trinchieri and perussia, ) . ifn-? also plays a role in regulating antibody production, promoting an igg a response through a direct effect on b-cells (snapper and paul, ) . importantly, ifn-? is a potent stimulator of expression of the antigen-presenting components of the immune system. mhc class i and ii molecules are dramatically upregulated in the presence of ifn-'y on a variety of cell types (reviewed in vilcek et al., ; benveniste and benos, ) . ifn-? is also a powerful effector molecule of the inflammatory response (reviewed in schreiber and celada, ) . ifnq,-stimulated macrophages release a number of inflammatory cytokines, including tumor necrosis factor-alpha (tnf-a), il- , il- , and il- . in addition, ifn-? increases the microbicidal activities of macrophages through the induction of the synthesis of hydrogen peroxide and nitric oxide . ifn-? is also a potent stimulator of the expression of the fc receptors for igg immunoglobins on macrophages (erbe et al., ) . all of these actions of ifn-? on macrophages serve to stimulate the cytocidal activity of these cells. multiple sclerosis (ms) is the most common human demyelinating disease that affects the cns. although the etiology of ms has not been established, it is widely believed that immunological mechanisms are involved (reviewed in martin et al., ; raine, a,b) . re cns is immunologically distinct owing to the presence of the blood~brain barrier (crone, ) , which inhibits entry of both immune cells and humoral factors. in ms patients, this barrier is disrupted (martin et al., ) allowing increased access of blood components (cellular and noncellular) to the cns. the enhanced permeability of the blood-brain barrier is thought to be a contributing factor in immunemediated demyelinating disorders (reviewed in poser, poser, , . importantly, there is an increased infiltration of t-lymphocytes (cd + and cd +) into the cns of ms patients (martin et al., ; utz and mcfarland, ) , with cd + (t-helper) t-cells being predominant at the sites of active demyellnation (booss et al., ; traugott et al., ) . t-cell responses to a variety of myelin antigens, including myelin basic protein (mbp) and proteolipid protein (plp), have been identified in ms patients (reviewed in miller et al., ) . experimental allergic encephalomyelitis (eae) is the primary animal model used in the study of ms (reviewed in alvord et al., ; zamvil and steinman, ; martin and mcfarland, ) . eae can be induced in a variety of laboratory animal species by immunization with either cns tissue, myelin, or volume i , myelin components. as demonstrated through passive transfer studies, eae is a t-cell (cd +, thl) mediated disease model (zamvil and steinman, ; voskuhi et al., ) . a relapsing/remitting form of eae is induced at high incidence in sjl mice immunized with an encephalitogenic epitope of plp (tuohy et al., a (tuohy et al., ,b, (tuohy et al., , mcrae et al., ) . this model of eae exhibits many of the clinical, pathological, and immunological feat~ares of ms. the t-cells that infiltrate into the cns in ms and eae are activated and produce ifn-i, which is believed to play a role in the pathogenesis of irnmune-rnediated demyelinating disorders (reviewed in hartung et al., ; olsson, ; panitch, ). there is considerable circumstantial evidence that suggests that ifn-~, is a key mediator of inflammation in ms and eae. many of the pathological events observed in ms and eae, such as increased mhc expression, macrophage activation, increased expression of leukocyte adhesion molecules on blood-brain barrier endothelial cells (olsson, ) , and reactive gliosis (balasingam et al., ) , are consistent with the known effects of ifn-~,. ifn- is detected in active ms lesions (traugott and lebon, ) , and increased numbers of ifn-~,-secreting tlymphocytes are present in the cerebrospinal fluid of ms patients (olsson et al., ) . beck et al. ( ff) reported a cor~iation between the onset of clinical symptoms of ms and mitogenstimulated ifn-y production. moreover, eae can be transferred to naive animals using ifn-~,-secreting tqymphocytes isolated from animals experiencing eae (zamvil and stelnman, ) , and a significant increase in the level of ifn-?, mrna is observed in the cns of animals with severe eae compared to animals with mild eae (renno et al., ) . perhaps the best direct evidence for the deleterious effects of ifn-y in ms came inadvertently from a small clinical trial in the early s. of ms patients receiving ifn-~/, seven experienced exacerbations during the -wk treatment period, which was significantly higher than the pretreatment exacerbation rate (panitch et at., ; panitch, ) . not only did this study clearly eliminate ifn-~/as a potential therapeutic agent for ms, but it also demonstrated that ifn-~, activity can lead to a wo~,ens of tlne disease course, suggesting a role for this cytokine in normal disease progression. there are, however, contradictory experimental data concerning the role of ifn-~, in eae. the administration of antibodies to ifn-~, e~anced the severity of eae in mildly susceptible mice (billiau et al., ; voorthuis et al., ; duong et al., ) , and resulted in eae susceptibility in resistant strains (duong et al., ) . moreover, treatment of sjl/j mice, which are very sensitive to eae, with ifn-~, results in enhanced survival (billiau et al., ) . nevertheless, the protective effect of ifn-~, is not observed when eae is passively transferred. voorthuis et al. ( ) reported that intraventricular administration of ifn-~/ into eae-induced rats resulted in complete suppression of clinical signs, and willenborg et al. ( ) , using a viral delivery system, suggested that ifn-y had no effect on disease. recently, ferber et al. ( ) examined the eae susceptibility of b .pl mice that contained an experimentally inactivated ifn-~, gene (dalton et al., ) . b .pl mice are normally very susceptible to mbp-induced eae, and the ifn-?, ka~ockout mice appear equally sensitive to disease induction. although morphological data were not presented, this work dearly demonstrates that ifn-~, is not essential for eae induction, at least not in b .pl mice. to understand better the effects of ifn-u on cns cells in vivo, in the absence of other t-cellderived factors, a variety of approaches have been used to deliver this cytokine to the cns. the direct injection of ifn-~, into various animal models has been shown to upregulate the volume , expression of both mhc class i and class ii glycoproteins on various neuronal celt types that do not normally express these proteins at detectable levels. wong et al. ( ) demonstrated a dramatic increase in the expression of the mhc class i glycoprotein in astrocytes, neurons, oligodendrocytes and microglia following the delivery of ifn-y to the cns of -dold mice. direct injection of ifn-y into the cns has also been demonstrated to increase the expression of mhc class i on rat endothelial and ependymal cells and class ii on microglia, ependymal, and perivascular cells (sethna and lampson, ) . continuous iv infusion of ifn-~f into lewis rats for a period of d induced mhc class ii expression on microglia, ependymal and endothelial cells of large blood vessels (steiniger and van der meide, ) . these findings support the earlier work of momburg et at. ( ) who reported that the iv infusion of ifn-y into mice resulted in class ii expression in many organs, including the brain, where "round cells in the vicinity of meningeal blood vessel," presumably microglia, were class ii immunoreactive. ifn-y has also been suggested to play a role in the breakdown of the blood-brain barrier and the recruitment of inflammatory cells into the cns. seth_ha and lampson ( ) reported that a single injection of ifn-y into the rat basal ganglia induced the infiltration of cd + t-cells into the perivascular space and monocytes/ macrophages, and presumptive natural killer cells into the brain parenchyma. these authors suggested that such cellular recruitment may be facilitated by ifn-y-induced changes in the endothelial cells, resulting in an alteration in the permeability of the blood-brain barrier. moreover, simmons and willenborg ( ) , using direct injection of ifn-~. into the spinal cord of rats, reported an inflammatory response similar in pattern to that observed in early eae with the accumulation of inflammatory cells in the meninges and around spinal cord veins also suggestive of a compromised blood-brain barrier. tjuvajev et ai. ( ) reported an ifn-y-induced breakdown of the blood-brain barrier in an in vivo brain tumor model, and huynh and dorovini-zis ( ) demonstrated that brain endothelial cells undergo dramatic morphological, functional, and permeability changes in response to ifn-y, suggesting that this cytokine alters bloodbrain barrier function. steffen et al. ( ) demonstrated increased expression of vascular cell adhesion molecules (vcam) and intercellular adhesion molecule- (icam- ) on the brain endothelium of sjl mice experiencing eae, and antibodies to these molecules inhibited binding of peripheral lymphocytes to inflamed brains. furthermore, mccarron et al. ( ) showed that ifn-y significantly increased the adhesion of mgp-specific encephatitogenic t-ceils to mouse cerebrovascular endothelial cells, and that this effect correlated with the induction of icam- expression by the endothelial cells. the studies discussed above consistently indicate a role for ifn-y in the inducement of expression of mhc and cell adhesion molecules, and the recruitment of inflammatory cells; however, the infusion of ifn-y into the cns has also been implicated in a variety of other processes. for example, yong et al. ( ) reported that the direct administration of ifn-~/ into the adult mouse brain following corticectomy resulted in an increase in trauma-initiated gliosis. brosnan et al. ( ) , using the visual pathway of the rabbit, demonstrated that the injection of ifn-y significantly reduced neural conduction and induced subtle axonal pathology. in addition, the simultaneous injection of ifn- and a myelin/oligodendrocyte glycoprotein antibody significantly delayed cervical somatosensory evoked potentials, and caused massive demyelination in the spinal cord (vass et al., ) . vass et al. ( ) also reported an ifn-y-induced increase in mhc expression, which accompanied spinal cord demyelination. in complement with various delivery systems previously discussed, we have used a transgenic approach to examine the effects of ifn-y on the developing nervous system, (corbin et al., ) . transgenic mice were generated in which the expression of ifnq was volume , i placed under the transcriptional control of the mbp gene (mbp/ifn-y transgenic animals). transgenic mice generated with tl-ds construct have a shaking/shivering phenotype that is similar to that observed in naturally occurring mouse models of hypomyelination (e.g., sb_iverer, jimpy, quaking), and these transgenic animals have dramatically less cns myelin than control animals. reactive gliosis and increased macrophage/microglia f / immunostaining were also observed. additionally, mhc class i and class ii mrna levels were increased in the cns of mbp/ifn-y transgenic mice, and the increase in mhc class i mrna expression was detected in both white and gray matter regions. surprisingly, cerebelfar granule cell migration was also disrupted in these animals. these results strongly support the hypothesis that ifn-~, is a key effector molecule in irnmtme-mediated demyelinating disorders, and suggest that the presence of this cytokine may also disrupt the cytoarchitecture of the developing nervous system. the mechanism by which ifn- exerts its action within the cns remains unresolved. one idea concerning the potential cellular site of action of ifn-y in cns demyelinating disorders is that ifn- activates macrophages/ microglial cells, which in turn mediate cytotoxic effects on oligodendr~ytes (fig. ) . tnf-(z, which has previously been shown to be toxic to oligodendrocytes in vitro, is expressed by ifn-y-activated macrophages/microglia (selmaj et al., ) . more recent in vitro experiments suggest that the microglial cell toxicity to otigodendrocytes is mediated through cellsurface expression of tnf-cz and the production of nitric oxide (merrill et al., ) . nitric oxide is very labile, such that to elicit their cytotoxic effects, the activated microglia need to be in intimate contact with the target oligodendrocytes. oligodendrocytes undergo necrotic cell death following exposure to nitric oxide (mitrovic et al., ) . in support of the hypoth~ esis that nitric oxide is important in the pathogenesis of immune-mediated demyelinating diseases, it has been shown that nitric oxide synthase mrna levels are increased in mice experiencing eae (koprowski et al., ) , and nadph-diaphorase histochemical staining, a marker for nitric oxide production, as well as nitric oxide synthase rnrna levels were found to be elevated in the brains of ms patients (b et al., ) . ifn-y may also have a direct deleterious effect on oligodendrocytes (fig. i) , which have been shown to express ifn- receptors (torres et al., ) . we have examined the effects of ifn- on oligodendrocytes using the moch- cell line, which was derived from an oligodendroglioma that developed in a transgenic line of mice and expresses a variety of features of oligodendrocytes (hayes et al., ; li et al, ) . when moch- cells are cultured in medium containing low concentrations of volume , serum ( % fetal bovine serum [fbs]) they extend multiple, thin, branched processes and have phase-bright cell bodies, which are typi ~ cal features of cultured primary oligodendrocytes (reviewed in popko et al., ) . furthermore, these cells are immunoreactive against antibodies to galactocerebroside, a glycolipid marker of mature oligodendrocytes, and abundantly express myelin protein genes (hayes et al., ) . mochq cells cultured in % fbs or chemically defined medium do not express detectable levels of the astrocyte marker glial fibrillary acidic protein (gfap). when moch- cells are cultured in % fbs medium containing ifn- . however, they transform to flat, astrocyte-like cells with enlarged cell bodies and appear more adhesive ~ in addition to these morphological changes, ifn- stimulates the expression of abundant levels of the astrocyte marker gfap, as well as slightly elevated levels of mbp and plp mrna. ifn- also induces an enormous increase of mhc class i expression, as well as a comparatively modest increase of mhc class ii mrna expression in mochq cells. the morphological and molecular changes mc)ch- cells undergo in response to ifn- suggest that ifn- may induce a direct effect on oligodendrocytes, or a subpopulafion of these cells, in vivo . recent studies further support the hypothesis that ifn-y has a direct, deleterious effect on oligodendrocytes. using a morphological assay, oligodendrocytes in vitro were shown to undergo apoptotic cell death at very high frequency following exposure to relatively low concentrations of ifn- (vartanian et al., ) . the addition of rnicroglia to the culture did not significantly increase the frequency of otigodendrocyte death, further supporting a direct effect of the cytokine. interestingly, vartanian et ai. ( ) also detected apoptotic oligodendrocytes, along with ifn- , in the advancing margin of active ms plaques, but not in the chronic portion of the plaque or in adjacent unaffected white matter. agresti et al. ( ) have also observed deleterious effects of ifn-y on cultured oligodendrocytes. in the absence of cell death, they observed a decrease in the ability of oligodendrocyte progenitors to divide and differentiate, as well as changes in myelin protein gene mrnas and a general metabolic depression. these effects were shown to be fully reversible following cytokine withdrawal (agresti et al., ) . the harmful effects of ifn-y on myelination might be mediated through the induction of mhc expression in oligodendrocytes, which normally do not express these molecules at an appreciable level. in support of this possibility, transgenic mice in which mhc class i expression has been targeted to oligodendrocytes are severely hypomyelinated (turnley et al., b; yoshioka et al, ; turnley and morahan, ) . this hypomyelh~ation occurs in the absence of immune celt infiltration, suggesting that mhc class i expression in oligodendrocytes is sufficient to disrupt myelination. recently; power et al. ( ) examined these transgenic animals in more detail and demonstrated that cell-surface expression of mhc class i in oligodendrocytes of these mice was minimal moreover, immunocytochemistry for f~ -microglobulin, which is required for cellsurface expression of mhc class i, revealed that expression of ~ -microglobulin was undetectable in oligodendrocytes and limited to microglia in lhe cns. power et al. ( ) suggest that the accumulation of mhc class i protein m the cytoplasm of the oligodendrocytes, owing to the absence of f~ -microglobulin, may interfere with normal myelin protein trafticking, leading to the observed hypomyelination in these mice. in vitro, oligodendrocytes at all stages of differentiation can be induced by ifn- to express cell-surface mhc class i (suzumura et al., ; turnley et al., a; massa et al., ) . nevertheless, it is unclear what the consequences of oligodendroglial expression of mhc class i are. as mentioned previously, cd + t-cells are present in the cns of ms patients (martin et al., ; utz and mcfarland, ) . it is, however, unknown whether oligodendrocytes in vivo can present antigen to these cells. in contrast, many investigators have searched for, but failed to demonstrate, ifn- mediated induction of mhc class ii on oligodendrocytes in culture (wong et al., ; suzumura et al., ; turnley et al., a; satoh et al., b) , suggesting that oligodendrocytes are refractory to mhc class ii induction. calder et al. ( ) demonstrated that oligodendrocyte precursors (o- a progenitor cells) can be induced to express mhc class ii by ifn-~/, but that maturation into oligodendrocytes leads to loss of inducibility. nevertheless, bergsteinsdottir et al. ( ) were able to induce mhc class ii expression in a subset of mature oligodendrocytes in culture with ifn- in the presence of the synthetic glucocorticoid dexamethasone. although gtucocorticoids are normal cns constituents (birmingham et al., ; kumar et al., ) , the in vivo significance of these in vitro observations is unclear. another activity of ifn-t that might be relevant to the pathogenesis of immune-mediated demyelinating disorders concerns the ability of this cytokine to stimulate the expression of mat-shock (stress) proteins. these are ubiquitously expressed proteins that are believed to play a role as chaperones in normal protein synthesis and degradation (reviewed in jindal, ) . the expression of these proteins increases following cell stress, and this response is thought to be protective. heat-shock proteins are highly conse~rved across divergent species and are very irnmunogenic. it has been demonstrated that there is increased heat shock protein expression in ms and eae lesions, and importantly, that there is an immune response to [he stress proteins in these disorders (selmaj et al., ; prabhaker et al., ; gao et al, ; van noort et al., ) . it has also been demonstrafed that fn-'f is capable of potentiating the induction of stress protein synthesis (morange et al., ; ferm et al., ) , such that this effect might facilitate the induction of the immune response to the heat-shock proteins in immune-mediated disorders. the characterlzation of the heat-shock response in these disorders should provide further insight into the role these proteins play in the pathogenesis of disease. a potential direct effect of ifn-? on oligodendrocytes in immune-mediated demyelinating disorders is consistent with recent studies that examined biopsy specimens from patients with acute ms (rodriguez and scheithauer, ) . they observed degeneration of inner myelin loops with preservation of outer loops and axons in early plaques, as well as areas in which well-myelinated and demyelinated axons were in close proximity. these observations suggest that the early pathological lesions that occur in ms are compatible with a primary disturbance in the myelinating function of oligodendrocytes, not a nonspecific inflammatory reaction that affects the myelin sheath, as might occur if microglia were activated subsequently to phagocytose the myelin sheath. astrocytes, which have been shown to express the receptor for ifn- (rubio and de felipe, ) , respond to the presence of this cytokine in a variety of ways. as mentioned previously, mhc class i expression on astrocytes increases substantially in the presence of ifn- (wong et al., ; mauerhoff et al., ; massa et al., ) . this expression appears functional in that astrocytes are susceptible to lysis by cytotoxic t-cells in an antigen-specific manner (skias et al., ) . ifnq,-induced mhc class ii expression by astrocytes has also been demonstrated in vitro and in vivo (fontana et al, ; fierz et al., ; pulver et al., ) , although fine in vivo levels are low relative to that detected on microglial cells (reviewed in benveniste, ) . the biological significance of the antigen-presenting potential of astrocytes remains to be resolved in vivo. there is also evidence that suggests that ifn- plays a role in the reactive astrogliotic response, particularly in immune-mediated disorders. reactive astrogliosis is the response in which astrocytes express increased levels of gfap, undergo hypertrophy, and proliferate (eng, ) . there are dramatically elevated levels of geap expression in the cns of the volume i , mbp/ifn- transgenic animals, although it is unclear whether this represents a direct response of astrocytes to i~- or an indirect response elicited by the myelin abnormalities that occur in these mice (corbin et al., ) . moreover, as mentioned, yong et al. ( ) showed that if n< , as well as a number of other cytokines (balasingam et al., ) , increased trauma-induced gliosis in mice. these investigators also demonstrate that ifn-? promotes the proliferation of adult human astrocytes in vitro (yong et al., ) . nevertheless, in a follow-up study, yong et al. ( ) were unable to reproduce these results using mouse astrocytes, suggesting that there are species differences in the astrocytic response to ifn- . ifn- has also been shown to increase the expression of intercellular adhesion molecules on astrocytes. human fetal (frohman et al., ) and adult (satoh et al., a) astrocytes, as well as mouse astrocytes (satoh et al., b) , have been shown to express icam- in the presence of ifn- . such expression might facilitate the interaction of these cells with lymphocytes. ifn-~,-stimulated human and rat astrocytes have also been shown to express vcam- , possibly suggesting a role in lymphocytic infiltration across the blood-brain barrier into the cns (rosenman et al., i ) . although ifn- readily induces the expression of mhc molecules in astrocytes, oligodendrocytes, and resident cns microglia, !fn- -induced expression of mhc molecules in neurons is under tighter control. early evidence demonstrating the ability of brain cells, including neurons, to express mhc molecules includes experiments in which ifn-y was either added to mixed brain cultures or i~ected directly into the cns (wong et al., (wong et al., , . as detected by immunohistochemical analysis, the addition of ifn- to brain cells in culture results in % of neurons expressing mhc class i, whereas direct injection into the cns results in approx % of neurons expressing mhc class i. moreover, the response of the olb neuronal cell line to ifn-? has been investigated (joly and oldstone, ) . the olb cell line was developed by retroviral-mediated oncogene delivery to cells from the olfactory bulb (ryder et al., ) . in these cells, ifn- stimulates the expression of mhc class i both at the mrna level and on the cell surface. furthermore, in the presence of ifn- , the peptide transporters ham and ham , which are involved in the processing of class i antigens, are induced in these cells, whereas an increase in the expression of ~ -microglobulin also occurs (joly and oldstone, ) . in other studies, cell lines derived from two medulloblastomas, which are immunoreactive against neurofilament antibodies, but unreactive against gfap and s- antibodies, have also been shown to empress mhc class i and class ii antigens following exposure to ifn-y (tamura et al., ) . although these studies further demonstrated that cells of neuronal origin are intrinsically capable of expressing mhc molecules and suggest that this expression may serve a functional role, the physiological conditions under which functional neuronal mhc expression can occur have not yet been fully elucidated. toward this goal, neumann et al. ( ) have combined whole-cell patch-clamp recording techniques with single-cell rt-pcr analysis to demonstrate that in the presence of ifn- , both mhc class i and -microglobulin are significantly more inducible on electrically silent neurons compared to neurons spontaneously firing action potentials. this result suggests that functional mhc class i expression may occur only in neurons that are no longer biologically active. this would then allow these cells to be recognized and killed by cytotoxic t-lymphocytes, thus permitting active virus to be cleared from inactive neurons. this result also suggests that active neurons may not possess the potential to express functional mhc, thus allowing neurons to survive at the cost of continued viral infection (joly et al., ) . future investigation in this area will likely yield additional insight into our understanding of the functional interactions between neurons and cells of the immune system. although the majority of the studies addressing the effect of ifn-~, on neurons have been directed at examining the expression of mhc glycoproteins, several studies have reported other ifn-~,-induced neuronal consequences. as previously discussed, brosnan et al. ( ) reported a significant reduction in neural conduction and modest axonal pathology in the visual system of the rabbit following injection of ifn-,f. recently, mcmillian et al. ( ) reported that the treatment of rat primary basal forebrain mixed cultures with ifn- decreased the number of choline acetyl transferase (chat) immunopositive neurons, whereas the number of chat immunonegative neurons was unaffected. this selective neuronal killing was shown to be mediated through ifn-~,-induced microglia activation. moreover, birdsall ( ) demonstrated that following exposure to ifn-~[, two human neuroblastoma cell lines, but not cultured human cortical neurons, expressed icam. ifn-y also enhanced tnf-el-induced binding of these cells to neutrophils. finally, collins ( ) demonstrated that susceptibility to viral infection of a human cerebral cortical neuronal cell line was dramatically increased following treatment with human ifn-~, and that this increased susceptibility was owing to membrane expression of hla class i molecules. ifn--f has also been shown to affect neuronal differentiation in vitro. chang et al. ( ) demonstrated that ifn-~, retarded the death of cultured rat sympathetic neurons following nerve growth factor (ngf) deprivation. moreover, ifn-y has been shown to potentiate ngfstimulated differentiation of pc cells (improta et al., ) . barish et al. ( ) also demonstrated that ifn-y increased the differentiation of cultured cortical and hippocampal neurons. nevertheless, it is unlikely that the developing nervous system is exposed to appreciable levels of ifn- under normal conditions, such that the in vivo relevance of these observations is unclear. in contrast, there is in vivo evidence that suggests that ifn-~, might have a detrimental effect on the developing nervous system. as mentioned earlier, transgenic mice in which ifn-y was targeted to the cns demonstrate a disruption in cerebellar granule cell migration (corbin et al., ) . during development, cerebellar granule cells migrate from the external granule cell layer (egl), through the molecular layer, to the internal granule cell layer (igl) along processes extended by radial gliai cells (chuong, ) . in transgenic mice expressing ifn-? within the cns, many granule cells are still observed in the egl and in the molecular layer. this observation is reminiscent of other mouse mutants (e.g., weaver, staggerer) in which the interaction between granule cells and radial glia is disrupted (chuong, ) . perhaps the ectopic expression of ifn-~, in the cerebellum of transgenic mice has a deleterious effect on lhe interaction between granule cells and radial glia, thus disrupting the migration of these cells from the egl to the igl. further investigation is warranted to characterize the potentially deleterious developmental effects of ifn-g on the cns in more detail. recently, meda et al. ( ) suggested a role for ifn-y in alzheimer's disease. the senile plaques that develop in alzheimer's patients are characterized by the extracellular deposits of [ -amyloid protein. meda et al. ( ) demonstrated that ifn-y acted synergistically with fj-amyloid in the activation of microglia, which produce tnf-o~ and nitric oxide. in coculture experiments, it was shown that microglia activated by ifnq, and ~-amyloid caused neuronal injury. clearly, these are exciting findings that lead to the speculation that ifn-~/might also be involved in other neurodegenerative disorders. ifn-? is a multifunctional cytokine involved in regulating a variety of immune responses. this cytokine also has a multitude of effects in the cns (table ) . although normally excluded from the cns, this cytokine is present during many disorders in which the cns is affected, including the most common human demyelinating disease, ms. the presence of table effects of ifn-? on the cns disruption of myelination exacerbation of reactive gliosis disruption of the blood-brain barrier recruitment of inflammatory cells from the periphery induction of mhc expression stimulation of macrophage/microglial cells disruption of cerebellar granule cell migration this cytokine in the cns leads to an abundance of pathological effects, including decreased myelination, increased gliosis, disruption of blood-brain barrier func~on, increased mhc expression, stimulation of macrophage/microglial activation, and disruption of cerebellar granule cell migration. taken as a whole, these observations demonstrate that ifn-? can either directly or indirectly mediate many of the cns pathological effects that are observed in disorders in which immune cell traffic in the cns increases. understanding the mechanism(s) by which ifn-~,r exerts its actions on cns function will undoubtedly prove useful toward generating rational therapeutic approaches for the treatment of cns disorders in which this cytokine plays a harmful role. we thank numerous colleagues for many helpful discussions that guided the formulation of the ideas presented here. we also thank kathy toews for assistance in preparing the manuscript. the ifn-? work in brian popko's laboratory has been supported by the national institutes of health (nih) research grant roi ns , and the national multiple sclerosis society research grant rg . j. d. is supported by a training grant hd from the nih. b. p. is a recipient of an nih research career development award (ns ) from ninds. reversible inhibitory effects of interferon-y and tumouar necrosis factor-o: on oligodendroglial lineage cell proliferation and differentiation in vitro molecular cloning and expression of the human interferon-? receptor experimental allergic encephalomyelitis: a usefid model for multiple sclerosis ligand-induced assembly and activation of the gamma interferon receptor in intact cells reactive astrogliosis in the neonatal mouse brain and its modulation by cytokines, f neurosci gamma-interferon promotes differentia* tion of cultured cortical and hippocampal neurons increased production of interferon gamma and tumor necrosis factor precedes clinical manifestation in 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interferon-like virus-inhibitor induced in human leukocytes by phytohernagglutinin cytokines and murine autoimmune encephalomyelitis: inhibition or enhancement of disease with antibodies to select cytokines, or by delivery of exogenous cytokines using a recombinant vaccinia virus system inducible expression of h- and ia antigens on brain cells interferon-gamma induces the expression of h- and la antigens on brain ceils r ( ) y-interferon promotes proliferation of adult human astrocytes in vitro and reactive gliosis in the adult mouse brain in vivo differential proliferafive response of human and mouse astrocytes to gala-interferon ~ ) try_ ~nsgewic mouse model for central nervo~is system demyelknation role of interferon-y in immune cell regulation the t lymphocyte in experimental allergic encephalomyelitis key: cord- - ttps nx authors: barlas, georgios; stamatatos, efstathios title: cross-domain authorship attribution using pre-trained language models date: - - journal: artificial intelligence applications and innovations doi: . / - - - - _ sha: doc_id: cord_uid: ttps nx authorship attribution attempts to identify the authors behind texts and has important applications mainly in cyber-security, digital humanities and social media analytics. an especially challenging but very realistic scenario is cross-domain attribution where texts of known authorship (training set) differ from texts of disputed authorship (test set) in topic or genre. in this paper, we modify a successful authorship verification approach based on a multi-headed neural network language model and combine it with pre-trained language models. based on experiments on a controlled corpus covering several text genres where topic and genre is specifically controlled, we demonstrate that the proposed approach achieves very promising results. we also demonstrate the crucial effect of the normalization corpus in cross-domain attribution. authorship attribution (aa) is a very active area of research dealing with the identification of persons who wrote specific texts [ , ] . typically, there is a list of suspects and a number of texts of known authorship by each suspect and the task is to assign texts of disputed authorship to one of the suspects. the basic forms of aa are closed-set attribution (where the list of suspects necessarily includes the true author), open-set attribution (where the true author could be excluded from the list of suspects), and author verification (where there is only one candidate author). the main applications of this technology are in digital forensics, cyber-security, digital humanities, and social media analytics [ , ] . in real life scenarios the known and the unknown texts may not share the same properties. the topic of the texts may differ but also the genre (e.g., essay, email, chat). cross-domain aa examines those cases where the texts of known authorship (training set) differ with respect to the texts of unknown authorship (test set) in topic (cross-topic aa) or in genre (cross-genre aa) [ , ] . the main challenge here is to avoid the use of information related to topic or genre of documents and focus only on stylistic properties of texts related to the personal style of authors. recently, the use of pre-trained language models (e.g., bert, elmo, ulm-fit, has been demonstrated to obtain significant gains in several text classification tasks including sentiment analysis, emotion classification, and topic classification [ , , , ] . however, it is not yet clear whether they can be equally useful for style-based text categorization tasks. especially, in cross-topic aa, information about the topic of texts can be misleading. an approach based on neural network language models achieved top performance in recent shared tasks on authorship verification and authorship clustering (i.e., grouping documents by authorship) [ , ] . this method is based on a character-level recurrent (rnn) neural network language model and a multiheaded classifier (mhc) [ ] . so far, this model has not been tested in closed-set attribution which is the most popular scenario in relevant literature. in this paper, we adopt this approach for the task of closed-set aa and more specifically the challenging cases of cross-topic and cross-genre aa. we examine the use of pre-trained language models (e.g., bert, elmo, ulmfit, gpt- ) in aa and the potentials of mhc. we also demonstrate that in cross-domain aa conditions, the effect of an appropriate normalization corpus is crucial. the vast majority of previous work in aa focus on the closed-set attribution scenario. the main issues is to define appropriate stylometric measures to quantify the personal style of authors and the use of effective classification methods [ , ] . a relatively small number of previous studies examine the case of cross-topic aa. in early approaches, features like function words or part-of-speech n-grams have been suggested as less likely to correlate with topic of documents [ , ] . however, one main finding of several studies is that low-level features, like character n-grams, can be quite effective in this challenging task [ , ] . typed character n-grams provide a means for focusing on specific aspects of texts [ ] . interestingly, character n-grams associated with word affixes and punctuation marks seem to be the most useful ones in cross-topic aa. another interesting idea is to apply structural correspondence learning using punctuation-based character n-gram as pivot features [ ] . recently, a text distortion method has been proposed as a pre-processing step to mask topic-related information in documents while keeping the text structure (i.e., use of function words and punctuation marks) intact [ ] . there have been attempts to use language modeling for aa including traditional n-gram based models as well as neural network-based models [ , , ] . the latter is closely related to representation learning approaches that use deep learning methods to generate distributed text representations [ , ] . in all these cases, the language models are extracted from the texts of known authorship. as a result, they heavily depend on the size of the training set per candidate author. an aa task can be expressed as a tuple (a, k, u ) where a is the set of candidate authors (suspects), k is the set of known authorship documents (for each a ∈ a there is a k a ⊂ k) and u is the set of unknown authorship documents. in closedset aa, each d ∈ u should be attributed to exactly one a ∈ a. in cross-topic aa, the topic of documents in u is distinct with respect to the topics found in k, while in cross-genre aa, the genre of documents in u is distinct with respect to the genres found in k. bagnall introduced an aa method [ ] and obtained top positions in shared tasks in authorship verification and authorship clustering [ , ] . the main idea is that a character-level rnn is produced using all available texts by the candidate authors while a separate output is built for each author (mhc). thus, the recurrent layer models the language as a whole while each output of mhc focuses on the texts of a particular candidate author. to reduce the vocabulary size, a simple pre-processing step is performed (i.e., uppercase letters are transformed to lowercase plus a symbol, punctuation marks and digits are replaced by specific symbols) [ ] . the model, as shown in fig. , consists of two parts, lm and mhc. lm consists of a tokenization layer and the pre-trained language model. mhc comprises a demultiplexer which helps to select the desirable classifier and a set of |a| classifiers, where |a| is the number of candidate authors. each classifier has n inputs, where n is the dimensionality of the lm's representation, and v outputs, where v is the size of the vocabulary. the vocabulary is created using the most frequent tokens. the output of lm is a representation of each token in text. if the token exists in vocabulary its representation propagates to mhc, otherwise is ignored (despite the fact that the representation is not further useful, the calculations that took place in lm to produce the representation are mandatory to update the hidden states of the pre-trained language model). if the sequence of input tokens is modified, the representation is also affected. the function of lm remains the same during training, calculation of normalization vector n and test phase. the mhc layer during training propagates the lm's representations only to the classifier of the author a which is the author of the given text. then the cross-entropy error is back-propagated to train mhc. during the test phase (as well as the calculation of normalization vector n explained below) the lm's representation is propagated to all classifiers. the mhc calculates the cross-entropy h(d, k a ) for each input text d and the training texts of each candidate author k a . the lower cross-entropy is, the more likely for author a to write document d. however, the scores obtained for different candidate authors are not directly comparable due to different bias at each head of mhc. to handle this problem, a normalization vector n is used which is equal to zero-centered relative entropies produced by using an unlabeled normalization corpus c [ ] : where |c| is the size of the normalization corpus. note that in cross-domain conditions it is very important for documents in c to include documents belonging to the domain of d. then, the most likely author a for a document d ∈ u is found using the following criterion: in this paper, we extended bagnall's model in order to accept tokens as input and we propose the use of a pre-trained language model to replace rnn in the aforementioned aa method. the rnn proposed by bagnall [ ] is trained using a small set of documents (k for closed-set aa). in contrast, pre-trained language models have been trained using millions of documents in the same language. moreover, rnn is a character-level model while the pre-trained models used in this study are token-level approaches. more, specifically, the following models are considered: -universal language model fine-tuning (ulmfit): it provides a contextual token representation obtained from a general domain corpus of millions of unlabeled documents [ ] . it adopts left-to-right and right-to left language modeling in separate networks and follows auto-encoder objectives. -embeddings from language models (elmo): it extracts context-sensitive features using a left-to-right and a right-to-left language modeling [ ] . then, the representation of each token is a linear combination of the representation of each layer. -generative pretrained transformer (gpt- ): it is based on a multilayer unidirectional transformer decoder [ ] . it applies a multi-headed selfattention operation over the input tokens followed by position-wise feedforward layers [ ] . -bidirectional encoder representations from transformer (bert): it is based on a bidirectional transformer architecture that can better exploit contextual information [ ] . it masks a percentage of randomly-selected tokens which the language model is trained to predict. we use the cmcc corpus introduced in [ ] and also used in previous crossdomain aa works [ , ] . cmcc is a controlled corpus in terms of genre, topic and demographics of subjects. it includes samples by undergraduate students as candidates authors (a), covering six genres (blog, email, essay, chat, discussion, and interview) and six topics (catholic church, gay marriage, privacy rights, legalization of marijuana, war in iraq, gender discrimination) in english. to ensure that the same specific aspect of the topic is followed, a short question was given to subjects (e.g., do you think the catholic church needs to change its ways to adapt to life in the th century?). in two genres (discussion and interview) the samples were audio recordings and they have been transcribed into text as accurately as possible maintaining information about pauses, laughs etc. for each subject, there is exactly one sample for each combination of genre and topic. more details about the construction of this corpus are provided in [ ] . in this study, our focus is on cross-topic and cross-genre aa. in cross-topic, we assume that the topic of training texts (k) is different from the topic of test texts (u ) while all texts (both k and u ) belong in the same genre. similar to [ ] and [ ] , we perform leave-one-topic-out cross-validation where all texts on a specific topic (within a certain genre) are included in the test corpus and all remaining texts on the remaining topics (in that genre) are included in the training corpus. this is repeated six times so that all available topics to serve exactly once as the test topic. mean classification accuracy over all topics is reported. similar to cross-topic, in cross-genre we perform leave-one-genre-out crossvalidation as in [ ] , where all texts on a specific genre (within a certain topic) are included in the test corpus and all remaining texts on the remaining genres (in that topic) are included in the training corpus. the number of available genres is also six like topics, and though we repeat the leave-one-genre-out cross-validation six times and report the mean classification accuracy. in both scenarios, crosstopic and cross-genre, the candidates authors set a consists of undergraduate students as mentioned in sect. . . all the examined models use a mhc on top of a language modeling method. first, we study the original bagnall's approach where a character-level rnn is trained over k. then, each one of the pre-trained language models described in previous section. in our experiments, all of the pre-trained lms was fine-tuned for the specific aa task with mhc as classifier without further training the language model , since our goal is to explore the potential of pre-trained models obtained from general domain corpora. in mhc, each author corresponds to a separate classifier with n inputs and m outputs, where n is the dimensionality of text representation, table , and m is equal to vocabulary size v . during training, each classification layer is trained only with the documents of the corresponding author. the vocabulary is defined as the most frequent tokens in the corpus. these are less likely to be affected by topic shifts and the reduced input size increases the efficiency of our approach. the selected values of v are , , k, k and k. each model used its own tokenization stage except from elmo (where ulmfit's tokenization was used). note that rnn is a character-level model while all pre-trained models are token-based. since rnn is trained from scratch for a corpus of small size, it is considerably affected by initialization. as a result, there is significant variance when it is applied several times to the same corpus. to compensate this, we report average performance results for repetitions. regarding the training phase of each method, we use epochs for rnn and examine four cases for the pre-trained models: the minimal training of epoch and the cases of , and epochs of training. table presents the leave-one-topic-out cross-validation accuracy results for each one of the six available genres as well as the average performance over all genres for each method. two cases are examined: one using the (unlabeled) training texts as normalization corpus (c = k) and another where the (unlabeled) test texts are used as normalization corpus (c = u ). the former means that c includes documents with distinct topics with respect to the document of unknown authorship while the latter ensures that there is perfect thematic similarity. as can be seen, the use of a suitable normalization corpus is crucial to enhance the performance of the examined methods. as concerns individual pre-trained language models, bert and elmo are better able to surpass the rnn baseline while ulmfit and gpt- are not that competitive. in addition, bert and elmo methods need small number of training epochs while ulmfit and gpt- improve with increased number of epochs. table also shows the corresponding results from previous studies on crosstopic aa using exactly the same experimental setup. these baselines are based on character -grams features and a svm classifier (c g-svm) [ ] , a compression-based method (ppm ) [ ] , and a method using text distortion to mask thematic information (dv-ma) [ ] . as can be seen, when c = u all of the examined methods surpass the best baseline in average performance and the improvement is high in all genres. it is remarkable that all models except ulmfit achieve to surpass the baselines (in average performance) even when c = k. from the aspect of vocabulary size, in contradiction to the state of the art [ ] , where the best results achieved for vocabularies that consisted of less than k words (most frequent), in our set up the most appropriate value seems to be above k. despite the gap between k and k words in vocabulary size bert and elmo have minor difference in accuracy indicating that above k words the affect of vocabulary size is minor. gpt- continues to increment the accuracy and ulmfit started to decrement for values above k words, table . experiments with values over k were prohibitive due to runtime of training, with k words the runtime was approximately days for each model running on gpu. from the aspect of training epochs, bert and elmo achieved their best performance in c = k case with minimal training. in c = u case their performance is slightly affected by the number of training epochs. this behavior raises the question of over-fitting. as mentioned in sect. the selection criterion eq. , is based on the cross-entropy of each text. mhc is trained on predicting the text flow and thus the cross-entropy decreases after each epoch of training. having in mind the cross-entropy, if we have a second look on fig. , the case of overfitting is rejected since the behavior of accuracy in relevance with the number of training epochs (indicated by the shape of point) do not have the characteristics of over-fitting (increment of training epochs decrements the accuracy). the experiments on cross-genre performed on the same set up as in cross-topic. table presents the accuracy results on leave-one-genre-out cross-validation for each one of the six available topics and the average performance over all topics, similar to table . based on the results of sect. . the most reasonable value of v in order to check the performance of each method is v = k. the case of v = k is very time consuming without offering valuable gain and below k the performance is not remarkable. for the experiments on cross-genre the values of k and k were selected for v . comparing the two cases, the results with v = k surpass in all experiments the results with v = k and thus we selected to present only the case of v = k on table . table . accuracy results (%) on cross-genre aa for vocabulary size k (v = k) and each topic (church (c), gay marriage (g), war in iraq (i), legalization of marijuana (m), privacy rights (p), gender discrimination (s)). the reported performance of the baseline models (only available in average across all topics) is taken from the corresponding publications. bert and elmo achieved high results as expected from their performance on cross-topic, with elmo achieving the highest accuracy result. unexpectedly, ulmfit which had the worst performance in cross-topic achieved the second best performance. gpt- performed lower than rnn baseline in both cases of c = k and c = u . comparing table and table is noticeable that elmo and bert are more stable in performance than gpt- and ulmfit. the main difference between the former and the latter is the directionality, the former two are bidirectional while the latter are unidirectional, we suspect that this is the main reason that affects the stability in performance. in this paper, we explore the usefulness of pre-trained language models in crossdomain aa. based on bagnall's model [ ] , originally proposed for authorship verification, we compare the performance when we use either the original characterlevel rnn trained from scratch in the small-size aa corpus or pre-trained tokenbased language models obtained from general-domain corpora. we demonstrate that bert and elmo pre-trained models achieve the best results while being the most stable approaches with respect to the results in both scenarios. a crucial factor to enhance performance is the normalization corpus used in the mhc. in cross-domain aa, it is very important for the normalization corpus to have exactly the same properties with the documents of unknown authorship. in our experiments, using a controlled corpus, it is possible to ensure a perfect match in both genre and topic. in practice, this is not always feasible. a future work direction is to explore how one can build an appropriate normalization corpus for a given document of unknown authorship. other interesting extensions of this work is to study the effect of extending fine-tuning to language model layers and focus on the different layers of the language modeling representation. author identification using multi-headed recurrent neural networks bert: pre-training of deep bidirectional transformers for language understanding learning stylometric representations for authorship analysis language models and fusion for authorship attribution authorship attribution using a neural network language model person identification from text and speech genre samples universal language model fine-tuning for text classification authenticating the writings of julius caesar distributed language representation for authorship attribution author identification on the large scale domain independent authorship attribution without domain adaptation surveying stylometry techniques and applications proceedings of the conference of the north american chapter of the association for computational linguistics: human language technologies language models are unsupervised multitask learners authorship attribution for social media forensics overview of pan' not all character n-grams are created equal: a study in authorship attribution domain adaptation for authorship attribution: improved structural correspondence learning cross-topic authorship attribution: will out-of-topic data help? a survey of modern authorship attribution methods on the robustness of authorship attribution based on character n-gram features masking topic-related information to enhance authorship attribution overview of the pan/clef evaluation lab attention is all you need key: cord- - tl f tt authors: liu, guangliang; wang, qun; tong, tiegang; xiao, yihong; bai, yu; liu, shengwang; wu, donglai title: construction and functional test of a chicken mhc-i (bf * )/peptide tetramer date: - - journal: vet immunol immunopathol doi: . /j.vetimm. . . sha: doc_id: cord_uid: tl f tt the major histocompatibility complex class i (mhc class i) peptide tetramer is a sensitive and valuable tool to evaluate antigen-specific cytotoxic t lymphocytes (ctls) of many animal species. to date, no chicken mhc class i peptide tetramer has been reported. in this report, we describe construction and functional evaluation of a chicken mhc-i (bf * )/peptide tetramer. to construct the chicken mhc class i peptide tetramer, genes of the chicken mhc-i α chain (bf * ) and β microglobulin (chβ m) were synthesized by rt-pcr from the total rna of pbmcs and the signal sequences were deleted. the bf * was then fused with the bira substrate peptide (bsp) sequence at the c terminus. next, the synthesized pcr products of bf * and chβ m were cloned into the expression vector pet- a (+) and expressed in escherichia coli strain bl (de ). highly purified bf * -bsp heavy chain and chβ m were obtained by a ni( +) nta column affinity purification, yielding approximately . mg of bf * -bsp and . mg of chβ m per g of the pelleted bacteria. the purified bf * -bsp heavy chain and chβ m were refolded with synthetic peptide originated from infectious bronchitis virus nucleoprotein (ibv n( – )) in refolding buffer to generate the monomer of bf * /peptide complex. the monomer was then biotinylated and tetramerized using pe-labeled streptavidin. upon functional evaluation of the construct by using flowcytometry, we observed that . % of ctls were specific to ibv nucleoprotein. this demonstrates that the ctl response of ibv-infected chicks could effectively be evaluated using the prepared mhc-i bf * /peptide tetramer. the classical mhc class i is a membrane surface protein found on virtually all cells in the body, and its main function is to bind antigenic peptides and present them on the surface of virally infected or tumor cells (kindt et al., ) . ctls recognize antigenic peptides, in the context of mhc class i molecules on the cell surface, viatheir tcr and, upon recognition, lyse these target cells. therefore, it is important to quantitatively measure antigen-specific t-cells accurately and promptly. until the development of mhc-i tetramer technology, the main techniques for functional and quantitative measurement of antigen-specific t-cells were limiting dilution assay (lda), enzyme-linked immunospot assay (elispot), and intracellular cytokine staining (ics) in conjunction with flow cytometry (taswell, ; jung, ; scheibenbogen et al., scheibenbogen et al., , . www.elsevier.com/locate/vetimm available online at www.sciencedirect.com veterinary immunology and immunopathology ( ) - altman et al. ( ) first described the use of hla (human leucocyte antigen)-peptide tetrameric complexes to directly visualize antigen-specific ctls by flow cytometry. this technique is not dependent on proliferation of the cells and therefore allows the direct quantification of antigen-specific ctls without in vitro manipulation (meidenbauer et al., ) . to date, this technique has been applied successfully to study cellmediated immunity on human (hla), mouse (h- , mouse mhc), macaque (mamu, macaque mhc), chimpanzee (patr, chimpanzee mhc), horse (ela, equine leucocyte antigen), and pig (sla, swine leucocyte antigen) (dunbar et al., ; donahoe et al., ; kalergis et al., ; oleksiewicz et al., ; skinner et al., ; meidenbauer et al., ; mealey et al., ) . the chicken mhc class i (bf and rfp-y) gene sequences have been previously reported (kaufman et al., ; briles et al., ; miller et al., ) . moreover, the genomic structure of leghorn chicken mhc has also been reported by kaufman et al. ( ) . within a kb dna segment, there are two loci-encoding class i heavy chains named bf and bf . the bf locus is dominantly expressed, whereas the bf locus is less expressed and has a lower mrna transcript (kaufman et al., ; miller et al., ) . however, despite this knowledge of the mhc-i sequence and genomic structure, no chicken mhc-i tetramer has been reported. in this communication, we report the first construction and functional evaluation of a chicken mhc-i/peptide tetramer. et al. ( ) reported the first defined coronavirus t-cell epitope which is located in the amino acid sequence - of the infectious bronchitis virus nucleoprotein (ibv n - ). the ibv n - (wrrqaryk) derived from ibv h strain was synthesized at gl biotech co. (shanghai, china) purified to purity > % by fplc. all bf * white leghorn spf (specific pathogen free) chickens used in this study were obtained from the experimental animal center of harbin veterinary research institute, and housed in isolator cages. the ibv h strain was titrated and stocked in our laboratory. . . cloning the bf * and chb m gene from chicken pbmcs total rna was extracted from pbmcs (peripheral blood mononuclear cells) with trizol (invitrogen, san diego, ca) from a spf chicken. the full-length bf * and chb m cdna were cloned by rt-pcr. oligonucleotide primers were designed according to the entire coding region of bf * and chb m cdna sequences reported in genbank (accession numbers: l and m ); the sequences of forward and reverse primers of the bf * and chb m were as follows: bf * forward: -tgc agc ggt gcg agg cga t- , bf * reverse: -tta ttt cac agg aag cag tgc- ; chb m forward: -aca gcg gag cca tgg gga a- , chb m reverse: -atc ccg ggc aca gct cag a- . reverse transcription reaction was conducted at c for h using amv reverse transcriptase xl (takara, dalian, china). pcr amplifications were performed using the la-taq dna polymerase system (takara, dalian, china) under the following conditions: starting at c for min for denaturation, followed by cycles at c for min, at c for min, at c for min, and then at c for min for extension. the pcr products were inserted into the pmd -t vector (takara, dalian, china) according to the manufacturer's instructions. the positive clones with the correct sequence were named as pmd-bf * and pmd-chb m. the signal peptides of bf * and chb m genes were predicted with signalp (bendtsen et al., ) and the sequence of the signal peptide in the bf * gene was deleted. in addition, a -amino acid residue substrate peptide for bira-dependent biotinylation was fused to the cooh terminus of bf * through the ecori and hindiii site adapters with the forward primer -gga att cat ggg gcc gtg cgg gg- and the reverse primer -gcg caa gct ttt aac gat gat tcc aca cca ttt tct gtg cat cca gaa tat gat gca ggatgg agg ggt tgc tcc cgg g- , using the pmd-bf * plasmid as the template. the signal peptide of the chb m gene was also deleted using ecori and hindiii site adapters with the forward primer -gga att cat ggg gaa ggc ggc ggc- and the reverse primer -gcg caa gct ttt aga act cgg gat ccc a- , using the pmd-cb m plasmid as the template. the amplified dnas were digested with ecori and hindiii, and ligated into prokaryotic expression vector, pet- a (+) (novagen, madison, wi) at the ecori/hindiii site. clones with the correct inserts were identified using pcr and restriction analysis of vectors carrying the recombinant dna. finally, the nucleotide sequences of the selected clones were confirmed by dna sequence analysis. the positive clones were named as pet-bf * -bsp and pet-chb m. for protein expression, recombinant plasmids pet-bf * -bsp and pet-chb m were transformed into escherichia coli strain bl (de ) and plated on lbagar supplemented with kanamycin ( mg/ml). the transformants were grown overnight at c in lb supplemented with kanamycin ( mg/ml) and the bacteria were subcultured the following morning at c. iptg ( mm) was added when the cultures reached od = . . four hours after induction, the samples were harvested by centrifugation at rpm (f rotor, beckman) for min and resuspended with mm tris-hcl (ph . ), sonicated, and spun down. the inclusion bodies were washed twice with mm tris-hcl (ph . ) and rinsed with mm hepes (ph . ), m urea, and mm nacl. the procedure used for the % tricine-sds-page has been described (schagger and jagow, ) . the protein bands were visualized by staining with coomassie brilliant blue (cbb). the quantity of the products was calculated by comparison of the density to that of the protein molecular mass standards (concentrations of the markers are described in the manufacturer's manual, ferments uab, lithuania). for western blot analysis, the proteins on the gel were blotted onto a nc membrane (pall-gelman, ny, usa). the membrane was blocked for h at room temperature with phosphate-buffered saline (pbs) with % bovine serum albumin (bsa) (sigma, louis, mo, usa), then incubated for h with : diluted anti-his monoclonal antibody (novagen, madison, wi, usa), followed by incubation with an hrp-conjugated anti-mouse igg reagent (sigma, louis, mo, usa). bound antibody was visualized using diaminobenezidene (dab). the inclusion body fraction which was solubilized in mm hepes (ph . ), m urea, and mm nacl was loaded directly onto ml ni-nta agarose beads (novagen), which had been equilibrated with binding buffer ( mm tris-hcl (ph . ), mm nacl, and mm imidazole). before loading the lysate onto the ni-nta agarose beads, the concentration of imidazole (ph . ) was added up to mm. the beads were rotated with the inclusion body fraction for h at c, unbound protein was collected, and the beads were washed with volumes of wash buffer ( mm tris-hcl (ph . ), . m nacl, mm imidazole, m urea, % glycerol, and . mm pmsf). his -tagged protein was eluted with the wash buffer containing mm imidazole. the concentrations of purified bf * -bsp and chb m were determined using the bradford method with bsa as a standard. construction of monomeric and tetrameric bf * peptide complexes were carried out according to the protocol described by dirk h. busch (http://www. mikrobio.med.tu-muenchen.de/forschung/projekte/ index.htm). briefly, bf * -bsp heavy chain and chb m were refolded with synthetic peptide ibv n - in refolding buffer, consisting of mm tris-hcl (ph . ), mm l-arginine, mm edta, mm reduced glutathione, . mm oxidized glutathione, and . mm phenylmethysul-fonylfluoride (pmsf) per ml. the refolding mixture was stirred and incubated at c for h and then directly concentrated and desalted using ultrafiltration system and micro spin tubes (amicon, usa). the folded product was then subjected to enzymatic biotinylation by bira enzyme (avidity denver co. usa) at c overnight. after biotinylation, the sample was run over a gel filtration column to purify the bf * /peptide fraction. the tetrameric complexes of biotinylated bf * /peptide were produced by mixing purified biotinylated monomer with pe-labeled streptavidin at a molar ratio of : . then the resulting bf * /peptide tetramer was further purified with kda mwco spin tube (amicon, usa) and stored at c in pbs with a cocktail of protease inhibitors: mm pepstatin, mm leupeptin, . % naazide, and mm edta. pbmcs from five infected or five uninfected spf chicks were used as the source of effector cells for flow cytometry assay. the chicks were inoculated at weeks by the nasal-oral routine with . egg infectious doses (eid ) of the ibv h strain. anticoagulated blood from chicks was collected at days post-inoculation (p.i.) as described elsewhere (seo and collisson, ) . pbmcs were isolated by centrifuging ml of the anticoagulated blood for min at rpm through ml of ficoll-hypaque gradient (histopaque, sigma, usa). viable cells were collected from the interface and washed three times with pbs (ph . ). the pbmcs, from each group, were pooled and subjected to flow cytometry to detect the antigen-specific ctl. for immunofluorescence staining, cells were incubated with anti-chicken cd monoclonal antibody (southern biotech, birmingham, usa) for min at c and then washed three times in pbs containing . % bovine serum albumin (bsa). an fitc-conjugated f(ab') fragment of goat anti-mouse igg (southern biotech, birmingham, usa) was used as the second antibody, and the cells were washed three times. the cells were then stained with ml of : diluted pe-labeled bf * -peptide tetramer at c for min and then washed twice. stained cells were analyzed with a facscan flow cytometer (becton dickinson, san jose, ca, usa). all flow staining and fcm assays were repeated in at least three independent experiments. the fluorescence intensity (fi) of cells was analyzed with cellquest software (becton dickinson). the rt-pcr products of bf * and chb mencoding genes were separated by electrophoresis in % agarose gel. specific bands at positions corresponding to about bp and bp were detected, which was in accordance with expectation. the bf * and chb m genes were sequenced and the results showed that they were c + g rich ( . % and . %, respectively). additionally, the first bp sequences of both genes, with . % and . % c + g, respectively, encode the signal peptides. also, of amino acid residues in each signal peptide were strongly hydrophobic. the sds-page analysis showed that the transformed cells with pet-bf * -bsp produced a large amount of the protein at a mass of about kda (fig. , lane ) . similarly, the analysis of chb m showed that there was a protein expression at position appropriate for a mass of about kda (fig. , lane ) . the soluble analysis showed that the bf * -bsp and chb m were located mainly in the insoluble fraction in the cells as inclusion bodies. the molecular mass, showed by the mobility of the recombinant proteins observed by sds-page, was in accordance with that predicted from the amino acid sequence. western blot assay showed that there was strong hybridized signal where the expressed proteins of each of the two constructions localized, while there was no such signal at the corresponding position in the negative control cells. using the pet- a (+) vector, we introduced a his tag at the nh terminus of bf * -bsp and chb m. thus, the protein can be purified using ni-nta affinity material. both bf * -bsp and chb m proteins were purified using ni-nta agarose columns under denaturing conditions. fractions of eluted proteins were analyzed by sds-page and identified after staining with cbb. the analysis revealed that single-step elution using standard conditions resulted in above % purity of both protein fractions and their molecular masses were about kda and kda, respectively, by a comparison of the protein molecular weight standards (fig. ) . typically, about . mg of bf * -bsp and . mg of chb m were recovered from g of pelleted cells measured by bca assay. . . detection of ctl with soluble bf * -peptide tetramer in vitro bf * -bsp heavy chain and chb m were refolded with ibv n - in refolding buffer to generate the monomer of bf * -peptide complex. determined by bca assay, mg bf * -peptide monomer was recovered after the gel filtration. the monomer was biotinylated, and tetramerized with pe-labeled streptavidin at a molar ratio of : . after further purification with kda mwco spin tube, . mg/ml pe-labeled bf * -peptide tetramer were obtained. in order to confirm whether the bf * -peptide tetramer is able to bind to specific ctls, the pbmcs from either ibv h infected or uninfected chickens were stained with the prepared pe-labeled bf * -peptide tetramer, and subjected to analysis by flow cytometry. the frequency of tetramer stained ctls days post-infection was . % ( fig. a) , while the uninfected control was only . % (fig. b) . these results demonstrate that the prepared bf * -peptide tetramer can be used for detection of specific ctls in chickens. cd + t-cells mediate defense against primary infection of microbial pathogens and also provide long-term protective immunity (serbina and pamer, ) . quantitative analyses of antigen-specific t-cells have provided important information on the course of immune responses (owen et al., ; maier et al., ; murali-krishna et al., ) . before the development of the mhc-i tetramer technology, the main techniques for functional and quantitative measurements of antigen-specific t-cells were lda, elispot, and ics in conjunction with flow cytometry (taswell, ; jung, ; scheibenbogen et al., scheibenbogen et al., , . lda requires at least - days in vitro culture of the t-cells of interest in conjunction with cytolytic testing (taswell, ) . this method is notoriously subject to variability, which probably reflected the frequency of specific cells, their expansion potential in vitro, and the ability to lyse appropriate targets (rehermann et al., a,b) . using elispot, some peptide-specific tcells, which either secrete cytokines levels below the assay's detectable limit or have cytokine profiles outside of those being assayed, may not be detected. lastly, intracellular cytokine staining of peptide-stimulated t-cells usually leads non-viablity of the t-cells being assayed (jung, ) . the advent of peptide-mhc tetrameric complexes heralds a new era in the study of antigen-specific t-cells and their role in viral infections. because of low affinity and fast off-rates of the mhc-peptide ligand to the tcr, direct staining of tcells seemed unachievable. altman et al. ( ) constructed soluble mhc class i/peptide molecules linked together to form tetramers that were able to bind to the matching tcr with higher avidity than the sum of the single monomeric affinities. this stable structure allows enumeration of antigenspecific t-cells without prior in vitro expansion, in addition, to phenotypical analysis using flow cytometry and functional studies on tetramer sorted t-cell clones (altman et al., ) . additionally, the mhc class i tetramer staining can also be used in situ to visualize antigen-specific cd + t-cells directly in tissues, leaving their spatial relationship to other cells intact (haanen et al., ; skinner et al., ) . to construct the chicken mhc class i peptide tetramer, the bf * -bsp and chb m were expressed in prokaryotic expression system and purified on the denatured condition. then the two subunits refolded to form a bf * /peptide monomeric complex by dilution method in the presence of an antigenic peptide ibv n - . we adopted a procedure established by john altman (nih tetramer facility, www.niaid.nih.gov/ reposit/tetramer/index.html) into our protocol. the mhc complexes, in the refolding buffer, were directly concentrated instead of dialysing. this approach allows us to reuse the flowthrough, which contains most of the peptide, for a second (or even third) refolding. the monomeric complex was tetramerized by binding to fluorescently labeled streptavidin to generate bf * / peptide tetramer. the results of flow cytometer analysis demonstrate that the prepared bf * -peptide tetramer can be used for detection of ibv nucleoprotein-specific ctls in chicks. in this paper we have described our construction of a chicken mhc-i tetramer, which can used to evaluate the antigen-specific ctls. when the t-cells interact with the target cells, the t-cells recognize both the antigen displayed in cleft and mhc molecules. so, in this experiment, the positive signal detected in the two-color flow cytometry assay was enough to illustrate the peptide-specific ctl. however, it could be better to understand the peptide-specific ctl if there are two more controls, that is, to use the chickens with different mhc types and chickens infected with a different virus. the chicken mhc-i tetramer constructed in this paper can be used in wide variety experiments. besides its potential use in the study of the overall evaluation of the t cell immune response, this tetramer can also be used to track the kinetics of the ibv-specific ctl response. also, the bf tetramer can be used for the evaluation on ctl response specific to other chicken viruses by replacing the peptide in the peptide-binding groove of bf during refolding procedure. additionally, few bf -restricted t cell epitopes have been identified, thus far. the prepared bf * -peptide tetramer can be used as an effective tool for mapping the bf -restricted t cell epitopes. once more bf restricted t cell epitopes have been mapped, this bf * -peptide tetramer can be more effectively used to investigate the ctl response to viral infection in the chicken. in summary, the generation of bf * /peptide tetramer lays the foundation for further study on the characteristic of antigenic peptide presented by bf * , and provides a valuable tool for the research on cell-mediated immune of virus-related diseases in chicks. phenotypic analysis of antigen-specific t lymphocytes localization of a t-cell epitope within the nucleocapsid protein of avian coronavirus a polymorphic system related to but genetically independent of the chicken major histocompatibility complex direct measurement of cd + t cell responses in macaques infected with simian immunodeficiency virus direct isolation, phenotyping and cloning of low-frequency antigen-specific cytotoxic t lymphocytes from peripheral blood in situ detection of virus-and tumor-specific t-cell immunity a simplified procedure for the preparation of mhc-peptide tetramers: chemical biotinylation of an unpaired cysteine engineered at the c-terminus of mhc-i different features of the mhc class i heterodimer have evolved at different rates. chicken b-f and beta -microglobulin sequences reveal invariant surface residues the chicken b locus is a minimal essential major histocompatibility complex kuby immunology limiting dilution analysis of proliferating and helper t cells in the in vivo immune response to klh: derepression of helper t cells at moderately increased frequencies early detection of dominant env-specific and subdominant gag-specific cd + lymphocytes in equine infectious anemia virus-infected horses using major histocompatibility complex class i/peptide tetrameric complexes direct visualization of antigen-specific t cells using peptide-mhc-class i tetrameric complexes two mhc class i and two mhc class ii genes map to the chicken rfp-y system outside the b complex nomenclature for the chicken major histocompatibility (b and y) complex. immunogenetics counting antigenspecific cd t cells: a reevaluation of bystander activation during viral infection development of a rapid in vitro protein refolding assay which discriminates between peptide-bound and peptidefree forms of recombinant porcine major histocompatibility class icomplex (sla-i) limiting dilution analysis of the specificity of influenza-immune cytotoxic t cells differential cytotoxic tlymphocyte responsiveness to the hepatitis b and c viruses in chronically infected patients quantitative analysis of the peripheral blood cytotoxic t lymphocyte response in patients with chronic hepatitis c virus infection tricine-sodium dodecyl sulfatepolyacrylamide gel electrophoresis for the separation of proteins in the range from to kda a sensitive elispot assay for detection of cd + t lymphocytes specific for hla class i-binding peptide epitopes derived from influenza proteins in the blood of healthy donors and melanoma patients quantitation of antigen-reactive t cells in peripheral blood by ifn-gamma-elispot assay and chromium-release assay: a fourcentre comparative trial specific cytotoxic t lymphocytes are involved in in vivo clearance of infectious bronchitis virus quantitative studies of cd + t-cell responses during microbial infection cutting edge: in situ tetramer staining of antigenspecific t cells in tissues limiting dilution assays for the determination of immunocompetent cell frequencies i. data analysis key: cord- -c i a f authors: moore, tamson v.; nishimura, michael i. title: improved mhc ii epitope prediction — a step towards personalized medicine date: - - journal: nat rev clin oncol doi: . /s - - - sha: doc_id: cord_uid: c i a f numerous neoepitope-based vaccination strategies are in testing for clinical use in the treatment of cancer. rapid identification of immunostimulatory neoantigen targets hastens neoantigen vaccine development. papers recently published in nature biotechnology describe two independent machine-learning-based algorithms that demonstrate improved identification of mhc class ii-binding peptides. herein, we outline the benefits of these algorithms and their implications for future immunotherapies. immunotherapies, including immunecheckpoint inhibitors (icis), can induce durable tumour regression and even disease remission in a diverse subset of patients with chemotherapy-refractory metastatic cancers. the efficacy of icis is generally greater in cancer types with higher median numbers of somatic mutations , which can generate neoantigens that are targets for specific cd + and/or cd + t cells . evidence indicates that cd + t cell responses to mhc class ii (mhc ii)-restricted antigens are required for robust responses to icis and that neoantigen vaccines can enhance cd + t cell responses . to develop effective neoantigen vaccines, it is essential to identify neoantigen epitopes (neoepitopes) that will bind to mhc ii molecules and be presented to cd + t cells. whereas the presence and expression of neoantigen proteins can be identified through sequencing of the tumour exome, the neoepitopes presented by mhc ii molecules must be either discovered empirically using expensive and time-consuming mass spectrometry (ms) techniques or predicted using software-based estimations of peptide-mhc ii binding affinity. smn align (p < × − ) and netmhciipan (p < . ), respectively , , which are two commonly used mhc ii-binding prediction algorithms. in both studies , , large (approximately , - , peptides) ms-based datasets of mhc ii-presented peptides were used to train independent algorithms to estimate peptide-mhc ii binding. however, the algorithms have different advantages. for example, maria incorporates tissue-specific gene-expression levels, in order to account for effects of the abundance of protein on the likelihood of peptide presentation by mhc ii molecules, whereas mixmhc pred does not. mixmhc pred, with modec, used a larger training dataset (~ , peptides compared with ~ , for maria), encompassing more cell types, and enables the identification of peptides that bind to the different mhc ii isotypes (encoded by the hla-dr, hla-dp and hla-dq genes), without retraining, whereas the maria benchmarks were established using versions of the algorithm trained independently for different mhc ii isotypes. the large datasets used to train these algori thms improved both the accuracy and specificity of mhc ii-binding predictions , . ms is becoming increasingly popular as a method of identifying the peptidome from a variety of tumour types and the resultant increased dataset availability for the training of mhc ii-binding algorithms will probably further improve the accuracy of these algorithms over time. nonetheless, a key caveat of using software-based modelling instead of empirical testing is the inability to identify outliers -mhc ii-binding algorithms model the average ways in which most peptides bind (thus identifying recurrent motifs) and are likely to exclude peptides that bind mhc ii molecules in unusual ways. with regard to the clinical goal of predicting cd + t cell reactivity, mixmhc pred identified a higher number of true immuno genic mhc ii-binding epitopes than netmhciipan, as demonstrated in vitro using cd + t cells in the november issue of nature bio technology, the authors of two independent studies , described novel machine-learning algori thms for identifying mhc ii-binding peptides. chen et al. developed the mhc analysis with recurrent integrated architecture (maria) platform, in which neural network-based models trained on large ms-based peptide datasets are used to generate a peptide presentation score, given inputs of a query peptide sequence and corresponding gene name in addition to mhc ii (hla-d) alleles. racle et al. developed modec, a motif decon volution algorithm with conceptual similarity to convolutional neural networks, to identify mhc ii-binding motifs, binding core offset preferences and peptide cleavage motifs from large ms-based peptidome datasets encompassing hla-dr, hla-dq and hla-dp alleles. the deconvoluted peptidomic datasets were then used to train a prediction algorithm, mixmhc pred, which returns an mhc ii binding score for a given peptide sequence and hla-d allele. when tested on known mhc ii-binding epitopes and decoy epitopes, both the maria and mixmhc pred algorithms had significantly improved predictive accuracy compared with nature reviews | clinical oncology isolated from two patients with melanoma . similarly, maria successfully identified patient-specific neo epitopes with reactive cd + t cells in two of three patients with mantle cell lymphoma . although these datasets are small, they indicate that both algorithms can accurately predict peptides that can stimulate cd + t cells. one remaining hurdle, however, is that only a minority of predicted mhc ii-binding peptides induced cd + t cell responses ( . % ( of ) with mixmhc pred and . % ( of ) with maria) , . notably, in the majority of previous studies, < % of potential neoepitopes were found to stimulate t cells, even after preselection for mhc binding . however, the absence of a t cell response should not be automatically attributed to the production of false-positive predictions by an algorithm. mhc ii-binding peptides can fail to activate t cell responses for several reasons. first, the development of immunity to an mhc ii-bound antigen is dependent on the presence of t cells bearing a cognate t cell receptor (tcr). t cells are able to recognize a large pool of antigens , made more numerous by cross-reactivity ; however, with the proteinogenic amino acids, . × - . × peptides comprising - amino acid residues (mhc ii-binding peptides can contain - resi dues) could potentially exist, exceeding the number of t cells in the human body (estimated to be < ). therefore, few or no reactive t cells might exist for some peptides, resulting in the absence of detectable cd + responses. second, the assays used to test peptide immunogenicity usually involve several million t cells, at most, meaning that t cell clones with a very low abundance might not be represented or their activity might not be detectable above background levels. finally, neoantigen-specific t reg cells have been detected in patients with cancer , and these cells could potentially suppress the activity of and thus prevent the detection of neoantigen-specific effector cd + t cells in the typical enzyme-linked immunosorbent spot (elispot) immunogenicity assays. therefore, factors other than mhc ii-binding they are returned to the patient to attack the tumour (nct and nct ). the efficacy of such neoantigen-based immunotherapies will be dependent on the identification of a sufficient number of mhc ii-binding peptides to stimulate cd + t cell responses. both maria and mixmhc pred have the potential to make personalized neoantigen-based therapies more accessible to patients, including patients with tumours harbouring fewer mutations, by identifying more mhc ii-binding epitopes to which cd + t cells can respond within each patient's pool of putative neoantigens. genomic correlates of response to immune checkpoint blockade an immunogenic personal neoantigen vaccine for patients with melanoma mhc-ii neoantigens shape tumour immunity and response to immunotherapy direct identification of clinically relevant neoepitopes presented on native human melanoma tissue by mass spectrometry robust prediction of hla class ii epitopes by deep motif deconvolution of immunopeptidomes predicting hla class ii antigen presentation through integrated deep learning application of mass spectrometrybased mhc immunopeptidome profiling in neoantigen identification for tumor immunotherapy the potential of donor t-cell repertoires in neoantigen-targeted cancer immunotherapy a direct estimate of the human alphabeta t cell receptor diversity cross-reactive cd + t cells against one immunodominant tumor-derived epitope in melanoma patients tumor-infiltrating human cd + regulatory t cells display a distinct tcr repertoire and exhibit tumor and neoantigen reactivity a poly-neoantigen dna vaccine synergizes with pd- blockade to induce t cellmediated tumor control the authors declare no competing interests. might dictate cd + t cell responses to predicted mhc ii-binding peptides, including -but not limited to -deficits in the tcr repertoire or suppression of t cells.maria and mixmhc pred both enabled enhanced detection of cd + t cellstimulating neoantigen peptides and reduced false-positive rates compared with prior platforms. therefore, both algorithms are usefully impro ved tools for identifying mhc ii-binding neoepitopes, as long as the low rate of cd + t cell response is taken into account and a sufficient number of peptides to induce a response are included in any experimental vaccines. historically, neoepitope-based vac cines have demonstrated clinical benefit as single agents, mostly in the adjuvant or pro phylactic setting . the limited efficacy of cancer vaccines in the treatment of unresectable metastatic disease has been largely attributed to tumour-mediated immunosuppression. combinations of neoepitope vaccines with icis that reduce immunosuppression have, however, shown promise in the treat ment of non-resected aggressive cancers in mice ; this combination strategy is currently being tested in multiple clinical trials (for example, nct , nct , nc t , nc t and nct ). neoepitope vaccines could potentially also be combined with adoptive t cell therapies. specifically, neoepitope vaccination of patients is being used to promote the expansion of neoepitope-specific t cells in order to facilitate the cloning of patient-specific neoepitope-specific tcrs, with subsequent ex vivo genetic modification of large numbers of autologous non-tumour-reactive t cells to express the neoepitope-specific tcrs before both maria and mixmhc pred have the potential to make personalized neoantigen-based therapies more accessible to patients… www.nature.com/nrclinonc key: cord- -g sum authors: hou, yanxia; guo, yingying; wu, chunyan; shen, nan; jiang, yongping; wang, jingfei title: prediction and identification of t cell epitopes in the h n influenza virus nucleoprotein in chicken date: - - journal: plos one doi: . /journal.pone. sha: doc_id: cord_uid: g sum t cell epitopes can be used for the accurate monitoring of avian influenza virus (aiv) immune responses and the rational design of vaccines. no t cell epitopes have been previously identified in the h n aiv virus nucleoprotein (np) in chickens. for the first time, this study used homology modelling techniques to construct three-dimensional structures of the peptide-binding domains of chicken mhc class Ι molecules for four commonly encountered unique haplotypes, i.e., b , b , b , and b . h n aiv np was computationally parsed into octapeptides or nonapeptides according to the peptide-binding motifs of mhc class i molecules of the b , b , b and b haplotypes. seventy-five peptide sequences were modelled and their mhc class i molecule-binding abilities were analysed by molecular docking. twenty-five peptides (ten for b , six for b , two for b , and seven for b ) were predicted to be potential t cell epitopes in chicken. nine of these peptides and one unrelated peptide were manually synthesized and their t cell responses were tested in vitro. spleen lymphocytes were collected from spf chickens that had been immunised with a np-expression plasmid, pcaggs-np, and they were stimulated using the synthesized peptides. the secretion of chicken ifn-γ and the proliferation of cd (+) t cells were tested using an elisa kit and flow cytometry, respectively. the significant secretion of chicken ifn-γ and proliferation of cd (+) t lymphocytes increased by . % and . % were monitored in cells stimulated with peptides np( – ) and np( – ), respectively. the results indicate that peptides np( – ) (pkktggpiy) and np( – ) (krgindrnf) are np t cell epitopes in chicken of certain haplotypes. the method used in this investigation is applicable to predicting t cell epitopes for other antigens in chicken, while this study also extends our understanding of the mechanisms of the immune response to aiv in chicken. the introduction into the human population of animal-derived influenza a viruses with a novel haemagglutinin (ha), or a novel ha and neuraminidase (na), and their subsequent spread could result in global influenza pandemics [ ] . since , the highly pathogenic h n avian influenza virus (aiv) has caused numerous cases of severe disease and death in humans [ ] . an influenza pandemic could ensue if this virus developed the capacity to spread easily among humans [ ] [ ] [ ] . migratory birds constitute the natural reservoir for aivs, but chickens may play a key role in the transmission to humans [ ] . epitopes can be used for accurately monitoring immune responses to aiv and for the rational design of protective vaccines. however, only two epitopes from the h n avian influenza a/vietnam/ / virus are included in the immune epitope database and analysis resources (iedb). the majority of t cell and b cell epitopes have been identified in mouse, human, or rabbit hosts. few epitopes have been described in chicken [ ] . the structural basis of peptide binding to mammalian major histocompatibility complex (mhc) class i molecules is well understood [ ] . the peptide-binding groove is formed by the a and a domains. each domain contributes four strands to an eight -stranded anti-parallel b-sheet. two long interrupted helices, one from each domain, pack against the side of this sheet in an orientation directed away from the cell membrane. there is a series of pockets (a-f) along the peptide-binding groove where highly polymorphic amino acids mediate recognition via haplotype-specific associations with antigens and t cell receptors. however, highly conserved residues are found at both ends of the peptide-binding groove that form a network of hydrogen bonds, which directly interact with hydrogen bonds at the peptide's nterminus and c-terminus [ ] [ ] [ ] . peptides that bind to mhc class i molecules are usually octamers or nonamers, where only one or a few residues can interact with polymorphic residues in the groove. these residues are known as anchor residues when they are found in an anchoring position [ ] [ ] [ ] [ ] . several public databases and prediction services are available for mhc molecular ligands and peptide motifs, including syfpeithi and rankpep [ , ] . unlike mammalian studies, the majority of investigations of mhc class i molecules in chicken remain limited to primary sequences and the structure of mhc class i molecule from the b haplotype was solved only recently [ , ] . the chicken mhc b system is located on chicken chromosome (chr ) and is composed of tightly linked polymorphic regions: bf (class i) and bl (class iib) and a large family of polymorphic ig-superfamily (igsf) genes called bg-the latter sharing sequence similarities with mammalian mhc butyrophilin, myelin oligodendrocyte glycoprotein, and trim genes [ , ] . the mhc b-f molecules are structurally and functionally similar to mammalian mhc class i molecules. the two class i genes are known as bf and bf , although the bf gene is mainly expressed. the mhc class i (b-f) molecules present antigen peptides to the cd + t lymphocytes, which have a central role in the immune system. the mhc class i molecules of different haplotypes have specific peptide-binding tropisms, and b-f-associated peptide-binding motifs have been determined for several haplotypes, including b , b , b , and b [ ] . thirteen peptides derived from the v-src gene of the rous sarcoma virus (rsv) prague strain were predicted to fit the peptide-binding motif of the mhc class i molecules of b haplotype and all synthetic peptides actually bound to the bf class i molecule in subsequent binding tests [ ] . this demonstrates that peptide-binding motifs can be used to predict the antigen peptides presented by chicken mhc class i molecules. however, no t cell epitope of h n aiv np has yet been identified in the chicken [ ] , while no information is available on the structure of chicken mhc class i molecules belonging to the b , b , b , and b haplotypes. for the first time, the current study reports the homology modelling structures of the peptidebinding domains of chicken mhc class i molecules for the b , b , b , and b haplotypes. potential t cell epitopes were predicted by molecular docking of peptides in the h n aiv np in chicken. np - and np - were shown to be t cell epitopes of h aiv np by analysing the cd + t cell proliferation and the interferon (ifn-c) expression. to our knowledge, this is the first report to describe the structure of chicken mhc class i molecules from the b , b , b and b haplotypes and the t cell epitopes of h n aiv np in chicken. all computations were conducted using the discovery studio . (ds . ) program developed by accelrys software inc and the sybyl . program developed by tripos inc on an sgi fuel workstation running red hat enterprise . and a dell server running the red hat enterprise . linux operating system. spf chickens were housed in hepa-filtered isolators. chicken lymphocytes collected from immunized chickens, which were approved by harbin veterinary research institute, chinese academy of agricultural sciences and performed in accordance with animal ethics guidelines and approved protocols. the animal ethics committee approval number is heilongjiang-syxk- - . dna vaccine pcaggs-np was constructed and assessed according to ma and jiang [ , ] . the synthetic gene for np of the isolate a/goose/gongdong/ / (h n ) with codons optimized for chicken usage was synthesized by pcr assembly of long single-strand dna templates ( bases in length) (the oligonucleotide sequences are available upon request). the synthetic optinp was cloned into the plasmid vector pcaggs under the control of the chicken b-actin promoter. the plasmid was named pcagg-np. the expression of the np protein from the plasmid was confirmed by indirect immunofluorescence assay and western blotting of plasmid transfected cef cells. nine of the predicted peptides (table ) were selected to test their t cell responses in vitro. they were synthesized to . % purity by the genscript corporation (nanjing, china): np [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] (krsyeqme), amino sequence of h n aiv(gd/ / ) np was parsed into octamers or nonamers according to the peptide-binding motifs of mhc class i molecules belonging to the b , b , b , and b haplotypes. the d structures of those peptides and the mhc class i molecules were predicted using homology modelling method and then the binding affinity between each peptide and mhc class i molecule was analysed using molecular docking. those peptides, which have correct binding conformation and high binding affinity, were predicted to be potential t cell epitopes in chicken. predicted peptides of were synthesized and used to stimulate splenic lymphocytes collected from np dna vaccineimmunized spf chickens. mhc class i molecule-restricted potential t-cell epitopes were confirmed by detecting of cd + t cell proliferation and interferon (ifn-c) expression. the amino acid sequences of chicken mhc class i molecules belonging to the b , b , b , and b haplotypes were obtained from genbank (b , genbankid: cak . ; b , genbankid: bag . ; b , genbankid: bag . ; and b , gen-bankid: bag . ). blastp was performed to search for homologous proteins in the protein data bank (pdb) using the blosum scoring matrix optimized with a gap penalty of and a gap extension penalty of . the modeler [ ] program was then executed in ds . to construct the d structures of chicken mhc class i molecules for the four haplotypes. the quality of the d models was evaluated with a ramachandran plot using the procheck [ ] program and the verify protein (profiles- d) program [ ] in ds . . to improve the quality of the models, unsatisfied loop regions in each model were refined using the loop-refinement protocol based on the modeler energy in ds . . all structures were then minimized using the charmm force field [ ] and an explicit solvent model (tip p water) with a steepest descent method for steps and a conjugated gradient minimization for a further steps. the cavity depth, lipophilic potential (lp), flexibility (fx), and electrostatic potential (ep) of the four structures was analysed using the molcad program in sybyl . . during this process, the gasteiger-hückel charges were assigned to all atoms, while surface maps were generated and visualized using sybyl . . the np protein sequence of h n isolate a/goose/gongdong/ / (h n ) was downloaded from the uniprot database (uniprotid: ncap_i a ) and automatically parsed as octapep-tides or nonapeptides using a computer program, which was developed in our laboratory, based on the peptide-binding motifs of chicken mhc class i molecules belonging to the b , b , b , and b haplotypes [ ] . the motifs were as follows: the structures of the octapeptides and nonapeptides were modelled using ds . . in total, seventy-five peptide structures were prepared for docking analysis. surflex-dock [ ] is used to dock ligands into a protein-binding site and it is particularly successful at eliminating false positive results [ ] while offering unparalleled enrichment in virtual highthroughput screening combined with state-of-the-art speed, accuracy, and usability [ , ] . to test the feasibility of using surflex-dock for docking mhc molecules and peptides, peptide-mhc complexes retrieved from the pdb database were separated and re-docked using the surflex-dock program. the rmsd was calculated for each modelled pair and crystal structure pair to evaluate the docking program. surflex-dock was used to dock the octapeptides and nonapeptides to their corresponding mhc class i molecule receptors, where the parameters of the threshold and bloat values of the program were optimized to . and . the interaction modes and binding energies of the docked complexes were analysed using ds . . peptides with a higher docking score and rational conformation were predicted as candidate t cell epitopes for each haplotype. for the dna vaccine immunizations, three-week-old spf chickens were separated into two groups, eight were immunized twice with mg of pcaggs-np in their leg muscle at threeweek intervals, while five chickens were injected with the same volume of pbs as controls. sera were collected weekly for detecting of np antibody. serum antibodies to h n aiv np were detected using an indirect elisa method as described by [ ] , which used prokaryotically-expressed np as the antigen. the testing steps as follows, after washing of the plates, ml of the test serum mixed in the test wells with ml of antigen diluted : in elisa buffer. after incubation at uc for h, ml horseradish peroxidase conjugate, diluted : in elisa buffer, was added to each well and plates were further incubated at uc for h. after two washing steps, ml of tmb substrate was added and incubated at room temperature for min. the reaction was stopped by adding ml h so m. the extinctions were measured at nm with a micro elisa reader (bio-rad). to prepare splenic lymphocytes, all eight pcaggs-np immunized chickens were killed by cardiac puncture blood collection. sterile spleens were collected and meshed through a sieve screen using a syringe plunger to obtain a single-cell suspension in tissue culture medium (rpmi , gibco brl ny, usa). cell suspensions were overlaid onto histopaque density gradient medium and centrifuged at rpm for min at uc. lymphocytes were collected from the interface and washed three times in rpmi, before cells were counted using a trypan blue dye exclusion assay. splenic lymphocytes collected from the immunized chickens were stained with mm cfse (invitrogen) in pre-warmed pbs for min, washed three times, and suspended in rpmi containing mm l-glutamine, iu ml - penicillin, iu ml - streptomycin, and % foetal bovine serum (r medium). cells were plated at well - in -well plates with rpmi medium, before stimulation for d with mg ml - of the nine peptides generated from np and the unrelated peptide n - . cells were then washed with pbs and stained using anti-cd -pe before flow cytometric analysis, which was performed on cytomics fc mcl(beckman) and analysed with its embedded software cxp. two repeats were performed simultaneously for each peptide during the flow cytometric analysis. splenic lymphocytes collected form the vaccinated chickens were plated at well - in -well plates with rpmi medium, before stimulation for h using mg ml - of the nine peptides generated from np and the unrelated peptide n - . cells were then collected and centrifuged at rpm for min. the cell culture supernatants were then analysed to determine the chicken ifn-c using chicken ifn-c cytosets tm (invitrogen), according to the manufacturer's instructions [ ] . the extinctions were measured at nm with a micro elisa reader (bio-rad). for each peptide, two copies of splenic lymphocytes from one chicken were treated and analysed simultaneously. results were expressed as mean s.e.m. for two replicates. statistical analyses were performed using spss . for windows. significant differences (p, . ) between means were tested by one-way anova, followed by tukey's honestly significant difference test. the four haplotypes selected in this study belonged to commonly encountered unique haplotypes (b , b , b , b , b , b , b , b , b , b , and b ) and the motifs of their binding peptides were previously reported [ , ] . the peptide-binding groove of the chicken mhc class i molecule contains a and a domains [ ] , so only those two domains were used for modelling and analysis in this study. blastp search results showed that the chicken mhc class i molecule bev (pdb accession number) belonging to the b haplotype had the highest sequences identity and similarity with the four target sequences. the sequence identity and similarity between bev and b , b , b , and b were . %, . %, . %, and . %, and . %, . %, %, and . %, respectively. the sequence alignments of the four target sequences and bev are shown in fig. . bev has been crystallized to a high resolution of . Å , so it was selected as the template for modelling the four protein structures. the initial crude homology models were built using mod-eller and refined using modules for loop refinement and charmm minimization in ds . . thus, the final structures were of high quality. the profiles- d scores of all amino acid residues in the four structures were greater than zero (fig. ) , while an evaluation of the stereochemical quality of the models using procheck showed that no residues were in the disallowed regions of the ramachandran plots (fig. ) . this indicated that the backbone dihedral angles, phi and psi, of the four structure models were reasonably accurate. the four models are similar to the previously reported b haplotype. the peptide-binding domain is formed by two helices at the top and an eight-stranded sheet at the bottom (fig. a) . the pairwise root mean-square differences (rmsd) between the ca positions of bev and the structures of the b , b , b , and b haplotypes are very small ( . , . , . , and . respectively). however, there are some variations around the peptide-binding grooves and these differences may determine differences in their peptide-binding properties. specific residues in the binding groove have an essential role in peptide binding. figure b shows the key residues comprising the binding site, when all the models and the template were superimposed. peptide binding to a given class i mhc molecule requires the presence of anchor residues that complement the physicochemical characteristics of the specificity pockets [ ] . anchor residues are usually found at peptide position (p ), where they interact with pocket b, but sometimes at position or (p or p ), where they interact with pocket c or e. a c-terminal residue (pc) also typically binds in pocket f [ ] . the anchor residues glu or asp (p ) in b have an electrostatically favourable interaction with the side-chain of the pocket b residue aspa , which points up from the b-sheet. similarly, anchor residues p arg in b and b provide an electrostatically favourable interaction with the negatively-charged residues aspa and glua , the side-chains of which point towards the binding groove from the a-helix. the presence of glu or asp as major residues in the c-terminal position of the b motif is unprecedented, and there are no previous mammalian examples of negatively-charged residues in the c-terminal anchor position [ ] . it is possible that the positively-charged arga residue of the f pocket is the key to the binding of anchor residues p glu or p asp. there is an anchor residue in the central cavity region corresponding to pocket c in b and b . this is very similar to the mammalian mhc class i molecules h- kb and h- db, and it may be related to the glya ( ) residue without a side-chain located in the a-helix (the residue number in parentheses is derived from mammals, whereas that without parentheses is from the chicken). the volumes of the binding grooves in b and b are . Å and . Å , respectively, which are greater than those of b ( . Å ) and b ( . Å ) (fig. ) . this may explain why the former have anchor residues in the middle region of their binding peptides. in general, anchor residues point downwards into the binding groove, which allows them to fill the pocket and maintain the stability of the peptide-mhc complex. the electrostatic potential of the peptide-binding groove of the b haplotype is highly positively-charged, whereas those of the b and b haplotypes are negatively-charged and that of b is neutral (fig. ) . the peptide-binding grooves are highly lipophilic in all models (fig. ) . the b pockets of the peptide-binding grooves . verification score plots of the models (plots generated using ds . ). the verification scores of all residues in b were greater than zero, whereas one residue in b and b and three residues in b were less than zero. doi: . /journal.pone. .g are more flexible than the f pockets in all structures (fig. ) , which suggests that the variation of the anchor residues in the b pocket is greater than in the f pocket. as described in the materials and methods, peptide-mhc complexes were selected from the pdb database to validate the docking process. all the peptides were extracted from the complexes, before they were re-docked to the corresponding mhc molecules using surflex-dock. when compared with the corresponding initial complexes, the average rmsd was . and the docking scores were all greater than . (table ) . docking was also successfully performed with v-src c-tail peptide - (lpacvlev) and an identified t cell epitope [ ] to the modelled b mhc class i molecule, where the docking score was . and the docking complex conformation was rational. thus, it was appropriate to use surflex-dock for screening t cell epitopes based on their homology-modelled structures. based on the assessment results, the criteria for t cell epitope prediction using surflex-dock were defined as follows: a) the peptide made close contact with the groove and it docked in the correct direction; b) the anchor residues bound to the anchor site in a rational conformation; and c) the docking score of the complex was greater than . . a total of potential t cell epitope peptides were predicted (eight for b , six for b , two for b , and seven for b ). nine peptides, which marked in bold in table , were selected to test their t cell responses in vitro. the binding energies of those complexes were also calculated. the results are shown in table . peptide stimulation experiments were conducted using splenic lymphocytes derived from np dna vaccine-immunized chickens to verify some of the predicted potential t cell epitopes. to assess the immune effects of the vaccine, serum np antibody was detected using the elisa method. compared to the control group, a significant increase (p, . ) in blood np antibody was observed in the immunized group two weeks after the first vaccination. after the boost, the blood np antibody level of the immunized group increased further and it remained at a high level throughout the duration of the experiment (fig. ). this indicated that the immune systems of the chickens were activated by the vaccine immunization and that the splenic lymphocytes of chickens were sensitized. activation of lymphocytes using peptides np to verify the predicted t cell epitopes in np, ten synthetic peptides (top nine of the predicted peptides from np and one unrelated peptide) were incubated with sensitized splenic lymphocytes for d. flow cytometry analysis showed that the proliferation of cd + t lymphocytes increased by . % and . % in cells stimulated with the peptides np - and np - , respectively (fig. ) . chicken ifn-c concentration in cells stimulated using peptides np - and np - were significantly higher than the control and unrelated peptide-stimulated cells (fig. ) . these results demonstrate that the peptides np - and np - are np t cell epitopes in chickens of certain haplotypes. the objective of this study was to predict and verify t cell epitopes in the h n aiv np in chicken. using a motif combined with a structure-based method, potential t cell epitope peptides were predicted in the h n aiv np in chickens of b , b , b , and b haplotypes. np - and np - were found to induce a significant proliferation of cd + t lymphocytes and they increased the secretion of chicken ifn-c in sensitized splenic lymphocytes. these data suggest that peptides np - and np - are np t cell epitopes in chickens of certain haplotypes. this study is important for the following two reasons. first, this is the first study to determine the structural characteristics of the peptide-binding domains of chicken mhc class i molecules belonging to the b , b , b , and b haplotypes using a combined motif-structure method to predict t cell epitopes in chickens. second, np - and np - are the first two t cell epitopes to be identified in aiv np in chickens of certain haplotypes. homology modelling is widely used in many areas of structure-based analysis and study [ , ] . however, there are few studies of chicken mhc class i molecules compared with those of human or mouse. the only chicken mhc class i molecule structure that has been solved is the b haplotype [ ] . thus, there is little information available on the structure and function of chicken mhc class i molecules. therefore, the current study used homology modelling to investigate the peptide-binding domains of chicken mhc class i molecules belonging to the b , b , b and b haplotypes. to the best of our knowledge, this is the first attempt to understand the peptide-binding properties of these molecules based on their structures. the assessment indicated that the models were of high quality in terms of their folding and they were suitable for structure-based t cell epitope prediction. the structural characteristics of the peptide-binding properties of these mhc class i molecules were described in the results section. only four haplotypes with known motifs were selected in this study. however, it is possible to use this solution to predict t cell epitopes of mhc class i molecules belonging to different haplotypes for other antigens in chickens, because the only difference would be the increased number of peptides required for molecular docking. this study found that out of peptides were potential t cell epitopes in the h aiv np in chickens of the four haplotypes. an analysis of previous results showed that some of those peptides have been identified as t cell epitopes in humans. there is evidence that np - (ktggpiykr), np - (rgvqias-nenmetme), and np - (rrsgaagaavk), which are derived from the h n , h n , and h n , respectively, are t cell epitopes, with mhc restriction alleles of hla-a , h- db, and hla-b , respectively [ ] . of these peptides, np - (pkktggpiy) and np - (gvqiasne) are completely conserved in all influenza virus strains, while np - (agaavkgv) is relatively conserved. some potential epitopes were also shared by several haplotypes. chickens used for meat and egg production are always heterozygotes, so the identification of these shared t cell epitopes will facilitate the development of broad-spectrum protective vaccines for chickens. furthermore, epitopes shared by birds and humans are of great importance in the design of rational vaccines for protecting humans and birds from aiv infection. this study used a dna vaccine plasmid expressing the np from a/goose/gong dong/ / (h n ). a dna vaccine expressing ha from a/goose/gong dong/ / (h n ) has been found to elicit antibody responses and protect chickens against challenge with hpai virus [ ] . methods used for producing the ha-expression dna vaccine were adopted when preparing the dna expression plasmid pcagg-np. np antibody responses were detected in immunized chickens, suggesting that this vaccine elicits successful immune responses to the np antigen in chickens. the t cell responses were not detected directly in this study, but it was inferred that a successful antibody response would have been accompanied by a successful t cell response based on our knowledge of the dna vaccine. nine of the peptides were synthesized and used to stimulate sensitized splenic lymphocytes to verify that they were epitopes. increases in the proliferation of cd + t lymphocytes and the secretion of chicken ifn-c demonstrated the antigenicity of these peptides. np - (pkktggpiy) and np - (krgindrnf) induced significant t cell responses in splenic lymphocytes. an analysis of the prediction results showed that np - was predicted to be a t cell epitope for both b and b , while np - also belonged to the b haplotype. thus, it is suggested that the major haplotype of the experimental spf chickens might be b . np - also overlapped with a previously identified hla-a restriction t cell epitope np - (ktggpiykr) of the h n aiv [ ] . this further verifies the significance of this epitope, which could be used in human and chicken vaccines to provide protection against different influenza virus subtypes. using in silico and in vitro approaches, this study identified two novel t cell epitopes np - (pkktggpiy) and np [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] (krgindrnf) in the h n aiv np in chickens of certain haplotypes. the method used in this investigation is applicable to predicting t cell epitopes for other antigens in chicken, while this study also extends our understanding of the mechanisms of the immune response to aiv in chickens. characterization of an avian influenza a (h n ) virus isolated from a child with a fatal respiratory illness avian influenza. h n moves into africa, european union, deepening global crisis emerging and re-emerging infectious diseases: influenza as a prototype of the host-pathogen balancing act pandemic influenza threat and preparedness highly pathogenic avian influenza (h n ): pathways of exposure at the animalhuman interface, a systematic review ab and t cell epitopes of influenza a virus, knowledge and opportunities the three-dimensional structure of peptide-mhc complexes complex assembly, crystallization and preliminary x-ray crystallographic studies of the swine major histocompatibility complex molecule sla- * complex assembly, crystallization and preliminary x-ray crystallographic studies of duck mhc class i molecule structural diversity of class i mhc-like molecules and its implications in binding specificities comment on ''characterizing the n-terminal processing motif of mhc class i ligands consensus motifs and peptide ligands of mhc class i molecules chemistry of peptides associated with mhc class i and class ii molecules characterizing the n-terminal processing motif of mhc class i ligands syfpeithi: database for mhc ligands and peptide motifs enhancement to the rankpep resource for the prediction of peptide binding to mhc molecules using profiles structures of an mhc class i molecule from b chickens illustrate promiscuous peptide binding complex of a b chicken mhc class i molecule and a mer chichen peptide the chicken b locus is a minimal essential major histocompatibility complex nomenclature for the chicken major histocompatibility (b and y) complex peptide motifs of the single dominantly expressed class i molecule explain the striking mhcdetermined response to rous sarcoma virus in chickens v-src oncogenespecific carboxy-terminal peptide is immunoprotective against rous sarcoma growth in chickens with mhc class i allele b-f construction of a recombinant vector expressing avian influenza virus nucleoprotein with an avian-derived promoter and detection of its immunogenicity enhanced protective efficacy of h subtype avian influenza dna vaccine with codon optimized ha gene in a pcaggs plasmid vector localization of a t-cell epitope within the nucleocapsid protein of avian coronavirus modeller: generation and refinement of homology-based protein structure models procheck: a program to check the stereochemical quality of protein structures assessment of protein models with three-dimensional profiles charmm: a program for macromolecular energy, minimization, and dynamics calculations surflex-dock . : robust performance from ligand energetic modeling, ring flexibility, and knowledge-based search surflex: fully automatic flexible molecular docking using a molecular similarity-based search engine comparative evaluation of eight docking tools for docking and virtual screening accuracy fast structure-based virtual ligand screening combining fred, dock, and surflex effects of reticuloendotheliosis virus and marek's disease virus infection and co-infection on ifn-gamma production in spf chickens refined structure of the human histocompatibility antigen hla-a at . ?resolution homology modeling in drug discovery: current trends and applications a homology modeling of ferredoxin-nitrite reductase from arabidopsis thaliana epitope-specific tcrbeta repertoire diversity imparts no functional advantage on the cd + t cell response to cognate viral peptides key: cord- -aa cf u authors: cassotta, antonino; paparoditis, philipp; geiger, roger; mettu, ramgopal r.; landry, samuel j.; donati, alessia; benevento, marco; foglierini, mathilde; lewis, david j.m.; lanzavecchia, antonio; sallusto, federica title: deciphering and predicting cd (+) t cell immunodominance of influenza virus hemagglutinin date: - - journal: j exp med doi: . /jem. sha: doc_id: cord_uid: aa cf u the importance of cd (+) t helper (th) cells is well appreciated in view of their essential role in the elicitation of antibody and cytotoxic t cell responses. however, the mechanisms that determine the selection of immunodominant epitopes within complex protein antigens remain elusive. here, we used ex vivo stimulation of memory t cells and screening of naive and memory t cell libraries, combined with t cell cloning and tcr sequencing, to dissect the human naive and memory cd (+) t cell repertoire against the influenza pandemic h hemagglutinin (h -ha). we found that naive cd (+) t cells have a broad repertoire, being able to recognize naturally processed as well as cryptic peptides spanning the whole h -ha sequence. in contrast, memory th cells were primarily directed against just a few immunodominant peptides that were readily detected by mass spectrometry–based mhc-ii peptidomics and predicted by structural accessibility analysis. collectively, these findings reveal the presence of a broad repertoire of naive t cells specific for cryptic h -ha peptides and demonstrate that antigen processing represents a major constraint determining immunodominance. cd + t lymphocytes orchestrate adaptive immune responses by secreting cytokines that promote multiple types of inflammatory responses in tissues and by providing help to b cells and cd + t cells (sallusto et al., ) . for antigen recognition, cd + t cells rely on the interaction with antigen-presenting cells (apcs) that take up, process, and present antigen in the form of short linear peptides bound to mhc class ii (mhc-ii) molecules (roche and furuta, ; unanue et al., ) . typically, only a small fraction of the multitude of potentially immunogenic peptides contained in a complex foreign antigen are able to induce a measurable t cell response, with some peptides recognized with higher magnitude and/or frequency and thus arising as immunodominant, and others that remain subdominant or even cryptic (sercarz et al., ; yewdell and bennink, ; yewdell and del val, ) . given the complexity and tight connection between antigen presentation and recognition, many factors may pertain to peptide and t cell immunodominance. some of those reflect the biochemical rules of antigen processing and mhc presentation, such as the molecular context in which the peptides are embedded (graham et al., ; kim and sadegh-nasseri, ; landry, ; mirano-bascos et al., ) , the affinity of the generated peptides for mhc-ii binding, the resistance to hla-dm-mediated editing of newly formed peptide mhc-ii (pmhc-ii) complexes mellins and stern, ) , or their kinetic stability on the cell surface of apcs (sant et al., ) . furthermore, the heterogeneous set of proteolytic enzymes and endogenous inhibitors that different kinds of apcs are equipped with (unanue et al., ) , as well as the interactions with molecular partners that facilitate antigen uptake, such as b cell receptors (bcrs) or soluble antibodies (simitsek et al., ; watts and lanzavecchia, ) , can affect the antigen processing and the composition of the mhc-ii-presented peptidome. other variables influencing t cell immunodominance depend on the architecture of the t cell repertoires and the mechanisms of antigen recognition (yewdell, ) , such as the availability of antigen-specific naive precursors (jenkins and moon, ; moon et al., ) , the interaction affinity of their tcrs with pmhc-ii complexes (malherbe et al., ) , or the occurrence of tcr cross-reactivity to similar antigenic peptides (campion et al., ; nelson et al., ; su et al., ) . in this study, we chose influenza a virus as a model infectious agent that triggers complex adaptive immune reactions comprising both humoral and cellular responses. every year, influenza viruses infect more than a billion people worldwide, and are the cause of prominent economic loss as well as significant morbidity and mortality, especially in children < yr old and adults > (krammer et al., ; lee et al., ; zens and farber, ) . despite great efforts in research, vaccines are only moderately effective against seasonal strains and are challenged by the rapidly evolving nature of influenza viruses that occasionally emerge as new strains causative of serious epidemics or pandemics (angeletti and yewdell, ; krammer et al., ; webster and govorkova, ; zens and farber, ) . we focused our attention on hemagglutinin (ha), which represents the main target of antibody response to influenza virus upon vaccination or infection (angeletti and yewdell, ; corti et al., ; krammer et al., ; lee et al., ; pappas et al., ) . the detailed and unbiased characterization of ha-reactive memory and naive cd + t cell repertoires, paralleled by a deep analysis of the naturally presented repertoire of mhc-ii-binding ha peptides by mass spectrometry (ms)-based immunopeptidomics, allowed us to shed new light on the factors governing cd + t cell clonal selection and immunodominance to influenza ha in humans. to capture the entire repertoire of memory t cells specific for influenza ha, we obtained multiple and large blood samples from a donor (hd ) after vaccination with the / seasonal inflexal v vaccine containing ha from the pandemic a/california/ / h n strain (h -ha). central memory (tcm), effector memory (tem), and circulating follicular helper (ctfh) cd + t cells were isolated by cell sorting, labeled with cfse, and stimulated with inflexal v. when analyzed on day , proliferating cfse lo t cells were detected in all three memory subsets from samples obtained and mo after vaccination ( fig. a) . to select h -ha-reactive t cells, the cfse lo t cells were sorted, relabeled with cfse, and stimulated with h -ha ( fig. b) . t cells proliferating in the secondary stimulation were cloned, and h -ha-specific clones were isolated (fig. c and table s ) and characterized for peptide specificity, mhc restriction (hla-dr, hla-dp, or hla-dq), and tcr vβ usage. strikingly, > % of the clones isolated ( of ) recognized two overlapping peptides (h -ha - or h -ha - ; fig. d) , thus defining, in this individual, a highly immunodominant region. t cells specific for the immunodominant h -ha - region were found in all three memory subsets ( clones in tcm, clones in tem, and clones in ctfh) and were hla-dr restricted, as shown by antibody blocking experiments (fig. s a) . several t cell clones specific for subdominant h -ha regions were also hla-dr restricted, with a minority being hla-dq or hla-dp restricted (fig. s , b and c). tcr vβ sanger sequencing performed on t cell clones showed that the response to h -ha was highly polyclonal, comprising distinct clonotypes, even when directed against the immunodominant region (table s ) . for instance, t cell clones specific for the immunodominant peptide h -ha - comprised different clonotypes, and t cell clones specific for the immunodominant peptide h -ha - comprised different clonotypes ( fig. e and table s ). of note, of the h -ha - -specific clonotypes used the trbv gene, suggesting a preferential tcr cdr and cdr usage that might facilitate cognate interaction with the peptide-mhc complex. tracking of h -ha-reactive t cell clonotypes within the cfse lo t cell population responding to inflexal (fig. a) showed that those against the immunodominant h -ha region were among the most represented and that some were also found in the tcm, tem, or tfh repertoire ex vivo (fig. f) . strikingly, several of these clonotypes were still detected in memory t cell subsets isolated from donor hd mo later (fig. g) . collectively, these findings indicate that in an inflexal-immunized donor, a polyclonal repertoire of h -ha-specific tcm, tem, and ctfh cells is highly focused on a small immunodominant region. memory t cells are focused against immunodominant regions, while naive t cells recognize multiple peptides spanning the entire h -ha sequence we next investigated whether the immunodominance observed in the memory repertoire is a general phenomenon and whether it is reflected in the naive repertoire of a given individual. to address these questions, we used the highly sensitive t cell library method (geiger et al., ) to screen naive and total memory cd + t cells from hd and three other immune donors with a diverse hla background (table s ) . for each donor, naive and memory t cells were polyclonally expanded in multiple cultures (each containing , - , cells) in the presence of phytohemagglutinin, il- , and feeder cells. for a broad and unbiased screening of t cell reactivity against h -ha, the t cell libraries were then screened using overlapping mer peptides covering the entire h -ha sequence. in all four donors tested, h -ha peptide-specific t cell clones were readily detected in naive and memory libraries although, as expected, their frequencies measured in the naive libraries were lower compared with that measured in the memory libraries (fig. , a and b) . epitope mapping of memory t cell clones confirmed in all four donors a skew toward one or two immunodominant regions, which for donor hd coincided with those detected by antigen-driven proliferation of memory t cell subsets (fig. c) . strikingly, however, epitope mapping of naive t cell clones showed that these cells covered a broad range of peptide specificities (fig. , c and d) . this pattern was particularly evident for donor hd , which was analyzed at high depth. in this donor, naive t cells with diverse tcr vβs recognized peptides spanning virtually all the h -ha sequence (fig. c and table s ). collectively, these findings demonstrate that the naive t cell repertoire has a very broad coverage of the h -ha sequence and that only a fraction of this repertoire is selected in the memory repertoire. naive and memory t cell clones show different functional avidities for peptide and naturally processed h -ha a plausible explanation for the selection of immunodominant peptides is their binding affinity to mhc molecules and/or tcrs. we therefore measured the binding affinity of dominant figure . clonally expanded memory cd + t cells target an immunodominant region of influenza h -ha. (a) memory cd + tcm, tem, and ctfh cell subsets were isolated from blood samples of donor hd and mo after inflexal v vaccination. cells were labeled with cfse and stimulated with the inflexal v vaccine in the presence of autologous monocytes. shown is the cfse profile and the percentage of proliferating cfse lo cells on day in the -mo sample. percentage of cfse lo in the -mo sample was % (tcm), % tem, and % ctfh. (b) the inflexal v-reactive cfse lo t cells were sorted, relabeled with cfse, and stimulated with recombinant h -ha in the presence of autologous monocytes. after d, cfse lo proliferating t cells were sorted and cloned by limiting dilution. shown is the experiment performed with the -mo sample; comparable results were obtained with the -mo sample. (c) a total of h -ha-specific cd + t cell clones were isolated from the tcm, tem, and ctfh cultures based on the proliferative response (stimulation index ≥ ) to a pool of overlapping peptides spanning the entire h -ha sequence. proliferation was assessed on day after a -h pulse with [ h]thymidine and expressed as counts per minute. the data are representative of at least two independent experiments. (d) epitope mapping of the h -ha-specific t cell clones. epitopes were identified by screening the t cell clones with the individual h -ha peptides in at least three independent experiments, with consistent results. the x axis indicates h -ha amino acid sequence; each color-coded segment represents the sequence recognized by individual clones isolated from tcm (black), tem (green), or ctfh (red) cultures. the numbers of h -ha-reactive t cell clones isolated from each subset are reported. the chart at the bottom indicates ha and ha domains colored in blue and red, respectively (fp, fusion peptide; tm, transmembrane). (e) rearranged tcr vβ sequences of h -ha-specific t cell clones were determined by rt-pcr followed by sanger sequencing. shown in the pie charts are the repertoires of rearranged tcr vβ sequences of t cell clones recognizing immunodominant h -ha - and h -ha - epitopes. each slice of the chart indicates a different tcr vβ clonotype (h -ha - , n = ; h -ha - , n = ); the number of sister clones bearing the same tcr vβ sequence are reported for each slice. the total number of clones sequenced is reported at the center. (f and g) to evaluate the clonal expansion of h -ha-reactive memory t cell clones, tcr vβ deep sequencing was performed on inflexal and subdominant peptides to the recombinant hla-dr molecules of donor hd (alleles drb * : or * : ). in vitro refolding assays performed in the presence of titrated peptides showed that the immunodominant h -ha - and h -ha - peptides bound to hla-drb * : molecules with an affinity that was comparable to that of subdominant peptides binding to the same hla-dr ( fig. s c) , indicating that immunodominance of these peptides is not explained by preferential binding to mhc class ii molecules. immunodominance to ha antigen may be also the result of repeated exposure through natural infection or vaccination that can select cross-reactive t cells. however, as shown in fig. s , t cell clones responding to immunodominant or subdominant h -ha peptides were both capable to cross-react, to variable but similar extents, to h -ha and/or h -ha from the widespread strains a/brisbane/ / (h n ) or a/brisbane/ / (h n ). a few clones cross-reacted to h -ha from the highly pathogenic subtype a/viet nam/ / (h n ). we then selected a large number of h -ha-specific t cell clones derived from naive or memory t cells of donor hd and determined their functional avidity by measuring the proliferative response to autologous monocytes pulsed with different concentrations of the h -ha peptides or h -ha protein, which need processing for presentation on mhc-ii molecules. functional avidity, measured in response to peptide stimulation and expressed as half-maximal concentration (ec ) value, was spread over almost logs for both types of t cell clones, with clones from memory t cells being enriched for high-avidity cells ( fig. a and fig. s , a and c), consistent with previous observations on the response to tetanus toxoid (geiger et al., ) . when the same clones were tested for their response to h -ha protein, several observations were made. first, clones from memory t cells responded to h -ha protein, and there was an overall correlation between ec values for protein and peptide, with the immunodominant clones, such as m specific for h -ha - , showing the highest avidity for both peptide and protein (red dots in fig. b) . however, there were a few notable exceptions. for instance, the subdominant clones m and m , specific for h -ha - and h -ha - , had high functional avidity for peptide, comparable to clone m , but showed , -fold lower avidity for the h -ha protein (fig. b) . second, memory t cell clones restricted by hla-dp (m specific for h -ha - ) or by hla-dq molecules (m and m specific for h -ha - and h -ha - , respectively) recognized peptides and protein with functional avidity lower than the median of the distribution (fig. , a and b) . third, several clones from naive t cells, including some with intermediate avidity for peptides, did not proliferate in response to h -ha protein (fig. c) . finally, and importantly, the differential recognition of dominant and subdominant epitopes in response to h -ha was also observed for clones derived from naive t cells. for instance, clone n , specific for the immunodominant h -ha - peptide, had high functional avidity for both peptide and naturally processed h -ha, comparable to that of memory clone m , while clones n and n (specific for the subdominant peptides h -ha - and h -ha - , respectively) had high avidity for peptide but -fold lower avidity for protein, similar to what was observed for memory clones m and m (fig. c) . collectively, these findings indicate that in both the naive and memory repertoires, immunodominant peptides are recognized with high avidity by t cells. they also reveal the presence of a broad repertoire of naive t cells for cryptic ha peptides. finally, they show that the functional avidity of peptide recognition is not predictive of the response to the naturally processed antigen, suggesting a major role for antigen processing in determining the abundance of the processed peptide generated. the mhc class ii peptidome defines immunodominant regions and reveals modulation for antigen processing by antibodies based on the above results, we hypothesize that cd + t cell immunodominance could be primarily related to the yield of peptides generated by antigen processing by apcs. we therefore used ms-based immunopeptidomics to identify peptides naturally presented on mhc-ii molecules by different types of apcs. briefly, monocyte-derived dendritic cells (dcs) from donor hd and ebv-immortalized b cell clones carrying surface bcrs specific for h -ha from all four donors were pulsed overnight with recombinant h -ha. mhc-ii molecules were isolated from lysed cells using a pan-anti-mhc-ii antibody, and peptides were purified by reversed-phase chromatography. using this approach, we identified thousands of total mhc-ii bound individual peptides derived from self-molecules as well as - h -ha-derived peptides (median ) with the expected length peaking at - aa (fig. , a and b) . remarkably, almost % of the h -ha-derived peptides presented by dcs of donor hd corresponded to a rich set of nested peptides overlapping the immunodominant regions h -ha - targeted by memory t cells, suggesting that peptides in this region are presented in high abundance and are recognized by t cells with high functional avidity (fig. c and table s ) . a similar correspondence between immunodominant peptides recognized by memory t cells and peptides presented on mhc-ii molecules was found in the other three donors analyzed (fig. c and table s ), although the breadth of the analysis in these donors was limited by the amount of peptides that could be retrieved from b cells. a notable exception, however, was observed for peptide h -ha - that was presented at high abundance by b cells of donor hd that lacked memory t cells specific for the same peptide. the antibody response to influenza a virus is directed against several regions of ha, such as the highly variable globular head or the conserved stem region, that can be the target of neutralizing antibodies with high breadth for multiple viral subtypes (corti et al., ; lee and wilson, ) . the mhc-ii peptidome of h -ha-pulsed b cell clones with distinct epitope specificity isolated from donor hd revealed that a clone specific for the ha globular head presented the h -ha immunodominant region in a similar manner as dcs, whereas a b cell clone specific for the ha stem region was a poorer presenter of h -ha-derived peptides (fig. s , a and b) . indeed, even if we resolved a comparable total number of mhc-ii presented peptides in the two kinds of b cells ( fig. s a) , from the anti-stem clone we did not detect any h -ha-derived peptides corresponding to the dominant h -ha - region, nor any subdominant peptides from the ha domain ( fig. s b) . consistently, presentation of the recombinant h -ha by anti-stem b cells resulted in a much lower activation and proliferation of t cell clones specific , calculated based on the number of t cell clones recognizing each particular h -ha peptide. inverse simpson index ( -d) ranges between and , and reflects the probability that two h -ha-specific t cell clones randomly selected from a repertoire recognize different h -ha epitopes. **, p = . as determined by two-tailed paired t test. for h -ha - or h -ha - (both hla-dr restricted), resulting in a -fold reduction of their functional avidity compared with antigen presentation by anti-head b cells (fig. s , c and d). conversely, hla-dr-restricted t cell clones specific for h -ha - or h -ha - , which were detected by mhc-ii immunopeptidomics on both anti-head and anti-stem b cell clones, showed comparable functional avidity when stimulated with either kind of apc (fig. s , c and d). taken together, these data show that the mhc-ii peptidome presented by professional apcs defines the immunodominant regions of ha recognized by memory cd + t cells. moreover, the spectrum of peptides naturally presented by b cells can be modulated by antibody binding to ha, thus potentially being an additional variable affecting b cell clonal selection during t cell-dependent immune responses. epitope prediction can be improved by combining mhc binding affinity prediction and antigen processing likelihood (apl) the binding of processed peptides to the groove of mhc-ii molecules follows precise rules that have been instrumental to the development of algorithms capable of predicting binding affinity with high accuracy (jensen et al., ; unanue et al., ; wang et al., ) . using the iedb tool for mhc-ii binding prediction, we found that virtually all the h -ha mer peptides predicted as good binders for the mhc-ii alleles carried by the four donors analyzed were recognized by t cell clones isolated from either the naive or the memory compartment (fig. s ) . nevertheless, it was surprising to note that the immunodominant h -ha peptides did not show stronger binding to mhc-ii compared with subdominant or cryptic peptides, by either in silico prediction or, as demonstrated before, in vitro measurement of mhc-ii binding. these data suggest that the binding to mhc-ii is a necessary, but not sufficient, feature for t cell immunodominance. we therefore set out to explore further parameters that might improve the in silico prediction of haderived t cell epitopes. the molecular context in which a peptide is embedded and its structural accessibility might influence the propensity of unfolding during the progressive ph acidification that occurs in the endocytic pathway, therefore affecting the exposition of denatured stretches of the antigen to the proteolytic environment of the late endosomes (graham et al., ; kim and sadegh-nasseri, ; landry, ) . to evaluate the role of structural constraints of ha in influencing the immunodominance observed in the memory repertoires, we adopted a recently developed algorithm that uses antigen conformational stability to estimate the likelihood of antigen processing (mettu et al., ) . in brief, an aggregate z-score of conformational stability was determined for each h -ha residue by integrating four structural parameters obtained from the d structure of postfusion ha resolved by x-ray diffraction (pdb codes: lzg for ha domain [xu et al., ] ; htm for ha domain in the postfusion conformation [bullough et al., ] ). the z-score statistic was then used to calculate an apl for each theoretical h -ha mer peptide (fig. a) , following the rationale that the liberation of antigenic peptides might be facilitated by surrounding unstable regions that are readily unfolded and targeted by endosomal proteases. as shown in fig. a, several peptides recognized by memory t cells in donors hd -hd were found in regions of high apl. we then evaluated the performance of the apl and mhc binding predictive algorithms by computing the receiver operating characteristic (roc) curves using the set of epitopes recognized by memory t cells of each donor as true positives; the performance was assessed using the area under the roc curve (auroc) metric (fig. b) . we also built a combined predictor by iteratively weighting the contributions of apl and mhc binding until we could maximize the auroc value of the predictor. with this approach, we found that in all donors the t cell clones specific for the immunodominant h -ha - epitope are reported as red dots. lines represent the median and quartiles. ***, p < . as determined by two-tailed mann-whitney u test. (b and c) scatter plots of reciprocal ec values of t cell clones from the memory (b) or naive (c) compartment, stimulated in parallel with recombinant h -ha (x axis) and synthetic peptides (y axis). ec values below the detection limit for stimulations with recombinant h -ha were set arbitrarily to µg/ml; the corresponding t cell clones are reported as white dots. spearman correlation was calculated based on ec pairs from t cell clones responding to both peptides and recombinant h -ha (b, n = ; c, n = ). thresholds of functional avidity were set arbitrarily at ec values of µg/ml, ng/ml, and ng/ml of antigen. n.d., not detected. cassotta et al. journal of experimental medicine combined predictor achieved a better auroc value, outperforming the predictors based solely on apl or mhc binding (fig. b) . as shown in fig. c, there are a number of ways to combine apl and mhc binding weights to achieve the highest auroc ( . in hd and hd , . in hd and hd ), giving an estimate of the maximum and minimum weight of the two scores for each donor. importantly, all the immunodominant peptides identified in the memory repertoire of the four donors analyzed were grouped within a peak of high apl (as defined by a threshold of . ; fig. d) . on average, the top-scoring peptides predicted by apl accounted for > % of the haspecific memory t cell clones, with a sensitivity comparable to the set of peptides measured by mhc-ii immunopeptidomics (fig. e) . thus, a combination of apl and mhc-ii peptide binding affinity may be helpful to improve prediction of immunogenicity and immunodominance in cd + t cell responses. in this study, we report the identification of influenza h -ha peptides recognized by naive and memory cd + t cells in individuals with different hla haplotypes. whereas naive t cells recognized a variety of peptides spanning the whole h -ha sequence, memory t cells were highly focused on just a few peptides. these immunodominant peptides were readily identified by ms-based analysis of peptides eluted from mhc class ii molecules isolated from dcs or h -ha-specific b cell clones and could be better predicted taking into account the ha structural accessibility to proteolytic cleavage. collectively, these findings indicate that processing of native proteins represents a major constraint determining the immunodominance to influenza ha and delineate new methods to identify immunodominant and cryptic t cell epitopes. the identification and characterization of antigen-specific t cells in the naive and memory repertoire is of both fundamental and practical relevance. the high-throughput t cell library method used in this study can rapidly identify, in different individuals, the range of peptides that can be recognized by t cells, thus determining, with a simple assay, both peptide binding to mhc-ii and the presence of specific tcrs (campion et al., ; geiger et al., ; latorre et al., ) . considering the small size of the naive t cell libraries analyzed ( × t cells) compared with the total naive t cell pool, we can estimate that the naive repertoire contains a large number of diverse ha-specific t cells, with frequencies ranging from − to − for each epitope and with a range of functional avidities. importantly, we showed that, in humans, naive t cells recognize multiple peptides spanning the whole h -ha sequence. the diversity of epitope recognition observed in the naive compartment indicates that a multiplicity of ha-derived peptides can potentially trigger t cell activation, underlining the redundancy of the mhc-ii system in accommodating and presenting a large variety of different peptides. nevertheless, many t cell clones isolated from the naive compartment recognized peptides but not naturally processed protein. this finding suggests that the naive repertoire retains t cell precursors recognizing peptides that fail to be generated and/or presented by professional apcs or that are produced in amounts insufficient to trigger priming of cognate naive t cells. it remains to be established whether these cryptic peptides can be generated through unconventional antigen processing, as suggested in some cases of tissue antigens (mohan and unanue, ; sadegh-nasseri and kim, ) , or by nonprofessional apcs, such as epithelial cells in the respiratory tract that are main targets of influenza viruses. epithelial cells readily up-regulate mhc-ii molecules in response to inflammatory cytokines or viral infection (gao et al., figure . immunodominant epitopes localize in h -ha regions predicted as promptly liberated by endosomal proteases. (a) apl based on structural accessibility was calculated for each theoretical h -ha mer peptide (upper panel). sets of h -ha epitopes mapped in the memory cd + t cell repertoire of each donor (lower panels); each segment indicates a peptide recognized by at least one t cell clone isolated from memory cd + t cells. note that apl could not be calculated in the n-terminal ha region, corresponding to ha transmembrane region, since this is not included in the h -ha crystal structures. (b) performance of in silico predictors was benchmarked by computing roc curves using the sets of h -ha memory epitopes reported in a as true positives; the performance of each method was assessed using the auroc metric. shown are the maximal auroc values achieved by single predictors based uniquely on apl (orange bars) or mhc-ii binding (green bars) and the optimized combined predictor (black bars). (c) contribution of apl (orange) and mhc-ii binding (green) in the different combinations all resulting in the highest auroc values for each donor (hd and hd , . ± . ; hd and hd , . ± . ). the relative contribution of apl and mhc binding would differ across donors, since each one has a distinct hla background and a distinct set of peptides recognized by memory t cells. (d) apl (black curve) and peptide specificity (blues bars) of memory cd + t cell clones of each donor. (e) percentage of memory t cell clones for which the cognate peptide was identified by ms-based mhc-ii peptidomics (ms pept., orange bar) or by apl at different thresholds (green bars). each symbol represents a different donor. ; reith and mach, ) and, although they have limited endocytic potential, they can generate peptide ligands for mhc-ii presentation through endogenous degradation pathways, such as autophagy and macroautophagy (dengjel et al., ; schmid et al., ) . the analysis performed on memory t cell libraries and on ex vivo-stimulated memory t cells revealed the presence, in each individual, of one or two immunodominant sites targeted by polyclonal and clonally expanded t cells as well as a few subdominant sites. based on the findings from the analysis of the naive repertoire, this immunodominance cannot be explained by holes in the repertoire. importantly, our study shows that the immunodominant peptides correspond to those that are found most abundantly in the mhc-ii peptidome from h -ha-pulsed dcs and identified also on h -ha-specific b cells, although in the latter case, the assay had a limited sensitivity. these findings point to a simple model whereby immunodominance is determined by the abundance of a given peptide-mhc complex generated by processing followed by selection and clonal expansion of high avidity t cells. the functional avidity of memory t cell clones was on average -fold higher compared with that of naive t cell clones, consistent with the notion that high-avidity t cells are selected in the memory pool, as originally reported for mouse cd + t cells (busch and pamer, ; mcheyzer-williams et al., ; savage et al., ) . the memory repertoire contains also subdominant memory t cell clones that showed very high functional avidity when stimulated by peptides, but not by naturally processed h -ha protein, suggesting that subdominance may be simply due to a lower abundance of the naturally processed peptide. collectively, our data reveal that only a small fraction of the peptides that bind to mhc-ii molecules and can be recognized by t cells are generated by antigen processing, and even in this case, the yield of processed peptide can vary ≥ -fold between immunodominant and subdominant peptides. previous studies using tetanus toxoid as a model antigen showed that antibodies can modulate antigen processing by enhancing or suppressing the generation of different t cell epitopes (simitsek et al., ; watts and lanzavecchia, ) . these findings are extended by the analysis of donor hd , where a b cell clone with a bcr specific for the h -ha globular head generates the same sets of immunodominant peptides as dcs, while a b cell clone specific for the h -ha stem generates a different set of peptides, as demonstrated by peptidomics and activation of specific t cell clones. at this stage, we do not have a mechanistic explanation for these findings, since the structural characterization of the anti-stem antibody and anti-head antibody in complex with ha is not available, and the antigen used was an uncleaved ha that is not fusion competent. we can only speculate that by stabilizing a protein domain or by locking ha in the prefusion conformation, certain antibodies might change processing of ha by endosomal cathepsins, leading to decreased production of relevant t cell peptides (corti et al., ; lee and wilson, ) . further experiments using native antigens and well-characterized antibodies or b cells will be necessary to address the impact of anti-head and anti-stem antibodies in ha processing and presentation to t cells and its physiological relevance in the context of the response to influenza virus infection or vaccination. the high-resolution epitope mapping of naive and memory t cells from donors with a diverse mhc background offered us the possibility to benchmark currently available in silico predictors of cd + t cell immunogenicity. indeed, we found that, although being a prerequisite for recognition by t cells, peptide-mhc-ii binding affinity, either predicted in silico or measured in vitro, is a weak correlate of t cell recognition and in particular of immunodominance. along with the protein antigen expression level and subcellular localization, the position within the d structure of the native antigen may profoundly influence the amount of processed peptides (abelin et al., ; graham et al., ) . recent reports have shown that t cell epitopes from viral antigens tend to localize adjacent to highly flexible, surface-exposed regions of the protein that could act as sites of initial proteolytic cleavage (koblischke et al., ; landry, ; mirano-bascos et al., ) , suggesting that the physical accessibility within the tertiary structure is a requirement for efficient peptide release by proteases. by analyzing the d conformation of ha, we found that immunodominant epitopes are embedded in regions predicted as readily accessible targets of endosomal proteases. furthermore, combined in silico analysis of both peptide-mhc-ii binding affinity and apl yielded higher predictive values for the set of ha memory epitopes here described, thus indicating a possible strategy to develop more accurate predictive algorithms for t cell immunogenicity of protein antigens. in conclusion, the findings here reported suggest a model for epitope selection by antigen processing based on a trade-off between multiple factors, including the structural accessibility to proteases and the binding affinity of liberated peptides for the groove of mhc-ii molecules. structural constrains might define regions prone to be liberated at a higher rate by protease cleavage, thus being a property intrinsic of the antigen tridimensional structure, whereas the mhc-ii allelic background of each individual would select the final sequences of high-affinity binder peptides. the implications of this model are that highly accessible epitopes could be presented at high abundance on the apc surface even if they are not strong mhc-ii binders, and conversely, potentially strong binder peptides could never be presented at relevant amounts if they are not accessible to proteolytic liberation or if they are destroyed by cathepsin cut (resulting in crypticity). the net result of such complex processes would be the differential abundance of some mhc-ii presented peptides, which might drive more prominent clonal expansion of cognate naive t cells, leading to immunodominance. perturbation of the antigen structure, for instance by bound immunoglobulins, might further alter the substrate for endosomal proteolysis and therefore influence the antigen presentation and interaction with cognate t cells. cell purification and sorting serial blood samples from healthy donor (hd ) vaccinated with inflexal v / (crucell) and from blood donors from the swiss red cross were used in compliance with the federal office of public health (authorization no. a / to f. sallusto) and approval from the ethical committee of canton ticino (authorization no. - /ce ). blood from hd , hd , and hd vaccinated with fluarix tetra / (glaxosmithkline) was obtained from the surrey clinical research centre (university of surrey, uk). these studies were conducted in compliance with relevant local guidelines, approved by the london-surrey borders research ethics committee (reference /lo/ ) and registered on clinicaltrials.gov (reference nct ). written informed consent was obtained from all subjects participating in the study. peripheral blood mononuclear cells (pbmcs) were isolated with ficoll-paque plus (ge healthcare). monocytes were isolated from pbmcs by positive selection using cd magnetic microbeads (miltenyi biotech). cd -depleted fractions were stained at °c for min with a primary anti-human cxcr (clone ; mab ) from bio-techne, followed by staining with a biotinylated secondary goat anti-mouse igg b ( - ) from southern biotech. after washing, cells were stained on ice for min with pe/cy -streptavidin ( ) from biolegend, and with the following fluorochrome-labeled mouse monoclonal antibodies: cd -pe-cy (clone b . ; a ), cd -pe-cy (clone b . . ; im ) from beckman coulter, cd -fitc (clone hib ; ) from bd biosciences, cd -pe-texas red (clone s . ; mhcd ), cd ra-qdot (clone mem- ; q ), cd -percp-efluor (clone dx ; - - ) from thermo fisher scientific, ccr -bv (clone g h ; ) from biolegend, and alexa fluor -conjugated goat anti-human igg ( - - ) from jackson immunoresearch. naive and memory cd + t cells were sorted to > % purity on a facsaria iii (bd) after exclusion of cd + , cd + , and cd bright cells. naive t cells were sorted as cd + cd ra + ccr + cd − ; the remaining cd + t cells were sorted as total memory cells. in some experiments with donor hd , total memory cd + t cells were divided in ctfh (sorted as cxcr + cells), tcm (sorted as ccr + cxcr − cells), and tem (sorted as ccr − cxcr − cells). igg + memory b cells and igg − b cells were sorted to > % purity after gating on cd + cd − cd − cd − cells. cell culture t cells were cultured in rpmi supplemented with mm glutamine, % (vol/vol) nonessential amino acids, % (vol/vol) sodium pyruvate, penicillin ( u/ml), streptomycin ( µg/ml; all from invitrogen), and % human serum (swiss red cross). for some experiments, medium was supplemented with il- ( iu/ml). b cells were cultured in rpmi supplemented with mm glutamine, % (vol/vol) nonessential amino acids, % (vol/vol) sodium pyruvate, penicillin ( u/ml), streptomycin ( µg/ml; all from invitrogen), and % fbs (hyclone, characterized, ge healthcare life science). sorted igg + memory b cells were immortalized with ebv and plated in single-cell cultures in the presence of cpg-dna ( . µg/ml) and irradiated pbmc-feeder cells, as previously described (traggiai et al., ) . wk after immortalization, the culture supernatants were screened by high-throughput elisa for binding to h -ha or h -ha as described (pappas et al., ) . ebv-immortalized b cell (ebv-b) cell clones that resulted positive for binding to h -ha and/or h -ha were isolated and expanded. igg − b cells to be used as apcs for t cell libraries were expanded with cd l according to an established protocol (zand et al., ) . autologous monocyte-derived dcs were generated by culture in complete medium containing % fbs (hyclone) supplemented with recombinant gm-csf (gentaur) and il- (immunotools), as previously described (sallusto and lanzavecchia, ) . sorted naive or memory cd + t cells were polyclonally stimulated with µg/ml phytohemagglutinin (remel) in the presence of irradiated ( gy) allogeneic feeder cells ( × per well) and il- ( iu/ml) in a -well plate. the size of the library (number of wells and initial input of cells seeded per well) depended on the number of cells isolated from each donor. t cell lines were expanded as previously described (geiger et al., ) . library screening was performed - d after initial stimulation, by culturing thoroughly washed t cells ( . × per well) with autologous irradiated b cells ( . × per well), untreated or pulsed with a pool of ha overlapping peptides ( µm per peptide) composed of mers ( peptides, mers overlapping of ) and mers ( peptides, mers overlapping of ) covering the h -ha sequence. proliferation was assessed on day , after incubation for h with µci/ml [methyl- h] thymidine (perkinelmer). data were expressed as counts per minute. stringent criteria were used to score positive t cell lines based on two cutoff values: ( ) a Δcpm value ≥ × (cpm with antigen and apcs − cpm with apcs only) and ( ) a stimulation index ≥ (cpm with antigen and apcs ÷ cpm with apcs only). this threshold was chosen based on previous observations made across multiple negative and positive samples assessed by the t cell library technique and with a variety of donors and antigens (campion et al., ; geiger et al., ; latorre et al., ; lindestam arlehamn et al., ) . the specificity of positive cultures was confirmed in subsequent independent experiments of epitope mapping. precursor frequencies were calculated based on numbers of negative wells, assuming a poisson distribution (geiger et al., ) , and are expressed per million cells within each subset. inverse simpson index of diversity ( -d) was calculated for each donor by considering the number of individual t cell clones recognizing each particular h -ha peptide (simpson, ) . the inverse simpson index ( -d) quantifies the richness and evenness of populations, ranges between and , and can be interpreted as the probability that two h -ha-specific t cell clones randomly selected from a repertoire will recognize different epitopes. isolation of h -ha-specific t cell clones sorted memory cd + t cell subsets from donor hd were labeled with cfse and cultured at a ratio of : with irradiated autologous monocytes untreated or pulsed with inflexal v / ( µg/ml). after d, cells were stained with antibodies to cd -pe (clone m-a ; ) from bd biosciences and icos-apc (clone c . a; ) from biolegend. proliferating activated t cells were facs-sorted as cfse lo cd + icos + and expanded in vitro in the presence of il- ( iu/ml). to select haspecific t cells, inflexal v-reactive cfse lo cultures were relabeled with cfse and stimulated with irradiated autologous monocytes untreated or pulsed with recombinant h -ha ( µg/ ml). after d, proliferating activated t cells were sorted as cfse lo cd + icos + and cloned by limiting dilution. in some experiments, positive cultures from t cell libraries were labeled with cfse and cultured at a ratio of : with irradiated autologous monocytes untreated or pulsed with h -ha peptide pool ( µm per peptide). after d, proliferating activated t cells were sorted as cfse lo cd + icos + and cloned by limiting dilution. t cell clone reactivity was determined by stimulation with h -ha peptide pool ( µm per peptide) or recombinant h -ha ( µg/ml) in the presence of irradiated autologous monocytes or b cells as apcs. in some experiments, h -ha peptides or recombinant h -ha were titrated by serial dilution. epitope mapping was performed by stimulation of t cell clones with irradiated autologous ebv-b clones, untreated or prepulsed for - h with individual peptides ( mers overlapping of or mers overlapping of ) covering the entire sequence of h -ha ( µm per peptide). to determine mhc restriction of t cell clones, autologous apcs were pulsed for h with recombinant h -ha, washed extensively, and then cultured with t cells in the absence or presence of blocking anti-mhc-ii monoclonal antibodies produced in-house from hybridoma cell lines (anti-hla-dr, clone l from atcc, hb- ; anti-hla-dq, clone spvl [spits et al., ] ; anti-hla-dp, clone b / [watson et al., ] ). in all experiments, proliferation was assessed on day , after incubation for h with µci/ml [methyl- h]thymidine (perkinelmer). data were expressed as counts per minute. ex vivo-sorted memory cd + t cell subsets and cfse lo fractions of inflexal v-stimulated memory cd + t cell subsets from donor hd were analyzed by deep sequencing. in brief, . - × t cells were centrifuged and washed in pbs, and genomic dna was extracted from the pellet using qiaamp dna micro kit (qiagen), according to manufacturer's instructions. genomic dna quantity and purity were assessed through spectrophotometric analysis. sequencing of tcr vβ cdr was performed by adaptive biotechnologies using the immunoseq platform. in brief, after a multiplex pcr reaction designed to target any cdr vβ fragments, amplicons were sequenced using the illumina hiseq platform. raw data consisting of all retrieved sequences of nucleotides or corresponding amino acid sequences and containing the cdr region were exported and further processed. the assay was performed at deep level for ex vivosorted total memory cd + cells (detection sensitivity, cell in , ) and at survey level for cfse lo inflexal v-reactive cultures (detection sensitivity, cell in , ). each clonotype was defined as a unique productively rearranged tcr vβ nucleotide sequence; data processing was done using the productive frequency of reads provided by immunoseq analyzer v . . sequence analysis of rearranged tcr vβ genes of ha-specific t cell clones from donor hd was performed as previously described (latorre et al., ) . briefly, cdna from individual t cell clones was obtained by reverse transcription of total rna from - cells per reaction. rearranged tcr vβ genes were pcr amplified using a forward primer pool targeting vβ genes and reverse primer pairing to c -c β-chain constant region. sequence amplification was assessed through agarose gel electrophoresis; successfully amplified fragments were sequenced by sanger method, and tcr sequence annotation was performed by using imgt/v-quest algorithm (lefranc et al., ) . hla typing and peptide-mhc-ii binding affinity measurement hla genotype of the patients was determined by reverse sequence-specific oligonucleotide probes dna typing (labtype; one lambda) performed at the irccs san matteo hospital foundation (pavia, italy). affinity measurements of h -ha mer peptides recognized by hla-dr-restricted t cell clones from donor hd to recombinant hla-drb * : or hla-drb * : molecules was performed by immunitrack (copenhagen, denmark), as previously described (justesen et al., ) . briefly, recombinant hla-drb isoforms were refolded in vitro in the presence of recombinant hla-dra and increasing concentrations of h -ha mer peptides. titrated pan-hla-dr-binding epitope was used as positive control. after -h incubation at room temperature and ph , correctly folded heterotrimeric pmhc-ii complexes were detected by elisa; data were analyzed using graphpad prism software. purification of mhc-ii presented peptides dcs generated from donor hd were pulsed for h with µg/ ml recombinant h -ha at a cellular density of × cells/ml and matured overnight with ng/ml lps (enzo life sciences) at a cellular density of × cells/ml. ha-specific ebv-b cell clones isolated from igg + memory b cells of each of the four donors were pulsed overnight with ng/ml recombinant h -ha at a cellular density of × cells/ml. mhc-ii complexes were purified from ∼ × ha-pulsed dcs or ha-pulsed ebv-b cells with a protocol adapted from bassani-sternberg et al. ( ) . briefly, the b cells were lysed with . % sodium deoxycholate, % octyl-β-d-glucopyranoside (sigma-aldrich), . mm iodoacetamide, mm edta, and complete protease inhibitor cocktail (roche) in pbs at °c for h. the lysates were cleared by -min centrifugation at , g at °c, and mhc-ii complexes were purified by immunoaffinity chromatography with the anti-hla-dr/dp/dq hb- monoclonal antibody produced in-house from hybridoma cell line iva (atcc, hb- ) and covalently bound to protein a sepharose beads (thermo fisher scientific). the cleared lysates were loaded three times into the affinity columns at °c and subsequently washed at °c with -column volumes of mm nacl, mm tris•hcl, ph . (buffer a); -column volumes of mm nacl, mm tris•hcl, ph ; -column volumes of buffer a; and finally -column volumes of mm tris•hcl, ph . the mhc-ii complexes were eluted at room temperature by addition of µl of . m acetic acid, in total five elutions for each sample. small aliquots of each eluted fraction were analyzed by % sds-page to evaluate yield and purity of mhc-ii complexes. sep-pak tc (waters) cartridges were used for further separation of peptides from mhc-ii subunits. the cartridges were prewashed with % acetonitrile (acn) in . % formic acid, followed by . % tfa, and subsequently loaded three times with each fraction eluted from the immunoaffinity column. after loading, the cartridges were washed with . % tfa, and the peptides were separated from the more hydrophobic mhc-ii chains by elution with % acn in . % tfa. the peptides were further purified using a silica c column tip (harvard apparatus) and eluted again with % acn in . % tfa. finally, the peptides were concentrated by vacuum centrifugation and resuspended in % acn, . % tfa, and . % formic acid for ms analysis. liquid chromatography-tandem ms and data analysis mhc-ii peptides were separated on an easy-nlc hplc system coupled online to a q exactive mass hf spectrometer via a nanoelectrospray source (thermo fisher scientific). peptides were loaded in buffer a ( . % formic acid) on in-house packed columns ( -µm inner diameter, -cm length, and . -µm c particles from dr. maisch) and eluted with a nonlinear -min gradient of - % buffer b ( % acn and . % formic acid) at a flow rate of nl/min and a column temperature of °c. the q exactive was operated in data-dependent mode with a survey scan range of - , m/z and a resolution of , at m/z . up to most abundant isotope patterns with a charge ≥ were isolated with a . -th-wide isolation window and subjected to higher-energy c-trap dissociation fragmentation at a normalized collision energy of . fragmentation spectra were acquired with a resolution of , at m/z . dynamic exclusion of sequenced peptides was set to s to reduce the number of repeated sequences. thresholds for the ion injection time and ion target values were set to ms and e , respectively, for the survey scans and ms and e for the tandem ms scans. data were acquired using xcalibur software (thermo fisher scientific). maxquant software was used to analyze ms raw files. tandem ms spectra were searched against the a/california/ / (h n ) ha sequence (uniprotkb: a a exw ), the bovine uniprot fasta database, the human uniprot fasta database, and a common contaminants database ( entries) by the andromeda search engine (cox et al., ) . n-terminal acetylation and methionine oxidation were set as variable modifications; no fixed modifications were selected; the enzyme specificity was set to unspecific, with a minimum peptide length of aa. a false discovery rate of % was required for peptides. peptide identification was performed with an allowed precursor mass deviation of ≤ . ppm and an allowed fragment mass deviation of ppm; "match between runs" option was disabled. the ms proteomics data have been deposited to the proteomexchange consortium via the pride (perez-riverol et al., ) partner repository with the dataset identifier pxd . in silico analysis mhc-ii binding affinity of each theoretical h -ha-derived mer peptide was calculated using the iedb tool for mhc-ii binding prediction (http://tools.iedb.org/mhcii/; paul et al., ) . donor-tailored analyses were performed using iedb's recommended method and considering the set of mhc-ii alleles carried by each donor at the following loci: hla-drb , hla-drb / / (if associated), hla-dqa /dqb in cis-or transpairing, and hla-dpa /dpb in cis-or trans-pairing. top scoring h -ha mer peptides for each donor were selected based on percentile rank calculated by comparison to a large set of random natural peptides. apl was computed as described in mettu et al. ( ) . briefly, an aggregate z-score of conformational stability was determined for each h -ha residue by integrating four structural parameters obtained from the d structure of postfusion ha resolved by x-ray diffraction (pdb codes: lzg for ha domain [xu et al., ] ; htm for ha domain in the postfusion conformation [bullough et al., ] ). the z-score statistic was then used to calculate an apl for each theoretical h -ha mer peptide, following the rationale that the liberation of antigenic peptides might be facilitated by surrounding unstable regions that are readily unfolded and targeted by endosomal proteases. for the optimization of combined predictors, we systematically performed peptide binding affinity predictions of each theoretical h -ha mer peptide using iedb for the mhc-ii alleles carried by each donor, considering for each peptide the best scoring affinity within each group of mhc-ii alleles. using the set of epitopes recognized by memory t cells of each donor as true positives, we computed roc curves for each predictive model and calculated the corresponding auroc. combined predictors for each donor were then built by iteratively weighting the contributions of epitope likelihood based on structural accessibility and peptide binding affinity to mhc-ii, until we could maximize the auroc value. statistical analyses were performed using graphpad prism software or r software v . . . ec (ng/ml) and k d (ng/ml) values were calculated by nonlinear regression curve fit ( pl with automatic outlier elimination) using graphpad prism software. significance was assigned at p < . , unless stated otherwise. specific tests are indicated in the figure legends for each comparison. online supplemental material fig. s shows in representative t cell clones blocking experiments with anti-hla-dr, anti-hla-dp, and anti-hla-dq antibodies to determine mhc-ii restriction. it also shows mhc-ii binding affinity of h -ha peptides recognized by hla-dr-restricted t cell clones from donor hd as measured in vitro. fig. s shows the ability of a panel of h -ha-reactive t cell clones to cross-react to has from different influenza a strains. fig. s shows the functional avidities for peptide and naturally processed h -ha of t cell clones isolated from the memory or the naive compartment determined by stimulation with titrated doses of peptides or recombinant h -ha. fig. s reports an analysis of mhc-ii eluted h -ha peptides measured by ms and of mhc-ii h -ha presented peptides to t cell clones of anti-head or anti-stem ebv-b cell clones. fig. s reports the theoretical h -ha mer peptide predicted to bind to selected mhc-ii alleles using the iedb tool and a the number of iedb-predicted peptides and mhc-ii eluted peptides measured by ms-based peptidomics found to be recognized by t cells. table s shows epitope mapping of h -ha-reactive t cell clones isolated from memory cd + t cell subsets of donor hd . table s shows tcr vβ sequence and epitope specificity of h -ha-reactive t cell clones isolated from cd + memory (tcm, tem, or ctfh) t cell compartment. table s shows hla class ii typing of the four hds included in this study. table s shows tcr vβ sequence and epitope specificity of h -ha-reactive t cell clones isolated from the cd + naive t cell compartment. provided online are six tables. table s shows epitope mapping of h -ha-reactive t cell clones isolated from memory cd + t cell subsets of donor hd . table s shows tcr vβ sequence and epitope specificity of h -ha-reactive t cell clones isolated from cd + memory (tcm, tem, or ctfh) t cell compartment. table s shows hla class ii typing of the four hds included in this study. table s shows tcr vβ sequence and epitope specificity of h -ha-reactive t cell clones isolated from the cd + naive t cell compartment. table s lists h -ha peptides identified by ms-based mhc-ii peptidomics in donor hd . table s lists h -ha peptides identified by ms-based mhc-ii peptidomics in donors hd -hd . figure s . peptide binding to mhc-ii is a necessary but not sufficient condition to define immunodominance. mhc-ii binding affinity of each theoretical h -ha mer peptide was calculated using the iedb tool for mhc-ii binding prediction (http://tools.iedb.org/mhcii/). personalized analyses were performed by considering the mhc-ii alleles carried by each donor (hla-drb , hla-drb / / , hla-dqa /dqb in cis-or trans-pairing, hla-dpa /dpb in cis-or transpairing). top scoring h -ha mer peptides for each donor were selected based on percentile rank calculated by comparison to a large set of random natural peptides. (a) sets of h -ha peptides predicted as mhc-ii binders at different thresholds for each donor. the x axis indicates h -ha amino acid sequence. the sets of top predicted mhc-ii binder peptides are reported as color-coded segments. the immunodominant regions targeted by memory cd + t cells of each donor are reported with color-coded shadows. (b and c) iedb-predicted peptides and mhc-ii eluted peptides measured by ms-based peptidomics defined discrete h -ha regions. the tables summarize the number of ha regions found presented on mhc-ii by ms-based peptidomics, or predicted as mhc-ii binders in different donors. the corresponding number of ha epitopes recognized by at least one t cell clone regardless of the subset of origin (b) or isolated from the memory compartment (c) are reported. identification of immunodominant epitopes is marked by an asterisk. (d) sensitivity was evaluated in terms of percentage of memory t cell clones for which the cognate peptide was identified by ms-based mhc-ii peptidomics (ms pept., orange bar) or by mhc-ii binding predictions (iedb) at different thresholds (iedb, green bars). each symbol represents a different donor. advanced immunization technologies). f. sallusto and the institute for research in biomedicine are supported by the helmut horten stiftung. author contributions: a. cassotta: conceptualization, methodology, validation, investigation, data curation, formal analysis, visualization, software, writing -original draft lewis: resources, writingreview & editing. a. lanzavecchia: writing -original draft, writing -review & editing. f. sallusto: supervision, conceptualization, project administration, data curation, visualization, funding acquisition, writing -original draft defining hla-ii ligand processing and binding rules with mass spectrometry enhances cancer epitope prediction is it possible to develop a "universal" influenza virus vaccine? outflanking antibody immunodominance on the road to universal influenza vaccination mass spectrometry of human leukocyte antigen class i peptidomes reveals strong effects of protein abundance and turnover on antigen presentation structure of influenza haemagglutinin at the ph of membrane fusion t cell affinity maturation by selective expansion during infection proteome-wide analysis of hiv-specific naive and memory cd (+) t cells in unexposed blood donors a neutralizing antibody selected from plasma cells that binds to group and group influenza a hemagglutinins andromeda: a peptide search engine integrated into the max-quant environment autophagy promotes mhc class ii presentation of peptides from intracellular source proteins human parainfluenza virus type up-regulates major histocompatibility complex class i and ii expression on respiratory epithelial cells: involvement of a stat -and ciitaindependent pathway human naive and memory cd + t cell repertoires specific for naturally processed antigens analyzed using libraries of amplified t cells antigen discovery and specification of immunodominance hierarchies for mhcii-restricted epitopes the role of naive t cell precursor frequency and recruitment in dictating immune response magnitude improved methods for predicting peptide binding affinity to mhc class ii molecules functional recombinant mhc class ii molecules and high-throughput peptide-binding assays determinants of immunodominance for cd t cells protein structure shapes immunodominance in the cd t cell response to yellow fever vaccination is it possible to develop a "universal" influenza virus vaccine? potential target antigens and critical aspects for a universal influenza vaccine three-dimensional structure determines the pattern of cd + t-cell epitope dominance in influenza virus hemagglutinin t cells in patients with narcolepsy target self-antigens of hypocretin neurons persistent antibody clonotypes dominate the serum response to influenza over multiple years and repeated vaccinations structural characterization of viral epitopes recognized by broadly cross-reactive antibodies imgt, the international immunogenetics information system memory t cells in latent mycobacterium tuberculosis infection are directed against three antigenic islands and largely contained in a cxcr +ccr + th subset clonal selection of helper t cells is determined by an affinity threshold with no further skewing of tcr binding properties evolution of antigen-specific t cell receptors in vivo: preimmune and antigen-driven selection of preferred complementaritydetermining region (cdr ) motifs hla-dm and hla-do, key regulators of mhc-ii processing and presentation cd + t-cell epitope prediction using antigen processing constraints antigen structure influences helper t-cell epitope dominance in the human immune response to hiv envelope glycoprotein gp unconventional recognition of peptides by t cells and the implications for autoimmunity naive cd (+) t cell frequency varies for different epitopes and predicts repertoire diversity and response magnitude t cell receptor cross-reactivity between similar foreign and self peptides influences naive cell population size and autoimmunity rapid development of broadly influenza neutralizing antibodies through redundant mutations development and validation of a broad scheme for prediction of hla class ii restricted t cell epitopes the pride database and related tools and resources in : improving support for quantification data the bare lymphocyte syndrome and the regulation of mhc expression the ins and outs of mhc class ii-mediated antigen processing and presentation efficient presentation of soluble antigen by cultured human dendritic cells is maintained by granulocyte/ macrophage colony-stimulating factor plus interleukin and downregulated by tumor necrosis factor alpha from vaccines to memory and back the relationship between immunodominance, dm editing, and the kinetic stability of mhc class ii:peptide complexes antigen-loading compartments for major histocompatibility complex class ii molecules continuously receive input from autophagosomes dominance and crypticity of t cell antigenic determinants modulation of antigen processing by bound antibodies can boost or suppress class ii major histocompatibility complex presentation of different t cell determinants measurement of diversity characterization of monoclonal antibodies against cell surface molecules associated with cytotoxic activity of natural and activated killer cells and cloned ctl lines virus-specific cd (+) memory-phenotype t cells are abundant in unexposed adults an efficient method to make human monoclonal antibodies from memory b cells: potent neutralization of sars coronavirus variations in mhc class ii antigen processing and presentation in health and disease peptide binding predictions for hla dr, dp and dq molecules detection of a novel human class ii hla antigen suppressive effect of antibody on processing of t cell epitopes continuing challenges in influenza structural basis of preexisting immunity to the h n pandemic influenza virus confronting complexity: real-world immunodominance in antiviral cd + t cell responses immunodominance in major histocompatibility complex class i-restricted t lymphocyte responses immunodominance in tcd + responses to viruses: cell biology, cellular immunology, and mathematical models a renewable source of donor cells for repetitive monitoring of t-and b-cell alloreactivity memory cd t cells in influenza proliferation was assessed on day after a -h pulse with [ h]thymidine and expressed as counts per minute. mhc-ii restriction was defined based on inhibition of t cell proliferation > %. shown are representative ( of analyzed) t cell clones specific for immunodominant (a) or subdominant (b) h -ha epitopes. data are grouped based on the epitope specificity of the t cell clones tested, reported on top of each plot. (c) summary of mhc-ii restriction of h -ha-specific t cell clones isolated from donor hd . the x axis indicates h -ha amino acid sequence; each color-coded segment represents the peptide recognized by t cell clones restricted by hla-dp (pink), hla-dq (blue), or hla-dr (green). the immunodominant h -ha - region identified in the memory compartment of donor hd is highlighted with a red shadow. (d) mhc-ii binding affinity of h -ha peptides recognized by hla-dr-restricted t cell clones from donor hd was measured in vitro. briefly, recombinant hla-drb isoforms were refolded in the presence of recombinant hla-dra and increasing concentration of peptides, at room temperature and ph . k d values were calculated by nonlinear regression fitting of pmhc-ii refolding curves we thank the blood donors for their participation in the study. we thank david jarrossay for cell sorting, sandra jovic and chiara silacci for technical assistance, and the servizio tipizzazione of the fondazione irccs policlinico san matteo, pavia, italy, for hla typing.this study has been carried out with financial support key: cord- -ntbvmssh authors: nan title: immunogenicity date: - - journal: j cell biochem doi: . /jcb. sha: doc_id: cord_uid: ntbvmssh nan ia moyecules with respect to their roles as peptide receptors and target structures for tcr interaction. particular attention has been paid to distinguishing between local and distant effects of amino acid substitutions on ia function and to determining which residues interact with peptide antigen and which (if any) with the tcr. this ex erimental approach has led to the identification of several regions of the pofvorphic amino-terminal domains of the a and p chains as playing critical roles in chain-chain association and quaternary ia conformation. the a and p l putative helical regions have been found to have distinct degrees of structural lability, with the a helix showing much greater susceptibility to conformational change due to allelic variation in other re ions of the molecule. allelically polymor hic residues in the a and p domainstave been shown to play important roles in &e activity of the assembly/folding control regions, and hence, analysis of local binding roles of specific residues in ia molecules must take this additional effect of substitutions at these positions into account. by controlling for large scale conformational effects, individual residues in the p chain have been assigned to desetopic ( eptide interaction) and histotopic (tcr interaction) roles. in the cytochrome c molel, a putative peptide bindin "pocket" involvin residues from both the postulated p l a helix and also the p-stran% floor has been defined, residues controlling both the extent of binding and the orientation of the bound peptide have been identified, and at least one residue with tcr interaction potential without obvious peptide binding properties has been localized. combining these data with those of other investigators leads us to propose a general model of class i mhc structure-function relationships. we have shown previously that memory b cells transferred into k-allotype distinct congenic rats in the absence of any priming antigen are deleted from the adoptive host within a matter of weeks (half-life of - weeks). in contrast co-injection of antigen with the cells facilitates their survival and the maintenance of a donor response for periods in excess of one year. in the experiments reported here we ask if the persistence of t cell memory is also dependent on antigen. . carrier (klh) primed t cells were transferred in the presence or absence of antigen into irradiated, k-allotype distinct adoptive host. a t various times after transfer these rats were injected with x ' hapten-carrier (dnp-klh) primed b cells together with ig of soluble dnp-klh. this limiting number of b cells makes a secondary type response only if carrier-specific memory t cells survive in the adoptive host. we found that already at weeks following transfer without antigen, no memory t cell help was available for these b cells. in contrast t cells transferred together with ~g klh provided help for secondary type donor responses at and weeks after transfer. we conclude that longterm memory at both the t and b cell levels does not reside with small, very long-lived, resting cells but. with active clones that are maintained by small amounts of antigen that may persist for long periods. once antigen is lost from lymphoid tissues both t and b cell memory wanes within a relatively short time. t cells recognize antigen in the form of short peptides associated to class i or class i mhc molecules. each mhc molecule has the ability to bind a large number of peptides and peptides with unrelated sequences can compete for binding to the same mhc molecule, as well as in vitro. in vivo competition strictly correlates with the capacity of the competitor peptide to bind to the mhc molecule presenting the antigenic peptide and its extent dependes on the molar ratio between antigen and competitor. competition among different peptides derived by processing of hen egg-white lysozyme (hel) appears to exert a major influence on the immunodominance of antigenic determinants recognized by t cells. thus, the h l peptides - and - are both generated by hel processing and are both able to bind to the i-e molecule but only - becomes immunodominant because it has th ability to compete in vivq with other hel peptides, such as - , for the available sites on the i-e molecule. however, two immunodominant t cell epitopes, such as those in hel peptides - and - , both interacting with i-ak molecules, do not compete with each other when injected together at equimolar concentrations. such a coexistance is anticipated between peptides that bind with relatively high affinity to the presenting molecule and thus have both the chance to occupy a number of binding sites sufficient for t cell activation. r v s e iii xen ic tr lantation. in v i m lnvesti$ation uslng mocloml a n t m i e s r e e -t e x e y skin gracs-val on m i c e w a s significantl pr:lrd l g anti-antihdy trea-t but n o t a b anti-antibody: w i d e r the saue animals but a n t i c d antibody did prolong minor a n t w -d i allqrdts. in v i m studies r m that primary proliferation a n f z . prcdwtion & -f i cells in response to mmkey stimulators was weak conpared to allogeneic reqonses. secondary responses t o xenogeneic stinulation were strong after in v i m priming but required the presence of responder nc's. assays for c y g t s t cell effectors in m i c e which had rejected monkey skin revealed few such cells. zhese results est that widely d i a t e xencgeneic processing and presentation, since xenogeneic antigens require that such presentation be in association with the.= antigens of regponder apc's, the xenogeneic r a f t s have a functional similarity t o aff leted allografts. shoved t h a t f e t a l r e n a l and f e t a l and p o s t n a t a l testis a l l o g r a f t s survived longer than corresponding a d u l t t i s s u e i n non-immunosuppressed outbred r a t hosts. the c u r r e n t study a s k s v h e t h e r t h e d i f f e r e n c e i n s u r v i v a l betveen r e n a l and t e s t i c u l a r g r a f t s and between g r a f t s of d i f f e r e n t ages is r e l a t e d t o d i f f e r e n t i a l t i s s u e expression of class i and class i mrna t r a n s c r i p t s or s u r f a c e antigens. and i f t h e s e p a t t e r n s change w i t h t r a n s p l a n t a t i o n . congeneic mice w e found t h a t prolonged s u r v i v a l of c bl/ f e t a l r e n a l (n= ; p< . ) and f e t a l (n= ; ~( . ) and p o s t n a t a l (n= ; ~( . ) t e s t i s mouse a l l o g r a f t s t r a n s p l a n t e d beneath t h e r e n a l c a p s u l e of a d u l t r e c i p i e n t bio.a mice and t h i s s u r v i v a l c o r r e l a t e s i n v e r s e l y w i t h t h e expression of class i and class i mrna (northern a n a l y s i s ) and p r o t e i n s (immunohistochemistryy) and t h a t both p r o t e i n and mrna increased throughout ontogeny f o r both t h e testis and kidney. after t r a n s p l a n t a t i o n t h e r e vas a marked i n d u c t i o n of mhc mrna t r a n s c r i p t s f o r both testis (n= ) and kidney (n= ). implanted f e t a l kidney t i s s u e t h a t survives. however. f a i l e d t o express d e t e c t a b l e mhc p r o t e i n , i n d i c a t i n g t h a t some p o s t -t r a n s c r i p t i o n a l modification i n t h i s t i s s u e occurs. t o a f f o r d it p r o t e c t i o n from r e j e c t i o n . implanted testis shoved i n d u c t i o n of both mrna and p r o t e i n v e l l above i t s much lower baseline. i n d i c a t i n g t h a t i t s r e g u l a t i o n , i n c o n t r a s t t o t h e kidney may be t r a n s c r i p t i o n a l . thus t h e f e t u s may lower t h e mhc burden as a s t r a t e g y t o escape r e j e c t i o n e i t h e r by p o s t t r a n s c r i p t i o n a l modification of p r o t e i n expression a s i n t h e kidney or by t r a n s c r i p t i o n a l modification of mrna as i n t h e testis. culture of thymus tissue in -deoxyguanosine ( dgua) is thought to reduce tissue immunogenicity by selectively depleting highly immunogenic, thymic immigrants of bone marrow origin. in the mouse dgua treated thymus tissue survival is markedly enhanced compared to untreated tissue when transplanted under the kidney capsule of allogeneic recipients. these experiments were repeated in the rat. as expected, strain da neonatal thymus tissue was rejected when transplanted under the kidney capsule of normal allogeneic strain pvg rats. surprisingly. acute rejection occurred even when the tissue was cultured for days in mm pdgua ( x the effective dose in mice). by in vitro criteria this dose was very effective in destroying thymocytes. to test whether residual marrow derived cells that escaped pdgua treatment were responsible for inducing rejection we "parked" the dgua treated da tissue in t cell depleted pvg rats. our working hypothesis was that the few remaining donor derived cells of marrow origin would be overgrown by host type cells. when pdgua-treated da thymus tissue was transplanted into t cell depleted pvg recipients graft rejection did not occur. however da pdgua treated thymus tissue, parked for as long as days in t cell depleted pvg rats, was acutely rejected when retransplanted into normal pvg recipients. we interpret these results to suggest that rat thymic epithelium devoid of marrow derived cells is innately immunogenic. c corinne amiel, violaine gugrin, thierry may, philippe canton, gilbert c faure, laboratoire d'immunologie and maladies infectieuses, chu de nancy, facult de mgdecine, vandoeuvre les nancy, france. lfal is a dimeric membrane molecule composed of a specific alpha chain (cdlla) and a beta chain (cd ) common to three members of the lfa family. lfal is physiologically expressed on all white blood cells, while other molecules of the lfa family (with cdllb and cdllc alpha chains) are restricted to cells of myeloid lineage. a defective expression of lfal has been described in some congenital immune deficiency and in aids. we investigated the lfal defect on peripheral blood lymphocytes from hiv+ patients. three different monoclonal antibodies were used, respectively directed to chain-specific epitopes of cdlla (spvl , sanbio) and cd (iot , immunotech) and to a conformational epitope involving both chains (iot , immunotech). cell suspensions were stained in indirect immunofluorescence and a flow cytometer (epics profile, coultronics) was used to assess the percentages of stained cell, the fluorescence intensity and the shape of fluorescent peaks. our data suggest that lfal expression is impaired in hiv+ patients both through the quantitative expression of each chain and through conformational alterations. the adhesion molecule lfa- is known to be important in antigen presentation. we have previously shown that both monocyte and t cell lfa- play a role in the interaction between these two cells (eji d; , ) . antibody to icam- (known to act as a ligand for lfa- ) also inhibits antigen presentation, although icam- is not thought to be expressed on resting t cells (eji : , ) . we have looked at the expression of icam- on t cells after incubation with cytokines and found that only il- consistently effects an increase in both the percentage of icam- positive cells and in the level of expression. in addition we have found that a proportion of resting t cells express very low levels of icam- . double labelling experiments have shown that these cells are part of the memory t cell population as defined by antibodies to uchli, lfa- and lfa- , and furthermore that icam- negative cells are unable to respond to to antigens such as ppd and flu but are able to respond to pha. this suggests that icam- represents an additional marker on the memory t cell population which more precisely defines the subset able to respond to recall antigens icam- expression on t cells, anne-marie buckle and nancy hogg, macrophage lab. icrf, lincolns inn fields, london, wc a px, u.k. immunization, francis r. carbone and michael j. bevan, department of immunology, research institute of scripps clinic, la jolla, ca . ctl recognize peptide forms of processed, foreign antigens in association with class i molecules of the mhc and are usually directed against endogenously synthesized "cellular antigens" such as those expressed by virusinfected cells. in vifro studies have shown that small exogenous peptides can directly associate with class i molecules on the cell surface and mimic the target complex derived by intracellular processing and presentation. we have recently generated ova-specific, h- kb-restricted ctl by immunizing c bl/ mice with a syngeneic tumor line transfected with the ova cdna. the ctl recognize the ova transfectant eg -ova and the synthetic peptide ova but fail to recognize the native protein. we reasoned that given the potential for direct peptide/class ?'&$%ation observed in vifro, ova s,- ra may induce ctl after in vivo priming. however, we found that this is not the case. ova,,,-,,, and peptides of increasing lengths up to which are all able to form the target complex in vitro, are inefficient at priming ~% -%~~ specific ?h%sponses following intravenous injection. this is also true for both native and denatured ova. in contrast to these results, the synthetic peptide ova g:z corresponding to a peptide in a partial tryptic digestion of ova can efficiently prime c bl/ mice in vrvo following intravenous injection. this peptide elicits ctl which appear identical to those derived from animals immunized with syngeneic cells producing ova endogenously. it is now well established that human t lymphocytes can be activated via the t cell specific cd antigen. in order to determine if a factor@) other than the single cd polypeptide is involved in cd mediated signal transduction, we have stamy transfected murine l cells with the human cd cdna. we report that such transfectants expressed hah levels of cd at the cell surface. formed sheep erythrocyte rosettes and expressed the three cd epitqm previously defined on human t lymphocytes, including the "activation associated' t i epitope. the latter observation unequivocally demonstrating that expression of the ti epitope, in contrast to a previous report, is entirely independent of t cell specific factors. combinations of cd mabs that are potent stimulators of human t cells. however, failed to elicit either an increase in the concentration of intracellular free calcium or augment [ h]-thymidine inmrporatbn in the transfectants. these results provide both formal identification of the cd cdna and dearly demonstrate that the single cd polypeptide expressed in an heterokgous cell system devoid of t cell specific factors, cannot alone transduce intracellular signals in response to stimulatory combinations of cd mabs. the results are therefore consistent with the notion. that the functional cd antigen expressed in human t lymphocytes, requlres the association of another, as yet, undefined factor@). this conclusion was based on several lines of evid nce incl ding the observation that mabs specific for the class i a domain of either h-zl or l d b interfered with t e generation of cd -dependent (low substitution at position in the a domain are not lysed by cd -dependent primary ctl but are lysed by secondary cd -independent (high affinity) ctl generated in the presence of antibody to the a domain. populations of ctl. we have isolated and characterized a c d w , cd -da-specific cpl line. this line is cd -independent and is capable of lysing the addition, we are currently generating clones from primary $-specific ctl cultures to obtain cd -dependent (low affinity) ctl. directed rnutagenesis are being tested with the cd -dependent and cd -independent clones to define additional residues important for cd recognition. the comparison of ctl clones with different cd dependencies will allow us to more precisely define the role of cd in t cell recognition. percolle from the buffy coat of one unit ot blood. these cells (= x ) are introduced into a curame elutriation centrifuge (rotor speed of rpm; loading flow of ml/min). nine fractions can be obtained. the first three containing > % lymphocytes; fraction ( rpm- ml/min) and fraction ( rpm- ml/min! contain both lymphocytes and monocytes and the next three fractions contain > % monocytes; the finat fraction (rotor off) contains monocytes + granulocytes. cells from each fraction ( x /well) are incubatee for five days with tetanus toxoid ( . lf/well) and an enriched population of t cells ( x /well). quadriplicate samples are then pulsed for hours with 'h methyl thymidine. maximum apc activity is found in fractions and representing to % of the mononuclear cells. apc activity for these two fractions can be further purified by selective absorption of the cells onto gelatin coated surfaces that have been preincubated with plasma. the non adherent lymphocytes are rgmoved after two hours. after overnight incubation spontaneously released cells ( - x ) can be harvested which have a higher apc activity than cells rotated by elutriation alone. these methods are now highly reproducible in our laboratory, so we can now begin to characterize and study these cells. the male s p e c i f i c h-y a n t i g e n h a s been shown t o behave as a minor histocompatibility a n t i g e n in man and mouse. i n t r a n s p l a n t a t i o n , male t i s s u e may t r i g g e r t h e c l o n a l expansion of h-y reactive hhc r e s t r i c t e d effector cells of female o r i g i n . although male epidermal cells (ec) can induce an anti-h-y t cell response in female mice, so far in v i t r o techniques have f a i l e d t o i d e n t i f y t h e cell-defined h-y a n t i g e n on murine ec ( ). here w e developed a cr release assay t o use human c u l t u r e d k e r a t i n o c y t e s (k) as t a r g e t cells for hla-a specific and ma-a r e s t r i c t e d h-y s p e c i f i c t cell clones. hla-a + but n o t h l a -a t k were l y s e d by anti-ma-a ctls i n a dose dependent manner. low but d e t e c t a b l e l e v e l s of anti-h-y k i l l i n g were found a g a i n s t ma-a + male k b u t n o t a g a i n s t h l a -a t male or hla-a + female k. both l e v e l s of a l l o r e a c t i v e and h-y s p e c i f i c l y s i s were d r a m a t i c a l l y enhanced after exposure of k t o ifn gamma. these r e s u l t s s t r o n g l y suggest t h a t h m a n male s k i n cells are d i r e c t l y s u s c e p t i b l e for h-y d i r e c t e d t c e l l k i l l i n g through t h e expression of f u n c t i o n a l h-y/hla complexes on t h e i r cell s u r f a c e . i n view of t h e s e f i n d i n g s , t o g e t h e r w i t h our r e c e n t s t u d i e s on t h e expression of h-y ctl determinants on h m a n hematopoietic p r o g e n i t o r c e l l s ( , t h e r o l e of h-y a s a t a r g e t s t r u c t u r e f o r c e l l mediated immunity i n o l i n i c a l t r a n s p l a n t a t i o n should be s e r i o u s l y taken i n t o account. . steinmuller d. and burlingham w.j. t r a n s p l a n t a t i o n @ , , , . . voogt p.j., goulmy e., fibbe w.e., e t a l . j . clin. invest. sept. . c diphteria toxoid (dt) presentation by hla dr transfected murine fibroblasts bismuth, laboratory of c e l l u l a r and t i s s u l a r immunology, chu p i t i e s a l p b t r i b r e , p a r i s , france and veterans medical c e n t e r , iowa c i t y , usa. l t r a n s f e c t a n t s e x p r e s s i n g s i n g l e type of human mhc c l a s s i molecules produced by dna conjugate formation has been studied with cloned t cell lines and a b cell hybridoma and with t cells and b cells from normal mice. resting t cells and b cells do not form appreciable numbers of conjugates but conjugates are formed between t cells stimulated with alloantigen for four days and b cells activated by hour culture with lps. irrelevant lymphocytes do not affect the rate of specific conjugate formation in suspensions of cells agitated by gentle rocking but impair conjugate formation when cells are allowed to settle in round bottom tubes. in further experiments, it was shown that the conditions for the induction of lymphokine secretion by the t cell were not indentical to the conditions for conjugate formation.the significance of these and other observations for the interaction of t cells and b cells in vivo will be discussed. of the primary mixed leukocyte reaction (mlr) and that this reaction occurs in multicellular dendritic cell-cd + t cell clusters [cellular immunology , - ( ) dendritic cells are able to contact, cluster, and retain allogeneic t cells and induce these alloreactive cells to proliferate and divide. tions labeled with a vital flvorescent dye, we show that only dendritic cells efficiently form stable clusters. labeled monocytes and b cells do not form clusters with t cells. when labeled monocytes and unlabeled dendritic cells are used to stimulate t cells, unlabeled clusters form. labeled monocytes do not move into the clusters until the third day of the mlr. significant levels of il- and a-ifn appear in the culture supernatant by the first or second day. blast transformation by the second day of the mlr as demonstrated by giemsa staining of cluster cytopreps. also been studied by immunoperoxidase staining. it is known that human peripheral blood dendritic cells are potent stimulators using purified dendritic cell popula- the distribution of certain adhesion molecules within clusters has c m microbiology and immunology, emory university, atlanta, ga . immunization of sjl/j mice with myelin basic protein (mbp) induces the t cell-mediated autoimmune central nervous system disease, experimental allergic encephalomyelitis. response against a dominant epitope (residues - ) leads to disease. lymph node t cells from mbp-immune mice react against several epitopes in addition to - indicating that the i-as molecule is able to form immunogenic complexes with several mbp peptides. the question asked in these studies was whether subdominant epitopes from the same molecule would compete with the dominant epitope for binding sites on the i-as molecule. to address this question two t cell clones, one specific for - (sp . ) and a second specific for a second epitope present in peptide - (sp . ) were tested for responsiveness when cultured with the dominant epitope alone or with mixtures of peptides containing dominant and subdominant epitopes. reactivity of sp . against peptide - was inhibited by peptides - and - . reactivity of sp . against peptide - was not inhibited by peptide - although peptides - and - were inhibitory. controls indicated that inhibitory reactivity was not due to toxicity at high concentrations of peptides. these findings imply that subdominant epitopes are able to compete with dominant epitopes of mbp for binding sites on i-as molecules. linda r. gooding, frances c . rawle, david i . kusher, w i l l i a m s . m. wold+ and barbara knowles*. department of microbiology and immunology, emory university school of medicine, atlanta, ga , 'institute f o r molecular virology, s t . louis university school of medicine, s t . louis, mo and *the wistar i n s t i t u t e , philadelphia, pa . i n several v i r u s systems e a r l y non-structural proteins localized predominantly i n the nucleus of infected cells are major t a r g e t antigens f o r cytotoxic t lymphocytes (ctl). whether early synthesis o r nuclear l o c a l i z a t i o n are important factors i n immunodominance is not known. w e have recently developed a system f o r studying the ctl response t o human group c adenoviruses i n mice. by us ng both transfected t a r g e t s and virus deletion mutants w e have shown t h a t , response t o wild type ad . there are two e a t r a n s c r i p t s , s and s . which both encode major e a r l y nuclear antigens d i f f e r i n g by a amino a c i d insertion: both antigens are recognized equally w e l l by ctl. the e encoded k glycoprotein (gpl k) of ad binds t o mhc c l a s s i antigens i n the endoplasmic reticulum preventing t h e i r translocation t o the c e l l surface and strongly inhibiting l y s i s by ad specific ctl. however, the presence of gpl k i n the priming v i r u s does not a f f e c t the s p e c i f i c i t y of the ctl generated f o r e l a , so the immunodominance of t h i s protein cannot be due t o the fact t h a t i t is the only major protein synthesized before gpl k i n the course of infection. using virus deletion mutants we are investigating whether ctl s p e c i f i c f o r other ad antigens can be induced i n the absence of ela, and whether e a is also the dominant antigen recognized i n mice of other mhc haplotypes. respond to antigens present on non-replicative virions. in contrast, we have obtained balc/c i-erestricted t hybridomas specific for the neuraminidase (na) glycoprotein of a/pr influenza which recognize infectious, but not non-replicative virus, closely resembling recognition requirements observed for most class i mhc-restricted responses to influenza. recognition correlated with the rte nova synthesis of viral na within antigen-presenting cells, but did not depend strictly upon the amount of na present in cultures, since high na concentrations could be achieved by addition of non-replicative virus without being stimulatory for na-specific t cells. recognition of a neo-antigen was ruled out, since, in high concentration, na isolated from purified virions, even if reduced and alkylated, was recognized by the t hybridoma clone. isolated na was recognized when added to pre-fixed apc, suggesting that this form of antigen was able to bypass the usual processing pathway of exogenous proteins. this suggests that endogenously-synthesized antigen may use different pathways to achieve class -associated presentation. t lymphocyte activation is a complex event which is influenced by a variety of distinct cell surface molecules. in order to determine the role of individual molecules in the activation process, we have developed an efficient methodology for generating cell variants in which expression of molecules is selectively inhibited by expression of anti-sense rna from an epstein-barr virus episomal replicon. in a previous study, we reported that marked inhibition of cd cell surface expression could be achieved in a human t cell clone using this approach. we have now extended this strategy to another t cell surface molecule, cd , as a first ste towards ascertaining its role in t cell activation. to this end, we s nthesized a &-mer oli onucleotide corresponding to a sequence in. the : end of the c d i n g re ion of human cd'i and inserted it in an anti-sense orientation into this replicon. this a-c% /rep construct was electroporated into jurkat cells. analysis of stable a-cd irep transfectants by immunofluorescence staining and flow cytometry demonstrated complete and selective inhibition of cd expression. in contrast to the nontransfected arent, this cd -variant demonstrated a partial loss in its ability to form conjugates a n to secrete interleukin when stimulated with anti-cd monoclonal antibodies. however, stimulation of the cd -variant with a and pma did result in interleukin secretion. several observations suggest that cd functions not only as an adhesion molecule recognising mhc class i on the adjacent cells but also potentiate the transducting capacity of the tcr/cdg complex. comparison of the mouse ly protein sequence with the homolog rat ox and human t sequences revealed most highly conserved regions in the membrane and cytoplasmic part of the molecule. the conservation of the transmembrane and cytoplasmic sequences in different species may be significant for the function of the cd molecule. in order to initiate the functional dissection of the cd -molecule we constructed mutations in different parts of the molecule. by transfecting the a and b chain genes donated by a cd dependent cytotoxic t cell clone(kb c ) into the mhc class i restricted agd cd t cell hybridoma do- . we were able to reconstitute the ability to respond to k only if the transfer was done with the ly molecules (gabert et al., . cell, . - ) . in this system surface expression of mutated and non mutated ly- molecules were checked by facs-analysis and the molecuar size of the proteins were analysed by immunoprecipitation with the anti-ly- monoclonal antibody /lj . finally functional effects of the mutations were investigated in response towards the k alloantigen. we have simulated graft versus host and host versus graft reactivity in vitro by studying primary anti-minor h responses in a limiting dilution culture system. the ability of bmm and peripheral blood mononuclear cells (pbm) to stimulate and respond in this system were compared by estimating the number of proliferating cells. in gvh-direction the combination of donor-bmm (d-beim) and host-pbm (h-pbm) was to times more effective in stimulating proliferation than any other combination; the same applied to the combination h-pbm/d-bmm in hvg-direction.-using these combinations the median frequency of proliferating cells in gvh-direction was / (range / c- / ) in pairs, in hvg-direction ( pairs) / (range / - / ). % of the proliferating cells had the phenotype of mature t-cells.-using the same combination of responder/stimulator cells we have also estimated the number of cytotoxic cells specific for the hla-identical target cell. in gvh-direction the median estimate (n= ) was / (range / o- / ), in hvg-direction (n= ) / (range / - / ). by split well analysis similar or higher frequencies of cytotoxic cells with specificity for nk-targets were detected (gvhr: / , hvgr: / ). it was however possible to identify a significant number of minor h-specific clones by segregation analysis; their specificity could be confirmed after clonal expansion. the clones had the phenotype of typical cytotoxic t-cells.-the relevance of the two cytotoxic subpopulations described above to clinical events such as gvhd, graft rejection and relapse needs to be clarified.- molecular cloning of murine icah- , k.j. horley, b. baker, and f. takei, terry pathology, university of british columbia, vancouver, b.c., canada. we have previously reported a novel cell surface antigen expressed on activated and proliferating murine lymphocytes. the antigen, termed hala- , is absent or present at low densities on thymocytes, lymph node cells, and fibroblast cell lines, indicating it is not a universal proliferation antigen. some cells of the spleen and bone marrow express mala- at a high density possibly representing in vivo proliferation in these tissues. apparent molecular weight of - kd under both reducing and nonreducing conditions, and is susceptible to endo f digestion. the monoclonal antibody yn / . that reacts with this antigen, profoundly inhibits mlr. a xgtlo cdna library was constructed from ns- cells that express a high level of mala- , and screened with synthetic oligonucleotides resulting in the isolation of a full length cdna clone (- . kb). the cdna sequence has high homology with the human icau- sequence, indicating that hala- may be the murine homologue of this characterized protein. hines, trudeau i n s t i t u t e , inc., p.o. box , saranac lake, ny a tumor c e l l l i n e , et- , has been derived from an apparent fibrosarcoma t h a t arose i n a c bl/ male mouse. antigens. mice t h a t have r e j e c t e d et- become imnune t o these minor h antigens, judged by accelerated s k i n g r a f t r e j e c t i o n , and t h i s imnunity can be t r a n s f e r r e d t o imnunod e f i c i e n t mice w i t h lymphoid c e l l s . however, spleen c e l l s from mice t h a t have r e j e c t e d according to the widely accepted view, cd (t , sheep erythrocyte receptor) is the first t cell-specific antigen to appear on differentiating thymocytes during ontogeny. it follows that cd should be expressed on all immature and mature t cells. using two-color cytofluorometry i have here identified subsets of cd -cd + t cells both in fetal human thymus or spleen and in adult peripheral blood. cd -cd + t cells constitute - % of fetal thymocytes and . - . % of peripheral blood t cells. il- -dependent longterm clones of cdi-cdj+ cells do not react with a panel of monoclonal antibodies (mab) directed against the t ll, tlll or t epitopes of cd and do not transcribe cd mrna. fetal tissue-derived clones react with the tigammaa mab and thus express a functional tcr gamma chain, while cd -cd + clones from peripheral blood are bha + and express a full-length . kb tcr c s .ria. the clones established here are currently being characterized with respect to functional capacities. i conclude that expression of cd is not an absolute prerequisite for the expression of the cd /tcr molecular complex on human t cells. if they are added after hours. these interactions are bidirectional. since both cdlla and cd . and t h e i r ligand i-cam . are expressed on the presenting c e l l s as well as the t cells. however, a l l such e a r l y adhesion related events are not bidirectional since anti-cdz and anti-lfa- . which are expressed d i f f e r e n t i a l l y on t c e l l s and presenting c e l l s respectively are also effective as inhibitors. antibodies, a n t i cd and a n t i cd antibodies do not i n h i b i t clustering but do i n h i b i t p r o l i f e r a t i o n , and t h i s i s seen irrespective o f when the antibodies are added i n t o the assay. our findings suggest t h a t there are two mechanisms involved i n dendritic c e l l -t c e l l interaction, f i r s t l y an inrnediate cell-cell adhesion step and l a t e r a secondary signal transduction process possibly mediated v i a cytokines. the q u a l i t a t i v e d i fferences between dendritic c e l l and b c e l l induced i m n o g e n l c i t y may thus l i e i n e i t h e r o f these two steps. king, department o f eathology, the bland-sutton i n s t i t u t e . university college and middlesex school i n contrast, a n t i class i m c lmmunogenicity c cultvred tissue is capable of stimulating an rggwwse when l " s p l m l e d ~e n e i c w y , robert j. ketchum and orion d. hegre, dept. of cell biology and neumanatoay, university of uinnesota, minneapolis hn . neonatal rat islets derived by culture-isolation have teen shown to k free of mlc class + cells, and are immunologically silent when transplanted to either syngeneic or allogeneic hosts. allogeneic transplantation of cultured neonatal non-islet pancreatic tissue, which is known to contain class + cells, results in rapid allograft rejection. unexpectedly, m i c transplantation of cultured non-islet ductal tissue also resulted in lononuclear lm,me cell (hnc) infiltration of the graft in % of grafts examined. highly purified syngeneic islets and ductal elements grafted syngeneically at remote sites display an i u n e response in the ductal element graft, while the islet graft is free of any imnme cell infiltrate. this syngeneic imune response does not result from the use of xenogeneic serum in the medium, since cultures carried out using syngeneic rat serum supplemented medium yielded identical results. uncultured neonatal pancreatic tissue grafted syngeneically does not result in iqk: infiltrate, thi i.rmne response to a syngeneic stimulus correlates with the presence of class + (antigen presenting) cells. in grafts free of class + cells (culture-isolated islet grafts) no i.rmne responrrc to syngeneic stimulus was observed, while a response was present when syngeneic ductal elements, known to include class + cells, were grafted. this indicates a need for cells capable of antigen presentation to stimulate this syngeneic rerrponne, and suggests that either a modified self antigen or a nomally sequestered antigen is being presented. this syngeneic imune response demonstrates many of the same characteristics of, and may be analagous to, the in vitro syngeneic, or autologous, mixed lymphocyte reactions. indicating this response is not to developnental antigen. c the presence of "self" mhc class i (ma-dr) antigens determines whether blood transfusions ihmunise or suppress. el lagaaij, a termijtelen. e goulmy, & jj van rood, leiden university hospital, the netherlands. blood transfusions can immunise the recipient, as well as induce prolonged allograft survival. it is not known what makes that some transfusions inrrmnise the recipient whereas others induce immune suppression. we investigated if certain mhc compatibilities or differences between recipient and transfusion donor and organ donor are required to induce the beneficial "transfusion effect" in man. we studied graft survival and blood transfusion induced changes in cellular and humoral immunity in different patient groups. the patients received a single blood transfusion of a randomly choosen donor. we found in all groups that to induce a beneficial "transfusion effect" compatibility for at least hla-dr antigen between recipient and transfusion donor is required. if the transfusion and recipient are mismatched for both ma-dr antigens, the recipient is immunised, resulting in an increased antibody production (p=o.ool), an increased cytoxicity (cml) (p=o.oos), an increased mixed lymfocyte reaction (mlr) (p=o.oos) and a decreased graft survival (p- . ). after a beneficial (ma-dr sharing) transfusion. the in vitro test remain unchanged or decrease. graft survival increases with the number of shared antigens between transfusion donor and organ donor (p=o.o ), suggesting that a donor specific suppression is induced. recent experiments have revealed a direct interaction between the cd molecule and hla-dr antigen. to address the nature of this interaction we have used a xenogeneic system in which a human cd cdna was expressed in the murine cd -and cd -negative hybridoma dt . . . the tcr of d . . recognizes the murine class i molecule od. a class i expressing dd-positive cell line was obtained by cotransfection of the human class i cdnas together with the murine od gene int. the murine fibroblast line dap . coculture of dt . . and dap expressing dp-dd resulted in a fold increase in il- production and in rosette formation only when both cd and dp were present on the responding t hybridoma and the presenting cell, respectively. we are using this system to map regions of the cd molecule that interact with the class i mhc ag. the cd molecule has also been shown to be the receptor for the human immunodeficiency virus (hiv) via the gp molecule. since gp and class i both interact kith cd , we have used our functional assay to verify if gp exerts an inhibitory function on cd class i interaction. recombinant gp inhibits the functional interaction and rosette formation in a concentration dependent fashion with maximal inhibition at about pg/ml.. this inhibition is specific since it can be reversed by recombinant soluble cd . the fact that recombinant. gp can inhibit the functional interaction between cd and its physiological ligand (class i ags) suggests that the use of gp on a vaccine against hiv infection could alter the immune response of such individuals. this work was supported by src, mrc and nci. t lymphocytes discern self from non-self molecules through the interaction of their antigen-specific receptors and proteins encoded by the mhc. although the nature of this association is not well-defined, a model has been proposed whereby the v-segments of the t cell receptor interact with residues along the alpha helices of the class i antigen (davis et al.; : , ) . we have recently shown that ctl generated against the class i molecule qiod crossreact on several unrelated murine class i antigens containing the shared qiod residues at amino acid positions , , and (mann et al.; j x : , ) . these residues contributed by the a- domain occur in the alpha helical portion of the class i molecule and amino acids and could interact directly with the t cell receptor. to further characterize the role of these amino acids, we are in the process of determining whether insertion of these residues by site-directed mutagenesis into a human class i molecule will allow for the antigen's recognition by anti- ctl. here the t cell repertoire becomes restricted, so that foreign antigen can be recognized only when associated with the mhc products of the host, and mature t cells are tolerized to self antigens, a process which also seems to be mhc-restricted. thus, t cells should be non-reactive to self antigens when they are associated with mhc products present on the tolerance-inducing thymic cells, whereas they may still react to the same self antigens when associated with different mhc products. to examine mhcrestricted tolerance in vivo, a model system must have: a) self antigen in the context of one mhc haplotype. and b) tolerance to both that and a second mhc haplotype. chimeras were prepared by aggregation of preimplantation embryos of two strains of mice, c bl/ (b ) and balb/c. the thymus of such chimeras should be composed of two distinct and completely intermixed populations of cells, one from each parental strain (isozyme analysis indicates no detectable fusion of cells). thus, t cells maturing in the chimeric thymus should be exposed to and tolerized to minor histocompatibility antigens (mhas) of one parental strain only in association with the mhc of that strain. for example, mice might be expected to express b mhas only with h- b (the mhc). however, our chimeras were fully tolerant to f skin grafts, which have "hybrid" combinations of mhas and mhc (e.g., b mhas with h- d). these results are most consistent with either, a) "wholesale" antigen processing and presentation of all mhas by the tolerizing thymic cells, and/or, b) functional sharing of mhc products between the parental thymic cell populations. many of the events critical to the maturation of t lymphocytes occur in the thymus. in the case of t-cell responses against viruses such response defects are associated with a marked increase in disease susceptibility as illustrated by class i mhc controlled susceptibility to lethal pneumonia induction by sendai virus. certain class i or class i mhc determined tc response defects (four out of six tested by us) can be restored by imunization in vivo and/or restimulation in vitro with dc. dc are the most effective apc. their superior apc capacity is due to ) a very high absolute number of class i and class i mhc molecules, and ) a low degree of sialylation of mhc and other surface molecules, reducing negative charge and facilitating access of the t-cell receptor to the mhc groove presenting the antigenic peptide and/or improved clustering with t cells. the more effective antigen presentation by dc allows a more prominent role for a cd + q cell independent pathway of cd + tc activation. it is postulated that the more effective direct triggering of cd + tc precursors lowers the threshold for il- production by cd + cells, reducing the requirement for il- production by the cd + cells. failure of dc to overcome certain mhc-linked specific tc response defects probably reflects complete failure of any foreign peptide derived from the processed antigen to interact efficiently with the mhc or a true tc repertoire defect. donald pious, department of pediatrics, university of washington, seattle, w mhc class i molecules bind inmunogenic peptides derivsd fro soluble antigen and the complex is recognized by specific t cells. ue have isolated eight independent mutant b -l u clones which are altered in their ability to present antigen. in standard proliferation assays using four different soluble protein antigens, the mutants are unable to stimulate the majority of t cell clones restricted to hlfl dr or dp. cllthough unable to present *hole hepatitis b surface antigen (hbdg), they effcctively present a hbdg peptide to a dprestricted t cell clone. the fact that both dr and dp restricted antigen presentation is abnormal in these mutants made it likely that the class i structurual genes are unaltered. this hypothesis is supported by the finding that dno sequences from the dr genes of one mutant are norml. however, two observations indicate that the uture class i di-expmsccd by the mutants are structurally altered. binding to the mutants with tuo polymorphic anti-dr antibodies and one anti-dp antibody is reduced, although the level of cell surface class i expression is normal. second, the class i diners from the mutants dissociate into no-rs under in vitro conditions (sds-pwe) which preserve dimers in the progenitor line. together, these functional and structural data suggest that the mutants are defective in a molecule that either associates with or post-translationally modifies class i molecules and is required for the physiologic formation of an twc/antigen complex. they do not function as restriction elements presenting foreign antigens to t-cells. to investigate the nature of this functional defect we have constructed different recombinant class i genes using dna segments from the b q (qa- ) or h- db genes. structural protein encoded by the recombinant genes is derived entirely from the q gene whereas the cis-acting transcriptional regulatory elements or the dna segment encoding the membrane anchoring domain is derived from the h- db gene. into fertilised cba/ca embryos by microinjection and transgenic lines were established. to date we have established transgenic lines. we have shown that the q (qa- ) antigen encoded by the recombinant genes behaves as a major transplantation antigen in skin grafts and provokes strong secondary cytotoxic t-cell responses in grafted animals irrespective of the tissue distribution or mode of membrane anchorage of the q antigen. at present, we are investigating whether the q antigens encoded by the recombinant genes are able to present influenza virus or mouse minor histocompatibility antigens to t-cells during immune responses and hence whether they can function as restriction elements. is a distinctive system because it permits an analysis of the activation requirements for antigen specific, resting t cells. been isolated following culture with anti-ig-sepharose and compared to dendritic cells as stimulators of cd ' t cells in the mix. i mhc products and independently stimulated the ' mlr and the production of several t derived lymphokines, including il- and - . however, the relative potencies of dendritic cells and anti-ig blasts as 'mir stimulators varied in a strain dependent fashion. times more active in stimulating hls-mismatched. mhc-matched t cells, relative to syngeneic t cells. anti-ig blasts when stimulating acroas an mhc barrier and were likewise more effective in binding iqic-disparate t cells to form the clusters in which the mix was generated. dendritic cell-t cell clustering was resistant to anti-lfa- mab, while b blast-t cell clustering was totally blocked. thus, anti-ig b lymphoblasts and dendritic cells, two cell types which differ markedly in phenotype, also differ in efficiency and mechanism for initiating responses in allogeneic t cells. only anti-ig blasts could stimulate across an mls barrier, being at least in contrast, dendritic cells were - times more potent than the as the lymphocyte f u n c t i o n antigen (lfa- ) and the i n t r a c e l l u l a r adhesion molecule (icam). we i n t e r p r e t t h i s as an increase i n the membrane expression o f these s t r u c t u r e s f o l l o w i n g incubation. the increase i s blocked by the t r a n s l a t i o n i n h i b i t o r , cycloheximide, implying t h a t p r o t e i n synthesis i s involved. helper t cell responses to soluble globular proteins require processing of the protein by ia-expressing antigen presenting cells (apc). antigen is internalized into acidic vesicles, proteolyzed, and peptides containing t ceu antigenic determinants are transported to the apc surface where they are recognized by the antigen-specific t cell in conjunction with ia. most ia-"pressing cells are competent apc, however, only b cells have antigen-specilic receptors on their surface auowing bound antigen to be processed and presented at /lw the antigen concentration required by nonspecific apc little is known about b cell antigen processing function during differentiation, or if ig-mediated apc function is altered at different maturational stages, thus allowing regulation of b cell-helper t cell interactions. neonatal acquisition of apc function was examined in mice ages day to day . splenic cells from d l to d mice process and present pigeon qochrome $, pg at - % of adult levels. by d neonatal spleen cells acquire the ability to process and present soluble pg at of adult levels. the ability to internalize antigen through ig rwptors was determined using an antigen-antibody conjugate, p$-&(ab')z. neonatal spleen cells acquire the ability to process antigen through ig simultaneously with the ability to process soluble antigen. lack of prowsing by neonatal spleen cells prior to d is not attributable to insufficient levels of surface ia. since d neonate spleen cells are able to activate t cell hybrids to % of adult levels when provided with p$ - , containing the t cell determinant. dlod neonate presentation of p@ - is indistinguishable from adult levels. b cell maturation into memory b cells was identified by the loss of the jlld differentiation marker. splenic jlld'o b cells increase from % following immunization and return to nonimmune levels after weeks. during antigen-induced b cell maturation, jllb b cells are indistinguishable from splenic b cells in the ability to present antigen introduced into the processing pathway either pinocytotically or via surface ig. p p antibody conjugates specific for mouse f(ab')z i n , igd, or igg are presented equally well by both splenic and jll@ b cells. thus, acquisition of b cell processing function appears to be developmentally regulated and may play an important role in b cell tolerance meshanisms. once b cells have acquired the ability to process antigen this function is maintained and is not regulated during maturation into memory b cells. we are currently investigating the role of ig isotype during neonatal acquisition of antigen processing. (supponed by nih grants ai- , ai- , and ai- ) as the preliminary studies suggested that carrier sc (apc) for ts vs. tcs activation might be distinct, studies were done to directly address this possibility by assessing tha ability of s coupled to various cell populations to activate ts and tcs. the results indicated that ts activation required that s be coupled to plastic adherent cells which bear both i-a and i-j determinants. these cells are nonadberent to anti-ig and nonfunctional in cyclophosphamide (cy) treated mice. i n contrast activation of tcs required coupling of s to plastic non-adherent and anti-ig adherent cells. these cells are functional in cy treated mice and bear the b cell markers jlld and i-a but not - . thus s -specific ts are activated by i-a+ i-j+ adherent cells (presumably macrophages) whereas tcs are activated when antigen is presented by b cells. (nih grant ca .) interleukin- activated killer (iak) lymphocytes (also known as lak cells) which destroy a broader spectrum of tumors invitro than nk cells have been used sucessfully in an adoptive immuno-therapy protocol for the treatment of patients with a variety of advanced cancers. the cell suface molecule(s) on tumor cells that a r e involved in specific binding to iak cells and in programming iak cells for cytolysis (iak acceptor molecules) have not been characterized. inorder to identify such acceptor molecules a crude membrane digest of the lung carcinoma cell line a was biotinylated and adsorbed to iak cells or to unstimulated human peripheral blood lymphocytes (upbl) (each from the same person). proteins from the washed solubilized cells were separated by page, western blotted and probed with streptavidin-alkaline phosphatase. several experiments demonstrated that different tumor membrane proteins bound to iak cells compared to upbl. the unstimulated cells bound one tumor membrance protein (about kd) not found on the iak-adsorded blot. the iak cells bound three tumor proteins (approximately , & kd) not found on the upbl-adsorded blot. three other proteins (about , & kd) were found to adhere equally well t o iak cells and upbl. utilizing a streptavidin affinity column, solubilized tumor membrane proteins that bound to iak cells could be separated from solubilized iak membrane proteins. the isolated tumor membrane proteins that adsorded to iak cells inhibited iak mediated lysis of a tumor cells by > %. these studies suggest that specific cellular adsorption techniques may be useful in isolating and characterizing tumor membrane proteins involved in interactions unique to cytolytic lymphocyte-tumor cell target binding and lysis. activation of human t lymphocytes occurs via the t cell receptor-cd complex but can also be induced through the non-antigen-specific cd molecule. selected combinations of mabs or the soluble cd ligand, namely lfa- and a unique anti-cdz mab (cd . ) induce human t cell activatlon. cd is an accessory molecule implicated in t h e activation of human t lymphocytes. this molecule may exert this function by increasing intercellular avidity through binding to mhc class i molecules and/or by transmitting intracellular signals. we have investigated the action of mabs directed against different epitopes on the cd molecules in the activation of human t cells via the cdz pathway. we show that anti-cd mabs inhibit cd induced t cell proliferation in an epitope-depe dent fashion. this inhibition does not appear to be linked to the lower cd mediated [ c a ' + ] response induced by anti-cd mabs, since [ca"] response is equally affected by anti-cd mabs whether or not they inhibi ed t cell proliferation. in conclusion, the partial inhibition of the cd induced [cb'] response of t cells by various anti-cd mabs suggest that : ) this inhibition does not totally account for the inhibitory effect of anti-cd mabs, ) and the proliferation induced by anti-cdz mabs may not be completely ascribed to the [ca +] response of t cells. rosenberg. and alfred singer, experimental immunology branch, nci, nih, bethesda, md . we have devised a model to study the in vivo generation of suppressor cells by using mice congenic at qa- , a class i-like molecule encoded to the right of h- d. disparate tail skin grafts (tsg) unless a second graft with additional helper determinants was also present. without any source of additional help, failed to reject their qa- graft, and were unable to reject them even upon the subsequent addition of exogenous help. thus, exposure to qa- disparate grafts, in the absence of additional help, either led to qa- specific tolerance or suppression. mice failing to reject qa- allografts revealed the presence of qa- specific suppressor cells that inhibited the in vivo activation of antigen specific effector cells capable of rejecting qa- bearing allografts. experiments using t cell subpopulations should allow for further characterization of these qa- specific suppressor cells. we found that b mice did not reject qa- however, animals engrafted with a qa- graft alone, in the thymus, the t-cell receptor genes are rearranged, the t cells learn to recognize their own major histoconpatibilitp complex "hc). and they learn to respond to foreing hec. these events seem to be linked to the interaction between t-cell precursors and the stromal cells of the thyms. thus increasing evidence points to an essential role for the thymus epithelial cells (te cells) in development of at least hec class i recognition by the t cells. to be able to study the importance of te cells in t cell maturation. we have developed a method for growing murine t cells in serum-free pediun with well defined constituents. the m d i u a allows far growth of te cells without concomitant growth of bone marrow derived cells as macrophages and fibroblasts. data obtained by en and lmmunocytochenistry showing the epithelial nature of the cultured cells, as well as autoradiographic data on the growth pattern, and characterization of tb cell supernatants will be provided in addition to results obtained from co-culture of te cells and t-cell precursors (cm-cds-thymytes). lmmunogenicity c branch, national cancer institute, bethesda, md the effector limb mediating skin allograft rejection is highly antigen specific, rejecting cells that express allogeneic mhc antigens while sparing those which fail to express allogeneic mhc determinants. disparate skin grafts are completely rejected in spite of the fact that only a small percentage of the cells within the graft express ia antigens. thus, it is possible that mhc class i disparate grafts are rejected by a mechanism that does not assess the expression of mhc determinants on each cell. we assessed the specificity of the rejection of ia disparate grafts by using allophenic skin grafts in an adoptive transfer system and concluded that skin grift rejection across an mhc class i disparity required recognition of allo-ia determinants expressed by every cell in the graft. therefore, we reasoned that mhc class i antigens must be induced on these ia negative populations. indeed, injection of mice with gamma interferon dramatically induced ia antigens on previously negative keratinocytes. we next tested whether the induction of allogeneic ia determinants on keratinocytes was necessary for graft rejection by engrafting parental strain mice with skin from f >parent bone marrow chimeras. such grafts failed to be rejected, in spite of the speciic rejection of the allogeneic langerhans cells, indicating that the failure of keratinocytes to express allogeneic class i determinants leads to graft preservation. conclusion, mhc class i disparate skin allografts are rejected in a highly antigen specific fashion, secondary to the induction of mhc class i antigens on skin cells that fail to constitutively express them. to address whether the extensive polymorphism characteristic of class i molecules influences cd binding, we have screened a panel of transfectants expressing individual class i mhc alleles. of alleles tested, only aw . did not bind. all other molecules dld bind, including a . and aw , which differ by and amino acids respectively from aw . . position in the alpha domain was identified by sitedirected mutagenesls as the critical residue differing between a . and aw . which determines binding. a mutant aw . molecule containin alanine at position bound cd , while a mutant of a . with valine at did not. alanine is found at position of all human and murine class i molecules sequenced to date except aw . and aw . , which have valine at that position. bulk cultures of a -allospecific ctl were also sensitive to this substitution, and preferenhally recognized both molecules with alanine at . this study shows that aw . differs from other class i molecules in its capacity to bind cd , and raises the possibility that aw . may not function as a restriction element as effectively as other class i alleles. t cell hybridomas derived from h-zd lnc recognked s and pres antigens in a i-ad restricted way, while t cell hybridmas from h- k lnc manifested a specificity for either pres in association w i t h i-ak or for s in association with i-!& the activation of lhese hybridomas by antigen and antigen presenting cells (af'c), as measured by il- secretion, was found to be sensitive to prostaglandines and could be completely inhibited by anti-lfa- moncclonal antihxlies. different aft populations were tested for their capacity to present spresz particles to these t cell hybridomas. various macmphage like populations such as resident, con a induced, thiiglycolate induced peritoneal exudate cells as well as splenic adherent cells were found to present efficiently the spres antigen. in conhast b cells and la+ b cell lines (ta , m . ) could not function as accessory cells in the spresz specific stimulation of these t cell hybridomas. the inability of these cells to present this antigen was not due t o inhibitory effects since these cells did not inhibit the presentation capacity of other potent apc's. funhcnnore addition of apc's of a different haplotype could not complement for the defective presentatiw of spresz by b cells and b ceu lines indicating that mhc independent accessory factors are not implicated in this process. hence it is clear that maaophage-like apc's and b cells d i f f a in their capacity to process and present spdz antigens. since spresz is a very stable particle composed of lipids and proteins it is conceivable that such antigen quires a smng degradation and swh plocessing might occur in cenain -phage-like apc's but not in b cells. recombinant human insulin biosynthetically labeled with k n d " s at several amino acids was used as an antigen and was exposed for varyillg lengths of time to ta mouse b cell apc. subcellular fractionation and hplc chromatography permitted several of the processed peptides distributed throughout the insulin molecule to be monitored. many insulin peptides localized to both the extracellular ( peptides) and intracellular ( peptides) compartments of ta cells were detected. membrane-associated peptides is in progress. many of the peptides processed by ta apc in situ co-elute with those obtained upon digestion i n vitro by the insulin-specific insulin degrading enzylg (ide). f o r the processing of -labelled human insulin suggest that insulin may be processed in b cell apc into immunogenic peptides by an enzyme(s) present on the plasma-membrane, intracellularly and extracellularly. (supported by mrc and cda) . we investigated the pathway of antigen processing itu in cell apc. hurine ctl clones specific for hia-a were generatef with the human cell line jy. four of five ctl clones were found to lyse a k transfected murine cells more effectively than a transfectants. anti-cd specific mab inhibited t lysis by these four clones, and this inhibition was more pronouncgd for a k one clone, which lysed a and a k transfectants equivalently, was shown to be insensitive to anti-cd antibody inhibition. these findings indicate that a -specific t r i n e ctl clones possess greater avidity for murine target cells expressing the a k hybrid molecule relative to those expressing the a molecule. this implies that a cd interaction with the same molecule seen by the t cell receptor is important for target cell recognition. hla class i antigens are highly polymorphic cell surface proteins involved in initiation and regulation of the immune response. allelic sequence variation primarily affects the structure of the first external domains of the a and j component chains. here we provide evidence for other types of allelic polymorphism for these genes. the sequences of two cdna clones corresponding to the hla-dqp mrnas from an hla homozygous cell line exhibit both alternative splicing and readthrough of polyadenylation. furthermore, the alternative splicing event is associated with only a subset of hla-dqb alleles, while the polyadenylation site readthrough is found in a larger subset. this suggests that polymorphic & acting elements within the hla-dqp gene control both processing steps. proteins, presumably encoded by the alternatively spliced mrnas lacking transmembrane exons, are immunoprecipitated with a monomorphic monoclonal antibody directed against hla-dq. these proteins are found in supernatants of cultured cell lines for which secretion is predicted, but not in those of cell lines which do not contain the alternatively spliced mrnas. such secretion class of i allelic products could profoundly affect interactions between effector and target cells in an immune response. departments of biology and chemistry and the cancer center, q- , university of california at san diego, la jolla, ca . we are studying the murine icam- gene and the effects of icam- on antigen recognition. using the human icam-i cdna, we have isolated cdna and genomic clones encoding the murine homologue. the murine icam- gene is a single-copy gene that consists of multiple exom spanning kb of dna and encodes a . kb mrna that is expressed at high levels in a wide variety of different cell types. sequence analysis indicates that murine icam- is % and homologous to human icam- on the protein and dna levels, respectively, and is a member of the immunoglobulin gene superfamily, consisting of several v-like domains linked tanddy. we are also studying the effects of icam- on antigen recognition. the t-cell clone d m s f ). this response is blocked with the anti lfa- antibody fd . when antigen is presented by blo.a( r) spleen cells but not when antigen is presented by dcek, a fibroblast transfected with i-ek. we are currently aansfecting the murine icam- cdna into dcek in order to determine if we can enhance the d response and to determine if the enhancement is lfa- dependent. wth an alp a beta tcr that recognizes moth the t cell differentiation anti en, cd , is expressed by mhc class i restricted t lymphocytes. mhc class i products. the association between cd expression and restriction by mhc class i rfioducts has led to the hypothesis that cd may interact with monomorphic determinants of a large body of experimental evidence suggests that cd interaction with mhc class i molecules leads to an increase in the binding avidity of t cell-stimulator cell interactions. a direct test for a functional cd -mhc class i interaction in t cell activation requires a separate evaluation of cd -ia interactions from tcr-a /ia recognition. however, a separate evaluation proves difficult since the t cell receptor and cb may interact with the same mhc class i molecule. in this report, we use a t cell activation protocol, where tcr-ag/ia recognition is replaced by tcr complex-antlcd antibodies interactions. using this activation protocol, we pave analyzed the effects of monoclonal anti-mhc class i antibodies on the activation o$a cd hybridoma in the absence of its tcr restr$tinj mhc class i molecule (ie ) but in the presence of unrelated mhc class i molecules (ie ,ia ). the data obtained clearly indicate a functional role for cd -mhc class i interactions in t cell triggering. we have targeted hen egg lysozyme (hel) to murine b cells using heterocrosslinked antibodies which specifically bind to surface igd or different mhc molecules. occurred more quickly with targeting to igd than to mhc structures as assessed by fixation and pronase stripping experiments. hel was internalized quickly into acidic compartments when targeted to igd but was detected much later when targeted to mhc molecules, as assessed by shifts of fluorescent signal of internalized fitc-hel. however, the data indicate that not all endocytosed hel entered low ph (< . ) compartments. degraded hel was released from b cells following endocytosis of -i-hel. this release was detected earlier with targeting to igd than to mhc structures. interestingly, the total amount of internal -i-hel decreased with time after endocytosis via igd, but the internal -i-hel was almost entirely whole undegraded hel at all times following endocytosis. these data and those of chloroquine and leupeptin inhibition studies indicate differences in the fate of antigen entering b cells via igd or mhc structures, and support the notion of a neutral ph storage compartment for antigen endocytosed via surface igd on normal splenic b cells. internalization and presentation of hel to hybridoma t cells laboratories, department of surgery, university o f iowa, iowa city, ia target cell lysis by cd ' ctl is a highly specific phenomenon in vitro, as we have confirmed repeatedly in reverse labelling tests by showing that admixed "third party" target cells are not lysed in the presence of specific ctl-mediated cytolysis. however, when mixtures of ctl and their specific targets are inoculated into the skin of hosts syngeneic to the ctl, host cells at the site of inoculation are destroyed, often to an extent that results in grossly observable, full -thickness necrotic lesions. we have evoked these "innocent bystander" reactions in mice with ctl directed against single and multiple non-h- antigens and tnphapten and influenza a virus-specific antigens. thus, the ability to trigger bystander tissue destruction appears to be a general characteristic of ctl-target cell interaction in vivo. our current evidence suggests that host inflamatory cells recruited and activated by factors stimulated by ctl-target cell recognition actually mediate the tissue destruction. these ctl-initiated bystander reactions may be the basis of the non-specific tissue destruction that contributes to allograft rejection and that is observed in many serious virus infections and in intense dth reactions and contact dermatitis. rong h a lin. baael i n s t i t u t e f o r i u m l o g y , baael, snitzerland. w e have investigated the b a d e for i n u n i t y or tolerance t o a m a e aezw proteinthe f i f t h caponent of c o l p l a c n t (a). i n c deficient nice t h i s protein is absent from s e r m and as a cnrwquence they are not tolerized t o cs. c deficient dca generate uy bearing t c e l l s which recognize c i n the c m t e x t of clasa . i n contrast, c s u f f i c i e n t mice i n which c protein is continuously probced d m t mtmt t c e l l reaponsea againmt u. we have tested i f t h i s self protein i s proceseed and presented with clase i i n n o m l mice and can be recognized by c specific t c e l l s i n the absence of exogenewsly added antigen. a l l clasa i bearing c e l l s fm c s u f f i c i e n t l i c e activated c specific t c e l l clonea without additional antigen. presentation was mt a cansaquence o f c secretion by macrophages i n culture but was a h t o be derived prom endqlenewaly generated cs/cl.ss i caplexea. thus t h i s self protein is e f f i c i e n t l y preaented ,inin and available f o r tolerance in&xtion. although c deficient dca cannot secrete c they s t i l l synthesize a precursor mlecule, pro-c , i n accumulating evidence from a number of models suggests that unique subsets of antigenpresenting cells a r e responsible for the induction of specific t cell-mediated responses. w e have previously described an age-dependent maturational defect in the ability of the sjl strain of mice to activate dth-inducer t cells to a wide variety of antigenic stimuli. none of the other strains tested exhibited a similar defect and all other accessory cell dependent responses were unaffected in the dth unresponsive sjl. w e have also shown that the adoptive transfer a macrophage from older dth responsive sjl or other dth responsive las strains can overcome this defect in dth responsiveness. w e have recently found that a subpopulation with the mac-i+, mac- ' and mac- -surface phenotype a r e able to transfer responsiveness. facs analysis indicate that the mac- phenotype is expressed on less than % of macrophages. titrations of the mac- ' cells isolated by facs indicate that adoptive transfer of o ly mac- ' cells can overcome the defect in dth responsiveness. by contrast, transfer of mac -or mac ' cells were unable to overcome the defect. our data suggest that the induction of cd ' antigen specific cells dth-inducer t cells is mediated by a phenotypically unique small subset of macrophage accessory cells. in our studies, we have examined the effect of administering fab' fragments of anti-l t moab (fabl-gk . ) on the inhibition of humoral immunity. treatment of klh-primed mice with . mg fabl-gk . depleted l t ' cells from lymph node tissue while leaving other lymphocyte subpopulations intact. after injection of klh in complete freund's adjuvant, these t,depleted mice were unable to produce anti-klh antibodies. long-lasting unresponsiveness against klh ( weeks) was observed despite the apparent regeneration of the t, population of the lymph node. the results obtained using either fab' or intact gk . antibody were comparable and suggest that a transient depletion of t, does not account entirely for the long-term humoral unresponsiveness. the aim of this study was to gain a more detailed insight into the molecular aspects of antigen processing during the imune response. as a first approach, endosomal vesicles were isolated from bovine alveolar macrophages and their proteolytic activity with respect to a model protein antigen, sperm whale myoglobin (mb), was characterized. during the first stage of digestion of mb by the endosomes, a limited number of fragments were preferentially released from the antigen. we have isolated and identified these fragments. the digestion of myoglobin is completely prevented by pepstatin, a specific inhibitor of aspartic proteinases, and only marginally by other proteinase inhibitors. when mb fragments preferentially released upon digestion with purified bovine cathepsin d, an aspartic proteinase abundant in macrophages, were identified, almost all coincided with the fragments released by the endosomes. to define in more detail the selectivity of cathepsin d under the mild conditions applied, other protein antigens were similarly treated with the enzyme and the peptides released were identified. the location of the preferential cleavage siteswhen related to known t-cell epitopessuggests a dominant role for cathepsin d in the processing of protein antigens to yield fragments for presentation to t-cells. possibly, the observed selectivity of the enzyme may account for the structural similarities among t-cell epitopes, noted by others. actively acquired tolerance in mice to the antigens of the mhc (h- ) is induced by exposure of the animals to allogeneic lymphocytes within hours of birth. actively acquired tolerance to the mhc in humans (hla) cannot be studied in the same way. however, we have evidence for the existence of actively acquired tolerance in humans in a study of highly sensitized patients waiting for a renal allograft. they had developed complement dependent antibodies to the hla antigens of almost all unrelated caucasoid donors. the sera of these highly sensitized patients were tested against a panel of lymphocytes that were mismatched for only one hla class i antigen. we found for these patients hla class i antigens that, although different from those present in the recipient, did not lead to a positive crossmatch. we called such antigens "permissible mismatches" and show that they often included those hla antigens of the patient's mother that the patient had not inherited (noninherited maternal antigens; nima). in of the patients, the permissible class i mismatches included the nimaa. the noninherited paternal antigens (nipas) were analyzed as a control; only two of the nipas tested were acceptable mismatches, which emphasized the preferential nonresponsiveness to nima. recent experiments indicate that what holds true for antibody formation also holds true for t cell activation. of hla class i and hia class i allospecific cd -positive ctl clones. monoclonal antibodies (mcab) directed against the cd structure were only found to inhibit antigen-specific cytotoxicity of a series of class i allospecific cd -positive ctl clones and not of a class i allospecific cd -positive ctl clone. however cytotoxicity induced by cd mcab (used at suboptimal concentrations) or cd mcabs in both types of ctl clone was blocked by cd mcabs. the absence of cd mcab blocking of antigen-specific cytotoxicity of the class- -specific cd positive ctl clone may be explained by assuming that it results from a triggering signal which is to strong to be overcome by the down-regulatory signal of the cd antigen. these combined findings clearly suggest a functional involvement of cd not only in tcr/cd activation, but also in tcr/cd controlled alternative activation routes, such as the cd activation pathway. moreover it shows that even an hla class i allospecific cd -positive ctl clone expresses a functional active cd antigen. the absence of hia class i expression on the target cells (daudi cells) used in the experiments described indicate that the cd antigens not act solely in an adhesion-like fashion, but exhibit also a more general regulatory function in t-cell activation. this regulatory role of cd may be explained by assuming the induction of a threshold for activation, which is triggered after binding of cd mcab or binding to its natural ligand, hla class i. in our view, cde-mediated regulation of t-cell activation could therefore prevent non-specific triggering of cytotoxicity by interactions of insufficient affinity. *this study was supported by a grant from the dutch kidney foundation. the cd t-cell surface antigen s felt to have the dual functlon of stabilizlng the interaction of the t-lymphocyte with the antigen presenting cell (apc) a s well as transduclng an independent signal that can potentiate the actlvatlon related alteratlons generated through the t-cell receptor. we have found that upon antibody-mediated cross-linklng of the cd molecules of cloned murlne t-lymphocytes there is a time and temperature dependent decrease in the abundance of the lymphocyte-speciflc tyroslne klnase p lok. this co-modulation is speclflc for cd and p lck slnce cross-linking of other t-cell surface antlgens (cd . t , thyl. ) does not result in detectable alteratlons in the abundance of the lck protein and slnce cd cross-llnklng does not induce any alteratlon in the abundance of p *.. another srcrelated tyrosine klnase highly expressed in t-cells. such data suggest that cd and the internal membrane lck protein are in close proximity within the cell. further analysls has revealed that slgnlflcant amounts of lck can be immunopreclpitated by antl-cd antibodies. in addltlon. cd can be speciflcally preclpltated by anti-lck antibodies. our data imply that cd and p ok are physically associated in cd + t-lymphocytes. the flndlngs that cd is msoclated to the lck proteln in either murlne or human t-cells and that cd is also complexed to p lck ln cd * t-cells suggest that the lck tyroslne klnase is involved in the functlon of the cd and cd accessory molecules. these apc do not appear to present processed klsa determinants. in light of these findings, of apparent interest is the issue of which cells types are responsible for hlsa-specific t cell tolerance induction. studies in mice treated from birth with anti-p antibodies suggest an important but perhaps not exclusive role for b cells in this process. we are currently pursuing the identity of other cell types which may be involved. in addition, lmmunogenlclty c university of texas southwestern medical center at dallas, dallas, texas qlo is a soluble class i-like major histocompatibility antigen produced specifically by the liver. previously, it has been shown that mice possessing soluble qlo can generate anti-q cytotoxic t lymphocytes (ctl), suggesting that this soluble molecule does not function as a tolerogen. we have recently constructed c h transgenic animals which express an exon shuffled q (al, p )/ld ( , tm) molecule. this qio/ld molecule is expressed specifically in the liver on hepatocytes but not on nonparenchymal liver cells, spleen, thymus, kidney or brain. the expression of qio/ld in the transgenic hepatocytes is equivalent to la expression on balb/c hepatocytes, suggesting the animals are expressing physiologic levels of the transgene. the presence of membrane bound qio/ld in c h animals has not caused anti- ctl precursors to be deleted, however, because primary in vitro ctl assays show these transgenic animals can specifically lyse qio/ld targets. histopathologic examination of the livers of these animals does not show extensive lymphocytic infiltration or inflammation. in addition, serum levels of alanine aminotransferase. aspartate aminotransferase, and alkaline phosphatase are also normal, confirming that these animals do not show overt signs of liver rejection. results are ampatable with a t least two different pathways of antigen hardling, a pathway for degradaticm of antigen, and a "pmcessiq" pathway for antigen presenbtim. my+ l monocytcq enes appear t o i n t e r f e r e with t h e processing pathway, either by i n h i b i t i n g production of antigenic material t h a t can associate with ia o r by i n h i b i t i n g putative intracellular event@) imr lvb-q the binding of ia to processed antigen and tmnsprt of annplexes to the cell surface the immunogenicity and antigenicity of synthetic peptides (sp) derived from the sequences of a streptococcal antigen were investigated in macaque monkeys. immunization with the free peptides of and residues failed to elicit serum antibodies or t cell responses. however, both serum antibodies and lymphocyte responses were elicited by immunization with the sp linked to tetanus toxoid (t) as a carrier. indeed, spl -lt and sp -tt elicited serum antibodies and proliferative responses of lymphocytes, not only to the sp but also to the native strtptococcal antigen. recall of sp -tt or sp - t immunized monkeys w i t h suboptimal doses of the native srreptococcal antigen resulted in a significant increase in antibodies, both to the sp and native antigen, confirming that the two sp share antigenic epitopes with the native antigen. the b and t cell epitopes were then determined and the b cell epitopes resides in residue - , whereas the t cell epitope overlaps and consists of residue - . the t cell epitope has an amino-terminal leucine and carboxy-terminal glycine and alanine added to residue - of the b cell epitope. in spite of the b and t cell epitopes being expressed in sp (residues - ), the monomer failed to induce serum antibodies without a carrier. however, immunization with dimers of peptide-linked or disulphide-linked residues - , without a canier, elicited both serum antibodies and proliferative responses of lymphocytes. the results suggest that the monomeric sp is not immunogenic, whereas the dimeric peptide elicits both antibodies and t cell responses. the minimal t cell-b cell structure required for immunogenicity is now being determined. lmmunogenlclty section a departments of immunology and rheumatology, mayo clinic, rochester, pin . susceptibility to collagen induced arthritis (cia) in mice maps to the i-a loci in h- mice. however, swr (h- ) mice are cia resistant, suggesting a role of non-mhc genes. we have recently shown gene complementation between h- from swr and tcr v genes from several non-susceptible strains. cia has been induced in b , c h.a and a fackcrosses with swr with similar high incidences; , and % respectively. c l shares a similar background with b and is h- b, but has the same v tcr mutation as swr. backcrosses showed a very low incidence ( %) of cia, and tte arthritis observed was of a much milder and transient nature. the invariant chain associated with hla class i molecules is a - kd glycoprotein implicated in antigen processing and assembly and intracellular transport of class i molecules. class i molecules and invariant chain are expressed primarily by b lymphocytes and antigen-presenting cells such as macrophages and can be induced by interferon-y in a variety of cell types. to define sequences involved in the human invariant chain gene regulation, bp ' to the initiation of transcription were subcloned upstream of the cat gene. transfection into invariant chain-producing cell lines and non-producing cell lines demonstrated that this ' region displayed tissue specificity and responsiveness to interferon-y. deletion mutants were constructed to ascertain the functional properties of specific regions of the invariant chain upstream regulatory regions. these deletion mutants have led to the identification of putative regulatory regions: to , to , and to bp ' to the cap site of the invariant chain gene. deletion of any one of these regions results in decreased cat activity. protein-dna interactions of these sequences have been characterized by mobility gel shift assay and dnase i footprinting. two regions have been identified that exhibit cell type dependent binding of nuclear proteins. two color flow cytometry was used to characterize the surface phenotypes of human bronchoalveolar lymphocytes (n= ). the cd /cd ratio was highly variable ( . - . , mean- . ). a high proportion of the t cells expressed hla-dr ( - %, mean= l%) indicative of t cell activation. however, detectable levels of the i+- receptor were expressed on < % of the cells. cd r was absent from cd cells in most preparations ( - % mean= %) suggesting that the cells are inducers of ig synthesis. uchl , a marker of memory cells was present on - % of lung t cells. uchll+ cd r-lung lymphocytes responded poorly to pha and cona but did respond to il- in the presence of accessory cells. together these data suggest that lung lymphocytes are recently activated memory cells. il- induced lung t cell lines were also characterized for antigen expression and w ( activity. high lak activity was obtained in preparations containing a high proportion of cd cells. these cultures appeared to be suicidal. in contrast, lines with a high proportion of cd + had low or absent lak activity but proliferated in the presence of il- for at least months expressing a cd cd r-phenotype. this abstract is a proposed presentation and does not necessarily reflect epa policy. the polymorphic second exons of the hla-dp, and dpd genes have been specifically amplified in vitro by the polymerase chain reaction (pcr) method, using the thermostable dna polymerase of aauaticus. sequence analysis of mi clones containing the amplified dp sequences from a panel of thirty-four df typed cell lines revealed only the two previously characterized alleles for dp, . fourteen allelic variants were defined for dpw eight of these are associated with the t-celldefined dpwl- types; two subtypes were found for both dpw and dpw . six additional dp alleles which were previously typed in the t cell assay as blanks were also idenlfied. based on this sequence information, non-isotopic sequence specific oligonucleotide probes have been developed and used to type a margarita betz, dominic dordai, brian e. lacy, and barbara s. fox. department of medicine, university of maryland school of medicine, baltimore md . murine type helper t cells (th ) secrete interleukin (il ) in response to antigen. despite the likely importance of these cells, little is known about their priming and expansion in vivo. we have demonstrated il production in response to a cytochrome p peptide following t cell expansion in vitro. this antigen has not previously been shown to induce th cells. bio.a mice were primed with a peptide fragment of pigeon cytochrome c in cfa. lymph node cells were restimulated in vitro with antigen for - days, ficolled and rested for days without antigen. cells were then tested by limiting dilution for the presence of antigen-specific il producing cells. il was detected using the il sensitive cell line ct s (provided by dr. w. e. paul, nih). the specificity of the response was confirmed by blocking with the anti-ll antibody b . following in vitro restimulation of the primed lymphocytes with antigen, il production was detectable from as few as cells per well. il secretion was antigen dependent and required both in vivo priming and restimulation in order to be detected. it is not clear why primed lymph node cells, placed in limiting dilution culture directly after removal from the animal, failed to secrete detectable amounts of il in response to antigen. suppression is an unlikely mechanism as fresh primed lymph node cells were unable to inhibit il production by restimulated cells. we are now investigating the factors that may regulate the development of il producing t cells. mark r. boothby, ellen gravallese, hsiou-chi liou. and laurie h. glimcher, department o f cancer biology, harvard school o f public health, boston, ma . regulated pattern, and normally expression is limited to certain cell types such as cells and macrophages. cells is accompanied by the loss o f class i mhc expression. these genes also respond to external stimuli such as the cytokine il- , which increases b cell ia. a region o f the aa mhc gene activated expression of a cat reporter gene in a b lymphoma cell line but not in a myeloma cell line. a nuclear protein that bound to two sites within this region was found. this binding activity was present in spleeiis that lack t cells and in b cell lines, but it was absent from all three myeloma cell lines tested. il- treatment of normal and athymic mouse spleen cells greatly increased the binding of this nuclear protein to its a a target sites, concomitant with increased aa transcription; "thus, cells contain a sequence-specific binding activity regulated both by il- and by differentiation. the differentiation o f b cells to plasma lmmunogenicity c peter van den elsen . ldepartment of immunology, the netherlands cancer institute, amsterdam, zdepartment of immunology, erasmus university, rotterdam, department of immunohaematology, academic hospital, leiden, the netherlands. human tcr y occurs in disulphide-linked (type ) or non-disulphide-linked (type ) forms, dependent on the use of the cyl or cy gene segment. the cyz gene segment can contain a duplication or triplication of exon , which gives rise to different protein forms (types bc or zabc). it is not known whether functional differences exist between these receptor types. protein chemical analysis of type and type bc receptors on functional human t cell clones derived from peripheral blood (pb) has indicated that not only the y chains, but also the chains have a molecular mass and charge which set apart type and q p e bc receptors. two sets of fifteen clones were randomly generated from pb of two normal donors after selection with the anti-tcr y - mab, which recognizes all receptor types. dna rearrangement and mrna expression analysis of y and genes allowed us to map the specificity of the anti-tcr y mabs tcs- and tiya to the v l and vy gene segments respectively. subsequently it could be concluded from the analysis of random clones that the majority of type receptors use vy , while this preference seems absent in type receptors. the great majority of type receptors do not use v l. while the majority of type receptors do. this was confirmed by fluorescence analysis of pbl of a large panel of normal donors. we conclude that vy and v gene segments in functional tcr y in pb are used in non random combination and that their expression is correlated with rearrangement of the y gene to cyl or cy . we have previously shown that polymorphic residues in the nh -tenninal half of the p domain (amino acids - ; hypervariable regions and [phvl and ) determine with which allelic or isotypic a chain a particular b chain can achieve efficient cell surface beterodimer expression. this result might be understood in terms of the current model for ia smcture which predicts that w v l would lie adjacent to region. therefore, to examine the role of ahvl residues in conmlling hetcroduncr expression. a mutant a d cdna was created in which the codon for amino acid was mutated to code for the auk residue at this position. in addition, recombinant a d and a& cdnas, in which the segments encoding the three a hypervariable regions were exchanged between the two alleles, were used to study the connibutions of other a chain polymorphisms to this process. interestingly, the polymorphic residues in ahv are predicted to lie in a region of the a a chain a-helix which is adjacent to the phv region of the p chain a-helix. allelic substitutions in this latter region of ad have been shown to similarly affect surface ia h e t e r o d i i expression. taken together, these results suggest that there are at least two spatially separate areas in which the a and p chains interact and that these interactions are affected by polymorphic rtsidues in both areas, conmbuting to the efficiency of heteroditner expression and, most likely, ia quartcmary conformation. the aim of this project is to identify contact residues of the t cell receptor (tcr) with antigen and/or mhc class i molecules. as a model system, a vp -containing tcr has been chosen since the majority of vb ' t cell hybrids react with ie molecules of the k,s,d, and b haplotype. t cell hybrids have been made which have a dual reactivity: they are vp + and recognize ie molecules but also show reactivity towards a known antigen, namely chicken ovalbumin (ova). one such hybrid has been mutagenized with ethyl methane sulfonate (ems). mutants were selected on the basis of their survival after stimulation by either antigen or ie. it was expected that mutations in all different kind of genes involved in t cell recognition and t cell activation would be found. mutants obtained fall into two major groups: ) loss variants of tcr a or fl chains,t or l t : ) mutants with point mutations in one of these genes. we are currently analyzing the mutants biochemically and functionally in order to identify the particular gene affected. point mutations in the a and p genes of tcr mutants will be localized using the polymerase chain reaction in combination with dideoxy sequencing. activation of t lymphocytes requires the intracellular fragmentation of foreign antigens and their presentation by class i or class i major histocompatibility complex glycoproteins. the direct binding of peptides to class i molecules has been shown in a number of experimental systems and its specificity compared to that of t cell activation. in contrast, direct binding of peptides to class i molecules has been difficult to detect; although peptide sensitization experiments and the crystallographic structure of hla-a persuasively argue for its occurence and importance. in this study, we demonstrate specific binding to hla-a of an influenza matrix peptide (flu-m residues - ) that has previously been shown to act as a target for certain hla-a restricted influenza-specific cytotoxic t lymphocytes. we estimate that less than . % of the purified hla-a molecules were able to bind the added peptide. we and others have shown that allorecognition by cytolytic t lymphocytes (ctl) is analogous to t cell recognition of foreign antigens in that both can occur via presentation of antigenic peptides by products of the major histocompatibility complex. we have used peptides corresponding to the alphal helix of selected hla molecules to analyze t cell recognition of this polymorphic region. the alpha alpha helix of hla-b and -b are identical, and show a high degree oh homology with those of hla-bw , -b , and -b . peripheral blood lymphocytes from normal donors were stimulated in vitro with targets expressing hla-b to derive allospecific ctl lines and clones. in some individuals, the allospecific response was almost totally directed against the alphal helix. the ability of peptides corresponding to the alphal helix of these hla molecules to inhibit and induce lysis as well as to modify other assays of t cell activation will be discussed. diseases and *national institute of child health and human development, d bethesda, md . classical transplantation antigens are constitutively expressed on cells of all tissues exce t brain. transaiption is regulated by the interaction of nuclear factors with ' flanking regions tiat include the class i re latory element (cre). previously, the cre has been divided mto re om on the basis of nuclear t%or blndin . several studies have implicated the nuclear protein (ri) wfich binds to the inverted repeat geggattcccca) of re 'on i as necessary for gene transai tion although region i is identicarin all se uencedouse $d and l enes, it is not conservefin qa r y genes. a comparison of the c& from h- ld with that of , a qa region gene expressed o y in the liver and fetal olk sac, shows that there are two changes within the inverted r eat se uence (tgaggactcc$a). these differences disru t the dyad symmetry. another nugotide diierence between h- l and qlo falls within re 'on ifof the cre. however, qlo can bind to the nuclear factor (rii) that binds to region ii of the fit ld cre, whereas qlo region i can not bind to the nuclear factor (ri) that binds to the region i inverted re at. to test whether the differences m region i contribute to the restricted tissue e ression of q l r w e have used site-directed in vitro mutagenesis to make the inverted repeat of]cglo region i like that of the classical class i genes. a change at either base enhances transcription as measured in a transient transfection system. either change also allows binding of the nuclear factor that binds to the classical r alterations in the cre regon i contribute to the limited tissue expression of . the presence of disrupted cre region i in other region genes likely contributes to their tissue restricted expression. on i sequence. thus, molecular analysis of t cell receptor structure/function in sperm whale myoglobin specific t cell clones. jayne s.danska, alexandra m. livingstone, toshi isihara and c. garrison fathman, stanford university medical school, stanford, ca. we have undertaken structural characterization of the t cell receptors (tcr) utilized by a well defined panel of murine dba/ t cell clones that recognize epitopes within the - peptide of sperm whale myoglobin (sp wmb) presented by i-ad or i e . only of independent clones show alloreactivity for whc haplotypes. using the polymerase chain reaction (pcr) and dna sequencing of the tcr a and fc chains from matched sets of clones bearing either whc restriction or epitope specificity in common, we are addressing structural relationship between tcr and mhc/antigen for this model system. among i-ed restricted t cell clones reactive with spwmb - , all have highly homologous tcr chains associated with a minimum of three different tcr a chains, some of which are derived from novel v gene families. to further characterized the specificity of these clones we are generating substituted peptides to identify residues within the epitope important for interaction with tcr or restricting mhc molecule. functional verification of the relationship between given tcr primary sequences, and recognition capability will be addressed by transfer of the a and/or chafns cdnas created by pcr amplification into t-cell hybridomas expressing endogenous tcr genes of known sequence and specificity. with mhc fine specificity. the differential impact of substitutions with the n-terminal and c-terminal portions of the apl domain is consistent with models of auap structure in which the n-terminus interacts with peptide while the c-terminus interacts with both peptide and the tcr. or aauapu-resmcted t cell clones. the antigens tested were l-tymsine-p-lmmunogenicity c expression of the q p gene, patricia m. day, katherine e. lapan and jeffrey a. frelinger, department of microbiology and lmmunolog university of north carolina at chapel hill, chapel hill, nc . generally, the transcription of class i genes tom the q a a region is limited to tissues of hematopoietic origin. previous work in our lab demonstrated widespread transcription of the q gene in the .p mouse, with high levels of mrna found in liver, lung, lymph node, spleen, testes and thymus. less rna was present in muscle and brain tissues. however,,it is not known whch individual cell types within these tissues are responsible for the transcription of the q gene. we raised polyclonal antisera against a synthetic peptide, derived from the predicted amino acid sequence of the q p transmembrane region. we selected this region since it is the most locus specific. this antisera immunoprecipitates a class lsized protein. a monoclonal antibody, directed against the same peptide, has also been produced. sv -transformed h- p fibroblasts show an abundance of q message. suprisingly, indirect immunoluorescent staining with the monoclonal antibody reveals a cytoplasmic localization of the protein with a perinuclear concentration. different patterns have been observed in examination of the h- b em onal carcinoma cell lines ax and pcc . qgspecific antibodies allow us to identify the cell ty es which express the gene product. the application of in situ hybridization techniques will correlate the cellular site ofmrna synthesis and protein detected by antibodies. understanding the paltern of expressim of the q gene is the first step in determining the so far elusive function of these mhc genes. and il r mrna a f t e r mitogenic stimulation. antibodies against cd r, but not against c common determinants, synergise with suboptimal doses o f mitogen t o induce il and il r mrna expression, suggesting t h a t cd r molecules are operative i n transmembrane s i g n a l l i n g i n immature thymocytes. there i s also an i n d i c a t i o n from northerns using cd probes t h a t cd p mrna i s not induced i n activated cd -thymocytes as i t i s i n mature t c e l l s . these r e s u l t s support the idea t h a t cd r+ molecules are essential f o r generating signals required f o r c e l l survival w i t h i n the productive intrathymic lineage. we examined a panel of thl and th t cell clones for the ability to induce antibody synthesis in a mishell-dutton culture system under cognate b-t cell conditions: our findings indicate that both thl and th t cells are heterogeneous, i.e., some but not all thl and some but not all th clones have the capacity to induce antibody under these conditions. we examined the effect of -irradiation or rnitomycin-c pretreatment of our thl and th clones on their ability induce antibody synthesis. asano et al. (j. immunol : ) have reported that th clones are exceedingly sensitive to -irradiation, with doses as low as rads abrogating the ability of th clones to induce antibody synthesis. we found that while the helper activity of th clones was very radiation sensitive, helper activity in thl clones was very radiation resistant. thl clones given rads of irradiation were as effective as unirradiated clones in inducing anti-tnp plaque forming cells (pfc). moreover, when used at higher t cell/b cell ratios in culture, irradiated thl clones were more effective than unirradiated clones in inducing antibody synthesis. the effect of irradiation on th clones was not simply due to inhibition of proliferation, since mitomycin-c pretreatment of the clones had little effect on helper activity. conservation, alexander l. dent, pamela j. fink and ste hen m. hedrick, department of biology, university of california, san diego, a . the p chain gene of the murine t cell receptor has been shown previously to have an alternative spliced form of message. this message contains a novel exon, termed c& which is inserted between the vdj and constant region exons. we have studi d expression of the cpo exon at the mrna level by rnase protection. we have found that about % or less of of p messages in normal t cell clones contain the cgo exon, whereas p m e s a es. in the thymus contain the exon at - fold higher levels. to address t i e importance of the c exon in the immune ,system, we have undertaken a phylogenetic approach. \y cloning and sequencin the rat analogue of cpo, we have found that while the rat exon is very simifar to the mouse exon, both donor and acceptor rna splice signals are defective in the rat cpo gene. this implies that rat cpo cannot be spliced into rat p m e s a s. furthermore, we have sequenced the analo ous region to mouse cpo in e human p chain locus, and have found no stretct of sequence remotel homolo ous to cpo. because cpo is not conserved evolutionarily, we beieve that ! he cpo gene element does not sewe an important function to the immune system of most vertebrates. ) . we found that one arrdno acid substitution at a vdjp juntional region position found to be highly conserved in pigcon cytochrome c-specific tcr's results in a change in antigen fine specificity, while an* change abolishes all detectable responses characteristic of the d tcr. we will present the results of mutagenesismansfection analyses of two other pigeon cytochrome c-specific tcr's. the murine ctl response to human class i molecules is - orders of magnitude lower than the response to murine alloantigens, due to structural differences between human and murine homologs. we investigated whether this discrepancy could be overcome by exposure of developing t cells to human class i molecules in transgenic c bl/ mice expressing hla-a . . ! c zhe bdixujiar basis of - . lbve digiustn ard ed p d l u b r , div- of s ch mice expressed hla-a .i in spleen, bone marrow and thymus at levels similar to those of endogenous h- molecules. however, the frequency of ctl specific for other human alloantigens remained similar to that of normal mice. the frequency of hla-a . restricted influenza specific ctl was - orders of magnitude less than the frequency of h- restricted ctl. these results indicate that the poor response of murine ctl to human class i antigens is not determined by selection in the thymus, but by species-specific constraints on the interaction of mhc antigens with t-cell recognition structures. while the mice are tolerant to hla-a .i expressed on murine cells, they still respond to hla-a . expressed on human cells. the epitopes defined by such clones are present on hla-a . positive human cells derived from several different tissues. such epitopej are not dependent upon the species of p m associated with the class i molecule, nor upon the structure of the attached carbohydrate. the results suggest that one or more highly conserved normal human proteins contribute to the formation of such epitopes, and provide an explanation for the failure of ctl raised against class i molecules on human cells to recognize the same molecules expressed on murine transfectants. this suggests that normal endogenously expressed molecules may also be important in the formation of epitopes on class i antigens recognized by allospecific ctl. we previously demonstrated t h a t several subclones derived from a c +, cd -/cd -t-cel i l i n e have undergone secondary rearrangements a t t h e t-cell receptor (tcr) a locus w h i l e maint a i n i n g i t s o r i g i n a l tcrb and igh d-j rearrangements (marolleau e t . al., i n press). these secondary rearrangements r e s u l t i n t h e j o i n i n g of germline va and j a gene segments which replace the p r e -e x i s t i n g va-jacmplexes of t h e parental t-cell line. i n an e f f o r t t o examine t h e molecular mechanism responsible f o r these va-ja gene replacements, t h e s t r u c t u r e s o f tcra cdnas prepared from both t h e parental and subcloned t-cell l i n e s were determined. i n addition, northern b l o t and southern b l o t analyses were performed on both t h e parental and subcloned t-cell l i n e s using a panel o f va and j a probes. our r e s u l t s i n d i c a t e t h a t : ) secondary rearrangements r e s u l t i n both productive and non-productive va-ja j o i n s , ) the mechanism whereby secondary rearrangements occur i s a d e l e t i o n event t h a t involves germline va genes ' t o t h e p r e -e x i s t i n g va-ja complex j o i n i n g t o j a the class ii major histocompatibility complex (mhc) antigens are a family of integral membrane proteins whose expression is tissue-specific and developmentally regulated. a pair of consensus sequences, x and y, separated by an interspace element, is found upstream to all class ii genes. deletion of each of these sequences eliminates expression of class ii genes in vitro or in transgenic mice ( - ). furthermore, the absence of a specific binding protein for the hla dr a x box in patients with severe combined immunodeficiency disease whose cells lack class ii suggests a critical role for these proteins in class ii gene transcription ( ). report the cloning of a agtll cdna encoding a dna binding protein (human x-box binding protein, hxbp- ) which, like the proteins in whole nuclear extract, recognizes both the x box and interspace elements of the human dra and murine aa genes. the hxbp- cdna hybridizes to two rna species, . kb and . kb in human, that are expressed in both class ii positive and class ii negative cells. hxbp- does not cross-hybridize to two murine aa x box binding cdnas recently isolated in our laboratory which also recognize the dra and a ax boxes. these observations provide evidence for the existence of multiple x box binding proteins which recognize a common or overlapping motif. chromosome mapping studies demonstrate that hxbp- arises from a multi-gene family two of whose members map to human chromosomes and . taken together, these data suggest a high degree of complexity in the transcriptional control of the class ii gene family. france . in an attempt to analyze positive or negative in vivo regulation of clonal expansion of cytolytic t lymphocytes (ctl), we immunized blo.br mice with the kb specific ctl clone kbs-cu). and we tested whether t cells obtained from such mice would influence the in vitro development of the ctl clone kbs-c?o. a clone-specific helper effect has k e n observed, which is mediated by cd + splenic cells from immunized mice. control immunizations of b o.br mice with ti negative variants of suggest that this growth regulation involves the recognition of the ti of kbsc . the precise nature of the antigen recognized on the ctl clone, the possible involvement of ti determinants with or without mhc products u e now under investigation. we have shown that thy-]+ dendritic cells present in the epidermis of mice (dec) express cd associated v and v gene products. we have produced a monoclonal antibody directed against v and found that a wave of cells appearing at the earliest stages of fetal thymic development express v . phenotypic and functional analysis of v + cells in the early fetal thymus indicates that they have characteristics in common with the v + dec. both populations express high levels of ly- and are ly- c+. neither express cd or cd . interestingly, the v + fetal cells express elevated levels of il- receptor, indicating that they may have been activated. functional analysis demonstrated that, unlike other fetal thymocytes, the v + cells can be stimulated to produce lymphokines and lyse a panel of target cells which are also lysed by the adult thy-l+ dec. these results raise the intriguing possibility that the first receptor-bearing component of the t cell system to appear in ontogeny might give rise to the thy-]+ dec. ( ritical to an understanding of the function of cells bearing the gamma-delta t cell receptor will be an understanding of when and where such cells function. in order to investigate this, we have used a variety of techniques (in situ hybridisation, cdna cloning, and pcr) to examine m a s of tcr gamma delta gene expression. one conspicuous site of expression is the intestinal epithelium, which is by contrast almost devoid of tcr alpha beta expression. interestingly, the v gene segment usage in this location is quite specific and is different to the specifcity that we have found in the spleen and in the thymus, and that others have found in the skin. this specificity suggests in turn that expression of the resmcting elements recognised by gamma delta may also be spatially nxticted. extensive analysis of junctional diversity can pmvide infomation on the diversity of antigen nxogniscd by gamma delta. the basis for selective expnssion of v gene segments may in part lie in different requkments of the v-gannna gene pmoters. to examine this, the wnscriptional capabilities of the various gamma gene promoters in t cells murine tcr gamma genes: distinct spatial restriction of v-gene segment usage, adrian thyday*, susan kyes*, simon carding#, charles a. janevay, being compared by linkage to the chloramphenicol acetyl transferase gene. biochemistry, university of wisconsin-madison, madison, . murine strain a sublines a/j and a/wysnj have a genetic polymorphism that regulates serum immunoglobulin responses to several protein antigens. strain a/j secondary igg responses to bovinv gamma globulin, oralbumin, hemocyanin and galactosidase l-ere -, l o -, i -and -fold greater, respectively, than a/wysnj responses. subline a/hej is a low responding strain like .l/wysnj. analysis of h- class and class i molecules provided no evidence for a breeding error to account for the genetic polymorphism. instead, an important immune response gene outside h- may hare been heterozygous when the sublines diverged, and the polymorphism resulted from sezregation and differential allele fixation. a mutation subsequent to subline divergence is also a possible source of the polymorphism, but is less likely. the high responder phenotype inheritance pattern in (a/wysnj x a/j)fl, f , and backcross mice was consistent with segregation of a single, recessive gene. we named this locus l a for the strain a sublines that define it; strain a/j represents the k a h allele and strains a/yysnj and a/hej represent the allele. hayes, keith d. hanson, faye nashold, and david j . miller, department of the secondary iggza responses were also affected. several different proteolytic digests of denatured seb have been tested for their ability to stimulate t cell hybrids to produce il- . a tryptic digest that retains activity has been fractionated by hplc and the stimulatory component is being analyzed. examination of the amino acid sequence of seb and the proteolytic cleavage sites has led us to synthesize several peptides for analysis. these peptides, and their analogues, will be tested for their function in vivo and in vitro. to understand the interactions involved in the famation of peptide-mhc complexes, an assay has been developed to detect dr specific binding of peptide analogues of t e l l determinants to cell surfaces. ebv msformcd b cell lines (bcls) w m incubated with biotinylated peptide followed by fltc conjugated streptavidin, and then anaiysed by flow cytomctg. a panel of bcls homoygous for diffmnt dr types bound analogues of peptide - from influenza virus haemagglutinin (previously shown to be a helper t cell determinant restricted through dr ) to varying degrees, w h m no binding was observed to the dr-bcl rj . binding could be specifically inhibited by the natural unbiotinylated t cell determinant or other drl restricted determinants. competition by a range of peptides revealed quantitative diffmnces in their ability to bind drl. the assay is currently being used to generate a detailed model of the complex formed betweenha - anddrl. and trp for leul . and hla-a . differs from hla-a .i by the substitutions of thr for ala glu fo??a and trp for zeu . residues and in the -sheet of the molecule, and residues , , and in the a-helix are thought to interact with bound peptide or the tcr. to evaluate the role of these residues on ctl-defined epitopes, two genes were constructed that encoded novel molecules which differ from hla-a . only at residues , , and , or at residue . the effect of a-helix substitutions on serologic and ctl-defined epitopes that varied between hla-a .i and hla-a . were evaluated by constructing genes that encoded the individual differences at residues , , and , as well as additional non-naturally occuring substitutions at these same positions. hla-a . specific ctl were found that were: ( ) insensitive to substitutions at either residues , , and , or residue , but were lost when all four positions were changed; ( ) dependent upon the residues , , , but not residue ; ( ) dependent upon residue , but not residues . , and ; and ( ) dependent upon residues , , , and residue . further epitope mapping with the a-helix mutants demonstrated that a substitution at residue often destroys an epitope not affected by substitution at residue . even conservative substitutions at position were more disruptive than nonconservative changes at residue . residue , while important in defining an mab epitope, had no effect on any ctl epitopes. these results indicate that spatially separate residues in the a-helix and -sheet of the molecule can contribute to the epitope recognized by a given ctl. furthermore, considerable complexity must exist in the spectrum of t cell receptors utilized to recognize hla-az, as ctl clones exhibited distinct fine specificity patterns. to follow the evolution of these class i types, to discern the chief selective pressures on its members and thus indicate the probable functional properties of the antigens. a cosmid library was screened for class i genes. clones were mapped and could be grouped into clusters of contiguous dna spanning , kb. by hybridisation studies, class i genes/ gene fragments could be distinguished. transfection analysis revealed that genes could be expressed as cell surface antigens: two genes, in a block of duplicated dna encoded serologically defined rt .c products, the other genes gave rise to novel class i antigens detected by the xeno-antibody x . using region specific probes, we could detect clear rat homologues of the mouse qa and h- genes, however there were only two rat genes with limited homology to the mouse tla genes. the analysis showed extensive remodelling of the class i region in the evolutionary gap between rat and mouse. while the immunological role of t cells bearing the ap t cell receptor (tcr) has been well characterized, much less is known about the function of t cells bearing the $ tcr. we investigated the role of tcr $cells in the immune response to complete freund's adjuvant (cfa). after immunizing mice with cfa, we observed a greater than -fold increase in the number of tcr $ cells present in lymph nodes draining the sites of immunization, compared to a - -fold increase in the number of tcr ap cells. there were at least three different species of tcr's expressed on these cells in the draining lymph nodes, including two protein products derived from the rearrangements of cyl and cp, and one product derived from cy . % of tcr ys cells from immunized lymph nodes expressed the il- receptor in vivo. and these cells constituted roughly % of the proliferative response of total lymph node t cells to - . tse, et al. (j.lmmun..vol.l , p. . ) have demonstrated that at least three cell types are involved in the t cell proliferative response to antigen, including an antigen specific-t cell, an antigen-presenting cell, and a t cell that is found in unprimed lymph nodes or spleen, which has been termed the recruitable cell. we have utilized their approach of analyzing the slope of log cell number-log response curves to examine whether tcr @ cells can function as "recruitable" cells. we found that tcr ys cells as well as tcr ap cells can function as recruitable cells in this system. these data suggest that tcr $ cells can participate in the immune response without being specific for the antigen. analysis of the membrane associated phosphoprotein profiles of b cells harvested from cultures of resting cells exposed to il- for - hrs reveals the presence of phosphoprotein with an mr in the range - , . destroy the autoradiographic signal from this phosphoprotein suggesting that it is phosphorylated upon tyrosine residues. appearance of this molecule, and lps also apparently fails to result in the presence of a kd structure in the phosphoprotein profiles. anti-il- antibody. , in the cultures prevents the appearance of the kd phosphoprotein. the genes for t n f -a and tnf-p are tandemly arranged on mouse chromosome , with only . kb separating the ' end of the tnf-p mrna from the ' end of the tnf-a mrna. yet, the two genes are independently regulated. in vitro transcription and nuclear run-on experiments indicate that the two genes are transcribed from independent promoters. in macrophages, which express tnf-a but not tnf-p, only the tnf-a promoter is active. in t lymphocytes, which can synthesize both proteins, both promoters are active. activation of either cell type results in a moderate (up to -fold) increase in the level of transcription, while mrna levels increase more than wfoid under the same conditions. interestingly, the tnf-p gene is aanscribed -fold less than the tnf-a gene in t lymphocytes, although the corresponding mrna is more abundant. these results indicate that the accumulation of both tnf-a and tnf-p mrna after cell activation and their relative steady state levels are controlled mostly at a post-transcriptional step. acanomycin d chase experiments reveal that tnf-a mrna stability in macrophages is not significantly altered after activation by lf's, and therefore that stabilization done cannot account for the observed accumulation of tnf-a mrna. in order to examine more closely which elements are required for the regulation of tnf-a and tnf-p mrna abundance, we constructed hybrid genes combining putative control regions of tnf-a and tnf-p with known constitutive control elements. results obtained from the transfection of these hybrid genes into various cell types indicate that elements located both ' and ' of the coding sequence are required for the proper regulation of tnf-a and tnf-p mrna abundance. celiac disease is characterized by small intestinal mucosal injury and malabsorption. disease is activated when a genetically susceptible host ingests wheat gliadin or similar proteins (i.e., prolamins) in rye and barley. d region specif icities -dr and -dqw . class i d-region haplotype associated with celiac disease is extended and also includes genes in the hla-dp subregion. chain gene with those encoding dr and dqw may indicate that the hla haplotype associated with celiac disease exhibits an unusual degree of linkage disequilibrium or, alternatively, that disease susceptibility involves the gene products of more than one hla locus. to characterize possible hla structural variants unique to celiac disease, the polymorphic second exons of the expressed dr, dq and dp genes were amplified from genomic dna of celiac disease patients, and their nucleotide sequences determined. our studies indicate the presence of a unique constellation of d region genes associated with the celiac haplotype, and exclude the presence of a disease specific dr, eq or dp structural gene variant in this disease. disease susceptibility is strongly associated with the hla class i we recently determined that the hla this same population of t cells contains a high frequency % ) of cells which will respond to a given allogeneic mhc protein, or to differences at two other genetic loci termed mls, in conjunction with mhc. we have transfered the a and b chain genes from a pigeon cytochrome c/el specific, alloreactive. and mis' specific murine t cell clone into an unrelated host t cell. we demonstrate that the genes encoding a single a b receptor chain pair can transfer the recogntion of self mhc molecules c m p l e x e d with fragments of antigen, allogeneic mhc molecules. and an m sc (hls- ) encoded determinant. in this case the transfer of antigen specificity and alloreactivity requires a specific a receptor chain combination, whereas mlsc reactivity can be transfered with the chain alone into a recipient expressing a randomly selected a chain. site directed mutagenesis of the ja region has also been performed in an attempt to identify sites involved in the alloreactivity of this t cell clone. in addition. we demonstrate that a single amino acid change in the v-j junction of the a b receptor can alter mhc restriction a s well a s antigen fine specificity. department of genetics, washington university school of medicine, st. louis, i( . the s tumor sublines are variants isolated from a sing parent balb/c tumor which demonstrate locus-specific shut-off of their kd, dd and l genes. four phenotypically different sublines were characterized at the dna and rna level. southern blot analysis indicated that no major chromosomal deletions have occurred, and treatment of the sublines with -azacytidine had no effect on class i expression. between loci are unlikely. none of the repressed class i antigens could be induced with interferon even though the expressed antigens were fully inducible. northern blot analysis revealed message only for the expressed antigens, showing that the repression mechanism is acting at the transcriptional level. rnase protection analysis confirmed this result and demonstrated that the transcriptional repression is exquisitely specific for the kd, dd and ld genes as other "class i-like'' messages are present in. all the cell lines. expressing class i antigens from both fusion partners, but the negative class i antigens originating from the s partner were not expressed. lymphokine gene expression was examined in a panel of short-term murine t lymphocyte clones derived by single-cell micromanipulation from allogeneic mixed leukocyte cultures. about % of clonable t cells, including both cd +cd -and cd -cdw cells, could be expanded for assay at an average of days after cloning. following stimulation with concanavalin a or anti-cd antibody, all clones secreted detectable granulocyte-macrophage colony stimulating factor (gi(-csf), interleukin- (il- ) and il- , but cd + clones on average secreted higher 'levels of each lymphokine than cd + clones. clones ( %- %) expressed detectable gm-csf, interferon-y and il- mrna and % expressed il- mrna. when the frequencies of co-expression of any pair of lymphokine mrnas vere determined, all were found to correspond to the values predicted for random assortment of the individual frequencies. for example, among il- -positive clones, also transcribed interferon-y, giving the frequency of double-positive clones expected for random association ( . % . %). expression of the four lymphokine genes therefore segregated independently among the clones and did not allow the division of t cells into subsets vith distinct patterns of lymphokine synthesis. greater than -fold in the adult liver cell line, to fold in the macrophage cell line and just slightly in l-cells. we have subcloned the region ' to the li gene which contains sequences that may be important to regulating expression of the li gene. this region includes a -mer (cctagaaacaagtga) which occurs ' to many ifn?i regulated genes. current research has been directed towards identifying and comparing proteins from nuclear extracts prepared from control and ifn- treated cells which bind to this region (- to - ). this data indicates the li molecule may be expressed in cells not known to be directly involved in the immune response. although there has been considerable interest in the recently identified gamma, delta t cell receptor, relatively little is known as to its function. during our studies of the human immune response to autologous b cell lymphomas, we generated cytotoxic t lymphocytes (ctl) specific for tumor idiotype. these ctl lysed only autologous tumor cells and none of a large panel of other autologous and allogeneic cells. inhibitable by anti-idiotypic and anti-immunoglobulin antibodies but not by a panel of classical anti-mhc antibodies. phenotypic analyses showed that these ctl were cd +, cd -, cd -, and express the delta, and presumably gamma! t cell receptor. such ctl can be used to gain new insights into the function of the gamma, delta t cell receptor and t cell recognition of immunoglobulin, and may prove clinically useful in adoptive immunotherapy. tumor lysis was for ebv-induced antigens. furthermore, lcl variant . , which does not express any hla -a, -b. or -c determinants. is killed by cultures primed to lcl-. . antibody blocking experiments suggested that this killing was mediated by t cells, and was not restricted by known class i antigens. depletion of leu positive cells from the effector population did not eliminate cytotoxicity on lcl-. . cold-target blocking studies further suggested that the class -nonexpressing lcl-. and the class i-nonexpressing lcl , share residual deterninant(s) other than hla class i or class i that can restrict cytotoxic t cell responses to ebv-induced antigens. national jewish center for immunology and respiratory medicine, denver, co it is uncertain to what extent lymphokines can be differentially produced by activated primary t cell populations. to determine if il and ifnr were differentially regulated in uncloned human t cells from adults (ad) and neonates (nt), these mrnas vere measured by in situ hybridization after maximal stimulation by ionoaycin and pma. il mrna was detected in . % of total (tl), . % of cd ', % of cd ' cd r-, and . % of cdb' ad t cells, but in none of the tl, cd +, or cd ' nt t cell populations (virtually all nt t cells were cd r'). in contrast, ipnr mrna was found in . % of tl, . % of c d ' , % of cd ' cd r-, and % of cd + ad t cells, but only . % of tl, % of c d ' , and % of cd ' nt t cells. these results agreed with other estimates of il and ifnr production based on ria of cell culture supernatants, rna blotting, and gene transcription assays. in contrast to il and ipnr, il was expressed in similar amounts by ad and nt t cell fractions, as well as the ad cd ' cd r' and cd r' subsets. thus, the capacity for increased il and ifnr production by ad t cells appears attributable, in large part, to the postnatal acquisition of the cd r' subset (putative memory t cell population). aowever, additional mechanisms exist which act transcriptionally to limit il production by both neonatal and adult t cells. such selective expression may be important for restricting the potentially pleiotropic effects of certain lymphokines t o appropriate responder cells. we observed significant inhibition (> % at ng/ml) of the presentation of wova and of ova - by the anti-ap - peptide mab's. exhibited significant inhibition. peptide mab's. after incubation with antigen +/-mab, indicate that the inhibition occurs at the level of antigen presentation. dg , was also observed for the anti-p chain peptide mab's and to a lesser extent by the anti-a chain peptide mab's. peptide sequences are capable of interfering with antigen presentation, in vitro. supported by nih grant, ai- . the ovalbumin (ova) i-ad restricted t cell hybridoma, do l. was used to the anti-i-ad mab, mkd , also much less inhibition was observed with the anti-% -experiments with glutaraldehyde fixation of the b d.p cells before or inhibition of i-ad allorecognition by the t cell hybridoma. these results indicate that mab's generated against class i rijllinghoff, institute for clinical microbiology, university of erlangen-nurnberg, erlangen, f.r.g. and the *institute for clinical immunology and rheumatology, university of erlangen-niirnberg, erlangen. f.r.g. recently we have shown that cloned l . major-specific l / t-helper cells of type (th cells), when stimulated with antigen, are able to induce polyclonal b-cell proliferation ( ). we here present evidence demonstrating that this process is dependent on a direct cellcell interaction between t-and b-cells. which in the effector phase, i.e. during stimulation of the b-cells by activated t-cells, can be mediated by a mechanism other than cognate interaction. this conclusion is derived from experiments, in which highly purified resting b-cells were polyclonally stimulated by l / t-cells triggered by an anti-t monoclonal antibody, in the absence of antigen. the triggering process was independent of the presence of the fc part of the antibody and occurred in cultures devoid of macrophages. thus, the well established cognate recognition does not appear to be the only way of b-cell induction by t-helper cells of type . studies show that a proportion of the peripheral blood cd ' t lymphocytes do not express cd or cd and are called double negative t cells. they normally have a tcr. however, another population of double negative t cells exists that expresses the a@ heterodimer. w e have purified and expanded such a population isolated from the peripheral blood of a healthy individual and studied i t s phenotypical and functional characteristics. the c e l l s are cd ' cd -cd -, positive for wt and negative for the nk markers. they express a and p mrna b u t lack ymrna. from surface iodinated cells were precipitated w i t h monoclonal pf two closely running bands ( & kd) . functional studies demonstrate that they proliferate to anticd and pha, t h i s response was blocked by cyclosporin a. there was no nk lysis b u t anticd induced l y s i s of target cells. the cells responded t o il- and il- as previously shown for other t c e l l s , b u t also t o il- , a lymphokine thought t o affect mainly stem cells and not previously shown t o stiaulate growth of mature cells. long term growth of these c e l l s was also maintained by these cytokines . roberto biassoni , silvano ferrini , rafck p. sekaly , and eric . long , laboratory of immunogenetics, national institute f allergy and infectious diseases, nih, beth-md , and istituto nazionale per la ricerca sul cancro , genova, italy. cd -cells grown in vim in the presence of il- acquire the ability to l~s e a wide variety of tumor cells in an mhc-unrestricted manner. we have previously shown that cd - clones expressed the cd epsilon gene but no functional transcript from cd gamma, cd delta, tcr alpha, tcr beta and tcr gamma genes. this result suggested that these cd - ' cells represented an early stage in t cell differentiation. to test for expression of the tcr delta gene in these cells, rna from a panel of cd - ' clones and from three highly enriched populations was hybridized with several dna fragments of the delta locus. abundant transcripts were detected with a c delta probe and a j delta probe in out of clones and in all three populations. at least four different transcripts were present with sizes similar to those found in cd ' tcr gamma-delta' cells. however, the tcr delta transcripts in cd - ' cells are most likely derived from unrearranged genes because no rearrangement could be detected in dna from an enriched population using a j delta probe, and because these aanscripts hybridized to a dna fragment corresponding to the unrearranged genomic sequence '-upstream of j delta . expression of unrearranged tcr delta genes in cd -cells provides further evidence that these cells belong to the t cell lineage. functional capabilities and by differential release of either il or il upon activation. we have produced a new monoclonal antibody to cd which has allowed us to separate normal murine cd + cells into two populations based on the density of expression of cd epitope. the separated populations seem to be analogous of subsets found in cloned t cell lines. cd + t cells with high density of cell surface cd after polyclonal activation produce il and mrna encoding ifw and il . it does not produce il or il mrna. cd low density population on the other hand transcribes mrna for il and secretes il protein. data will be presented to demonstrate that the two subsets of normal cd + cells also differ in their proliferative response to mitogenic stimuli and to exogenously added growth factors. the substitution of v to l at was the only change that could be discriminated by of allospecific ctl lines. suggesting that those ctl lines recognize a . plus a peptide whose presentation andlor binding is affected by the v to l substitution in the floor of the peptide binding site. in contrast, the l to w substitution at (but not the other substitutions) abolished the ability of the a molecule to present the viral peptide to out of peptide-specific a . -restricted ctl lines, suggesting that this substitution alters the presentation of the influenza matrix peptide but does not inhibit the ability of the peptide to bind to the a molecule. although y tcr.s have a great potential for diversity, it remains to be determined whether this potential is realized in terms of expressed y tcrs. preliminary studies in several laboratories have indicated that y tcrs expressed in earlg t h r c y t e s and adult epithelial tissues are more restricted in diversity com ared to adult tc expressin thymocytes. we have derived a panel of cloned dendritic epigrmal t cells (jetc, lines and ybridomas that express at least three types of y receptors -c , cy and c n . immunoprecipitation, northern and southern blot analyses, and sequence anazses of l gt cloned cdna or olymerase chain reaction ( k r ) amplified cdna segments have been used to anal ze in detail &e extent of diversit in the expressed y and chains and whether restricted airin o?y and chains occurs. our resu& indicate that for this panel of cloned cell lines and ! irkg is nonrandom and that variability in certain types of receptors appears to be restricted. %owever, we have observed significant chain diversity in these cells that is obtained by the use of multiple v-regions, and n-region and junctional diversity. we are investigating whether the observed y and chain pairing, and pattern of chain diversity are present in other $tcr bearing cells or whether they are only characteristic of detc. activation of ctl precursors from murine unprimed spleen cells with ril- or ril- results in distinct lytic spectra, depending on which lymphokine is present. we have used allo-stimulation in limiting dilution analysis with subsequent testing on an allo-specific target (a ) and an mhcdeficient, non-specific target (rle). in the presence of ril- exclusively allo-specific ctl are generated, while ril- supports a proximately equal numbers of precursors that k~ll a and rie targets. dose response analysis of ril- -supported killing activity indicates that the lytic spectrum is independent of the amount of ril- used, and therefore this il- effect is intrinsic in its activity on unprimed spleen cells. mixing experiments indicate that ril- can partially override the effect of il- on the generation of non-specific killer cells. split well analysis and cold target inhibition experiments are in rogress to ascertain the actual proportion of specific killer cells which can be generated with ril- . be. are also testing the ability of cofactors, such as il- and il- , to optimize the response of il- generated ctl. we conclude that il- , not il- , must be used when ctl are generated from unprimed spleen cells in mice. t r a n s c r i p t s i n y/ tcr populations. i n t e r e s t i n g l y , these same v genes, as well as a further+crosshybridizing v gene previously designated va . , are expressed by peripheral a& tcr c e l l s as . kb tcra transcripts. these data suggest t h a t b a -dn th represent a developmentally unique subset i n which both v and vg segments are non-randomly expressed. furthermore they i n d i c a t e t h a t there i s considerable overlap between the v a and v gene repertoires . indianapolis, in in order to detect the small amounts of lymphokines generated in vivo following antigen stimulation, we developed a co-culture system which allows for detection of il- / , il- /csf and tnf from ln cells stimulated in vivo with picryl chloride (pcl). utilizing thb system in combination with facs analysis and receptor binding studies, we examined the production of these lymphokines in primary and secondary immune responses. during a primary immune response, the production of il- was not readily detectable on dl, peaked on d and was gone by d . at no time were we able to demonstrate the presence of il- . alternatively, the presence of il- /csf and tnf was w i l y detected on dl, but olso peaked on d . in comparison to primary responses, secondary immunization lead to at least two alteraticns. (i) peak production of all lymphoki es shifted towards dl. ( ) although most lymphokines did not demonstrate increasea in the amount produced/lo cells, the amount of lymphokine generated/ln was vastly increased due to an increased number of cells. utilizing single and dual color facs analysis we also examined the ln cells for alterations in t cell subpopulations. during the course of the primary response: (i) the percentage of thy i+ and l t + cells decreased until d and then began to recover, ( ) the percentage of thy-i+, t -,t -cells peaked at the time of greatest lymphokine production (i.e.-d ) and ( ) the il- receptor was expressed solely on thy-i+ cells, was detected on both t + and t + subsets and peaked on d . most of these alterations also occurred during the secondary response, but their timecoune was shifted so that maximal effects occurred earlier (e.g., dl). finally. the maximal binding of radiolabeled il- by the ln cells following both primary and secondary sensitization correlated with the expression of the il-zr as detected by facs analysis. in addition, binding of radiolabeled il- demonstrated similar patterns except for the detection of significant binding on dl. these results demonstrate that (i) an ordered timecourse of lymphokine production occurs in vivo following exposure to antigen and ( ) the secondary immune response to pcl is characterrd by an accelerated tempo of lymphokine production, rather than an increased level of lymphokine production/lo cells. activation as direct g-protein activation by a f pi-hydrolysis using phorbol diester stimulation of pkc restores the inhibi$$ble phenotype and the ability to upregulate c-fos. even more interesting, sig-linked ca responses by vs . -c . are equivalent to those observed in the wildtype wehi- . resul$g suggest that contrary to current thought, sig-generated signals may not be coupled to ca fluxes via inositol phospholipid hydrolysis. thus, vs . -c . is a new and powerful tool with which to analyze signalling through sig at the molecular level. unlike the wildtype, crosslinking of sigm on vs . -c . did the signaling defect in vs . -cl. -appears to be proximal to phospholipase c triggers pi-hydrolysis and bypassing these latter lmmunogenicity c analysis of t cell receptor chains from adult cd -,cd -thymocytes mark w. moore, i. nicholas crispe and michael j. bevan, department of immunology, research institute of scripps clinic, la jolla, ca . the role of tcr genes in t cell development has not been determined. to extend out understanding of the repertoire of tcr expression, we prepared a cdna library from cd -,cd -adult balb/c thymocytes and cloned and sequenced tcrq genes from this cdna library. we found that clones were transcripts of the unrearranged c , gene and that clones terminated in the j , region. nine of the remaining clones were v , -j c genes and five of these were in frame. only one clone corresponded to c and was v -jy c jofned'in frame. sds-page analysis of the -chain proteins from the surface of both balb/c anzcdbl) adult cd -,cd -thymocytes did not detect the , mw vy c protein, but did detect the , mw v c protein. these results suggest that despite the abundanc$%f pull-length, functionally joined, v c transchpts in the thymocyte subset, the protein product is not expressed on the cell surface as the prehfcted , mw protein. finally, our analysis of the v-j jointing of the genes reveals both flexibility at the v-j junction and extensive n-region nucleotide addition that lead to diversity of the predicted protein sequence. il in response to the same stimuli. e identification of these two subsets of cd ' helper cells is mostly based on studies performed with long-term cultured t cell lines and it is not clear whether these two subsets exist in vivo and represent distinct lineages of t cells. in particular, the frequency, tissue distribution and ontogeny of cells capable of secreting il in vivo is not known. these studies have been hampered by the fact that freshly isolated t cells from unprimed animals failed to secrete detectable amounts of ila and il when stimulated in vitro by lectins or alloantigens, whereas iu is readily detectable in these same cultures. data presented here indicate that freshly isolated t cells from unprimed animals can be induced to produce il in a receptor-de endent, antigen-independent manner upon stimulation by anti-cd antibodies. our results also stow that only cd ' and not cdst cells can be induced to secrete il and that cross-linking of the receptor is required for o timal activity. we believe that this approach will be useful in identifying in vivo cells recomittefto the th pathway and study their ontogeny, activation requirements and tissue distriiution. hlb, brussels, belgium. we have studied the murine tcr repertoire against the c-terminus of cytochrome c in association with certain alleles of the mhc class i molecule, eakepk (iek) and eakepb (ieb). for mice possessing these alleles, the majority of responsive t cells utilize one member of the variable val gene family in conjunction with a limited set of vp genes. as an extension of these studies, we have examined ie specific, alloreactive hybridomas derived from ie non-expressing (eab) cytochrome c non-responder mice to determine their usage of va and vp genes. tion assay showed that fourteen utilized the same val gene segment used by the majority of cytochrome c specific, ie restricted t cells and eight utilized a closely related val gene that also is associated with this antigen response. element most commonly used by cytochrome c-specific t cells was not found among the alloreactive hybridomas tested, @ genes less frequently used in the cytochrome response were expressed by seven of the alloreactive hybridomas whose va segments were defined by rnase protection. determining recognition of ie molecules both in mhc-restricted, antigen specific immune responses and in alloreactive responses. the t-helper cells of seven mouse strains, representing class i haplotypes (ias, ia , iab, iakiek, iadied) were responsive to immunization and restimulation with parent peptide. the ied determinant was shown to be a presenting element by monoclonal antibody blocking and by use of l-cell-transfectants as af'cs to purified t cells and to t cell hybridomas. a series of overlapping synthetic peptides identified two minimal t-cell sites within the parent peptide: mice expressing ia and ie responded to a fragment at the n-terminus of the parent peptide (site ) while mice expressing only ia responded to a distinct but overlapping fragment at the c-terminus (site ) . these minimal sites identified in vitro could be used to immunize mice in vivo in an mhc-restricted manner. the human tcr locus is strategically located within the atcr complex between the cluster of va/v region and the ja segments. which can be spliced to ca in pre t cells, separates from the ja segments. pulse field gel mapping as we as molecular cloning link diversity (ds), j g , c, and tea within kb. considerable tcr diversity is generated despite the predominant use of one v and j segment. d and d are and bp long, are frequently recombine as d, /d ? and reveal exonucleolytic trimning with extensive "n" segment addition. specialized ' and ' deleting elements, rec and p j a , separate the locus from the a locus. cells with rec/$b ja recombinations comprise most deletion events although rec recombines with other major acceptor sites in fetal and post-neonatal thymic dna. the ' deleting element ( rec) is evolutionarily conserved in the mouse and functional comparisons are underway. delete the locus may prove to be the pivotal event establishing separate y and ae lineages. to study the mechanism of t-cell tolerance, transgenic mice were generated that expressed the mlsa reactive t-cell receptor (tcr) o-chain vb on - % of peripheral t-cells. in transgenic mice bearing mlsd, the numbers of high tcr expressing thymocytes and of thy . + peripheral t-cells were reduced. the cd /cd ratio of peripheral t-cells was decreased fourfold compared to negative littermates. both mlsa and mlsb tcr &transgenic mice were able to mount a t-cell dependent antibody response against viral antigens whereas the capacity to generate alloreactive and virusspecific cytotoxic t-cells was impaired in tcr &transgenic mlsa, but not in transgenic mlsb mice. rna analysis and immunof luorescence with tcr vb-specific mab further revealed, that the expression of endogenous tcr -genes in these mice was suppressed. tolerogen-reactive lymphocytes, as measured in the mlr, in spite of their long-term acceptance of a skin graft bearing the tolerated antigens. lymphokine production by mlr+ tolerant lymphocytes is different from that of syngeneic normal lymphocytes. normal lymphocytes produce only il- in primary response to tolerogen, while tolerant lymphocytes produce il- and il- . using limiting dilution analysis, we have to estimated the frequencies of pil- and pil- (precursor) cells in these cultures. after primary k vitro stimulation, normal responders have a low but measurable frequency of pil- cells, while tolerant responders have a much higher pil- frequency. however, following subsequent & restimulations, the pil- frequency of normal responders rises and begins to approach that of the tolerant responders, such that the two populations are indistinguishable based on pil- frequencies following the third round of in vitro stimulation. these data suggest that the high frequency of il- producers (presumably t,, cells) among the tolerant lymphocytes resembles unexpectedly a "primed" state, rather than "unprimed"as in nontolerant responders (where th , dominate the early response). the existence of "primed" t cells in phenotypically tolerant animals raises the possibility that precocious activation of tr (by neonatal exposure to tolerogen?) suppresses the later emergence of t,,, which would be expected to contain the cells responsible for graft rejection. a large number of cd + t-cell clones, obtained from peripheral blood t lymphocytes by direct limiting dilution, allowed us to address the question whether functional heterogeneity exists within the human cd + t-cell subset. six out of cd + clones were able to lyse daudi or p cells in the presence of anti-cd antibodies. the remaining cd + tcell clones tested did not acquire this cytotoxic capacity during a culture period of weeks. in the absence of anti-cd mab, no lytic activity against daudi. p and k target cells was observed under normal culture conditions. these two types of cd + t cells showed high reactivity with anti-cdw ( b ) mab and no reactivity with anti-cd r ( ) mab. the cd + clones without anti-cd mediated cytotoxic activities (th ) consistently showed a higher expression level of cd antigens. th cd + clones did produce il- , ifngamma and tnf-alpha.beta. whereas the th t-cell clones produced minimal amounts of il- . ifn-gamma and tnp-alpha. beta in response to anti-cd mab and pma. not all cd + clones did release il- , but there was no correlation with cytotoxic activity. moreover, as compared to the th cd + clones, tr cd + clones proliferated moderately in response to anti-cd mab. however, anti-cd mab induced proliferation of only the th cd + t-cell clones was enhanced by anti-cd mab. both cd + subsets provided help for polyclonal b-cell activation with anti-cd mab. our data suggest that the human cd + subset, in analogy to the murine system, comprises two functionally distinct t-cell subpopulations. in the mouse, when la antigens are isolated immunochemically, the predominant species isolated are the isotypic matched pairs, aaap and eaep. however, when la ap dimer expression is studied using an l cell transfection model, it is found that the isotype-mismatched dimer apdea is readily expressed at the cell surface. these results suggest that differences in assembly andl or transport of different la pairs may be most readily visualized in a competitive environment where multiple distinct la chains are available. to investigate this possibility, the relative efficiency of inter-and intra-isotypic dimer formation and expression was evaluated using a sequential l cell transfection system. l cells already expressing an ap dimer on the cell surface (apdea or apdaad) were supertransfected with a third la gene (aad or ea, respectively). synthesis of this second a or p protein led to competition for the unique partner chain. individual clones were scored for cell surface expression of the distinct dimers (e.g., apdea vs epdea or apdaad vs apdea) using facs analysis with chain specific monoclonal antibodies. in addition, each species of mrna was quantitated by northern blot hybridization using bcus specific probes. our results indicate that in the h- d haplotype. isotype-matched dimers are expressed with - x the efficiency of isotype-mismatched dimers. this result suggests that, regardless of the cell type studied, if each of the four murine la genes is expressed at equivalent levels, intraisotypic dimers will be expressed to the virtual exclusion of the interisotypic dimers. however, if chain synthesis asymmetry occurs, the isotype mismatched pairs may be expressed at immunologically relevant levels. differential we have identified a series of discrete stages among the cd -double negatives which seem to form a sequence, with tcr gene rearrangement and rna expression gradually progressing, but with potential for expansion and repopulation of irradiated thymuses diminishing along the series. on this pathway cd must be expressed late or after the acquisition of cd and cd . cell cycle analysis shows the highest rates of cell division to be among the rsa+ il- r-pgp- -population which probably precedes the transition to cd +cd + and tcr expression. thus it seems unlikely that tcr/antigen interactions play a role in cellular events occurring among the double negative cells which lead on to mainstream t-cell development. egr-l is a murine early growth factor inducible gene which encodes a protein with zinc fingers. its expression was investigated in murine b-lymphocytes stimulated through their antigen receptor (sig) with anti-recptor antibodies (anti-ig) . rapid (by minutes) upregulatlon of egr-l mrna expression was observed at doses of anti-ig sufficient to drive the majority of go cells into cell cycle. agonists and inhibitors of protein kinase c (pkc) showed that expression was coupled to the pkc component of receptor immunoglobulin transmembrane signalling. interestingly, signalling through sig on the murine b lymphoma wehi- did not upregulate egr-l expression even though similar signalling pathways are associated with this receptor in these cells. southern analysis showed that egr-l is not deleted or translocated in this cell line. importantly, cell growth and proliferation of wehi- is inhibited by anti-ig stimulation suggesting a relationship for egr-l expression and differential processing of receptor ig signals. this notion is further supported by the finding that murine b lymphomas whose proliferation is not inhibited by anti-ig showed receptor immunoglobulin coupled egr-l expression. reeulation of exdression of a class wc transeene. dinah s . the expression of the transgene product. the patterns of expression of the transgene parallels that observed in situ, indicating that regulatory elements necessary for normal patterns of expression are contained within the injected kb dna segment, and that trans acting factors involved in its regulation function between species. included among these elements are those specifying preferential expression in b cells relative to t cells. in vivo treatment of transgenic mice with a/@-interferon results in increased expression of the transgene in a number of tissues. the response parallels that observed for the endogenous h-zkb, but differs markedly from qa- . analysis of the chromatin structure of the transgene reveals a single constitutive dnase i hypersensitive site present in both spleen and thymus, which is not altered by interferon. both a novel negative and positive regulatory elements have been identified in the 'flanking region of the transgene. the negative regulatory element reduced the activity of both the homologous class i promoter and a heterologous viral promoter. in vivo competition experiments indicated that the functions of the positive and negative elements are mediated by distinct cellular trans-acting factors. the negative regulatory element requires the presence of a positive regulatory element to function. this interaction between elements represents a novel mechanism for regulating gene expression. mcdevitt. department of microbiology and immunology, stanford university school of medicine, stanford, ca . published data show that encephalitogenic h-zu murine t cell clones with specificity for the n-terminal eleven amino acid peptide of myelin basic protein display a restricted fine specificity when tested on substituted analogs of the native peptide. for example, substitution of alanine at certain positions in the peptide totally abolishes the response of each clone (acha-orbea et al.. . cell : ) . recent experiments also have shown that the ability of some peptide analogs to bind to h-zu i-a gene products does not always correlate with their ability to stimulate the t cell clones (see accompanying abstract by david c. wraith and hugh . mcdevitt). this suggests that h- u mice may lack a t cell repertoire capable of recognizing these peptides complexed to h- u i-a gene products. to test this possibility, h- u mice were immunized with a panel of peptide analogs, as well as the native peptide. the in vitro t cell proliferative response to each of the peptides then was measured. the results show that in vivo immunogenicity of the peptide analogs also does not strictly correlate with their capacity to stimulate the t cell clones. in this way, the polyclonal t cell repertotre of h-zu mice for the myelin basic protein peptide analogs was examined, and could be compared with the i-a binding characteristics of the peptides. terms of antigen and mhc recognition. this response involves a limited repertoire of t cells which crossreact on species variants of the antigen. in addition, t cells specific for the antigen in association with syngeneic mhc can recognize antigen on similar allogeneic mhc molecules. the groupin of clones by functional phenotypes defined by these crossreactivities allowed us to corrcfate tcr gene usage with either antigen or mhc recognition. some of the pigeon cytochrome c-specific clones within one functional phenotype use receptors that differ by as few as two amino acid residues. other clones e y s s very different tcrs but exhibit similarities in antigen/mhc reco nition. the efect of these tcr differences on recognition was assessed using a pane? of anti en analogs with single amino acid substitutions presented on different mhc molecufes. each clone exhibited a unique pattern of res onse to the antigen analog panel, even clones with very similar receptors. also, eace residue in the antigenic region of the peptide was critical for interaction with at least one t cell receptor. therefore, the antigen must either be a linear molecule with each residue available to interact with the tcr or be able to assume several conformations to interact with mhc and the tcr. lmmunogenicity c thy- + cd + ly- (b )+ cd -cd -tcrx- ' helper cells. anne i. we have found that these cells can be preferentially stimulated to proliferate when cocultured with the b lymphoma, ch . one to % of nylon wool non-adherent, ia-, jlld-, and cd -lymph node cells from normal unimmunized mice have the phenotype thy-l', cd ', cd -. and cd -. these cells proliferate when co-cultured with a syngeneic surface ig' lymphoma, ch , even in the absence of any added antigen, mitogen, or fetal calf serum. prior to stimulation we find that approximately % of thy . ' cd ' cd -cd -express the marker ly- (b ), however after culture with ch the majority of cells with this phenotype express the marker ly- (b ). after ch dependent proliferation the ly- (b )' t cells are able to provide help for secretion of ig by fresh ch b cells. surface labelling and precipitation of t cell receptor molecules reveals that most of the thy- ' cd ' ly- (b )* cd -cd -cells express tcr(r- ). furthermore, cd precipitation shows that as many as four different - heterodimers are utilized within the entire responding population. this suggests that a heterogeneous population of double negative tcri- cells are involved in the response to ch . college of kedicine at east tennessee state university, johnson city, tn interferon-producing (t ) and interleukin (il ) producing (t ) clones were assayed for their ability to diregtly induce cytostatic activity in macro:hages generated from splenic myeloid precursors (m -c). in the presence, but not in the absence, of antigen, t clones activated the m -c to inhibit the growth of p tumor cells in vitro. th cjlones were not able to activate such effector activity in the i -c. effectively present antigen to the t clones as evidenced by the proliferation of t cells cultured with antigen in the pfesence, but not in the absence, of m -c. thereyore, although both t and t were activated by cognate interaction with antigen presenting (ba) or nippostrongylus brasiliensis (nb). spleen cells from these mice were cloned at limiting dilution with alloantigen stimulation, and every two weeks, lk production in response to con a was measured. clones derived from, and stimulated with, cells from unimmunized mice initially tended to secrete low lk levels, with few clearly defined th or th clones. by days after cloning, some clones had acquired th or th patterns. cfa, ba and nb-imnunized mice gave rise to clones that were mostly th or th even at early times. cfa and ba immunizations induced almost exclusively th clones, whereas nb induced more th clones. these results are consistent with a model in which resting, previously unstimulated t cells produce low amounts of lks, and progress through stage(s) where they secrete both th and th lks before finally differentiating into th and th cells. the results with cfa, ba and nb-primed mice suggest that this process occurs in vivo as well as in vitro. strains as carriers of melioidosis antigens to the immune system, deja tanphaichitra, mahidol university, p.o. box - , bangkok , thailand the attenuated gale mutant, salmonella typhi strain, tyzla, served as the recipient in a conjugal dna transfer experiment. conjugal dna transfer was obtained by the mating procedure on an appropriate blood agar medium. were examined serologically. one selected strain was found to have the serological characteristics of the recipient s . typhi, tyfla strain and also expressed the pseudomonas the donor strain was a pseudomonas pseudomallei mu . the resulting antigen clones were repurified by restreaking on the medium and pseudomallei antigen. the s. typhi transconjugant strain is due to the presence of the pseudomonas pseudomallei plasmid. a group of subjects when received four doses of this bivalent vaccine strain in this study it appears that pseudomonas pseudomallei synthesis in developed antibodies against pseudomonas pseudomallei up to %. pseudomallei, an intracellular pathogen, produces a characteristic antigen probably to be plasmid coded, we considered that the gale salmonella typhi tyzla oral vaccine strain, highly effective against typhoid fever, might be modified so as to be protective also against melioidosis due to pseudomonas pseudomallei. terminal deoxynucleotidyl transferase (tdt) is a lymphoid-specific nuclear enzyme present in early lymphocytes. to investigate the regulation of tdt gene expression, pre-b and pre-t cells were treated with phorbol -myristate -acetate (pma) o r three analogs, and tdt steady-state mrna levels were determined by northern blot analysis. treatment of early lymphocytes with pma results in a rapid and reversible decline in steady-state tdt mrna levels within six hours. this rapid decline can be blocked by pretreatment of the cells with a protein kinase c inhibitor, implicating protein kinase c activation in the decline of tdt mrna. nuclear run-off studies demonstrate that tdt transcription is rapidly down-regulated within minutes after pma treatment, indicating that this regulation occurs mainly at the level of transcription. furthermore, cycloheximide blocks the decline in tdt in rna showing that new protein synthesis is required for transcriptional inactivation. the nucleoprotein gene from the influenza virus a/nt/ / was stably cloned into the attenuated aroa-strain of salmonella typhimurium sl . nucleoprotein purified from pnp - was tested for the ability to generate virus-specific immunity. immunization with recombinant derived nucleoprotein induged immunity to all type a influenza tested but not against type b viruses. cd helper t cells were primed but no evidence was found for priming of class i restricted ctc. mice immunized with recombinant nucleoprotein were protected against a subsequent challenge of influenza virus. the information obtained from the study of the immunity and protection generated by the purified recombinant protein was then used to design experiments to investigate the possibility of using the attenuated salmonella vector to deliver the nucleoprotein molecule to the immune system by the parenteral or enteral routes. we characterized the extrachrom- circular i n i s in -day-fetal and -week-old m u r i n e thpmcytes and -week-old m u r i n e splenocytes. f popllation of circular chias was clone into the kgtll phase vector. we screened ca. tna cl-by plaque hybridizations with all far kirds of tcr gene probes derived from jal , val , db , db , jyl , j and loci. cut of , cna cl-from fetal and -week-ld thymocytes, hybridized with tcr aprobes and hybridized with tcr &probes. positive cl-with tcr yand probes were to in fetal thymocyte erived library, but few in -week-old thymocyte. of fetal tcr clcnes analyzed, cl-had dd or vd reciprocal joints and clcne had vd ar dd d i n g joint. relative frequencies of circular dna clones for four different tcr genes are consistent with the order of the expression of the genes the t cell developnent. of , tna cl- signalling could be studied. llzmambxane signalling was maasured by ability to t?z nslocate fkc frcrm the cytcplasa to the nw leus after surface i-a was banrl by a or p dxdn specific monoclcnal antibody. i(pmwing either or amino rids fmn the a chain cvtoplasnic (cy) damin did not affect the ability of tkse i-a r m l d e s to trarslocate pkc to the nucleus. normal splenic b c e l l s were rendered non-responsive t o subsequent challenge w i t h lps, as measured by a decreased a b i l i t y t o generate antibody forming c e l l s (afc), by incubation overnight ( - hours) w i t h ug/ml a n t i -i g . both i n t a c t and f(ab)', a n t i -i g , as well as monoclonal anti-igm (bet and b- - ) , were able t o induce c e l l non-responsiveness t o subsequent lps challenge, suggesting t h a t sig/fcr i n t e r a c t i o n s are not necessary i n the induction o f lps non-responsiveness. i n contrast, induction o f nonresponsiveness t o subsequent challenge w i t h fitc-prucella abortus required i n t a c t a n t i -i g . the a b i l i t y o f mitogenic a n t i -i g (rab f(ab)', o r - - northern blot analysis and bioassay data were used to analyze separate lymphokines as well as the il- receptor (murine tac). northern blot comparison of fresh and primedt enriched rna revealed that primed t cells produced -fold more lymphokine than the fresh t cells. the only lymphokine that showed equal amounts of mrna for both fresh and primed t cells was il- . a time course of fresh and primed t + cell lymphokine production was also analyzed. the primed cells produced a short burst of lymphokine mrna that peaked between . and hr after con a stimulation and declined after hr. the fresh t cells produced a longer burst of lymphokine mrna that peaked - hr after stimulation. the il- receptor @- r) mrna time course from activated primed cells showed different kinetics than lymphokine mrna. this suggested that molecular regulation of the il- r might be different than lymphokine regulation. to further examine molecular regulation in the primed t cells polysome profiles were evaluated for lymphokines, l r , and other cellular genes. the recently developed method of gene amplification by the polymerase chain reaction (pcr) has proven to be particularly suited for the analysis of t cell receptor (tcr) genes. we adopted existing methods for the preparation of cytoplasmic rna from as little as cells and used this material as template for first strand c-dna synthesis. pcr amplification of this c-dna, using v-and c-specific oligonucleotide primers yielded enough material to produce single-stranded dna in a second pcr which could then be sequenced without cloning. in case of unknown v-usage, the pcr was employed for screening for v-beta elements by sequential reactions with different v-beta specific primers. we have used this method to reinvestigate the h- b restricted cytotoxic t cell response to tnp in c b mice. beta chain sequences of ctl clones obtained by direct cloning of immune spleen cells were compared to sequences of clones obtained by cloning of individual short-term in vitro ctl lines. it was found that a) in vitro bulk-stimulations reduced the heterogeneity of the beta-chain responses to tnp, b) similarities between different tcr-beta-chains concentrated on the usage of certain jb-elements ( jb . , . , . ) rather than v-region or nid-region sequences, and c) the majority of jb . containing beta-chains was associated with alpha-chains expressing v-segments of the val family. these expression of genes which encode the t cell antigen receptor is cenval to the generation of the t cell repemire. our labomtory has been investigating genes for both the alpha and beta chains of this receptor in inbred strains of runus norvqicus (the laboratory rat), a species in which several autoimmune disease models have been developed. and which is used extensively in transplantation studies. using genomic southern blots and mouse probes specific for five different v a subfamilies, we have estimated the size of the v a repertoire in ten inbred strains of rat. results show a significant increase in the size of one subfamily and suggest increases in two others in all ten strains. the rat v a l subfamily has about twice as many members as the mouse, while the va and vu subfamilies, depending upon the enzyme used, show a similiar duplication. the va and va subfamilies have a comparable number of members in both species. these data are most easily explained by a single duplication event in the rat invoking at least one and perhaps three subfamilies, but not encompassing the entire v a locus. this implies that the val subfamily (perhaps together with va and va subfamilies) is regionally clustered and not interspersed with either the va or va subfamily. based on restriction fragment length polymorphisms, we find evidence for six distinct v a haplotypes in the ren strains tested. we have also cloned eight unique germline v a l gene segments. one of these has been sequenced. and has a coding region % identical to the most closely related mouse v a l sequence. this degree of relatedness is similiar to ra#nouse vg homologues. which share % nucleotide sequence similarity. we are using these clones to generate angle copy probes from flankiig regions to further map the v a l locus. current approaches to mhc-peptide binding studies require either large quantities of highly purified mhc protein and/or the use of sophisticated detection apparatus. i n order to simplify detection of peptide-mhc interactions we have investigated the use of photosensitive-crosslinkers. two reagents have been successfully tested. a benzophenone derivative of peptide - from rat myelin basic protein (rmbp) was only effective after the introduction of a glycine spacer residue between peptide and crosslinker. an azido-nitro-benzoyl derivative of peptide . , a heteroclitic analog of rmbp - ( ). had a high affinity and bound specifically to the peptide binding site. the . photoaffinity probe has been used to test the binding properties of other analogues of rmbp - and is currently being used to define (a) the kinetics, (b) ph and (c) temperature dependence of the binding event. this particular photoaffinity conjugate retains both the mhc binding and biological properties of the original peptide and is helping us to define the roles of "determinant" versus "t cell repertoire" selection in the mhc linked autoimmune response to mbp the antigen-specific t cell repertoire is diverse in its ability to recognize a wide universe of foreign antigens. this t cell repertoire is composed of a set of clones each of which is specific for a given foreign antigen. therefore the precursor frequency of t cells specific for any give foreign antigen is extremely low. however, two prominent exceptions to this general rule exist, and these are the t cells present at high precursor frequency which are specific for foreign hhc products or for the products of the minor lymphocyte stimulatory (mls) genes in the mouse. the present studies were undertaken in order to examine factors involved in t cell repertoire formation by assessing the relationship between t cell repertoire for conventional foreign antigens and for mls products. studies indicate a striking degree of overlap between the set of t cells specific for pigeon cytochrome c and the set of t cells specific for mlsc gene products. demonstrate that the basis for this overlap lies in the predominant expression of one tcr vp gene, vbs, by those t cells which recognize mlsc. involvement of specific tcr afl dimers in recognition of mlsc and further suggest that t cell reactivity to these gene products may play an important role in establishing the t cell repertoire for foreign antigens. conclude that, rather than destruction of some essential apc structure, ecdi fixation prevents the apc from actively responding during the encounter with the t cell. this results in a failure to express new structures (probably located on the apc plasma membrane) that appear to be essential for stimulating t cell proliferation. these structures are distinct from ia or il . the induction of these structures during t-apc interaction occurs in six hours, requires protein synthesis, and can be elicited by il , il or lps, but not ifn-gamma. in the absence of these induced structures, the apc stimulates a partial t cell response, il release, but the t cells fail to proliferate. these induced structures on the apc may be either adhesion molecules that stabilize the t-apc interaction, or they may provide additional stimuli to the t cell. were not c m n t o the three s t r a i n s o f mice (balb/c, regions , , , and ; c h/he, regions , , , , ', and '; and c bl/ , regions , , , and ). immunisation with type i collagen (cii) leads to development of arthritis in mice with certain mhc haplotypes and is associated with an immune response against cii. we have been studying the t-cell response in the arthritis susceptible strain dbm (h- q) . analysing the proliferative response in cultures of lymph node cells from immunised mice a s well as t-cell lines and clones established from such cultures it was found t h a t li the t-cell response after immunisation with heterologous cii was preferentially directed against foreign determinants on the cii molecule with little o r no crossreactivity against autologous cii. ' both the primary response and the reactivity of established lines and clones were directed against the cbll fragment of the cii molecule, using c b l l fragments prepared from chick, bovine or rat cii. / pepsin present in cii preparations after using pepsin digestion for solubilisation of the collagen is strongly immunogenic even in very small amounts and it was therefore necessary to use cii prepared from lathyritic cartilage without pepsin digestion for immunisation. in contrast to the pattern in lymph node cultures from immunised mice we found that when culturing spleen cells from unimmunised mice there was a t-cell response against collagen that was preferentially directed against autologus cii. since we earlier have found that autologus cii may induce an immune response and also arthritis in dbn mice we conclude that there exist t-cells capable of reacting with autologus collagen and inducing an immune response as well as arthritis but that these cells are under regulation so that they not readily can be activated into proliferation but may be induced to perform certain effector functions. tested. the characterization of these two cd rdsas will be presented. analysis of hla polymorphism using sequence specific oligonucleotide probe hybridization to amplified dna, lee ann baxter-lowe, jay b . hunter, and jack gorski, the blood center of southeastern wisconsin, milwaukee, wisconsin . hla polymorphism plays a key role in antigen:mhc interaction. the polymorphism of the first domain encoding exon of the hla-dr p chain has been studied by in vitro dna amplification and use of sequence specific oligonucleotide probe hybridization (ssoph) to detect polymorphic sequences. a bp segment of genomic dna was amplified and hybridized with synthetic oligonucleotide probes ( - bases) under conditions that detect single base pair mismatches. identification of these mismatches can be used to predict micropolymorphism in the protein products, including single amino acid changes. haplotype specific patterns of oligonucleotide probe hybridization were defined for a panel of homozygous typing cells. analysis of family data demonstrated the expected inheritance patterns. most known serological specificities are encoded by multiple allelic forms of dr p chains and ssoph can identify these differences. this was exemplified by detection of unique ssoph profiles for subtypes of dr , drw and drw alleles. this procedure was also used for analysis of hla-dr polymorphism in large numbers of heterozygous individuals, including an hla-deficient scid patient. the ssoph data were correlated with serological specificities and will be useful for delineation of hla restriction in alloand autoimmunity. different cell membrane receptors have been shown to be involved in human t lymphocyte activation induced by either monoclonal antibodies or mitogenic lectins. these t cell surface molecules can be divided into two categories : a) the t cell antigen receptor (tcr) associated with the non-polymorphic cd antigen b) t cell differentiation molecules not linked to cd /ti such as cd (t ) and tp ( . ). monoclonal antibodies directed against these t cell surface structures triggered different t lymphocytes functions : mitogenesis, il- receptor expression, il- secretion. our knowledge about early events involved in t cell membrane activation is not complete, especially involving the transduction mechanism mediated by gtp-binding proteins ; nevertheless, numerous authors have demonstrated that cd /ti complex triggering induces the activation of phospholipase c, leading to the phosphoinositide cascade associated with an increase of free cytoplasmic calcium ions. in the present report, we show that different activating cell molecules (con a , pha and pma) can trigger oxygen free radical liberation when incubated with the human jurkat tumor t cell line. since membrane oxidative metabolism has been shown to be related to the stimulation of the phospholipase a , and to be the final consequence of a membrane nadphoxidase : this could represent a previously undescribed pathway of t lymphocyte activation. high affinity monoclonal antibodies (mab), specific for staphylococcal nuclease (nase), were produced and characterized. competitive inhibition assays were conducted resulting in a series of complementation groups that define eight overlapping epitopes. it is estimated that these epitopes account for % or more of the accessible surface of nase. mutagenesis of the coding sequences for nase was carried out to produce a series of variant molecules (each differing from wild-type. nase and from each other by a single amino acid) that will enable mapping of nase epitopes, determination of residues involved in antibody binding, and the contribution of various physical and chemical factors to affinity and fine specificity. screening some of these mutants with the panel of mab enabled us to map several nonoverlapping epitopes and further subdivided some of the mab complementation groups. oligonucleotide-directed mismatch mutagenesis has been done on codons encoding the original amino acid residue and other surface residues in its immediate vicinity. determination of enzyme activity and structural analysis by cd spectropolarimetry of several of the mutant proteins suggests that any structural changes that may occur are local and not global. supported by grants ai , l ca and s rr from the national institutes of health. activation of t cell proliferation is believed to occur via the hydrolysis of inositol phos holipids, which, through the second messengers inositol- , , -tris hosphate and diacylglycerof(dag), promotes the elevation of intracellular calcium levels anjactivation of protein kinase c (pkc), respectively. the role of pkc in t cell activation was investigated by comparing the effects of stimulation by - -tetradecanoyl phorbol acetate (tpa), and the dag, oleoylacetyl glycerol (oag), on a > % pure population of t cells cultured in rpmi medium containing % autologous serum. treatment with either tpa or oag caused down-regulation of the t cell rece tor, a consequence of its hosphorylation, but only tpa, in syner leuiin receptor (il -r), expression and, sgsequently, proliferation. immunohistochemical staining with antisera specific for the pkc subspecies a, pi, pii and shows that restin t cells express a, pi and pii pkc subspecies which are diffusely distributed throughout the celt. after minutes treatment with either oag or tpa all three subspecies are redistributed to a focal area within the cell. the redistribution is transient in oag stimulated cells, where the pkc distribution is similar to that in untreated cells after hour of treatment. in tpa stimulated cells, however, the pkc redistribution is prolonged, becoming more marked until mitosis occurs after - hours of treatment. these results suggest that transient intracellular redistribution ofpkc causes phosphorylation and down-regulation of the t cell receptor, but that prolonged redistribution is required or t cell proliferation. sm is a nucleoprotein complex associated with small rna molecules in eukaryotic cells. the spontaneous generation of anti-sm antibodies is specific for patients with systemic lupus erythematosus (sle) and develops in % of mrl mice. the response has been shown to be t-cell dependent in mrl/lpr mice. t-cells specific for sm are found only in mrl (h- k) mice and mice bearing h- s and h- f haplotypes (which do not develop anti-sm antibodies). we are currently working to define the variable regions of the t-cell receptor genes used in the sm response. a series of t cell hybridomas from mrl mice has been generated and are being screened for sm positivity. a technique has been designed to amplify specific alpha and beta chain tcr genes using the polymerase chain reaction allowing for a more rapid sequence analysis. it is also our intention to locate the sm specific epitopes of the t-cell hybridomas. d. bloom, p.l. cohen, and s.h. clarke, department medical institute and experimental immunology branch, nci. nih, bethesda. md . to determine whether prior activation history affects t cell receptor mediated activation of t cell clones, the murine type i helper clone ae was maintained in tissue culture by stimulation every ten days with either ( ) antigen (cytochrome c), irradiated h-zk spleen cells. and il- or ( ) il- alone. ae cells grown with antigen and antigen presenting cells (ae -ag) proliferated and produced t cell growth factor activity (tcgf) in its culture supernatants following stimulation with immobilized anti-t antibody. the tcgf activity was shown by bioassay using indicator cell lines and specific blocking antibodies to be almost entirely due to gm-csf with little or no il- activity detectable. detectable il- mrna levels. (ae -ilz) displayed substantially greater anti-t induced proliferation than did ae -ag cells. in contrast to ae -ag cells, ae -il cells produced large quantities of il- in response to anti-t stimulation. furthermore. one cycle of stimulation of clone ae -ag with il- in the absence of antigen and irradiated spleen cells was sufficient to cause this clone to produce substantial amounts of il- upon subsequent anti-t stimulation. these data suggest that t cell receptor mediated stimulation of t cell clones by specific antigen and antigen presenting cells inhibits subsequent anti-t induced il- production. t cell proliferative responses and sera antibody levels of myasthenic patients to several synthetic peptides representing different epitopes of the human achr were examined. we detected significant differences in the humoral and cellular responses of mg patients compared to healthy controls to peptides of the human achr alpha-subunit with sequences p - , p - and ~ - . proliferative responses of lymphocytes from myasthenic patients to p - and to p - correlated significantly with hla-dr and with hla-dr , respectively. in order t o investigate further the immune responsiveness to selected sequences of the human achr, t cell lines and clones specific for peptides p - and p - were established from lymph nodes of c h.sw mice. the recognition specificities of these lines were tested by examining crossreactivity to a series of shortened and/or extended peptides of the above sequences. deletions of amino acids in positions and ( =p, =l) resulted in a decrease of the peptides' stimulatory activity on the ~ - specific t cell line, whereas deletion up to position on the n-terminal end had no effect on the triggering potential of the peptides. similar results were obtained when deleting residues and ( =v, =p) in stimulation assays of the p - specific t cell line. help in determining important t cell epitopes on the human achr. the role of guanine nucleotide binding regulatory proteins (g proteins) in the regulation of phosphorylation of the y subunit of the cd antigen has been examined. cd y chain phosphorylation in isolated t cell microsomes or permeablised t cells was stimulated by the g protein activator, guanosine '- thiotriphosphate (gtpys), but other nucleotides such as camp or gdpbs were ineffective. dependent. these data are consistent with the involvement of a g protein in the signalling mechanisms that regulate the phosphorylation of the cd y chain. the regulatory effects of calcium and gtpys were compared in normal peripheral blood derived t cells and jurkat cells. there were differences regarding g protein regulation of cd y chain phosphorylation in normal t cell and jurkat cells and current models explaining these differences will be described. expression of the gamma-delta t cell receptor has been thought to first occur in a population of thymocytes shortly after their precursors populate the thymus between and days of gestation. in the course of our studies investigating the ontogeny of t cell receptor expression in the mouse embryo we have identified an extrathymic site of gammadelta expression in a population of cells present at distinct times of gestation. evidence will be presented demonstrating two periods of activity of the murine gamma locus in the developing embryo. are colonizing the thymus from the liver and the gene segment useage detected is different to that first expressed in cells of the developing thymus. around the time of birth) involves the functional rearrangement and expression of a gamma gene segment corresponding to the initial functional rearrangements detected earlier in gestation in the thymus, which can occur independently of thymic influence. demonstrate a new site of gamma-delta receptor expression in the liver of newborn mice that can occur in the absence of any thymic influence. the primary (in vivo) response of cs bl/ animals to the class i antigen qa- is a helper (th) dependent event as indicated by the requirement for copriming with a distinct antigen capable of activating helper cells. in contrast, the secondary (in vitro) response to qa- demonstrates no need for costimulation with the helper antigen. in attempts to more closely examine the helper requirements for activation of primed ctlp, we have observed that depletion of l t cells from spleens of qa- primed mice abrogates the in vitro generation of anti-qa- effectors. the response is restored by the addition of concanavalin a induced supernatant (cas) or by the addition of syngeneic but not qa- allogeneic l t cells. indeed, even in the presence of cas, l t cells expressing the qa- alloantigen specifically suppress the activation of anti-qa- ctl in a manner reminiscent of that seen with lyt- veto cells. although the mechanism whereby l t cells exert suppression is unclear, we have determined that ctlp are susceptible to veto only within approximately the first hours of culture, after which they resist suppression. results from further studies of the nature of suppression and the l t veto cell will be presented. group i proteins induce ige ab responses in - % of mite allergic patients. murine mab and human igg and ige ab. unrelated. crossreactive epitopes on gpi and gpii allergens from different mite species. in contrast, igg ab in balb/c mice immunized with loug specific" epitopes and < % was a n t i -u i (a gpi homologue, with - % amino acid sequence homology to i). four non-over lapping epitopes were defined by mab, with one species specific immunodominant site on each gpi allergen. cross-reactive gpi epitope and this mab could inhibit human ige ab binding by - %. specificity of the murine anti-gpi response was not h- restricted, but could be altered by immunizing balb/c mice with lower ag doses (lug) in alum or . dertussis. using these regimes, up to % of the murine igg ab responses was gpi cross-reactive. responses to gpii allergens appear to be strain dependant. unresponsive to gpii. however, balb/b, a/j, cba, c h c b all produce gpii cross reactive igg ab. anitgenic sites on gpi allergens are conformational, whereas those on gpii may be sequential. known to affect ige expression in mice may also affect the epitope specificity of igg ab. we have compared the b cell epitopes on these allergens using panels of however, ag binding ria on sera showed that human igg and ige ab recognize the gpi and gpii allergens are antigenically i in cfa was directed against "species murine ab balb/c are completely thermal denaturation and reduction and alkylation expts suggest that the results with the gpi allergens suggest that immunization regimes which are c this report demonstrates for the the exclusive recovery of - -specific t cell clones c further molecular analysis should identify and characterize achr reactive autoimmune clones and/or suppressor cells. cohplex, mogens h. claesson, p e t e r bkams a n d s t e e n d i s s i n g , l a b . e x p . h e m a t . immunol., d e p t . med. anatomy a, a n d d e p t . g e n e r a l p h y s the ly- alloantigens represent a family of phosphatidylinositol anchored proteins that function as accessory molecules in the process of t lymphocyte activation. the expression of these alloantigens is often induced on t and b lymphocytes after activation by mitogens or antigens. previous studies have shown that the induction of ly- alloantigens in t cells is at least in part due to the action of ifn-a/b or ifn- . in the present study, we have demonstrated that ifn- also induced ly- molecules on b lymphocytes and bone marrow cells. furthermore, we now show that tnf also participates in the induction of at least one of the ly- proteins, ly- a/e. tnf was found to synergize with ifn- to induce ly- a/e expression in thymocytes, t lymphocytes, and bone marrow cells, but not b cells. for t lymphocytes, the synergistic induction of ly- a/e by tnf was restricted to cells from the ly- . haplotype whereas ifn- was sufficient to fully induce ly- a/e expression in cells from the ly- . haplotype. this result is consistent with the notion that there is more complex regulation of the ly- afe molecules in t cells obtained from the ly- . haplotype. for t cells from balb/c (ly- . ) mice, ly- a/e, but not ly-cc, molecules were induced by ifn- and tnf. furthermore, when compared to ly- a/e, the regulation of mhc class molecules in these t cells by tnf was minimal. the induction of ly- afe molecules on balbfc t cells resulted in an enhanced capacity to activate these cells through the ly- t cell activation pathway. one transformed t cell line, . . . was also identified whose ly- a /e molecules were synergistically induced by ifn- and tnf. optimal expression of ly- a/e molecules on . . cells required continuous culture of this cell line with these two cytokines and resulted in the detection of optimal levels of cytoplasmic ly- afe mrna by northern blot analysis. this latter result suggests that ifn- and tnf regulate ly- a /e at the level of transcription and/or mrna stabilization. ut southwestern medical center, dallas, texas . an igm antlcd mab ( . ) was found to modulate cell surface cd on highly purified human t cells within hours in the absence of a secondary antibody or accessory cells. inhibition could be overcome with accessory cells or il . the inhibitory effects of . could be mimicked by briefly pulsing cells with the calcium ionophore, ionomycin. . or ionomycin pulsed cells were inhibited in their subsequent capacity to resp nd to pha even when exposures were carried out in the presence of egta to prevent increases in [cap*]. from extracellular sources. inhibition was not the result of an inability to respond to pha 'by increasing [ca +] .. moreover the newly expressed cd molecules were capable of generating increases in [ca *].' after reacting the cells with anti-cd + a cross-linking secondary antibody. these studies dem'onstrate that a state of nonresponsiveness in resting t cells can be induced by modylating cd with an anti-cd mab in the absence of co-stimulatory signals. a brief increase in [ca ' . resulting from the mobilization of intracellular calcium stores appears to be sufficient to induce'this state of t cell nonresponsiveness. cd . laurie s. davis, mary c. wacholtz, and peter e. lipsky, dept. of internal medicine, lmmunogenicity c a central lab.blood transf.service, lab. of exp. and clin.immunology of the univ. of amsterdam, amsterdam. the netherlands monoclonal antibodies (mab) directed against the human cd molecular complex induce a strong proliferation of t cells, when immobilized on microtiter wells. this activation system, that was shown to be independent of accessory cells, accessory-cell derived factors or lfa- mediated intercellular adhesion ( ). allows one to study the requirements for t-cell proliferation and differentiation in a well defined manner. il- and ifn-gamma but no il- could be detected i n culture supernatants of coated anti-cd stimulated t cells. the addition of ril- or ril- had only a moderate effect on t-cell proliferation, vhereas helper activity for ig production was strongly enhanced in the presence of these factors. in this system differentiation of precursors to cytotoxic t lymphocytes (ctl), as measured in anti-cd mediated cytotoxicity, could be demonstrated within days after initiation of the activation. allospecificity of the induced effector ctl was demonstrated using a panel of hla class-i p -transfectants. in this system the regulatory role of the cd molecule in tcell activation and differentiation was studied. addition of anti-cdz mab to t cells stimulated with coated anti-cd mab enhanced il- production, proliferation as well as ig production. interestingly, pctl differentiation was also enhanced by anti-cd mab. this system seems valuable for the analysis of requirements for differentiation of human t cells subsets. . van noesel et al., nature , - , analysis of the requirements for human t-cell differentiation, rolien de jong. vivienne rebel, g i j s van seventer. miranda brouwer, frank miedema, rene' van lier, the newly described t cell receptor (tcr) locus is located inside the tcr a locus between va and ja . despite this unique situation, a highly efficient regulatory mechanism results in the complete independence of these two loci. we have recently described, in humans, a site specific recombination which joins a ' deleting element (srec) to the send of the ja's (yja) resulting in the deletion of the tcr- locus in t lymphocytes expressing the a/b tcr. rearrangements of the tcr as well as immunoglobulin genes are mediated by a unique recombination machinery and therefore, the specificity of these rearrangements is thought to be the result of a differential accessibility of the dna involved in the recombination process. as a consequence (and/or cause) of the opening of a segment of dna, the region involved is fxst transcribed as a sterile transcript prior to the rearrangement. in that regard, we have found that the kb of dna u p s a u~n of yja are actively transcribed ('t early a" transcript, tea) early during fetal development. the presence of the tea transcript presumably reflects the opening of the tea sequence prior to the tcr- deletional rearrangement. in order to better understand the mechanisms involved in the dna accessibility model, we started to look for dna-binding proteins which might play a putative role in the opening or blocking of the tea sequence. by the technique of "gel shift assay" we found such a negative regulatory protein in the nuclear extract from a non-lymphoid cell. the binding activity appeared to be specific as it was competed out by an excess of unlabeled autologous dna and not by an excess of irrelevant dna. further studies are now in progress to determine first wether the presence of this binding activity can be correlated with a "closed" configuration of the tea region and second to determine the precise location of the dna binding region. cohen, laboratory of chemical biology, niddk, national institutes of health, bethesda, md . mabs retard lymphoproliferation as well as autoimmunity. interesting, so does the adminisuation of a mab to l t , thus suggesting that the t helper subset, which is not part of the unusual expanding population, is required for initiating the pathology in these animals. as a means of characterizing the expanding population of abnormal cells as well as the phenotypically mature (l t +) cells that may be associated with them, i have generated a series of t cell hybridomas from the enlarged lymph nodes and spleen of m p r mice. in parallel, i have derived a series of control (non-lprflpr) hybridomas from mrulpr x balb/c f animals (which show no sign of pathology), and a series from mrl, mice (which have a delayed onset of autoimmunity without lymphadenopathy). very few hybridomas ( - ) were obtained in the non-lpr derived fusions. when i con a stimulated the lymphocytes from non-lpr mice prior to fusion however, many more hybridomas were obtained(l - ). this is in contrast to the fusion efficiency obtained from lprnpr mice which did not require in viuo lymphocyte stimulation to obtain a comparable number of hybrids. this result suggests that the m u p r lymphocytes are activated in situ. in addition, while less than % of the lymphoid mass is comprised of t helper (l t +) cells , over % of the hybrids are l t +. the fact that a dispropomonate number of t helper cells are rescued by fusion suggests that the cells activated in situ may be autoreactive t helper cells. currently i am characterizing these t helper cells for their lymphokine production, t cell receptor gene usage and auto-specificity and will compare them to the hybridomas obtained from non-diseased animals. goodnow, s. gilfillan, h-j. garchon, j. erikson and m. davis. stanford university, stanford, ca . we have made transgenic mice bearing gene constructs encoding the t cell receptor a and p chains from a cytochrome c-reactive t cell hybridoma. despite a lack of tissue-specificity in mrna expression, cell surface expression of uansgene-encoded protein was limited to t cells, presumably because both chains require cd proteins in order to assemble on the cell surface. in mice carrying only the a chain consuuct, the transgene was expressed in the thymus as early as day of fetal life, - days before endogenous a chain mrna. the first detectable cell surface expression of a transgene was on % of day fetal thymocytes. this vast increase in up-bearing cells in fetal thymus was due to pairing of transgenic a chains with endogenous p chains, of which a substantial number are. normally rearranged by day of fetal liie. the balance between ap-expressing t cell supopulations was grossly disturbed in these mice, the most marked abnormality being an increase in the number of l tnyt- -cells both in thymus and in peripheral lymphoid organs. it therefore appears that premature expression of surface ap t cell receptor may disturb t cell differentiation pathways by allowing t cells to leave the thymus without expressing l t or lyt- . mice carrying the p construct showed no increase in surface expression of t cell receptor in fetal life, since endogenous a chain rearrangement was limiting. in mice carrying either the a or p chain mansgenes, the number and surface phenotype of t cells expressing y& t cell receptors was unaffected in early fetal liie. suggesting that the a p and y t cell lineages diverge before thc rearrangement and expression of the appropriate subset of t cell receptor genes. department of microbiology and immunology, institute and the university of pennsylvania, philadelphia, pa. .the thymic stroma plays a major role in initiating the colonization, organization and differentiation of precursor stem cells into functionally mature t cells. a variety of cell types including thymic nurse cells, cortical and medullary epithelial cells, nonepithelial dendritic cells, and macrophages, combine to form the thymic stroma. the differential role of such cells in thymic development is unclear. we have isolated a number of morphologically distinct stromal cell lines from the thymuses of sv transgenic mice. several of the cell lines are of epithelial origin, while others have features consistent with non-epithelial "dendritic" cells. we have focused on one of these cell lines, bearing the phenotype of a cortical epithelial cell, for its ability to support the growth and differentiation of stem cells from the fetal liver and fetal thymus, and cloned pre-t cells obtained from adult mice. the cortical epithelial cell line produces factors that induce the dramatic proliferation of fetal liver and thymic stem cells . in addition, fetal liver cells cocultured with this cell line are induced to rearrange and express their t cell receptor (tcr) genes. a cloned pre-t cell line is also induced to rearrange its tcr oenes in resoonse to sianals mediated bv this cell line. gugrin, marie c. b n , corinne amiel, nadia coniglio, jacques leclsre, laboratoire d'immunologie and clinique endocrinologique, chu de nancy and facult de mgdecine, vandoeuvre les nancy, france. the lfal molecule, an adhesin of the lfa family involved in cell-cell interactions, is physiologically expressed on all white blood cells. it is absent in some congenital immune deficiencies ((id), and is expressed on a decreased number of peripheral blood lymphocytes (pbl) in aids. we investigated its presence on pbl from patients with auto-immune disorders of the thyroid. a monoclonal antibody (iot , immunotech) directed to a conformational epitope involving both chains of lfal was used in indirect immmunofluorescence on pbl from blood drawn at a similar time in all patients. a calibrated flow cytometer (epics profile, coultronics) was used to measure the percentage and numbers of positive cells, as well as the mean fluorescence (mf) and shape of the fluorescent peak. data were correlated with clinical information,therapeutic, and other pbl features such as the cd icdb ratio. the percentage of lfa + cells was significantly decreased in patients with graves' disease, hypothyroidism and hyperthyroidism. the mf was lower and the shape of the fluorescent peak seldom displayed the bimodal characteristic noted in controls. these data suggest the participation of the altered expression of lfal in the pathogenesis or evolution of auto-immune diseases. pt=ciilat.pd that excess hla r l a s s tt expression, ciinmonly found i n a i t i v e human nutoinimiine tlisrases maintdin:? the *rctivatinn of dutnreactivr t c e l l s which in turn prnducr mediators which maintain r.la:;s i t expression. this hypothesis has been tested i n many ways i n t h y r o i d i t i s . crj t i c a l l y autoreactive t cell:? are €nilrid i n thyroid autoinnline tissues which a r e rrstimnlatrd hy thyroid f o l l j c u l a r r r l l s . more rrcently we have been exploring the s p e c i f i c i t y of the autoantigen reactive t c e l l s in hashinoto's t h y r o i d i t i s where thyroy-lobulin s p e c i f i c clones have been found, i n contrast t o graves' disease, where thyrocyte recognizing clones do not react wi.th tliyroglnhulin. tn rheumatoid a r t h r i t i s , collagen type i clones have been found, persistently in the activated (il- r') t c r l l pmil over several years i n t.he same p a t i e n t . to verify t h a t antigen present,ation is involved i n rhrumatoid art.hritis ( r a ) a disease i n which, unlike thyroi.dj t i s the nature of the major antigen presenting c e l l (apc) is unknown, thc e f f e c t of ,~nticl.ass i dntibodies a t -oncentration which block *ari;j vation of t r-el is mi the synthesis n € rla-dr mrna wa:j waluat.rd. the inhibitory effect supports d i.rifira role nf an d s yet iiriknown apc i n maintaining the i:hronj.ci t y o f r a conversely, all the clones were unable to respond to a substitution at (tyr to asp). nase mutant proteins were constructed with the same single amino acid substitution and t cell responses to peptides and mutants were compared. preliminary evidence suggests that the mutant proteins like the peptides, substituted at residue and , will not induce t cell clone responsiveness. these data suggest that the overall structure of the protein will not compensate for the lose of a particular amino acid which is necessary for t cell recognition. medicine, baltimore, md . to explore the variables important in t cell priming, an adjuvantfree immunization regimen was developed. bio.a mice were primed subcutaneously with syngeneic spleen cells that had been pulsed with high concentrations ( pm) of the peptide - , a cnbr cleavage fragment of pigeon cytochrome e. the t cell response was assessed using a sensitive limiting dilution assay that measures lymphokine production with the ctl-l cell line. the precursor frequency of antigen-specific cells in the draining lymph nodes of mice primed with antigen-pulsed spleen (aps) was in , indistinguishable from the frequency of in found in mice primed in the footpads with nmol of - in complete freund's adjuvant (cfa) (data are given as geometric means, n= , s.e.m = x/t . and . , respectively). despite the apparent similarity in the t cell compartment of mice primed using these different regimens, antibody induction was strikingly different. mice primed with - in cfa developed serum igm and igg responses against the peptide, with antibody detectable in an ellsa assay at a : dilution. mice primed with - owaps, however, produced no detectable anti-peptide antibodies. maximal t cell clonal expansion therefore appears to be possible in the absence of antigenspecific b cells. these data argue against the hypothesis that antigen-specific b cells play an obligate role in t cell proliferation in vivo. the reasons for the lack of antibody induction are currently under investigation. cell receptor (tcr) complex of jurkat cells. the coprecipitation of these peptides with tcr requires treatment with monoclonal antibodies (mabs) directed against tcr (c or r ) prior to cell lysis and immunoprecipitation. treatment of jurkat cells with mabs directed against cd ( - or . ) or hla (w ) does not not induce the association of these peptides with tcr. the signal-transduction mutant cell lines, j.cam and j.cam , have previously been described ( ,z). these cell lines, derived from jurkat, fail to activate the inositol-phopholipid second-messenger pathway in response to anti-tcr mabs. treatment with mab c induces the association of the and kd peptides with tcr in j.cam cells but not in j.cam . j.cam modulates tcr normally in response to anti-tcr mab treatment ( ). hence, these observations suggest that the two peptides are involved in the signal-transduction pathway of the t cell receptor complex rather than receptor internalization. sle is an autoimmune disorder associated with several different hla class i antigens. we studied a large sle patient population by sequencing of the pcr amplified first domain of the dqb and dqa chains and by sequence specific oligonucleotide probes to further define these associations. shared dqb sequences at amino acid positions = eu, =tyr, and fasp may predispose some individuals with hla dr , , , or to develop sle. a novel dqb sequence found in two drw dqwl sle patients shares these amino acids in the dqb hypervariable regions. the association of the drz dqwl.azh gene was greatly increased in the sle patients with lupus renal disease. the hla association may be directly due to structural aspects of the hla genes. when parent -+ f chimeras are prepared with supralethal irradiation ( rad + rad), the donor-derived cd + cells differentiating in the chimeras show partial tolerance to host-type h- determinants, despite the apparent absence of host-type apc. donor-derived cd + cells give only low proliferative responses to host-type apc in primary mixedlymphocyte reactions (mlr); furthermore, in i-e-+ i-e+ combinations, the donor cd + cells show molecules. this finding implies that tolerance is induced intrathymically, presumably through contact with a non-marrow-derived component of the thymus, e.g. epithelial cells. in support of this possibility, thymectomized & + ( a x b)f chimeras given strain j? marrow cells and a strain thymus graft (irradiated) show no detectable tolerance to host-type strain b determinants: the strain & cd + cells differentiating in these chimeras give strong mlr to strain b and do not show deletion of v + cells. = % deletion of cd + cells expressing i-e-reactive v t cell receptor b measured by these two parameters applies not only to lymph node (ln) cd + also to cd + cells recovered from the thymus. interphotoreceptor retinoid-binding protein) is a glycoprotein of residues (bovine) which localizes in the retina and pineal gland and induces inflammatory changes in these organs (eau and eap, respectively) in immunized animals. the experimental disease is considered a model for certain uveitic condiiions in man. we have recently shown that irbpderived synthetic peptides can also induce eau/eap in lewis rats. the present study compared two such peptides, "r " (residues - ) and "r " ( - ). peptide r was found to be immunodominant, shown by its being recognized by lymph node cells (lnc) or line cells sensitized against whole irbp. in contrast, peptide r was not recognized by the whole irbp-specific lymphocytes and is considered nondominant. in addition, lnc sensitized to r , but not to r , responded to intact irbp. r was superior to r in producing eau/eap and cellular immunity (minimal doses: . vs pg/rat). on the other hand, the two peptides were comparable in their capacity to stimulate presensitized lymphocytes. moreover, lnc sensitized against r were similar to those sensitized against r in their capacity to adoptively transfer eau/eap to naive recipients. this study thus provides a unique system in which both immunodominant and nondominant peptides produce autoimmune disease and can be compared for their immunological features. the age-related diminution in immune responsiveness has been shown to result from increased regulatory mechanisms and not from a paucity of immunobgical recruitment (aging: immunology and infectious disease , , ). we present evidence based on "libraries" of monoclonal antibodies (mabs) omained from young and aged donors that there occurs with aging an increase in autoimmunity which is possibly the result of the accumulation of liielong "original antigenic sins". the resultant increased connectivity of the immune system is represented by mabs obtained from aged donors which are multiply anti-self cross-reactive. furthermore increased connectivii is supported by the evidence that anti- . , -trinitrophenyl mabs are ad positive, d positive as determined by i n h b i i n studies using mabs anti-idiitypic reagents. analysis of the vh and vk region genes utilized by these mabs indicate a nonrandom gene usage. life bng stochastic immunological events lead to a pattern of cross-reactiities and non-random usage of vh genes. these immnological events lead to the emergence of the patterns which are partially elucidated by the data presented. these patterns mimick those seen early in ontogeny, but indicate a possible convergence to an ever-increasing connectance of the idiitypic repertoire expression. in other words, life-long immunological experiences contribute to a down-regulation resulting in both paucity of drimaw immune reswnses and an increase in autoimrmnity which are both the earmarks of immunity in aging. (supported by usphs grants ag- to eag and al to cab) lmmunogenicity c stimulation of these cell lines in suspension with saturating levels of mab okt produces total and fractional inositol phosphate accumulation linearly related to receptor number, (r > . ). this technique also allows an approximation of the minimal number of reccptors which must be engaged for second messenger generation in this system, which we estimate as . ~ receptors per cell. or terminate t cell activation. since this molecule plays an important role in human t cell development we sought to identify the murine homologue of cd in order to determine its expression on murine t cell and its role in activation. we have used a human cd cdna clone to isolate a full length cdna encoding the murine equivalent of cd from an el t cell lymphoma library. this clone shows similar domain organization and a high degree of homology to the human cd molecule. the murine cdna clone has been used to examine mrna expression of cd in normal and activated murine t cells, and in various t cell tumors. peptides generated from the translated sequence will be used to produce antisera to correlate the surface expression of cd with mrna expression, and to biochemically and functionally characterize this molecule. pat happ and ed palmer, basic sciences division, dept. of pediatrics, national jewish center for immunology and respiratory medicine, denver, co . we have attempted to determine the frequency of rearrangement and expression of the individual a and g chain v gene segments that make up an unselected, untolerized t cell repertoire. in order to do this we generated over t cell hybridomas from freshly-isolated thymocytes of newborn c blllo mice and subjected rna from these hybrids to northern dot blot analysis using va, vp. cy and c probes. comparison of the expressed repertoire of vp gene segments in this newborn thymocyle population with similar data previously generated from an adult peripheral t cell population reveals two vg genes, vp and vpl , whose expression is decreased in the periphery, possibly due to the effects of tolerance. two additional vp gene segments were expressed more frequently in the peripheral population than in the newborn thymus, vp ( . times higher in the periphery) and val ( times higher). it is possible that these represent ewo instances of positive selection of t cells which is determined primarily by the receptor's vg gene segment. va gene segments were expressed in only % of newborn thymocyte hybridomas (compared to % expressing vp) and determination of va rearrangement frequencies was complicated by the unexpectedly large number ( %) of hybrids expressing cs mrna. further examination revealed that several va gene probes were actually detecting rearrangements to cs. the most notable of these was va , which accounted for approximately % of the expressed va repertoire but was rearranged exclusively to cs. barbara bergman, brenda bradley, kevin lafferty and mary portas. barbara davis center for childhood diabetes, u. colo. health sci. ctr., denver, co . we have produced a panel of islet-specific t cell clones by culturing lymphoid cells obtained from non-obese diabetic (nod) mice in the presence of nod islet cell antigen and antigen-presenting cells (apc). these clones were selected to the panel on the basis of (a) their antigen-specific reactivity to islet cells and apc in an in vitro proliferation assay and (b) their ability to mediate islet graft rejection in vivo in a tissue-specific manner. we have further characterized these lines for cell surface phenotype, - production, and proliferative response to non-nod islet antigen. all of the clones tested to date are of the cd phenotype and make il- in response to islet antigen and nod apc. nearly all of the clones we have tested also make good proliferative responses to islet cell antigen obtained from mouse strains other than the nod or to a mouse beta cell tumor line. preliminary results indicate that at least one of these clones can lead to islet cell damage in a disease transfer experiment in which the cloned t cells are injected into a non-diabetic nod f recipient. we are currently carrying out tests to further characterize lymphokine production by these cloned cell lines, to analyze differences in antigen recognition and mhc restriction requirements among the clones, and to determine their effectiveness in mediating the disease process in nondiabetic animals. in an attempt to identify the epitopes on class i mo ecules recognized by alloreactive cytotoxic t lymphocytes (ctl) we have examined k -specific ctl for tpir recognition of synthetic peptides with sequences derived from the native k molecule. co secutive overlapping peptides molecule were tested for their capacity to inh&bit k -specific ctl clones in their recognition of cells expressing the native k molecule. in these studies inhib ion by peptide was found to be an extremely rare event, although one peptide (k - ) did inhibit recognition by a particular ctl clone (clone ). in a separate set of experiment it was observed that clone could recopize kblll- when presented by h- class i molecules. as clone was of h- origin, this finding led to conclude that inhibition may be due to class i-restricted recognition of the k pegtide on the surface of the ctl clone, peptide and native kb for the t cell receptor. we present evidence in favor of this conclusion. the pepscan method is used for the systematic identification of sequential b cell epitopes i n protein molecules (geysen, meloen, barteling. pnas : ; ) . it was designed for the synthesis and subsequent testing for antibody binding of large numbers of overlapping peptides directly on their solid supports. the mhc dependent presentation required for t cell recognition seemed prohibitive for the use of pepscan to identify t cell epitopes. however we now have shown that by a novel modification the peptides can be recovered from their solid supports and used in t cell assays. holmdahl, department of medical and physiological chemistry, box uppsala university. s- uppsala, sweden. both autoreactive t cells and autoantibodies play an important role in the pathogenesis of type i collagen (cii) induced arthritis in mice. we have earlier reported that only strains with h- q , h- w , h- w and h -r were responders to autologous mouse cii and only these strains developed arthritis after immunization with autologous or heterologous cii. however, heterologous cii induced a more acute and severe disease and a more pronounced autoantibody response. this findings indicate that ) the ability to mount an immune response against autologous cii is a prerequisite for the susceptibility to collagen arthritis and ) that a crossreactive autoantibody response after immunization with heterologous cii may further enhance development of arthritis. we have now studied activation of autoreactive b cells after primary immunization of den mice with rat cii. in hybridoma collections, obtained - days after immunization, . % of the hybridomas produced igg reactive with autologous cii, - % produced multispecific igm and a significant number produced igg rheumatoid factors. the anti-cii antibodies recognized at least different epitopes on the cii molecule and originated from many different vh and v kappa gene families. furthermore, none out of investigated anti-cii hybridoma expressed cd rna message. we therefore suggest that the primary anti-cii autoantibody response involves activation of memory b-cells. these memory b cells have most likely been earlier activated by cii autoreactive t cells. in these aspects the origin of the anti-cii autoantibody response is principally different from the origin of "natural" autoantibodies. t cell receptors (tcr) recognize antigen in association with self mhc molecules, usually following processing to smaller peptides. the t cell repertoire to an antigen, therefore, reflects not only the ability of a given mhc molecule to interact with antigen, but also the affects of initial repertoire selection by self mc. we have t#tn analyzing the tcr repertoire specific for beef insulin ( ) in balb/c mice (h- ), which are high responders to the antigy. these studies revealed that vp. is dominantly used in the tcr's specific for bi/a and our preliminary data suggests that the vgb. chain may be involved in mhc restriction. we have now obtained several t cell hybridmas specific for bi from (balb/cxa/j) f , animals. a/j mice (h- a) are low responders to bi while the f m ce a e high responders. most of the balb/cxa/j hybridmas were restricted to the hin the balb/c hybridmas. interestingly, the analysis of v gene usage demonstrated that vg . was not used in the balb/cxa/j hydridonas. the relevance of these results to the development of the tcr repertoire in different mouse strains will be discussed. this work was supported by the mrc of canada. ctl specific for the q k molecule were generated from normal splenocytes by & vitro culture with a k bearing stimulators. these ctl have been shown to lyse transfected targets expressing hla-a regardless of their murine haplotype, and they specifically kill a bearing human target cells. furthermore. the effector function of these ctl can be inhibited with an hla-a specific monoclonal antibody. thus, the transgene product functions correctly as a tolerogen and is recognized directly as a class i antigen. although transgenic mice have been shown to be tolerant to a k expressed by murine cells, transgenic ctl specific for hla-a on the surface of human cells have been generated. these the major virulence factor of m.pneumoniae was shown t o be a kda protein which is located in the tip structure membranes of these cells. beside the adhesin function this protein is also involved in first massive humoral and cellular responses of the human host during the acute phase of upper respiratory tract infections and interstitiel pneumonia. intranasal inoculation of guinea pigs with the isolated kda protein led to lympho-histiocyte infiltrations around bronchi and small vessels of the lungs which are characteristic infiltrations after an infection with live m.pneumoniae cells. furthermore one peptide ( amino acids long) which was synthesized according to the amino acid sequence of the adhesin, showed a proliferative activity to in vitro cultivated t-cells of bronchial washings, whereas synthetic peptides with th e sequences of the direct neighbourhood showed no in vitro activity. most interestingly this t-cell proliferative activity is located on a surface loop of this protein which is also responsible for the adhesin f uction. christopher a. smith, gwyn t. williams, rosetta kingston and john j.t. owen. department of anatomy, university of birmingham, medical school, vincent drive, birmingham tj, uk. rearrangement of t-cell receptor a and b chain gene segments during t-cell development results in a diverse array of receptor specificities. to avoid auto-immune responses, cells that have generated self reactive receptors are thought to be eliminated or inactivated, to produce self tolerance. recent studies have provided compelling evidence that clonal deletion of immature receptor bearing cells within the thymus makes an important contribution to this process, although the mechanisms involved are not uderstood. of immature mouse thymocytes with anti-cd antibodies added to thymus organ cultures, induces dna degradation and cell death through the endogenous pathway of apoptosis. is in marked contrast to the activation of mature t-cells by the same anti cd preparation and is specific to the extent that apoptosis is not induced by either anti-cd- or anti-thy- . to organ cultures suggesting a role for changes in intra-cellular c a w levels in the signalling pathway leading to the induction of apoptosis in immature cells binding. thus activation of the process of apoptosis in immature cells binding self antigens may be the mechanism responsible for the selective deletion of cells that could generate an auto-reactive response if allowed to mature. we have now obtained evidence that engaging the cd /t-cell receptor complex this in addition, calcium ionophore (ionomycin) also causes apoptosis when added anderson cancer center. houston, tx immunization of patients with bcg and irradiated tumor cells induces specific delayed-type hypersensitivity (dth) to tumor cells and not to normal colon cells. since igg antibodies may require t-cell help, we wished to characterize the igg-defined tumor-associated autoantigens (taaa) of human crc so as to define a subset of the t-cell repertoire for crc. western blots of detergent extracts of primary and metastatic human colorectal carcinomas and paired normal tissues were probed with autologous igg. nine taaa were recognized by % or more of the sera: , , , , , , , . and kda. these taaa may be normal colon differentiation antigens, since they were present in extracts of normal colon. autoantibodies are more frequently present to the kda antigen in patients with metastases ( %) than in primary tumors ( %. p<o.o ) while there is more reactivity to the kda antigen in primary tumors ( %) than in metastases ( %, pso. ). only of patients sensitized with bcg and irradiated autologous tumor cells, developed antibody to new taaa; whereas of control patients developed antibody to new taaa without sensitization. the taaa in the western blots that stimulate autologous lymphocyte proliferation are greater than kda. finally, immunization with a human crc failed to induce dth in mice to a syngeneic murine colon carcinoma that expresses at least of the igg-defined human taaa. thus, taaa that presumably require t-cell help in patients do not appear to be part of the t-cell repertoire for dth to human crc. in several rodent species and strains, the emergence of specificity to a discrete immunodominant epitope of guinea pig basic protein (gpbp) results from selection of cell lines with whole gpbp. these epitope specificities have been found to be strain-specific and define the specificity of the t helper cells which transfer experimental autoimmune encephalomyelitis (eae) in adoptive transfer experiments. line from aci-strain rats which was encephalitogenic and specific for a new t cell epitope of gpbp. the cell line responded in vitro to the peptide cokkesponding to the region - of gpbp but not to peptides corresponding to other regions of gpbp. this cell line is restricted by i-a but not i-e and transfers a dth response to gpbp as well as eae into naive recipients. these results provide support for the hypothesis that the in vitro immunodominant epitope specificity defines the specificity of gpbp-specific encephalitogenic t cells. the definition of a new encephalitogenic epitope in the aci strain also suggests that the mechanism of eae in rats depends on strain-specific extrisic properties of antigen recognition such as the mhc class i genotype or the composition of the t cell antigen-receptor repertoire. we have produced a gpbp-specific t lymphocyte medical center, stanford, ca . a wide variety of t lymphocyte surface antigens have been isolated and shown to play important roles in the t cell response. we outline here a strategy to isolate and characterize surface proteins unique to activated t cells. a rabbit anti-serum was generated by immunizing with allo-activated human t cells and subsequently absorbing with the t cell tumor hpb-all, a tumor that expresses all functionally relevant t cell surface antigens previously described. this absorbed antiserum was able to recognize an uncharacterized group of surface proteins on t cells that was absent from hpb-all. furthermore, the absorbed anti-serum was able to inhibit i n virro cytotoxic and mlr responses; an effect that was abolished by carrying out an additional absorption with the t cells used for the initial immunization. monoclonal antibodies to activation antigens were generated by immunizing with complexes made by adding the absorbed anti-serum to solubilized ctl. the absorbed anti-serum was also used to screen a lcgtll cdna library. one cdna clone so isolated is expressed on ctl as evidenced by northern analysis. dna sequence data indicates no similarity to other sequences in available databanks. childrens hospital of orange county, orange, ca , and university of california, irvine, ca . we examined the ability of cytokines to stimulate the proliferation of pbm from new-onset iddm, long-term iddm, non-diabetic pediatric control, type i diabetic, and non-diabetic adult control subjects. initial results demonstrated the ability of human pbm culture supernatants containing peak natural il- activity to stimulate a significantly higher proliferative response from pbm of new-onset iddm than from pbm of long-term iddm, type i diabetics, or non-diabetic controls. more recent results have shown that human recombinant il-la, -lb, - , - and - , interferon gamma, lymphotoxin, and tumor necrosis factor, as well as con p.-stimulated rat spleen supernatants, also stimulated a significantly higher proliferative respcnse from pbm of new-onset iddm than from pbm of controls. however, human recombinant il- stimulated equivalent levels of proliferation from pbm of both new-onset iddm and controls. these data indicate that, in new-onset iddm, there .e present pbm which are in an early stage of activation not revealed by increased responsiveness to recombinant il- . in the case of t cells, this may represent a stage prior to an il- -dependent stage of activation. this would be consistent with the active induction of newly responsive cells during a stage of iddm when islet cells are still present. this response is not the result of the metabolic aspects of iddm but a reflection of the immunologically activated state of t cells, b cells andlor monocytelmacrophages in iddm. houghten, c. david pendleton, james l. comette, charles delisi and jay a. benofsky, metabolism branch, national cancer institute, national institutes of health, bethesda, md . analysis of known helper t cell m) cpitopes suggests that th epitopes tend to be amphipathic alpha helices, that is, helices with hydrophobic residues on one side and hydrophilic residues on the other side. based on this hypothesis, a computer program "amphi" was generated which may aid in prediction of th epitopes. within residues to of spermwhale myoglobin (swmb), a th epitope is known to exist but has not yet been found. as an approach to further test the hypothesis, this undetermined th epitope was sought both by an unbiased test of ten evenly overlapping -residue peptides that span the region, and by computer analysis. of the ten peptides, only one was able to stimulate two clones that are specific for the - region. this peptide gave remarkably high stimulation index. analysis of this region by the computer program also predicted the site covered by this peptide (residues ) to be the most likely site for th epitope.. therefore, unbiased in v i m testing and the computer prediction correlated well. the peptide was able to prime t cells of b o.br mice in vivo for in v i m response to the native swmb as well as to the peptide fragment of residues - . immunization of mice with swmb showed that, of the ten overlapping peptides, the major site of response is to the identified peptide. finally, a peptide of residues - was made to increase the amphipathic score. this extended peptide induced greater proliferation of the clones. thus, this study has identified a new dominant epitope of swmb and confirmed the utility of the amphipathicity hypothesis in a prospective test. amino acids a t a selected site; ) t-cell antigenicity, that is analysis of prirrary structures with -and -amino acid templates obtained from database of knawn y-epitopes; gly, hhydropkbic, pplar or charged amino acid ( ); ) prediction of the secondary structure according to gamier ( ) to exclude sites having high turn or coil forming ptential. ccanparison of t h i s set with published program ( , ) shows that it has high predictive f r . so we used it fo search for probable t-epitopes i n hepatitis a virus capsid pro- and for mediators of delayed hypersensitivity (tdh ~ assayed by ear swelling induced by co-injection of responder cells with irradiated tumor cells). lymph node cells recovered days post-inoculation contained tdh cells and ptc (freq / , ) , but tc effector cells. by contrast, graft-infdtrating cells not only contained tdh and ptc (l/l,oao), but direct tc effector cells ( lytic units). all three sets of t cells were antigen-spedfic. the finding of significant frequencies of ptc within the lymph nodes and the graft site, but tc q& at the latter, suggests that differentiation of tc from unprimed ptc may be a multistep process, the first of which occuis in the draining lymph nodes. the data further suggest that the terminal step@) in this process take place after the donally expanded ptc have disseminated to the graft site. we suspect that the final step requires help provided uniquely by tdh cells within the graft. the fmal step may not occur (as might have been expected) in the lymph node because priming of ptc and tdh take place at noncontiguous anatomic sites. interference & seed, pnas a, , ) . the n-terminal amino acid sequence of normal t cell cd was found to match the hpb-all sequence except in residue and (...ts...) . in principle this might reflect somatic mutation in hpb-all or cd -gene polymorphism: erroneous residue assignment in the gas phase sequencing cannot be excluded entirely. to investigate the significance of this sequence variation a genomic cd dna clone of a normal human t cell clone was analysed around residues . and found to be identical with the hpb-all cdna sequence. sumtic mutation in hpb-all cd therefore can be ruled out. the molecular characterization of t cell receptor (tcr) structure in a panel of mouse t cell clones specific for sperm whale myoglobin (spw mb) has shown that -ed-restricted clones specific for the spw mb region - use highly homologous v bchains. four of these clones are indistinguishable in their response pattern to a panel of overlapping synthetic peptides spanning the spw mb sequence - , yet use different v a gene segments. we are currently trying to find fine specificity differences among these clones, in order to correlate them with tcr expression. t cell hybridomas have been made from these clones, and are being screened on panels of substituted peptides. we hope that conservative substitutions at positions important for interaction with the tcr will reveal fine specificity differences among these clones. sloan-kettering institute, new york, ny we previously showed that clonal deletion, non-suppressor mediated immunological tolerance neonatally induced to mls allo-determinants was specific in that the frequencies of t cells responding to a variety of other stimuli were unaffected while the frequency of t cells responding to mls were drastically reduced. here we show that mls tolerance is detectable in the thymus prior to a detectable positive response in the periphery (spleen and lymph node). tolerance induced by spleen cells from mlsa congenic mice in balb/c mice is as "potent" as that induced by whole background incompatible dba/ spleen cells, indicating that incompatibility at the ml s locus ( the ly- alloantigens have been shown to participate in the process of t cell activation based on the ability of anti-ly- monoclonal antibodies to induce il- production and proliferation of t lymphocytes. in the present investigation, we have demonstrated that peripheral t lymphocytes from a strain mice exhibited abnormally low proliferative responses after stimulation through ly- a/e and ly- c molecules when compared to responses of t cells from numerous other mouse strains. the abnormal activation of the ly- pathway of a strain t cells was not due to ineffective fc receptor cross-linking of the anti-ly- monoclonal antibodies, to inappropriate cellular expression of the ly- a/e alloantigen in a strain t cells, or to an active suppressive phenomenon. identification of this defect provided an opportunity to compare t cell activation by the ly- and tcr pathways. interestingly, t lymphocytes from a strain mice proliferated normally when the cells were activated by monoclonal antibodies to thy-i or the cd /tcr complex suggesting that this defective activation was selective for the ly- t cell activation pathway. cell separation studies of t cells and accessory cells demonstrated that this defect was quantitative rather than qualitative and that it was complex, residing at both the t cell and accessory cell levels. these results suggest that activation of t lymphocytes via the ly- molecule may involve unique signalling pathways and that activation via cd /tcr can proceed normally in the absence of a fully functional ly- pathway. the h-u)d transfected l cells were recognized by the cil when they were infected with either influenza a virus or a vaccinia recombinant virus encoding the pb . thirty-five synthetic peptides derived from pb , which had either an amphipathic suucture or contained a rothbard' epitope, were tested for their ability to render uninfected targets susceptible to lysis by influenza specific effectors. three epitopes were identified, each of which contained a rothbard epitope. department of microbiology and inununolag, albany medical college, albany, ny our laboratozy has developed a new, powerful technique for hvestigating the inmplne ~n s = to peptide epitopes, involving the covalentcoupling of peptide to phosphatidylethanol-, follmed by amplexing with additional lipids and phospholipids to form a peptide-phoqholipid complex. these cmplexes can be used to inununize mice in the absence of protein carriers or adjuvants, thus facilitating the study of the hmne response to a mall chemically defined antigen. use of this technology has allmed us to identify two t helper cell epitopes in conserved regions of kiv gp not previously identified by computer algorithims, defined by amino acids - and - . inununization with these peptides in peptide-phoqholipid mmplexes results in the prduction i g and igg antibodies, which truss react with cloned fragments of the whole protein. us& this tecfinolosy we have begun to characterize the irmnune reqonse to individual peptide antigens. ?he response of h -k mice to amino acids - of gp of hiv, has been analyzed. lhe optimal dose of a peptide containing both b and t cell epitopes was found to be - ug, depending on the mute of administtation. im mhization required less antigen for optinarm antibody response than did ip. anchorage in the phospholipid cmplex is a strict requirement for an antibody response. additional variables, such as phospholipid m i t i o n and method of cross-linking have been studied and will be discussed. we believe that the use of this peptide-phospholipid ccnnplex technology will be significant both for studying the i n m u m response to single epitops and for vaccine developrent. virology, national bacteriologica; laboratory, stockholm, departments of neurosurgery, virology and immunology, karolinska institute, stockholm, johnson & johnson biomedical research, la jolla, california, pentadecapeptides, sequentially overlapping by a.a., were syntesized based on the htlv-i sequences of gag and env proteins and used as antigens in igg-subclass elisas andt-cell stimulation assays. sera and cells were obtained from asymptomatic, hiv-infected homosexuals. reactivity in all subclasses could be found to parts of the gag-protein. extensive areas of iggl and reactivity were indentified mainly in the p and n-terminal half of p with an additional region in the n-terminal end of p . the highest iggl and reactivities were detected in a.a. - , rich in glycine, arginine and lysine and thereby hydrophilic with a high segmental flexibility; iggz and epitopes were found in the hydrophilic cooh-terminal of p j; the second highest iggl and reactivities were found in the hydro-?hilic n-terminal of p . for gp , the iggl epitopes identified were in the putativeloopregion (a.a. - ) and in the hydrophilic cooh-terminal end of gp (a.a. - ). the immunodominant region of gp (a.a. - ) showed some igg , and responses inaddition to igg . t-cell proliferative responses were detected against many peptides usually in areas not binding igg. still, simultaneously t-and b-cell reactive peptides could be detected, and the showed a distinctly preference for igg . the variation between different patients was considerable regarding both t-cell reactivity to peptides and the subclasses of reactive igg. a mapping of subclass restricted epitopes allows an elucidation of anti-viral imunological mechanisms. kristin komschlies, laboratory of experimental immunology, brmp, dct, and bcdp, program resources, inc., national cancer institute-frederick cancer research facility, frederick, md and jackson laboratories, bar harbor, me . "double positive" (dp) thymocytes are precursors for cd + and cd mature subsets have been based on indirect evidence with in vivo anti-cd or anti-cd treatments. we have approached this question directly by isolating subsets of dp thymocytes from fetal or adult thymus and have shown that only a subset of dp cells ( % of adult thymocytes) can generate donorderived thymocytes after i.t. injection into irradiated hosts. low-density dp cells are injected i.t., up to - % donor-derived cells can be seen at to days after transfer. cd ' cells can constitute up to % of these donor-derived cells at day . in contrast, an equivalent i.t. transfer of dull ly-l/cd (dly- ). early precursor thymocytes would not generate cd ' cells until days - but would produce some cd ' cells by days. thus we have detected differentiation of cd + cells and not cd ' cells from adult dp cells. depleted host and recovered donor-derived thymocytes to days after i.t. transfer of dly- cells, when most of the donor-derived cells are dp. when retransferred, these dp cells displayed a more rapid progression of thymic localization patterns than dly- cells, but were unable to produce enough donor thymocytes to analyze subsets. thus cd ' cells appear to differentiate via a very minor (low density) subset of dp thymocytes. may not be generated from these intermediate dp precursors but directly from dly- cells. the intrathymic differentiation process by which bone marrow-derived progenitor cells develop into mature, immunocompetent t lynyhocytes remains poorly understood. the majority of all intrathymic lymphocytes are cd "double positive" cells that have no well-documented function in thymic differentiation, but have been proposed to be either a critical intermediate between cd - precursor cells and mature t cells, or, alternatively, a "dead-end" cell type. furthermore, although signals mediated through the tcr complex are critical for t cell tolerance induction and repertoire selection, the functions of the cd and co differentiation antigens expressed by the vast majority of thymocytes are unknown. in the present study we addressed the possibility that cd functions as a signalling molecule during t cell development. we engaged cell surface cd on murine thymocytes by in vivo injection of anti-cd mab, and monitored the effects of that engagement on distinct thymocyte subsets. c d ' + cells respond to cd cross-linking by dramatically increasing their cell surface expression of cd and tcr, and decreasing their cell surface expression of cd . in contrast, mature cd ' -cells respond to cd engagement by decreasing their cell surface expression of both cd and cd . these results provide clear evidence that cd engagement regulates cd /tcr expression on developing thymocytes, and, most interestingly, results in increased tcr expression by cd + + double positive thymocytes. thus, cd engagement is a membrane event to which c d ' ' thymocytes respond significantly, indicating that cd may be a signalling molecule in t cell development and may promote t cell differentiation by inducing increased cd /tcr cell surface expression on cd + + thymocytes in vivo. f.g. terpstra. p.th.a. schellekens and r.a.w. van lier, central lab.blood transf.service, lab.exp.clin. immunol. of the univ.of amsterdam, the netherlands t cells can be activated through several membrane molecules that may use distinct intracellular signalling pathways. t-cell activatiep threygh both the cd /tcr complex and cd is accompanied by riaea in free intracellular ca (ca ) and pkc activation. in contrast, evidence from our laboratory suggests that triggering h a cdz occurs through a pkc-independent route. in this study we used mab to various t-cell membrane antigens to localize the activation defect in t cells from asymptomatic hiv-infected men that we previously showed to have an intrinsic defect in their proliferative response to soluble anti-cd nab. compared to hiv-homosexual controls, the monocyte-dependent response of t cells from hiv+ men to soluble anti-cd and a mitogenic combination of anti-cdz mab was decreased as was the monocyte-independent resgpnse to immobilized low-dose anti-cd . in t cells from both groups a normal rise in (ca ) . was induced by anti-cd and anti-cdz mab. however, t cells from hiv-infected men respondad poorly to direct pkc activation by pma in the presence of healthy donor monocytes, whereas control t cells responded vigorously. in contrast, induction of proliferation by anti-cd mab was comparable in t cells from hiv+ and hiv-men. our results indicate in hiv+ men a selective qualitative t-cell defect in activation routes that are dependent on pkc activation, whereas pkc-dependent alternative activation appears to be unaffected. these findings will be discussed with repect to hiv immunopathology and normal t-cell physiology. we have studied the role of the protein kinase c (pkc) in the activation of a prototype th- cell, d .g . . blocking of this enzyme with the drug h- [ -( -isoquinolinyl-sulfonyl)- -methylpiperazinet leads to an increase in cellular proliferation of d cells activated with specific antigen or mitogen. n addition, an increase in the amount of il- and il- mrna is seen under these conditions compared to the level observed in mitogen activated cells in the absence of h- . this superinduction of il- and il- lymphokine mrna is also detected in mitogen activated d cells that are depleted of pkc protein by pretreatment of the cells with phorbol esters. in contrast, mitogen activation of s lenic t cells measured by proliferation is blocked in the presence of h- . these data indicate that jkc has a regulatory role in th- cells by exerting a negative feedback on the events that occur after cell activation avoiding an uncontrolled response. this effect is opposite to that seen in a bulk t cell population that represents mainly th- type cells. the mechanism of signal transmission is, therefore, distinct in the two types of cd + t cells. aberrant expression of class i mhc proteins on non-immune somatic tissue has been implicated in several autoimmune diseases, but it was unknown whether this expression was involved in the initiation of disease or resulted from induction by the immune reaction. we ha e engineered several transgenic mouse lines expressing tissues using eith r the pancreatic elastase promoter or the class i kb gene promoter. the i-as on the pancreas is fully capable of presenting peptide antigens to i-ad restricted hybridomas. h-zb mic expressing i-ad on pancreatic acinar cells but not in thymus are not tolerant to i-as in mixed lymphocyte reactions in vitro, but do not spontaneously produce an immune respon e to the pancreas in vivo. peripheral antigen presentation, i-a restricted immune response, i-a expression by t-cells, and widespread somatic i-ad expression. expression, whereas another has only medullary expression. the effect that these different patterns have on mhc restriction is being examined. ctr., stanford, ca . a transient increase in the intracellular cat+ ion concentration has been shown to occur rapidly following activation of t lymphocytes. this response i s a more immediate and direct indication of t cell activation than either production of interleukins or proliferation. we examined ca++ flux analysis using indo-l and the facs to measure ag-speclfic t cell activation and also to sort for an ag-specific t lymphocyte subpopulation. human t cell clones were exposed to elther allostlmulatory lymphoblastoid cells (which cause proliferation of the t cells) or nonstimulatory lymphoblastoid cells. in both cd + and cd + clones, we observed an ag-specific cat+ flux response similar t o an lonomycin positive control. we then attempted to enrich for antigen specific t cells from a mixed population. two phenotypically different human t cell clones were mixed in equal amounts. after coculture of the cell mixture w i t h lymphoblastoid cells shown to stimulate only one of the t cell clones, the cell mixture was sorted on the facs based on specific cat+ flux. a ten fold enrichment was observed for the ag-specific clone. this approach allows rapld observation of t cell activation and provides a means of sorting antlgen reactive t cells from a heterogeneous cell population. previously attemps to characterize the function of beta- -microglobulin (beta- -m) have used anti-beta- -m antibodies, to block the functional activity of the protein. we now demonstrate that human beta- -m and the proteolytic derivates beta- -m lys and beta- -m deslys added in nanomolar concentrations to allogeneic, pha and con-a stimulated mouse lymphocyte cultures modulate the response induced. thus beta- -m and especially beta- m deslys when added day and augment the generation of specific cytotoxic t lymphocytes in mlc, problably due to stimulation of endogeneous lymphokine production in the culture. both the con-a and pha induced response were modulated. in the presence of beta- -m and its proteolytic derivates the proliferation was optimal at hours, as opposed to the hours optimum in the abscence of beta- -m. these results strongly suggest an active role of beta- -m or proteolytic derivates hereof in the t cell activation and generation of specific cytotoxic t lymphocytes. a ( . fold) . the increase peaked at min and returned almost to baseline by mi?. igm b cells showed random x inactivation, while more mature igg and iga b cells had only the normal x active. we conclude that the xla gene product is required for b cell development, while the xscid gene product is required for both t and b cell maturation. gene mapping has placed xscid in xq - . , linked to phosphoglycerate kinase (pgk), lod . , = . mouse xid, which affects only b cells, is also linked to mouse pgk, suggesting the possibility that xscid and xid belong to related gene families. c institute at denver, national jewish center, denver, co . mapping data for the bxd and akxd recombinant inbred strains shows that the mhc and the products of at least two other genes control the expression of vp . one of these is linked to ly- , and the second for the akxds is linked t mtv- on chromos me . a backcross of b o.br x (c h x b o.br)fl (mls- x ( m l~- ~ x mls- ) ) has been used to confirm the linkage of one of these controlling genes to ly . using the vp -specific monoclonal antibody, exceptions have been found to the general rule, that strains which have eliminated t cells expressing a particular vp specific for a defined self antigen in the thymus, also express this self antigen on their spleen cells, and so these cells are capable of stimulating t cell hybridomas bearing the particular vp in quest ion. the t cell receptor (tcr) of the alloreactive t cell clone c recognizes the h- ld specificity. the cloned a (va . ) and p (vg . ) tcr cdnas have been inserted into plj retroviral vectors containing ltr and regions of the momulv and neomycin resistance genes. three different constructs have been used to transfect v cells. g -resistant clones showing the highest titer of viral particle psoduction have been selected, and injected subcutan ously lacking the h- l vt cells producing retroviruses carrying the a gene have been examined for the expression of the new specificity on the infected t cells. northern blot analysis using the va and the neo-probes showed, in the lymphoid tissues, a transcript with an expected size of . kbs hybridizing with both probes. in the spleen of treated balbfc mice, but not in dm mice, the expression of the exogenous va resulted in proliferation of infected t cells, as assessed by an auto-mlr proliferative assays. into newborn balb/c mice (h- l +) o r into the congenic strain dm loose most of their lytic activity as well as the ability to form conjugates with specific tc. preliminary experiments show that spdp-anti-cd -derivatization of specific tc overcomes the trypsin effect, so that both conjugation and lysis are observed between trypsin-treated pel and sdpd-anti-cd -derivatized specific tc. many investigators, the ti/cd complex on pel surface is central to specific lysis. the observed inhibition of trypsin may be due to its action upon it. since trypsin-treated pel could be triggered toward conjugation with and lysis of tc by anti-cd , the cd at least must have remained intact and is the important molecule for both the critical conjugation step and lysis. trypsin treatment possibly disturbs the interaction of tc with ti/cd of pel so as to prevent proper engagement of the cd . concanavalin a (con a) treatment of tc appears not able to bring back the lytic activity or the ability to form conjugates of trypsin-treated cells, indicating a dichotomy in the mechanism of killing by derivatization with anti-cd versus con a mediation. comparing pel and their blasts ipeb). unlike pel, peb do not show ldcc or lytic activity against spdp-anti-cd -derivatized nonspecific tc. ldcc by con a has been proposed to function via ti/cd interaction. unlike pel, peb also do not show fluorescence. it may be that the ti/cd complex on peb differs from the one on pel. at the same time, total lack of cd is contraindicated for peb kill specifically. the region important in ti interaction for ldcc and that recognized by anti-cd may be within the aberrant area of cd , so neither the con a nor the anti-cd trigger could be transmitted on peb. the importance of the cd is indicated further by immunolabeling both cells with anti-cd revealed that c combination of cd + and cd + islet specific t cell clones are pathogenic in the nod mouse, eva-pia reich and charles a. janeway. jr., department of pathology, section of imunobiology, yale university school of medicine, cedar st., new haven, ct . although diabetes in the nod muse is thought to be initiated by t lymphocytes, thus far no t cell clone has been derived from nod islets. we therefore isolated islets from acutely diabetic nod mice and islet derived lymphocytes were propagated in il- and stimulated every weeks with fresh mitomycin-c treated nod islets to maintain islet specificity (i.e. a proliferative response to nod islets). t cell clones were generated by limiting dilution followed by recloning by single-cell manipulation. facs analysis showed that all clones were positive for thy-l+ and cd +, confirming that receptor bearing t cells had been cloned. cd +cd -and cd -cd + clones were isolated that proliferated in the presence of nod islets. respond to nod but not balblc or b o.br islets, while the two c d ' clones respond to nod and balb/c islets but not blo.br. islet-specific, i-anod restricted, while the c d e clones are islet-specific and dd restricted. injection of individual cloned lines into young, irradiated nod mice leads to no overt or microscopic disease. a syndrome of dehydration, rapid deterioration and death about - days after transfer. mice sacrificed at this time show lymphocytic infiltration around the islets. this suggests that the cd + clones are however, mixture of c d ' and cd + cloned lines causes these cloned lines may be useful in the search for islet cell autoantigens. dusseldorf (frg). autoantibodies to nucleolar components are a common serological feature of patients suffering from scleroderma, a collagen vascular autoimmune disease. while animal models which spontaneously develop abundant anti-nucleolar antibodies have not yet been described, high titers of such antibodies may be induced by treating susceptible strains of mice with mercuric chloride. we have identified the nucleolar autoantigen against which the hgci -induced igg autoantibodies from mice of strain b o.s are directed. it is a protein of apparent molecular weight of kda and pi value of about . which is associated with the nucleolar snrna u , and by these criteria, is identical with a polypeptide called fibrillarin. it is striking that scleroderma patients spontaneously form autoantibodies against the same u rnp protein. the hgc -induced murine and the scleroderma-specific human anti-u rnp autoantibodies were indistinguishable in their reactivities towards fibrillarin. they further resemble each other in so far as both recognize epitopes on the -kda protein which have been highly conserved throughout evolution. our results provide a basis to investigate at the molecular level whether similar immunoregulatory dysfunctions may lead to the preferential a n t i rnp autoantibody formation in the animal model and in scleroderma patients. forty bp-specific cd + t-cell clones were isolated from the peripheral blood o f a patient with multiple sclerosis. studies with a panel of xenogeneic bps of known amino acid sequence and with laree fragments of the bp molecule demonstrated the existence of at least epitopes recognized by human t-cells. at least of these epitopes were potentially clustered in the middle or at the c terminus of the molecule. in studies with synthetic peptides corresponding to residues - and - of human bp (hbp), clones proliferated in response to - and recognized - . ctl assays were carried out with targets consisting of an autologous b-cell line coated with hbp. nineteen of the clones demonstrated bp-specific ctl activity; these were the same that had recognized peptide - i n proliferation assays. these clones also killed targets coated with peptide - but not targets coated with - . clones that proliferated in response to peptide - or to epitopes located outside of these two regions failed to kill hbpcoated tarpets. to a lack of binding of - to the target cell, as the same b-cell line successfully presented peptide - , in proliferation assays, to clones that had previously recognized this peptide when presented by e-cells. activity against bp is more restricted than is the specificity demonstrated for proliferative activity. the lack of killinp of targets coated with - could not be ascribed prystowsky, department of pathology and laboratory medicine, university of pennsylvania, philadelphia, pa as an approach to studying regulatory factors important during t cell proliferation, the expression of genes induced during il -stimulated t cell growth was examined. to identify genes induced by il , a cdna library was made from il -stimulated cloned t cells in late g of the cell cycle and was screened by differential hybridization, selecting those clones which had higher steady-state mrna levels after il stimulation. of , clones originally screened, positive clones were identified, which represented different genes. when partially sequenced and compared with sequences in the nih and embl databases, clones were identified as cdnas encoding glycolytic enzymes, were cdnas of cytoskeletal genes, were cdnas coding for proteins important in protein synthesis, and clones were not identified. the maximal steady state mrna levels generally occurred during late gl/early s phase, and the induction of most m a s was not inhibited by cycloheximide at a dose that inhibited cell proliferation. nuclear run-on transcription demonstrated an increase in the rate of transcription for gapdh. in addition, there is post-transcriptional regulation of expression, as some mrnas were stabilized upon il stimulation. having cloned these mrnas, we can now begin to study the regulation of expression of selected genes. supported by nih grants. cyciosporin a (csa). a potent immunosuppressive drug, caused organ-spedfic autoimmune disease, such as gastritis with anti-parietal cell autoantibodies or oophoritis with anti-oocyte autoanliies, in balwc mice when the drug was administered daily for one week to newborns. administration to adult mice did not. csa abrogated the production of l t ' t cells and lyl- ' t cells in the thymus. consequently, these t cells were substantially depleted from the peripheral lymphoid organs, especially when the drug was administered from the day of birth. autoimmune disease was prevented when csa-treated newborn mice were inoculated with splenic t cells from normal syngeneic mice. on the other hand, removal of the thymus immediately after neonatal csa treatment produced autoimmune disease with a higher incidence and in a wider spectrum of organs, i.e.. thyroiditis, sialoadenitis of the salivary gland, gastritis. insulitis of the endocrine pancreas, adrenalitis, oophoritis, or orchitis. each autoimmune disease was accompanied by the development of autoantibodies specific for the corresponding organ antigens. it is likely that csa caused organ-specifc autoimmune disease, when administered to newborns, by depleting certain regulatory t cells (suppressor t cells) controlling self-reactive clones. (this work was supported by a grant from the lucille p. markey charitable trust.) artur scherf, philippe dubois, marika plat and luis perelra da sllva, instltut pasteur, - , rue du dr. roux. paris, 'u inserm, hopltal st. louis. , place du dr. alfred fournier. paris, france. we isolated from a genomlc expression library of the malarla parasite p. falciparum a clone coding for an antigen associated with the membrane of infected erythrocytes. intensive sequence analysis of this gene, called - , showed that the major part of this molecule consists of at least repeats of nine amino acids. the consensus sequence of these degenerated repeats is e-e-v-v-e-e-v-v-p and secondary structure analysis predicts t h a t they have a high tendency t o form amphipathic alpha helices. the -and t-cell response t o the two most frequent types of repeat were analysed in five different h- congenlc mice strains using two synthetic peptides (pqa: y-p-(e-e-v-v-e-e-v-v-p)n-k: pqb: the -cell response t o both peptides is restricted to the h- d and h- k xaplotype. pca and pqb specific antibodies do not dlscriminate between the two peptides (measured by elisa). the mhc restriction was confirmed by anaiyslng the tcell response to both peptides. t-cell lines speclfic for each of the peptides pqa/pqb were derived from h- d responding mice. although the peptides differed in four positions of the hydrophoblc part of the helices (valine replaced by isoleucine and leucine) no signiflcant dlfferences were observed in the antigen presentation between peptide pqa and pqb. in contrast, both peptides were unable to induce a proliferation of the heterologous t-cell ilne. suggesting t h a t the variable hydrophobic amino acids of the repeats are involved in tcell recognition. we are currently investigating if other variant repeats of the - antigen are restricted t o a different mhc haplotype and that the totality of repeats may be subjected to no restriction. glven the type of amino acid replacements within the two repeats tested, the t-cell receptor appears t o discriminate between very similar amino acids. auphan, claude boyer, annick guimezanes, claire langlet, cenae dimmunologie inserm-cnrs de marseille-luminy, case , marseille, cedex , france. the yinterferon gene is activated upon cell surface triggering of the aansmembrane t cell receptor (tcr)/cd complex or via phosphatidylinositol anchored thy- molecules cell clones. both these activation events are inhibited by antibodies directed at the lyt- jlyt- molecules and are lost on tcr a-chain deletion variants ( , ). biochemical analyses failed to reveal any association between the tcwcd complex and either lyt-wyt- or thy- molecules. however, such studies consistently revealed a non-covalent, cis-type association between lyt-ulyt- and h- class i products as well as between thy- and h- class i products. the structural features of the molecules required for such interactions are being determined using hybrid engineered molecules and possible implications of such interactions for t cell activation are being evaluated. ( ) schmitt-verhulst, a.-m. et al. nature , - ( ) . ( ) guimezanes, a. et al. cellular immunol. , - ( ) . i t i s increasingly apparent that the association of both chains is critical to reconstitute t,he g-mhc binding site. combinational mechanisms for generating diversity play a major role in increasing the total size of the repertoire. however, evidence for preferential associations have been described between other heterodiaeric proteins of the immunoglobulin gene superfamilg. i t is therefore important to determine if association between different sets of vas and ybs occur at random or if, alternatively, preferential associations exist. we have devised a system which should enable us to determine, by dna mediated gene transfer of cloned cdnas encoding different vas o r vbs, if such preferential associations do exist. we hare also used a panel of monoclonal antibodies specific for a particular va or vb to derive t cell clones expressing these vas or vbs. in a polyclonally activated t cell population expressing a specific va or vb chain, we will present data indicating that the association between these chains is preferential or random. !nteractions between t cells and mhc molecules are thought to select a t-cell repertoire skewed towards recognition of antigens in the context of self mhc molecules. in addition, t cells which react strongly to self mhc molecules are eliminated by a process called self-tolerance. we have recently described generation o f transgenic mice expressing the a t-cell receptor (tcr) from the cytotoxic t lymphocyte c (sha et al., nature : ) . the clone c was derived from a ealf .e (h-zb) anti-eale/c (h- d) mlc and is specific for the ld class i mhc antigen. in transgenic h- b mice, a large fraction of t cells in the periphery expressed the c tcr. these t cells were predominantly cd -cd + and were able to specifically lyse target cells bearing ld. in the periphery of transgenic mice expressing ld, functional t cells bearing the c tcr were deleted. this elimination of autoreactive t cells appears to take place at or before the c +cd + stage in thymocyte development. in addition, we report that in h- s mice, a non-autoreactive target haplotype, large numbers of cd ' t cells bearing the c tcr were not found providing strong evidence for the positive selection o f the c tcr specificity by h- molecules. the requirements for cd expression during t cell differentiation in fetal thymus organ culture woc) were investigated by analysis of the effects of addition of anti-cd mab. control etoc cult& for days contained predominantly cd +cd + cells as well as cd or cd single positive cells expressing high levels of cd . t cells from ftw cultured for days in the presence of anti-cd m a b expressed markedly diminished cd expression. when anti-cd m a b was removed hours prior to phenotypic analysis, it was found that cd was recxpressed on cd + cd + t cells. after days of culture, control roc contained high frequencies ( - ) of cd +cd and cd -cd + thymocytes expressing high levels of cd . in contrast, anti-cd mated cultures, while containing cd -cd +, cd + t cells, were well depleted of cells which reacted with anti-cd antimy. when anti-cd mab was removed hrs prior to analysis, cd was reexpressed on cd +cd + cells, and low frequencies ( %) of cd + cd -t cells were detected, but these cd single-positive cells did not express high levels of cd or detectable ap heterodimrs. while normal frequencies of cd -cd +,cd + cells expressed vg determinants, the a n t i -w mated cultures were relatively enriched in cd -cd +cd and cd -cd -cd -t cells compaml to control day eoc. these data demonstrate that while cd +cd + t cells can differentiate in the presence of anti-cd mab, differentiation of cd +cd + cells is inhibited and cell surface cd is down-modulated. cd +cd + cells can develop in the presence of anti-cd m a b and do express t cell receptors. these results suggest that cd expression, while not required for development of cd +cd +cd + t cells, is necessary for differentiation of mature cd +cd -cd + t cells, and that the interaction of with its ligand is critical for thymus selection of class-ii specific t cells. uppenkamp, and alfred singer, experimantal immunology branch, nih. bethesda, md . in order to investigate the role of the t cell receptor (tcr) in thymocyte maturation, we have studied thymocytes from the immunodeficient c.b-l /scid mouse, which due to a genetic defect, is unable to express tcr genes. despite this mutation, scid thymocytes were functional as they produced lymphokines and proliferated in response to stimuli (pma, ionomycin, il- , il- ). phenotypic analysis revealed that, like normal immature thymocytes, sci+dd'&mocytes were large, thy . +, cd -, cd -, tcr-and were enriched in cd , il-zr+, pgp-lf, and hsa+ cells. however other tcrpopulations present in normal mice, (i .e. cd - +tcr-and c d ' ' t c r -cells) were absent from scid mice. in order to determine if tcr expression was required for cd and cd expression, we analyzed thymi from the occasional scid mouse which possessed small numbers of tcr-bearing thymocytes. interestingly, expression of cd or cd was detected only in those scid thymi that possessed tcr' thymocytes, although cd and cdb expression was not confined to those tcr-bearing cells. further, the introduction of tcr' cells from thy congenic normal mice into tcr-scid mice consistently promoted the expression of cd and cdb (but not tcr) by scid thymocytes. moreover, scid thymocytes from these chimeras possessed cd + + cells, however no mature, single positive thymocytes of scid origin were detected. together, these results suggest that tcr-bearing cells within the thymic milieu are required for the expression of cd and cd and for the differentiation into cd + + cells. however, final maturation into mature single positive thymocytes requires that the thymocyte itself express tcr. tcr in thymocyte differentiation: evidence from the immunodeficient the cd t-cell surface receptor has been implicated in the stabilization of lntercellular interactlons. in addition, it is felt to mediate the transduction of an independent lntracellular signal. we have recently shown (veillette et al.. in press) that the cd molecule expressed in t-lymphocytes is complexed to the internal membrane tyrosine kinase p 'ck. suggestlng that alterations in the properties of thls enzyme mediate the cd related signal. to gain further insight into the structure and function of the cd -lck complex, we have generated nih t flbroblasts that co-express the cd and lck proteins. nih t flbroblasts expressing a murine lck cdna were transfected by calclum-phosphate preclpitatlon with a construct containing a murine cd cdna and the neomycin resistance gene. stably transfected clones were selected for resistance to the amlnoglycoslde c . we have now demonstrated that the cd and ~ '~~ expressed in these non-lymphoid cells can form a stable non-covalent complex. the structure and function of the cd -lck complex expressed in murine fibroblasts is currently being evaluated. h- b t c e l l s from h- b+hh- bxd responder animals fail to generate one (type a) of these two major clonotypes. environment i s permissive for the t cell specificities of both poly- clonotypes (type a and b) and also rovides the determinants for t h e i r selection. the absence of type a clones in the h- b+h- gxd chimeras can therefore only be attributed to the failure of h- b t c e l l s to generate the appropriate t cell receptors. these results suggest a genetic hole in the t cell recognition repertoire of h- b mice for the lysine-containing epitope of the poly- antigen. we have used oligonucleotide-primed gene amplification to study the association between hla class i alleles and autoimmune disease. pemphigus vulgaris (pv) is a severe autoimmune skin disease highly associated with dr and drw . we have shown that the nucleotide sequence of the first (and most polymorphic) domain of a new dq allele is found in / israeli drw pv patients but only / or& healthy israeli controls. this allele differs at only position from two other dq alleles not associated with pv. further, we show that a shared setuence in the third hypervariable region of the first domain of two common subtypes o f dr and drw cannot be the sole determinantdrgf susceptibility to pv. none of dr /drw + pv patients had this epitope. we also report a new dr first domain sequences from a drw pv patient. the frequencly of t h ! $ allele in patients and controls is being assessed. similar studies to identify disease associated class i sequences are underway in patients with multiple sclerosis and rheumatoid arthritis. the frequency of these autoimmune responses is determined genetlcally. and is essentially % in the projeny derived in crosses d transgenic mice to some inbred strains of mlce [e.g. those of h- d haplotype). and yet only in others (e. g. h- b) . recently. we initiated an analysis of the i m m~~l~g i d nonresponsiveness in two lineages of insulin/t antigen transgenic mice with early onset of expression of the transgene (rip- tag # and rir tag # ). while the presence of the transgenic protein durlng the prenatai/neonatal period is the primary requirement for the nonresponsive phenotype, it further appears that the levels of t antigen expression durlng embrydnic development quantjtatively influence the subsequent nonresponstvmess in adult me. an additional modification is exerted by a genetic component whlch seems to map to the mouse major histocompatibility complex [h- ). interesiingly, it appears that the h- d haplotype. whlch is associated with the most complete impairment of immune responses when t antigen is expressed prenatally. is also assodated with the highest frequency of autoimmune responses when the developmental expression of the transgene is delayed. this correlation is considerably strengthened by the observation that outcrosses to c b / j m-zb) produce mlce with either a weaker tolerance in the case of the early expressers. or a less hquent and weaker autoimmune response in lines with a delayed onset of expression of the transgene. thus. the two distinct biolcgical consequences of the presentation of this antigen (tolerance or autolmmunity) are &ly to result from differences of the developmental maturity of the immune system, and both effects appear to be similarly mhc restricted. lmmunogenicity c mapping the pzb geneti contribution t o lupus-like autoimmune disease. i-division o f basic science, national jewish center for immunology and respiratory medicine, jackson street, denver co ; -the jackson laboratory, bar harbor, me. nzb mice carry a geneb) that contributes to the lupus-like autoimmune disease seen in (nzw x nzb) fi hybrids. however, the precise locus of this gene has yet t o be determined. to map i t s location, we are breeding a panel of nzbxsm/j recombinant inbred (ri) with nzw mice. by determining which of the ri strains produce fi animals that develop autoimmunity, the location of this nzb gene can be deduced based on the strain distribution pattern of the known genetic markers among the nzbxsmlj ri strains. cells were then fractionated on a percoll gradient to obtain small dense 'resting" b celb and large 'activated' b cells. the small dense b celb demonstrated increased inhibition at every dose of ic examined, when compared to large b cells. for instance, pulsing with ug of ic resulted in a % reduction in anti-fl, but not anti-trinitrophenyl, plaque forming cell (pfc) response for small dense b cells. in contrast, the pfc response of large "activated' b cells was reduced only %. in addition, when subtolemgenic doses of ic were used ( ng) prostaglandin € ( x - y) could function as a potent secondary negative signal rendering these b cells susceptible to negative signalling. we concluded that small dense b cells are more sensitive to ic induced unresponsiveness and that prostaglandin € may further sensitize b cells along this pathway. . were developed in the mouse against antigens present on humans, hamster and rat nk and t lymphocytes. pre-incubation of lymphocytes with moab did not block the ability of the nk cells to bind to targets but did block their ability to lyse nk sensitive targets. the pre-incubation media contained lytic factors that lysed nk sensitive targets in a h chromium release assay. interestingly pre-incubation of thymus cells or resting peripheral t lymphocytes with the moab induced the t lymphocytes to release lytic factors and acquire nk activity against nk-sensitive targets. lytic factors and acquired lytic activity were unable to lyse lak targets. signalling events did not involve cross linking or fc receptor participation since factors were immunoprecipitation and western blot analysis using the moabs defined a molecule of kd on the surface of thymus or lung mononuclear cells. isolation and amino acid sequence analysis is being done to assist in the characterization of the g . we have been studying in vitro activation of cd -, cd -thymocytes. in the present studies, highly purified double negative thymocytes were cultured with il with or without il . following a hour culture with il + il (in the absence of mitogens) significant proliferation was detected, although all cells remained cd -, cd -. the frequency of cells staining for cd cell surface protein increased from % to % and tcr vbeta from % to %. facs sorted cd -cells from the cd -, cd population showed similar increases in cell surface tcr suggesting differentiation. northern blot analyses showed an increase in c -m rna at hours, no change in cd delta or epsilon expression at , , and hours, and a marked increase in the ratio of . to . tcr beta transcript between and hours. these data suggest that il + il can induce cd cell surface protein expression on progenitor thymocytes. furthermore, this induction is not associated with increased cd transcription, but rather is associated with an increased proportion of functional tcr beta transcripts. the p antigen of schistosoma mansoni has been shown to be able to induce protective immunity against schistosomiasis in rodents and primates. synthetic peptides were used to localize the major b and t-cell epitopes of this protein. the primary structure of the p molecule was analyzed using various predictive algorithms : surface localization, hydrophilicity, flexibility, secondary structure, amphipathicity of predicted a-helical regions, rothbard pattern. according to these criteria, several peptides were synthesized and tested in immunological assays in vitro and in vivo. correlations between these results and the physico-chemical predictions will be shown. simian virus (sv ) t antigen is recognized by cytotoxic t lymphocytes (ctl) in association with class i major histocompatibility antigens.we have identified four distinct antigenic sites on sv t antigen defined by h- db restricted ctl clones. we have therefore attempted to map these antigenic sites precisely by using overlapping synthetic peptides corresponding to sv t antigen amino acids. the synthetic peptides of - amino acid length were incubated for five hours with lcr labelled-non-sv transformed h- b cells in the presence of ctl clones specific for each of the four antigenic sites; and the released radioactivity was used as a measure of specific recognition of synthetic peptides by the ctl clones. the results demonstrated that the antigenic site i resides between amino acids - ; sites ii and between amino acids - . site can be separated from site ii as it was found to reside between amino acids - . the antigenic site v was localized between amino acids - . these results show that sv t antigen is the target protein for ctl and the regions of t antigen recognized by ctl are tightly clustered in the amino terminal half. attempts to identify amino acids that selectively interact with the class i antigen and the t cell receptor are in progress. peripheral blood lymphocytes of cancer patients demonstrate an early humoral and specific response to application of tumoral extracts. the response is measured as a change in the spectrum of fluorescence polarization of fluorescein molecules. this is probably a measure of sol-gel transformations induced in the cytoplasm upon cellular activation of resting lymphocytes. this measurement is carried by epifluorescent microscopy on single lymphocytes, sitted on a regular holes matrix that is scanned under computer control. it is possible to follow changes within each cell in a population of upto ten thousand cells. since the cells are sitted safely in their holes, it is possible to rinse them with various reagents and follow the kinetics of individual responses by repeated scanning. further studying of the stimulative process might be useful in identifying the responding lymphocytes and the tumoral reagents. this test of the immune system is intended for early and specific detection of cancer and for follow-up of immunotherapeutic manipulations as well. c katharine s. ullman, j.p. shaw, elizabeth emmel, and gerald crabtree. stanford university, stanford, ca . following antigenic stimulation, t cells undergo an orderly series of changes that result in cell division and immunologic function. in hopes of working backward in the cascade which links the membrane events to the genes essential for these differentiated functions, we have begun an analysis of the nuclear proteins responsible for gene activation in stimulated t cells. in previous studies, using a panel of internal deletion mutations of the l- enhancer, we found that when the region from - to - was mutated, the level of induction dropped -fold. this region is protected in a dnaase footprinting assay by a protein, nf-ilz-a. although the protection is seen with nuclear extracts from both stimulated and resting jurkat cells, in vivo this is a site of dnaase hypersensitivity found only in activated jurkats and peripheral blood t cells. a tetramer of the protected site directs transcription from an unrelated promoter in jurkat cells only when the cells are stimulated by lectin or antibodies to the antigen receptor. furthermore this activation can be blocked by ng/ml of cyclosporin a. as a means of detecting more subtle changes in protein binding following stimulation. the methylation-interference patterns of stimulated and non-stimulated jurkat cell nuclear extracts were compared and found to be identical. we propose that though the dna-binding protein, nf-il -a, is present constitutively, it becomes functional only following specific signalling through the antigen receptor. further characterization of this protein will help to clarify its particular role in the activation of il- expression following t-cell stimulation. the negative e f f e c t of f l a n k i n g sequences on t c e l l lmmunogenicity c central lab. blood transf.service, lab. exp. and clin. immunology of the univ. of amsterdam, the netherlands the hydrolysis of phosphatidylinositol-diphosphate and the formation of inositol-triphosphate (ip ) and diacylglycerol (dg) re among the first events that can be monitored after t-cell triggering. ip mobilizes c a ' from intracellular stores, whereas dg is the physiological activator of protein kinase c (pkc). although it is suggested that activation of pkc is essential in the induction of mitogenic t-cell responses, no direct evidence for this hypothesis has been presented yet. in this study we used two novel pkc inhibitors, .e. -alkyl methyl-glycerol (amg) and staurosporine to assess the role of pkc activation in human t-cell stimulation. we observed that the inhibl$ors did not interfere with the anti-cd or anti-cdz monoclonal antibody (mab) induced ca rises in peripheral blood t cells, whereas cytoplasmatic alkalinization initiated by the same stimuli or the phorbol ester pma was strongly reduced. amg as well as staurosporine gave a dose dependent inhibition of both interleukin (il- ) production in jurkat cells and t-cell proliferation. in contrast, the induction of il- receptor expression was highly resistent to the action of both compounds. it was shown that anti-cd mab were able to partly overcome the action of both inhibitors. these studies support the important role of pkc-activation in human t-cell triggering. moreover, since anti-cd mab were able to counteract the action of the pkc-inhibitors, it is suggested that mitogenic stimuli generated via different t-cell membrane molecules may use distinct intracellular signal transduction pathways. a f t e r incubation w i t h ndlal- o r al- , t a r g e t s o f @ phenotype were lysed by a n t i -a k i l l e r c e l l s . the a n t i t h e s i s was a l s o observed: a phenotype t a r g e t s were lysed by a n t i - t c e l l s a f t e r incubation w i t h ndlgl- . ndla we have developed a monoclonal antibody that recognizes an apparently t cell-specific antigen, ltac, expressed only in the late stages of t cell activation. when peripheral blood lymphocytes are activated with allogeneic stimulators or pha and examined by facs, ltac is expressed only after day or . ltac is expressed by all t cell clones tested, both cd ' and cd '. a wide range of cell types were tested and found negative for ltac expression, including b cells, fibroblasts, endothelium, and neural cells; thus ltac expression seems to be restricted to the t cell lineage. immunoprecipitation of cell-surface labelled proteins and sds-page revealed a kd band under reducing conditions and three bands of , , and kd under non-reducing conditions. ltac is distinct from all other reported t cell activation antigens on the basis of its pattern of expression and molecular weight. we have purified ltac protein using wheat germ agglutinin-agarose chromatography followed by monoclonal antibody-agarose chromatography, and have used it to produce an antiserum suitable for screening bacterial cdna expression libraries. the regulation and function of molecules expressed in the late stages of t cell activation is an important frontier in understanding the immune system, and reagents such as monoclonal antibodies may have important diagnostic and therapeutic uses. balb.h.p mice were capable of responding to all antigens tested but the magnitude of the response to some of these antigens ( of antigens) was reduced - fold when compared with balb/c mice. responses to the remaining antigens were either comparable ( of antigens) or occasionally even enhanced ( of antigens) compared to balwc mice. these results suggest that jp and dg elements are required to maintain the strength of the t cell repertoire and that, in the wild, mice carrying this deletion a u l d be at a significant selective disadvantage. the identification of - was carried out in radiolabeled murine macrophages by immunoprecipitation and sds-page, using antibodies to were predominately of mr . and . , respectively. a s assessed by estimating radioactivity in each band, -larwas about fold more abundant than - . most of the cell associated - activity, as measured by the d .g . proliferation assay, could be inhibited by anti -iswhereas no inhibitory effect was observed with anti - b. after removal of -ltc from cell lysates by immunoadsorbtion the residual activity accounted for only about % of the total - activity, and was completely neutralized by anti - b. however, in culture supernatants anti i - r reduced the - activity by approx. % whereas a combination of both, anti -leand anti - completely neutralized the activity to background levels. interestingly, only one radiolabeld polypeptide of m, . was precipated from the extracellular medium by the anti - antibody. these data therefore suggest, that externalization is required in order to generate bioactive - molecules, but apparently a proteolytic processing step of the precursor is not involved. moreover, in view of previous reports, demonstrating that cellular interactions during antigen presentation can be mediated via a membrane form of - , o u r experiments substantiate the hypothesis that membrane-associated, biologically active - is predominantely i - q. david l. brandon and joseph w. corse. usda agricultural research service, western regional research center, albany, ca in order to prepare antibodies which would recognize -glucosides of cytokinins (purine phytohormones), we investigated an alternative to carbodiimide reagents. carbodiimides induce the formation of amide bonds, and thus can be used to couple haptens containing carboxyl or amino groups to proteins. the use of water-soluble carbodiimides is limited by the sparing solubility of some haptens in water and by the formation of the intermediate -acylisourea, which can be less soluble than the hapten and which can itself act as an unwanted hapten. to overcome these problems, we have used -morpholinoethyl isocyanide and a suitable catalyst to effect amide bond formation. these reagents are the basis of recently developed chemical procedures for peptide synthesis (aigner et al.. ) . the reaction conditions permit coupling compounds which are soluble or insoluble in water to proteins in a simple, homogeneous system. we have demonstrated that this procedure is widely applicable for preparing enzyme-labeled and immunogenic conjugates of cytokinins and other biologically relevant compounds with an available carboxyl group. we hypothesize that conjugates made with these reagents will not induce the allergic responses and unwanted crossreactivities associated with the use of carbodiimides. supported, in part, by bard grant us- - . mlman r i g , not uwuse rig , uxluces a furctional y-btut-e subset of mdlse thymqtes to. secrete - , shichin turns iniuces cells w i t h i n this subpapllation to becane cytotcouc. nolmal mxls~ thymocvtes beaw ctl in to con a * il- , and that the cplresponses induced by-agentsm blocked canpletely by the addition of the anti-- nyib lb . in addition, normal muse thymxytes produced il- in response to con a f i t . the fijmtionally-ture lobster agglutinin (mgl)-positive t h y r e p subset responded to con a f i g in a similar mmer. ylhen the functionally-nunature subset of mgl-negative thymzyk subsets was incubated with con a f muse r i g , they did not becane ctl and &d not prcduce il- . haever, when wl-negative thymcytes were cultured w i t h con a f human r ~ , cells w i t h i n this subpsxllatim developea gccd ctl activity and also secreted 'ihese data qmnstrate that cpl generatim and i g pmauction occur mnccanitantly i n dl thymqte m a t i o r r s studied. momever, stirmli which f a i l to induoe cn generation also f a i l tostinulate erd qencu.s iir praduction. immunity to the typhus group of rickettsiae is largely dependent on the effector function of several classes of t lymphocytes including those which produce gamma interferon. since the surface protein antigen (spa) derived from typhus group rickettsiae has been shown to be an effective immunogen in animal models, human t cell clones specific for the spa of rickettsia tvoh were isolated and tested for their antigenic specificity as well as for their ability to produce gamma interferon. eighteen cm-positive clones specific for the spa of r. t v p b exhibited considerable diversity in their response to the spas derived from two strains of p. orowa zekii and from r. c a n a b . the vast majority of clones also recognized the spa's from & or w w but not from r. c w . two heteroclitic clones demonstrated significantly higher proliferative responses to the spa's derived from one or both of the of r. orowazek i i strains than to the spa of r., and one clone demonstrated a significantly higher response to the spa of r. t v o k as compared to the other spas. all clones produced gamma interferon in response to spa stimulation. one of the clones demonstrated significant proliferative responses to a fragment of the spa produced in e. coli infected with a recombinant lamda gt phage containing a kb fragment of the spa gene from r.. we conclude that the spa's from typhus group rickettsiae can elicit both a diverse t cell response in humans as well as the efficient stimulation of gamma interferon-mediated immunity. a monoclonal anti-mouse rbc antibody (g- ) has been prepared which appears to represent a pathogenic autoantibody related to those that arise spontaneously in aging nzb mice and which cause autoimmune hemolytic disease (aihd). this autoantibody recognizes native erythrocytes from mice but not from other species, and northern blot analysis revealed strong hybridization with the j probe but not with the other vh gene family probes tested. thus, g- is clearly distinct from autoantibodies that recognize bromelain-treated mrbcs and which belong to a unique vh gene family. when g- -producing hybridoma cells were grown as tumors in balb/c mice, the mice developed aihd characterized by a decrease in the number of erythrocytes (hematwrit) and the development of coombs-positivity. an anti-idiotypic antibody (e ) was prepared against g- and was found to recognize an idiotypic determinant present on most autoantibody forming cells derived from old (coombs-positive) nzb mice. previously, we demonstrated that treatment of spleen cells from young nzb mice with g- + c or with anti-ly antibody + c resulted in the elimination of suppressor cells and the generation of afc specific for mrbc. treatment with a control nzb igm mab (g- ) + c or with a rabbit anti-mrbc antiserum + c had no effect on the afc response to mrbc. similar deletion of suppressor cells was obtained following treatment of spleen cells from balb/c mice with g- + c. greater than % of the resulting afc expressed the g- idiotype. the results indicate that the response to self-erythrocytes is comprised of autoantibodies that express a recurrent idiotype. in an attempt to determine the optimal molecular parameters for the synthesis of anti-pathogen vaccines, we have studied the murine antibody responses to two types of model vaccines. the vaccines were: ( ) purified polysaccharide type derived from pneumococcal bacteria, and ( ) synthetic conjugates made from a peptide region of the circumsporozoite coat protein of the murine malaria pathogen, p. berghei, coupled with dextran of high molecular weight. in both cases the molecular size was systematically vaned, and an optimal size was found to be important in determining the level of immune responsiveness. in the case of the malaria peptide-dextran conjugate, epitope multiplicity (epitope density) was also found to be an extremely important parameter, immunogenicity increasing with increasing epitope density. the james p. allison. cancer research laboratory, university of california, berkeley, ca. . we have produced an antiserum to a synthetic peptide corresponding to a portion of the murine cd gene product. this antiserum reacted specifically with cells expressing cd . flow cytometric analysis of normal lymphoid tissues revealed that thymocytes were heterogenous in the level of cd expression while splenic t cells expressed cd at uniformly high levels. cd expression with that of other t cell differentiation antigens (il- r, j l l d , cd and cd ) in the murine young thymus and during fetal ontogeny was analyzed using three-color flow microfluorometry. our results indicate that cd expression is correlated with the stage of thymocytes maturation and demonstrate that il- r expression in the thymus is not induced via stimulation of the cd pathway. klinische argeitsgruppen fur rheumatologie/immunologie,schwabachanlage , erlangen. accessory cell depleted resting (la) t cells are not able to produce autocrine growth factors when stimulated via the t cell receptor complex (tcr), although il- receptor expression takes place. the addition of accessory cells restores the proliferative capacity. we have tested a series of recombinant lymphokines and growth factors for their ability to substitute this accessory cell function for human cdb and cd t cells. triggering via the tcr was achieved by a combinatorial stimulation with anti-cd and either anti-cd o r anti-cd that gives rise to il- receptor expression only on the corresponding t cell subpopulation. ll- , tnfa, if+, il- , il- , ll- and gm-csf were either negative or induced only a minor proliferative response. however, the combination of il- with il- was nearly as efficient as il- in cd t cells. cd t cells were much less stimulated by il- + il- . effect, which is in contrast to some reports on accessory-cell depleted mouse t cells whose function require costimulation with il- . this observation might be important for the understanding of t cell proliferation in chronic inflammatory tissues in which il- as well as il- is produced in large amounts. structural and serological studies of these secreted effector molecules have suggested that they are disulfide-linked dimers bearing t cell receptor u and chain d terminants. a t cell hybridoma, mts . , constitutively antibody staining indicated mts . expresses a t cell receptor containing a vg element. northern and southern analyses indicated mts . used v and va genes to encode the t cell receptor. the secreted mts . suppressor molecule was bound by monoclonal anti-v antibodies and by antibodies specific for constant region determinants of the u chain. to assess the role of a and chain genes in encoding the secreted suppressor molecule, we have generated v -and vu -negative variants from the mts . parent. experiments performed to date indicate that the loss of the v gene results in the inability to produce the suppressor molecule. the results are consistent with the idea that the t cell receptor u and chain genes are used to encode these hapten-specif ic/mhc-restricted secreted suppressor molecules. producing a dnp-specific/h-zk -restricted suppressor molecule was generated and studied. sensitivity to clonal deletion and selection for mhc restriction a m to be a feature of a particular stage of thymqte &el*, specifically l a t e mrtical cw+ + cells. in mcent studies we have slwm that early events i n signal trarrwluction i n respnse to antigen r e c p x crosslinkiq differ in the bmdture cw+ + p p l a t i o n and the mature cw' -or a>rl- p p l a t i o n . m t s indicate that antigen receptolg on hath inmature and mature -itive t cells tmnsdwe signals via calcium mobilization, h-er the maqnitu e of fnflux of e&acellular q'+ wfiich follckfi birding of antireceptor a n t q d i f f e r s tetmen these pqulaticns. specifically, imnature cells shcw a m& reduced q influx espcnse carpared to mature cells. we dmw here that dligation of ~~n p has different amepexe w i t h regard to q'+ nnbilization in mature and inmnture cells, no sucfi difference is seen f o l l a d r q ligaticm of the receptnr's transducer, a. ihe zpsults suggest that the signall* cascade leading to the influx of extracellular is intact hen c d~ is ligated, but is inccnplete w i e i i m p , the physiological liw, is ligated. in addition, ligation of cw or cd on bmdture t cells i n a~~ influx of extracellular a ' + canparable to that sem i n mature t cells. a clonal population has been isolated frcm inmnture thymocytes whir has the characteristic signal tramdmtion pxqerties of the tulk of inmnture thymdcytes. 'ihese f i r d h p suggest that "signal" transfer frcm xpnp to may be inefficient in cd + + cells. struchual analysis of the xpnp/cd canplex in hnature and mtum t cells is in progress. flood and alan friedman, dept. of pathology, yale university school of medicine, new haven, ct, . protective immunity to the ultraviolet (w) light-induced sarcoma -re is directed toward a single tumor-specific transplantation antigen expressed by the -re tumor cells. termed the a antigen. a progressive variant line of -re, termed -pro , lacks only the expression of this a antigen. immunization of mice with -re tumor cells haptenated with trinitrophenyl (tnp- -re) leads to the subsequent rejection of tnp-haptenated progressive tumors and to increased delayed-type hypersensitivity and ctl responses to tnp in normal, immunocompetent syngeneic mice. however, little or no humoral immunity to tnp is seen in animals injected with tnp- -re tumor cells. pro did not exhibit tnp-specific tumor protective or cell-mediated immunity, but rather exhibited tolerance to subsequent immunizations with more immunogenic forms of tnp. biochemical and molecular genetic studies have revealed the a antigen to exist on the cell surface as a complex of class i mhc-like molecules. transfection studies with dna encoding each of these molecules into -pro reveals that the expression of one, and only one, of these three molecules mediates this increased immunity. these experiments suggest that an mhc-like antigen expressed on the -re sarcoma acts as a natural adjuvant to increase cellmediated but not humoral immunity to linked antigens. the mechanism of this increased immunity is discussed. efforts to immunize cattle against economically important gastrointestinal nematodes showed that inrmunity is manifested by: ) a response that reduces the fecundity of established and subsequently acquired worms. and/or ) a reduction in the number of worms developing upon challenge infection. however. the ability of individuals to mount such immunity is highly variable. extending these studies to naturally infected populations indicate that there is a great difference in the number of eggs excreted by individual young calves on pasture. to delineate whether these differences were the result of host genetics and to begin to elucidate the mechanisms of resistance to parasite infection, a genetically defined cattle herd was assessed for parasite levels by determining fecal eggs per gram. three years of sampling of the calves during their first grazing season indicates that: ) certain individuals in the herd will consistently excrete high or l o w numbers of parasite eggs, ) the high or low phenotype is significantly controlled by the genetic make-up of the calf, and ) the high or low phenotype is highly heritable (heritability - ). susceptibility is currently under investigation. calves have been determined and mhc class i typing is currently in progress. information is being used to assess the role of the bovine mhc in controlling immunoresponsiveness to parasite antigens. we have been studying the differential effects of il + il versus il on the growth and differentiation of cd -, cd -thymocytes. culture of highly purified cd -, cd -thymocytes with il ( u/ml) + il ( u/ml) resulted in marked proliferation and increased cell size without change from the cd -, cd -phenotype. culture with il or il alone did not cause proliferation. a substantial contribution to the proliferation was secondary il release: addition of anti-il ( b ) blocking antibody inhibited proliferation induced by culture with il + il . il mrna was demonstrated by northern blot analyses after and hours of culture with il + il whereas none was detected at culture initiation and very little was present at hours. effects of il + il on il transcription rate will also be reported. despite a marked inhibition of proliferation with anti-il , there was no affect on expansion of cd + cells following culture with il + il (increase from % to % in hours). thus, in this system, il enhances proliferation of progenitor thymocytes but does not contribute to induction of t cell receptor. carplex. lhese cwplexes can be used to inmmize mice in the absence of protein carriers or adjuvants, thus facilitating the study of the inmum to a sml ckmically defined antigen. use of this tecfinology has allawed us to identify two t helper cell epitopes in cclllserved regions of hn gp not previcusly identified by computer algorithims, defined by amino acids - and - . inummization with these peptides in pptide-@nqhlipid cwplexes results in the prpauction i* and i* antibodies, which cioss react with cloned fragmnts of the w l e protein. us& this &logy we have begun to characterize the innume response to individual peptide antigens. ?he reqonse of h -k mice to amino acids - of p of hiv, has been analyzed. lhe optimal dose of a peptide containing both b and t cell epitopes was found to be - ug, depending on the route of administration. im d z a t i o n requir& less antigen for opthum antibody resporrsf, than did ip. ment for an ant-response. additional variables, as phosfholipid carpasition and method of cross-linking have been studied anl will be . webelievethattheuseof this peptide-phospholipid canplex tehmlogy will be significant both for studying the innwe response to single epitcpes and for vaccine develolment. based on assays in which t cell proliferation was induced via oxidative mitogenesis and exposure to mhc alloantigens, it has been reported that langerhans cells (lc) isolated from normal mouse skin aquire maximum capacity to activate t cells only after hours of culture. we have studied lc from balb/c mouse skins for their capacity to present ovalbumin (ova) and ia doantigens to unprimed t cells and to antigen-specific t cell hybridomas. the data reveal that both fresh and cultured lc presented ova and alloantigens with equal efficiency to previously primed responders (and with fold greater efficiency than spleen cells or the b cell lymphoma a . - ). by contrast g& cultured lc displayed the capacity to present antigen to ynorimed t cells. we propose that the antigen presenting potential of freshly prepared and cultured langerhans cells, respectively, reflect the in vivo functional properties of intraepidermal lc and of lc that have picked-up antigen in the epidermis and migrated via dermis to the regional lymph node. if so, these data suggest that resident epidermal lc are fully prepared to present cutaneous antigens to memory/effector t cells (efferent limb), whereas resident lc must leave the influence of the epidermis in order to develop the capacity to meet the more stringent conditions required for antigen presentation to porimed t cells (afferent limb). we have investigated the structural restrictions placed on residues contained within a minimal t cell determinant, using the balb/c class i restricted t cell response to the site determinant of the influenza hemagglutinin molecule as a model system. to delineate which of the residues comprising the site determinant are involved in interaction with the t cell receptor, we have determinaed the response of a large panel of site specific t cell hybridomas to a collection of peptide analogs differing by single conservative or non-conservative substitutions at positions. the fine specificity patterns of the t cell panel is extremely diverse; t cells varied in both the location and number of residues within the antigenic peptide that effected recognition. our results implicate at least out of residues within the antigenic pepetide as being involved in interaction with the t cell receptor. this result suggest that peptides comprising the site determinant do not form alpha helical structures when in association with mhc molecules. rubella-specific isotype and igg subclass responses were evaluated using elisa techniques in rubella ha seronegative adult females undergoing rubella immunization (ra / strain). responses were evaluated prior to immunization and at , , , , , , and wks post-immunization. pre-immunization sera showed detectable levels of rubella-specific antibody in the igg class ( / ); iga class ( ) and in one or more of igg subclasses ( / ). post-immunization, i subjects failed to develop igm class responses by the ha (sdg) technique while / developed igm antibody by elisa techniques. iga responses were detected at low levels in all vaccinees beginning at - wks and declining by wks post-immunization. antibody in iggl and igg subclasses by - wks post-vaccine with sustained iggl levels but significant decline in igg levels noted between and wks post-immunization. no seroconversion was noted in the igg subclass although individuals had detectable pre-immunization iggz rubella antibody present. igg levels were detected in all vaccinees post-vaccine with a delayed and progressive rise over the study period. subsequent correlation was then performed between rubella-specific antibody responses and the presence or absence of adverse joint reactions occurring in association with rubella vaccine administration. all individuals produced detectable c t lymphocyte responses to varicella zoster virus. anthony hayward, abbas vafai, roger giller & eileen villanueba. departments of pediatrics and microbiology, university of colorado school of medicine, denver co university of iowa school of medicine, iowa city i the proliferative response of blood lymphocytes from varicella zoster virus (v v)-immune donors to live vzv, extracted vzv antigens or purified glycoproteins is predominantly by cd +, hla-d restricted t cells but little is known of the specificies of the responder cells. we restimulated t cells cloned by limiting dilution from vzv-stimulated cultures with purified vzv glycoproteins gpi, gpii and gpiii and found that t cell clones with specificity for each of these mediated both help for antibody responses and hla-dr restricted vzv-specific cytotoxicity. polypeptides of to amino acids length corresponding to predicted amphipathic sequences in the primary structures of g p i, gp i and gp iv were synthesised. proliferative responses were observed to of these peptips (one from each glycoprotein) with responder cell frequencies in the :lo blood t cells range. the gp i peptide additionally defined an epitope recognised by serum antibody. an immunomodulatory approach to treating hsv- corneal disease, hendricks rl, departments of ophthalmology, and microbiology/immunology, university of illinois school of medicine, chicago, il herpes simplex virus type i (hsv- ) corneal infections are a leading cause of blindness worldwide. we and others have demonstrated that the cellular immune response to hsv- contributes to the elimination of virus from the cornea, but in doing so causes the tissue destruction that is responsible for the blinding complications of the disease. we have demonstrated that specifically suppressing the cytotoxic t lymphocyte (ctl) response to hsv- renders mice resistant to corneal disease following topical corneal hsv- infection. in agreement with this observation was our recent finding that in vivo depletion of wt ' (t helper/inducer, and most dth effector cells) neither reduced susceptibility to corneal disease, nor increased susceptibility to disseminated disease. the corneal lesions in wt depleted mice contained numerous lyt- (t suppressor/cytotoxic) cells, and no l t cells. the wt depleted mice exhibited normal hsv-specific ctl precursor frequencies. experiments designed to determine the effect of in vivo lyt- depletion on susceptibility to corneal disease are in progress. our goal is to identify cellular immune responses to hsv- that maximize protection, while minimizing immunopathology in the cornea, and identify hsv- epitopes that preferentially activate those responses. supported we found that affinity purified antibodies to bsa, klh and diptheria toxoid all contain a substantial amount of specific anti-idiotypic activity. against bsa react with mouse anti-bsa antibodies, which suggests that we are dealing with internal image antibodies. mrl-lpr/lpr mice develop spontaneous autoimmunity. we found that these mice make anti-anti-(self h- ) antibodies prior to making appreciable amounts of pathological autoantibodies such as anti-dna, anti-rnp.sm, and rheumatoid factor. the anti-anti-self antibodies are detected using an inhibition of antibody mediated cytotoxicity assay, that also detects anti-anti-(self h- ) in ordinary allogeneic anti-sera. the antibodies are not rheumatoid factors, although the animals do make rheumatoid factors later in the development of the disease. anti-self activity is fully developed at months, when the other autoantibodies are typically barely detectable. important role in the etiology of the disease. the anti-we conclude that anti-anti-self antibodies could play an c feedback regulation of - synthesis in monccytes by t cell products: dual effect of - . mikko hurme, tessa palkama and marja sihvola, department of bacteriology and immunology, university of helsinki, sf- , helsinki, finland. il-i production of human monocytehnacrophages is regulated by several cytokines some of which are themselves able to activate the il- production (e.g. tnf and il- ) while others (e.g. ifn-y) modulate the production activated by other signals. we have now examined the effect of - on the - synthesis. - alone did not induce any - bioactivity or il-la or - mrna expression in freshly isolated peripheral blood adherent cells. in contrast, il- effectively suppressed the lps induced - production. this suppression took place without any decrease in the steady-state levels of il-la and il-i mrna, suggesting that this downregulative effects is posttranscriptional. monocytehnacrophages are known to rapidly loose their ability to produce il-i when cultivated in vitro. if ifn-yis present in the culture fluid, the cells remain capable of producing - . as ifn-yand have been reported to have similar "priming" effects on macrophages (e.g. increasing the tumoricidal capacity and mhc class ii antigen expression) we cultivated monocytes for h in the presence of either ifn-y or - , and after washing the cells they were stimulated with lps. il- activity could be detected both in the ifnyand il- preincubated cultures (but not in the cultures preincubated with medium alone). these data suggest that il- can also display a similar upregulatory function in il-i production as ifn-y. gahreston, tx development of immunity to members of the spotted fever group of rickettsiae is a t-cell dependent response. we have used t-cell hybridomas and cloned t-cell lines from immune animals and convalescent humans to identify the rickettsial antigens that induce antigen-responsive t-cells. in these studies we found that the kda antigen of rickettsia tickettsii. the causative agent of rocky mountain spotted fever, is one of the immunodominant tcell antigens. t-cells from immune animals and humans were responded in culture to a recombinant kda antigen. both sources of t-cells were of the t-helper type (l t * and ' respectively) and produced l- and interferon. it was found that soluble antigenic material of b. rlckettsii obtained by extraction with hypotonic buffer maximally stimulated the t-cell lines. this material was enriched far the high molecular weight polypeptides of s kda and kda. also. ethirwwi ' will induce a long-lived immunity against infection with r. rickettsii. infected guinea pigs develop a minimally cross-reactive antibody response to b. nckettsii. in contrast. a strong cross-reactive t-cell proliferative response is produced. studies are in progress to determine the nature of the common protective antigen of r. humans infected with the parasitic nematode ascaris lumbricoides vary considerably in antibody responsiveness to a kda component of the parasite. this molecule is secreted by the parasite, and is also abundant internally. this heterogeneous reactivity has been modelled in laboratory rodents, and the antibody response to it is h- -and rt -restricted in mice and rats, respectively. using inbred and congenic animals, only mice of h- ' and rats of rtiu were, so far, found to be responders, and this restriction only operated in the context of infection. the specificity of the ige response in these animals was assayed by passive cutaneous anaphylaxis, and in an ige-specific elisa assay. the data show that the above mhc restriction also applied to the specificity of the reaginic antibody response, although animals of all mhc haplotypes responded to other ascaris allergens. amino acid analysis of the kda equates it to a previously identified "allergen a" of the parasite, and we now have its sequence available. these findings have implications for the genetic control of allergic responses in general, and, in particular, to the hypersensitivity responses which are such a feature of infections with parasitic nematodes. there are also implications for the generation of hypersensitivity responses by recombinant vaccines involving certain parasite antigens. the cns. immunohistochemial analysis of both frozen sections prepared from the brains of animals immunized in this manner and of highly enriched glial cell subpopulation cultures for viral gp expression indicated that oligodndrocytes and possibly a subset of astrocytes were the targets of this infection. further, microscopic analysis of frozen sections failed to reveal any overt signs of gross pathologic changes associated with the viral infection. we have been able to demonstrate the presence of virus specific antibody in the serum of these mice as well as virus specific cytolytic t cells in the peripheral lymphoid organs. ments are currently underway to determine whether the lack of pathology associated with wb infection in light of the previously shown virus specific immune responses in these mice is due to a failure of antigen presentation within the cns or some other form of immunoregulatory phenomena. the t lymphocyte proliferative response to pigeon cytochrome in bio.a mice is restricted to the egk:e,k ia molecule and specific for the c-terminal determinant comprised of residues - . blo.a( r) and blo.a(sr) mice are nonresponders to pigeon cytochrome nonetheless, the t cell repertoire of blo.a( r) or ( r) contains some t cell clones capable of recognizing and proliferating to pigeon cytochrome c w h e n presented by bio.a antigen-presenting cells (aft). therefore, one would expect to stimulate such clones in allogeneic bone marrow chimeras of the type bio.a +blo.a( r) or ( r) b o.a apcs and a blo.a( r) or ( r) t cell reperto ! ~.~espectively. ,en(isea c rzave were primed with pigeon cytochrome cytochrome , they showed a good antigen specific proliferative response in vitro. surprisingly, however, if pigeon cytochrome was used for priming, no response was detected, even at priming doses as high as nmol per mouse. - could only be achieved by treating the allochimeras with an anti-cdb monoclonal antibody in vivo during the priming step. clones specific for purified protein derivative (ppd) in the same chimera. thus the regulation which involves cd positive cells is antigen specific. transfer of pigeon cytochrome - primed lymph node cells from the chimera into naive bio.a mice prevented priming of the recipient for a t cell proliferative response to pigeon - , but not priming to the moth synthetic fragment. chimeras of an antigen-specific suppression mechanism involving cd positive cells. faculty of medicine, kyoto university, kyoto , and department of oncology, nagasaki university school of medicine, nagasaki , japan. sera from b mice immunized with a syngeneic ctl specific for fbl- tumor of b origin blocked the cytotoxic activity of only the immunizing ctl clone. therefore, a monoclonal antibody (mab) n - was produced by fusion of the b spleen cells immune to a syngeneic fbl- -specific ctl clone (no. ). the specificity of the mab n - was confirmed by immunoprecipitation, blocking of cytolytic activity, stimulation of proliferation, and induction of tcr-mediated nonspecific cytolysis of the ctl clone no. . in some b mice, - % of the anti-fbl- mltc cells were positive for this n - -defined idiotype, and formed a well demarcated population upon examination by flow cytomehy. even in mice in which no such population was observed some ctl clones established by limiting dilution culture were also positive for this idiotype ( out of clones from mice). the cytotoxic activities of these ctl clones were blocked by n - , which in turn induced the nonspecific cytolysis in redirected assay. however, no positive cells were detected in non-cultured normal or fbg -immune spleen and lymph node cells. this indicates the presence of cross-reactive (dominant) idiotype in the b anti-fbl- cytotoxic t cell responses and may provide a potent tool for analyzing the idiotype-mediated regulation of the anti-tumor immune responses. slade andsylvie gillard, max-planck-institut fur immunbiologie, d- freiburg, federal republic of germany t cells play a n essential role in t h e protective immune response to malaria and a r e associated with s o m e of t h e pathological consequences of t h e disease. however, t h e n a t u r e of their responses and t h e antigens t o which they respond a r e not well defined. w e have developed a limiting dilution assay system in which specific t cell responses to malaria antigens c a n be monitored a t t h e clonal level. i t is possible to determine t h e nature of t h e responding t cell by t h e growth f a c t o r s they s e c r e t e and by their ability to a c t as helper cells for t h e antibody response to malaria antigens. our d a t a suggest t h a t t h e t cell response changes during t h e primary infection and in hyperimmupe animals. o n e to t w o weeks a f t e r initiation of a blood s t a g e infection t h e major cd + t cell which proliferates in response t o parasite antigens s e c r e t e s il- and ifn-y but is not a n efficient helper cell for antibody responses. in c o n t r a s t l a t e r in infection and in immune animals t h e r e is an e f f e c t i v e helper cell response and many of t h e s e cells a r e distinct from those secreting ifn-y and il- . we a r e currently investigating whether these cells retain these phenotypes when grown in long-term in vitro culture and whether defined antigens of t h e erythrocytic parasite elicit different t cell responses. we have localized linear neutralization epitopes on the coronaviruses ibv, mhv, fipv and tgev. the results can be summarized as follows: . linear epitopes of the spike proteins ( - residues) could be mapped to a resolution of a single residue by expression of gene fragments in the prokaryotic pex plasmids and/or pepscan peptide synthesis. . the length the epitopes varied from to at least amino acid residues. we present evidence that the larger epitopes, although conformation-independent according to operational criteria, are nevertheless discontinuous. . in ibv, we localized several overlapping but different epitopes within an immunodominant region of residues. this region is recognized by all polyclonal antisera tested. we propose that its immunodominancy is a consequence of its structure and function and does not depend on antigen presentation or idiotypic networks. an immune response against the mouse testis-specific antigen ldh-c reduces fertility by percent in female baboons. an immune reaction to human ldh-c would be expected to be more effective in primates. since the human testis enzyme is not readily available in large quantities, recombinant dna technologies were uscd to create a source of human ldh-c . antibodies to mouse ldh-c were used to screen a xgtll human testis cdna expression library. a full length human ldh-c clone was identified, sequenced, and the ldh-c cdna was engineered for expression in e.coli. the ' and ' untranslated sequences were removed by restriction enzyme digestion, and synthetic linkers were added adjacent to the start and stop codons of translation. the modified cdna was subcloned into the prokaryotic expression vector pkk - and introduced into w l a c iq cells. cells were grown to mid-log phase, and induced with iptg for positive regulation of the strong hybrid tac promoter. induced cells overexpressed the kd subunit which spontaneously formed the enzymatically active kd tetramer. human ldh-c was purified -fold from liter cultures of cells by two step affinity chromatography to a specific activity of i.u./mg. the n-terminal amino acids sequenced were identical to those predicted from the nucleic acid sequence. antibodies to synthetic peptide epitopes of human ldh-c cross-reacted with the enzyme produced in e.coli. two mg human ldh-c were expressed per liter of bacterial cells. the purified protein is now available for innunogenicity and fertility studies. it is now generally accepted that the principal effector mechanism in the host's defence against leishrnaniasis is gamma-interferon (ifn-y) which activates infected-macrophage to eliminate intracellular parasites. mice by prior sublethal whole body irradiation or treatment with anti-igm or anti-cd antibody. protection can also be induced by repeated intravenous or intraperitoneal immunisation with killed parasites or purified antigens. ly immunised mice produce little or no il- or il- but substantially elevated levels of ifn-y when stimulated with leishmania antigens in vitro. lymphoid cells from balb/c mice with progressive disease can inhibit the maf (macrophage activating factor) and leishmanicidal activities of the culture supernatant of lymphoid cells from mice recovered from l. major infection. maf appears to be ifn-y, whereas the maf inhibiting factors are il- and il- . system can be reproduced with recombinant ifn-y, il- and il- and the maf inhibiting activity of the suppressive supernatant can be reversed by specific anti-il- and anti-il- antibodies. the disease by influencing the ability of macrophage to kill the intracellular parasite. the development of efficacious vaccines against malaria requires an understanding of the mechanisms involved in protective immunity. f'revious studies with plasmodium berghei demonstrated that sporozoite immunity is dependent upon antibody responses specific for the repeat region of the circumsporozoite (cs) protein and cell mediated mechanisms involving cd + t cells. in this study we analyzed the splenic t cell repertoire directed against epitopes on the cs protein of p. berghei and determined whether sporozoite-immune cd + and cd + t cells respond to shared or distinct epitopes. sporozoite-immune spleen cells, cd + and cd + enriched t cell populations of balb/c (h-m), c h @i-%), and c bv (h- b) mouse strains were cultured in the presence of irradiated sporozoites or synthetic peptides representing % of the complete cs protein. surprisingly, none of the cultures proliferated to any of the peptides tested, although proliferative responses to sporozoites were observed in unfractionated spleens and cd + t cell populations. cd + t cells did not respond to any of the antigens tested, even in the presence of exogenously added - . titration of cd + cells into proliferating cd + cell cultures did not suppress the anti-sporozoite response. the lack of anti-peptide reactivity contrasts with uniform responses to sporozoites and may be the result of the context in which cs antigens are presented to t cells. functional analysis of accessory splenic b cells and macrophages revealed that while the anti-sporozoite proliferative responses were not affected by the removal of macrophages, sporozoite-primed b cells were essential for the responses. these data suggest that the cs protein on sporozoites is not processed extensively by macrophages to yield many potential t cell epitopes, but instead is presented by immuncdominant b cells that resmct responses to a limited number of t cell clones. of the primary infection and is also required for optimum protection against reinfection. current studies have demonstrated that relatively few of the viral antigens tested to date ( viral envelope glycoproteins or nonsmctural nuclear proteins) are recognized by hsv- immune ctl populations generated in several different strains of mice (h haplotypes h b, h d. or h k). this failure of hsv specific ciz to recognize the cloned gene products in in v i m assays was demonstrable at the clonal level and could not be attributed to a peculiarity of the recombinant vaccinia conshucts used because studies with adenovims vectors or tranfected l cell constucts yielded the same results. surprisingly, despite their inability to be recognized by hsv specific ciz in vim, when used to immunize mice several of the vaccinia virus constructs would induce memory ciz populations capable of lysing hsv- infected autologous cells. for example, hsv- glycoprotein c (gc) was recognized by h b restricted but not h k restricted hsv specific ctl. however, immunization of either haplotype of mice with a vaccinia gc recombinant induced ctl populations which upon in v i m restimulation with hsv- would lyse histocompatible cells infected with hsv- . this demonstrates that despite the presence of suitable epitopes (intrinsic factors) the context of the immunogen (extrinsic factors) will also influence it's ability to induce ctl. the results of further studies into the nature of these extrinsic factors will be presented and discussed with relevance to future sub-unit vaccine design. w d ~upponrd by public ~~l t h service g~mu, ai md ai fran the ti-^ ~n,litu= md lnrcniour d ,~~~~~~. infection of mice with hsv- induces a brisk ciz response which is necessary for the subsequent resolution we have investigated the structural basis for antigen mimicry by anti-idiotypic internal image antibodies. two mouse monoclonal antibodies (mabs) that bear internal images of a well-defined protein epitope, i.e., the rabbit immunoglobulin (ig) a allotype, were produced and the variable region sequences were determined by rna primer extension sequencing. the results showed that the mab light chains did not contain any allotype-related residues; however, both heavy chain v regions contained a unique sequence homologous to the nominal antigen but in opposite orientation. this reversed sequence was expressed within cdr of both mabs. synthetic peptides corresponding to the putative antigenic regions of rabbit ig and the mab internal images, respectively, were tested for the ability to mimic the al-like determinant. although the homologous residues were presented in opposite orientations, both peptides completely inhibited at similar concentrations the binding of rabbit ig to anti-a antibody. a paired thr and clu was necessary for expression of the a epitope as revealed by conservative substitutions in the peptide sequence. computer-generated, energy-minimized models of rabbit ig and the mabs revealed that the critical a residue side chain placements could be almost superimposable in either context. thus, it appears that an antigenic epitope can be determined solely by md . proliferation of murine type i cd + t cell clones quires simultaneous occupancy of the t cell antigen receptor and delivery of an accessory cell-duived costitnulamy signal in contrast, isolated t cell receptor occupancy induces the cell into a state of reduced proliferative responsiveness to antigen. based on the observation that pkc-activating phorbl esters can at times substitute for the p s e n c e of accessory cells in t cell proliferative response. to mitogens or anti-cd mnodonal antibodies, we investigated the requkment for accessory cells in the antigen-and con a-induced hydrolysis of p m and activation of pkc. the presence of normal accessory cells was found to be unnecessary for the development of pkc-dependent phosphorylations and the addition of normal accessory cells had no effect on the activity of pkc. cell il- synthesis and proliferation presents a paradox. we have studied the effects of ueatment with a calcium ionophore and p h h l ester on t cells and find that increased [ca +]i and pkc activation are in fact insufficient biochemid second messengers in the induction of proliferation. while pliferation was induced at high t cell density in response. to these stimuli, incubation of t cells at decrrased cell density drmonsuated markedly reduced proliferation, and single t cells failed to divide. this suggested that cellular interactions were. q u i r e d in the response. additions of either il or normal accessory cells allowed p l i f d o n at low density, consistent with a requirement for an accessory cell-derived costimulatoq signal in the induction of i l synthesis, even in the plifcrative response to ionomycin and pma. this result underscons the importance of an accessory cell-duived costimulatory signal, acting independently of t cell receptor-mediated increases in [caz+] i and pkc activation, in the induction of t cell proliferation. we describe experiments designed to determine the molecular requirements for recognition by fluorescein-specific ctlps and ctls derived both from n a i v e and from immunized mice. we the production of prostaglandin e, a major immunesuppressor secreted by the macrophages was inhibited by the addition of . m indomethacin to the cultures of monocytes harvested from patients suffering from pulmonary tuberculosis and those from equal number of normal controls. the il-i activity was estimated i n the supernatants of these cultures by their ability to proliferate mice thymocytes. it was found that the supernatants from cultures with indomethacin showed a greater il-i activity than the ones without it( % p . ). this indicates the possibility of pge offering a negative feedback control over il-i production. the defective cell mediated immunity i n patients with pulmonary tuberculosis may be explained through the inhibition on il-i production by pge whose enhanced production is reported i n our earlier studies. the results and our hypothesis on the autoregulation of il-i production w i l l be presented and discussed. in variant viruses which differ from the parental virus (gv) at specific epitopes recognized by monoclonal antibodies directed against the env gene product, gp . biological clones isolated from gv express the gv phenotype suggesting that the loss of specific epitopes is the result of selective de novo processes in the immunocompetent host. additionally, inoculation of adult mice with a biological clone expressing the gv phenotype also results in similar variant viruses. however, inoculation of gv into neonatal or nonlethally irradiated mice results in a population of viruses expressing only the gv phenotype suggesting that the emergence of antigenic variants may be influenced by neutralizing antibodies and/or cellular host res sds-page analysis of immunoprecipitates of sg~s~~,elled lysates of fibroblasts infected with clones expressing gv or variant pehnotypes shows a size difference of the gp precursor. additionally, the recognition of a neutralizing epitope (e- ) associated with gp by mab is dependent on the appropriate native conformation of the epitope which appears to require glycosylation for expression. experiments are in progress to further examine the immunogenetic basis for the generation of these variants and to determine the molecular changes in the virus genome responsible for changes in epitope expression. investigated the capacity of murine splenict cells depleted of acceso cells ( ac ) t o proliferate in response t o stimulation by con a, @ cd ab and activated t cells. the zepletion procedures consisted of carbonyl iron treatment, x "panning " on anti-ig coated flasks, x anti-la cytotoxic treatments and percoll gradient purification of small resting t la-cells. the appropiate concentration of con a ( ng/ml ) and plastic-bound (pb) @cd lg or its f (ab)' fra ments induce proliferation, r expression and (but not secretion in t la-cellscultured for % at x cells/well . responsiveness of tlacells t o con a and dcd in low density cultures ( x cells/well) is restored by the addition of irradiated th cloned cells but not thl ,splenic cellsor r l l l + rll + r . likewise, responsiveness t o non activating doses of con a (lnglml) or soluble @cd is restored by the addition of irradiatedth costimulatory cells . these experiments demonstrate that the ability of t cells t o proliferate in the absence of ac is critically dependent on t-t interactions. t cell subsets prepared by either negative (l t -and ly 'cells) or positive selection proliferate in response t o pb @cd lg . although the proliferative responses of both l t -and ly -cells are maximal at h, the l t -cells require l o x more pb @cd lg for maximal stimulation and their res onses decline much faster than those of ly cells. in addition, l t -cells are not stimulated by pb &cd f(ab)' fra ments and their responses t o @cd i are inhibitable by anti fcr as well as anti-lfa abs . re onsesof%oth l t -and ly -cells are !nhibita%le by @i and @i r abs but not by @l t , @ly or b l l . these experimentsdocument interesting differences in the triggering requirements of l t -and ly 'cells. supported by nih grants po ca , t m- and gm . the k glycoprotein encoded in the e region of ad and ad (gpl k) binds to class i mhc antigens in the endoplasmic reticulum and prevents their translocation to the cell surface. this has been proposed as a mechanism by which virus infected cells can avoid recognition by the host cytotoxic t lymphocyte (ctl) response. we have shown that gpl k can inhibit target cell lysis by adenovirus specific ctl, but the effectiveness of this inhibition varies greatly between different mouse strains. this is due in part to differences in the affinity of gpl k for different mhc class i molecules, but this cannot account for a l l the variation observed. i t has been shown that cd + + immature thymocytes fail to secrete il- or express il- receptors in response to activation signals. furthermore. they cannot induce il- gene transcription. several tumor lines have now been characterized which have a cd + + phenotype and fail to secrete i l - or express il- receptors in response to stimulation with ionomycin plus pma. these cells also do not express il- mrna after stimulation, as determined by northern blotting and rnase protection. to determine the molecular mechanism for this lack of transcriptional activity, nuclear extracts were analysed for the presence of the d n a binding factor nfat-i. this nuclear factor i s present only in ac- we have undertaken an mhc analysis using the polymerase chain-reaction (pcr) and dot blot analysis of the amplified lyme arthritis patients dna with allele specific oligonucleotide (aso) probes. genomic dna for the first domain of the dq beta chain and of the dr/pi from patients with lyme arthritis has been amplified and we are analyzing the distribution of dridrr and w a l l e l e s in this population to test the hypothesis that the mhc class i genes might be involved in presentation of selected spirochete epitopes whose recognition by t lymphocytes leads to lyme arthritis. most inbred strains of mice do not respond to porc insulin (pins). experiments were conducted to elucidate the mechanism of the non-responsiveness in h-zk mice: ) purification of cd- ' t c e l l s from pins-immune b o.mbr mice revealed pins-specific t helper (th) cells, ) these pins specific th cells could be activated by i-ak and i-ek expressing l -fibroblasts. therefore, both i-ak and i-ek molecules can present pins in an immunogenic manner and activate pins-specific th cells. by means of different cell-fractionation procedures, it was found that antigen-specific t suppressor (ts) cells regulated the pins immune response. these ts cells were of the fcr-, cd- -, cd- '. thy- ' phenotype, and they were present in normal mice. we believe that these experiments indicate that antigen-specific ts cells exist and are important regulators of immune and autoimmune responses. the possibility of functional inactivation of cd + clones by ts-cells was investigated. m leprae-responsive cd + clones were preincubated with ts cd + clones, apc and antigen for ; hours. after which the cd + cells were removed from culture. the cd + clones were then restimulated with e. leprae and apc. cd + clones incubated with cd + cells and antigen were unresponsive to restimulation by antigen, although they were not killed and could respond well to il- . addition of il- in the preor post-incubation culture neither prevented the induction of unresponsiveness nor reversed it. earlier models of tolerance have suggested that receptor occupancy in the absence of second signals induces tolerance in b-and t-cells. we would suggest that in the presence of ts-cells. a second signal may be negated leading to th-cell unresponsiveness. university of texas southwestern medical school, dallas, tx graft versus host disease (gvhd) poses a serious threat to the survival of patients with bone marrow transplants. the state of immunosuppression established in gvhd results in a variety of immunological abnormalities at the humoral and/or cellular level. we have developed a murine model of chronic gvhd across a minor histocompatibility (mh) barrier. in this model, immunosuppression develops. spleen cells from mice undergoing this type of gvhd are unable to respond to the polyclonal activators lipopolysaccharide and concanavalin a . however, the response against the b cell leukemia bcll remains intact. the protective immune response against bcll is directed towards the mh antigen h- and is mediated by cytotoxic t lymphocytes. thus, the specific t cell response against a mh antigen can occur in the presence of chronic gvhd despite the absence of a polyclonal b and t cell response lymphocytic choriomeningitis virus (lcmv), a member of the arenavirus family, has a biseqmented rna genome which encodes at least three polypeptides. the smaller rna segment encodes two virus structural proteins, the slycoprotein (gp) and the nucleoprotein (np). upon infection of mice with lcmv a cytotoxic t cell immune response directed against these proteins is measurable in vitro and in vivo. it can be demonstrated that depending on the haplotype of the mice, one or the other protein may play a major part in the immune response. in order to define the immunogenic epitope(s) of the nucleoprotein which are recognized specifically by the t cell receptors of cytotoxic t cells, stepwise ' truncated qene fraaments encodina the nucleoprotein were cloned and expressed in vaccinia virus. with these recombinant vaccinia viruses, protection experiments in mice aqainst lcmv infection were performed in parallel with in vitro studies, namely specific recoonition of target cells expressing truncated fragments of the nucleoprotein by lcmv primed spleen cells. cdna clones encoding the mouse and human t cell il- receptors have been isolated and expressed in mammalian cells. the recombinant receptor binds il- indistinguishably from the natural il- receptor, and is functional in signal transduction. deletion of the cytoplasmic portion of the receptor abolishes its signal transduction abilities. sequence and secondary structure analysis suggest that the cytoplasmic segment of the il- receptor binds a nucleotide. experiments designed to test this hypothesis and to examine the mode of signal transduction will be presented. also to be discussed are the mechanism of triggering of the receptor by il-i. and the nature of il- receptors expressed in other cell types such as b cells. it became evident that these subsets reflect different stages of helper t cell maturation before and after activation. therefore, these t cell subsets have been designated as naive t cells (cd r/ h +, cdw [ b ]-) and memory t cells (cd r/ h -, cdw [ b ]+). we analysed the expression of these antigens in dermal lymphohistiocytic infiltrates from different benign skin diseases and cutaneous t cell lymphomas (chronic contact dermatitis (n= ), parapsoriasis en plaques (n= ), lymphomatoid papulosis (n= ), mycosis fungoides (n= ), sezary's syndrome (n- ), pleomorphic t cell lymphoma (n= ) and high grade t cell lymphomas (~ )). in almost all cutaneous t cell infiltrates memory t cells were preferentially found whereas in the peripheral blood both subsets are equally distributed. this implicates, that t cells infiltrating the skin already have had contact with their respective antigen. where the switch from naive to memory t cells takes place can not be answerded by our findings, as we have investigated rather longstanding skin diseases. however, these memory t cells, which can be activated more easily, make diseased skin more e f f e c t i v e in the nmdlification of an immune resnonse. organs and after several days of stimulation with antigen or mitogen and lymphokines. we find that fresh th synthesize and secerte -iu,ifng,il and gmcsf but very little il or il. within - hours. this pattern resembles the pattern of lymphokines secreted by thl cell lines. the th responsible for this secretion are cd positive t cells which are long-lived since they disappear very slowly following adult thymectomy. they are also sensitive to the in vivo administration of ats(antithyrn cyte serum) and they express high levels of pgp- . the kinetics of lymphokine secretion and the phenotype of the cells suppon the hypothesis that lymphokine secretion from fresh lymphoid cells comes from a population of memory cells. in contrast we fmd that we can also stimulate a separate, ats-resistant population to become lymphokine-secreting cells after four days of in v i m priming.these primed cultures rapidly synthesize an secrete large amounts of i u and il in addition to ifng , il and gmcsf( a phenotype which could be combination of both thl and th helpers), when they are restimulated with ag or mitogen. the cells whic are responsible are cd positive and have a shorter liespan since they decline considerably after adult thymectomy. we suggest that the lymphokine secreting cells detected after priming come from a population(s) of helper t cell precufiofi which have differentiated to become effectors. this generation : effectors requires lymphokines, especially e- andlor ilz and apcs. thus the development of helper th appears to follow a similar pathway as that of cells of the b cell lineage developing into ab-secreting cells and cd positive t cells which develop into cytotoxic effectors from precursors. we have studied the secretion of lymphokines by helper t cells freshly obtained from lymphoid interleukin- production by t cells has been shown to be required for both humoral as well as cell-mediated imune responses. thus, il- production was measured in syphilitic rabbits as a function of their imune response. maximal il- production induced by con a at - days post-infection was only l/ that observed for uninfected rabbits and this correlated well with a decrease in t cell proliferation ( < % that of normal rabbits) upon stimulation with con a . this decrease in il- production in infected rabbits was restored upon removal of most of the adherent cells. furthermore, the il- production by - day infected spleens was restored above normal levels upon addition of indomethacin. this decrease in il- levels was not due to an increase in the ability of infected spleen cells to adsorb il- . finally, studies assessing il- production at various times postinfection indicated that at days post-infection il- levels were higher than normal, however as early as - days after infection il- levels decreased below normal levels and continued to be depressed as late as days post-infection. these results may explain why all organisms are not eliminated during primary infection w i t h ' . pallidum and why secondary and tertiary phases of the disease may develop. has been shown that especially the antigenic presentation of the fusion protein is important for eliciting a functional immune response. to study the immunolo ic properties of the f protein, we expressed the f gene in e.coli as a $galactosidase-f-fusion protein after insertion in a pex vector. we constructed deletion mutants with fragments generated with restriction enzymes and with the polymerase chain reaction method. using a panel of monoclonal antibodies a rough epitope mapping has been performed. two arears were found on the protein, with one area two monoclonal antibodies react and with an other area four monoclonal antibodies react. both area's were found in f , the c-terminal part of the protein. the pepscan method was used to fine map the epitopes of the monoclonal antibodies reacting with the second area on the primary sequence. in at least one viral system, cd + effector cells can be induced in animals lacking cd + t cells. since cd + effector cells are important in immunity to malaria sporozoites, we wished to know if they,too, could be generated without help from cd + cells. we depleted balb/c mice of their cd + t cells by injection of an anti-cd monoclonal antibody, and then tried to immunize them with irradiated plasmodium yoelii sporozoites. when challenged with infectious sporozoites, these mice were not protected against malaria infection. although they did not make antibodies to sporozoites, passive transfer of hyperimmune serum into these animals still did not protect them against a sporozoite infections. cd + t cells from these animals functioned normally in in vitro assays against tnp labelled targets. it appears that, unlike viral systems, the generation of cd + effectors in malaria requires cd + helper cells. thus both cd and cd epitopes should be included in any synthetic vaccine against malaria sporozoites. univ. pennsylvania, philadelphia, pa migrate from fetal liver or bone marrow. rearrange t cell receptor (tcr) genes, express tcr. undergo thymic selection and finally emerge as mature single positive t lymphocytes. most studies of thymic t cell development have been performed by using polyclonal populations of t lymphocytes, which have made the interpretation of the results complicated. cells ( clone) from nude mice by culturing nylon wool non-adherent cd -cd -spleen and lymph node cells in the presence of wehi supernatant and con a supernatant. clone was thy- -cd -cd -cd -il r(il receptor)-and they have been maintained more than months without changing phenotype. when the c clone was stimulated with il . ili/il . illnl . gm-csf, the cells were induced lo express thy-i, tcr and il r proteins. however, culture of the cells with gm-csffll did not induce the expression of these molecules. southern blotting of the dna isolakd from gm-csffll culture suggested that they have undergone partial db -jp rearrangement. the cultured cells were then recloned twice by limiting dilution. the cloned cells were again shown to induce expression of cd complex by the stimulation of il enriched medium. therefore. we have established a system in which to induce differentiation of cloned pre t cell line into tcr+ cells in vitro. the human lymphocyte differentiation antigen cd is encoded by a single gene which gives rise to a kda glycoprotein expressed on the cell surface as a dimer, and in higher molecular weight forms. we demonstrate that the mrna is alternatively spliced such that an exon encoding a transmembrane domain is deleted. that is secreted and exists primarily as a monomer. messenger rna corresponding to both forms is present in peripheral blood lymphocytes,(pbl), con a activated pbl and three cd + t cell lines with the membrane form being the major species. ratio of mrna for membrane cd (mcd ) and secreted cd (scd ) exist. in addition, the splicing pattern we observe differs from the pattern found for the mouse cd gene. this mrna is also alternatively spliced, but an exon encoding a cytoplasmic region is deleted giving rise to a cell surface molecule which differs in its cytoplasmic tail from the protein encoded by the longer mrna. neither protein i s secreted. this is one of the first examples of a different splicing pattern between two homologous mouse and human genes giving rise to very different proteins. this represents one mechanism of generating diversity during speciation. cd ' t cells in the rat can be divided into two non-overlapping subsets by theii reactivity with the monoclonal antibody mrc ox- which binds some of the high molecular weight forms of the cd antigen. recent work, to be described has shown that the two subsets represent different stages of t cell maturation, with distinct t cell functions. the lymphokine repertoire of the memory t cell pool will be discussed with reference to the antigenic environment fmm which the cells are obtained. pancreatic islet allografts, gill, ronald g. and lafterty, kevin j., barbara davis center for childhood diabetes i univ. colo. health sciences center, e. th ave, box b- , denver, co. we studied the cellular interactions requkd for the rejeaion of cultured mhc class i-dispiuate islet allografts. this model was suitable for studying t-t collaboration in that islet allograft immunity is cd dependent but rejection of the cultured islet graft is mediated by the cd cell. recipient c by (b ) mice were grafted with mhc class idisparate b .c-h- bml (bml) islets beneath the mal capsule. islet grafts wen preaated for days in % oxygen culture to reduce immunogenicity. thirty days after grafting, recipient mice wen immunized with oe live spleen cells from the strains indicated below. rejection of the established graft was not trig-by challenge with donortype bml spleen cells, indicating that the mhc class i stimulus was insufficient to initiate all@ immunity. further, immunization with a mixture of m bml and oe mhc class ii-disparate b .c-h- bm (bml ) spleen cells failed to trigger host immunity. however, challenge with loe (bml x bml )fl spleen cells t r i g g d acute rejection of the established bml islet grafts. the requirement for l i i presmtatidrecognition of class i and class all* antigens to trigger allograft immunity indicates that the antigen-presenting (apc) plays an essential role for t-t collaboration in vivo. uature hla-dr complexes are purported to spontaneously internalize from and recycle to the plasma membrane of b but not t lymphocytes. using a neuraminidase protection assay, we have radiolabeled surface class i antigens on intact cells and cultured these cells under conditions which permit or prevent endocytosis; subsequently, surface glycoproteins on viable cells were desialylated and class i molecules were analyzed by iamunoprecipitation and two-dimensional gel electrophoresis. a panel of buman b lymphoblastoid cell lines and activated tonsillar b cell blasts failed to exhibit any internalization of class i complexes; control transferrin receptor molecules were endocytosed as ascertained by insensitivity to neuraminidase digestion. class ii+ pba blasts and sezary cells of the t lineage were also deficient in detectable hla-dr internalization. results did not vary regardless of the time allowed for efficient endocytosis ( ok ~saethionine). or the addition of anti-class i monoclonal antibody during the chase period for endocytosis. therefore. within the limits of sensitivity of this assay, class complexes do not appear to be internalized, either spontaneously or uben crosslinked by antibody. recycling represent a dynamic pathvay for regulating surface expression of class i antigens or a means of associating with and presenting foreign antigenic peptides. supported in part by usphs grants # t cao - and # eo a - . in previous studies of antigen-specific t cell responses in two distinct models of autoimmune tubulointerstitial nephritis (tin), viiia villosa lectin binding (vv+) t cells have been shown to be necessary for effector t cell expression and mediation of tin. in anti-tubular basement membrane disease, antigen-specifi vv+ t cells direct the phenotypic selection of cd + nephritogenic t cells in susceptible mouse strains (j. immunol. , nov. , ) . this function is mediated by an antigen-binding, i-js+ soluble protein factor. current studies investigate the role of the t cell glycoprotein which binds vv lectin in mediating w+ t cell function. using the previously described effector t cell induction assay, we found that n-acetyl-d-galactosamine (galnac) (at mm but not . mm) inhibits vv+ t cell function and cd + effector t cell selection. when cd + effector t cell differentiation occurs in the presence of soluble factors derived from antigen-primed vv+ t cells, galnac is not inhibitory. these studies suggested that soluble gal nac may competitively bind to a soluble protein which stimulates vv+ t cells, in part by binding to the w lectin receptor, to synthesize andlor secrete their biologically active soluble factor. as an additional test of this hypothesis, we prepared detergent solubilized membranes from vv+ t cells and purified vv lectin binding proteins by affinity chromatography. like galnac. these membrane derived lectin binding proteins also inhibit vv+ t cell function and cd + effector t cell selection. inhibition by soluble 'lectin receptors' is dose dependent and is demonstrable with lectin binding glycoproteins derived from - x lo cells, in an assay utilizing x vv+ cells. we are now further characterizing the lectin receptor and its endogenous ligand. elementary bodies and outer membranes of chlamydia trachomatis produce a high-titered igg response in rabbits and mice as measured by elisa and microscopic immunofluorescence assays. western blot analysis of total elementary body protein identifies a kd major outer membrane protein (momp) as the predominant antigen. to identify the cbmical structure of the epitope, purified momp was subjected to chemical and enzymatic fragmentation and the resulting peptides were purified by hplc and assayed for immunoreactivity. an immunoreactive kd cyanogen bromide peptide was amino-terminal sequenced and a series of overlapping synthetic peptides were synthesized and assayed for immunoreactivity. sequential single amino acid deletions at both the nhz and cooh termini allowed us to identify the precise epitope as a amino acid peptide spanning residues - of momp. two amino acid substitutions at positions (phe-gly) and (pro-gly) completely eliminated antibody binding. the -amino acid synthetic peptide is a potent immunogen producing high-titered antibody responses that are specific for the momp molecule. analysis of an independently derived mutant harboring the same defect has shorn that this trans-acting gene is not required for transport of class i molecules. class i heavy chain is synthesized in this cell line and associates with b m. transport of the class i appears to be blocked in the er or cis olgi as the majority of the class i glycoproteins are not processed to the endo b resistant form. the ability of the cell to significantly increase expression of surface class i when the incubation temperature is lowered from oc to oc suggests that this gene may function to stabilize a particular conformation of the protein. consistent with this is the increased sensitivity of class i molecules in the mutant as compared to the parent to degradation when cell lysates are incubated at elevated temperatures. the inability to immunoprecipitate class i antigens in the mutant is possibly due to the action of endogenous proteases present in these lysates. two complementing approaches are being employed to isolate this gene and further analyze its role in class i biosynthesis. the first involves inactivation of the trans-acting gene by insertion of a retroviral vector and subsequent pcr amplification of regions flanking the vector. in another approach a cdna library will be introduced into the mutant cell line and the cdna will be reisolated from cells reexpressing surface class i. houston. tx. we have induced a panel of highly immunogenic (imm+) vanants of the murine fibrosarcoma mca-f using i-methyl- niml-niuasoguanidine (mnng). -aza- '-deoxycytidine ( -azacdr). and uv radiation. these tumors grew m immunosuppressed mice. but were complelely rejecled by normal syngeneic hosts. mice thac had rejected large numbers of imm+ also developed a smng, tumor. specific immunity to the parental mor. lmmunizalion with low numbers of lmm+ engendered only variant-specific immunity. the frequency of imm+ variant g e n e d o n was similar for the three induction different protocols ( % to %), suggesling lhat generauon of imm+ was more closely relalcd to the cell line used than to the inducing agent however, the swngth of the imm+ phenaypc was related lo ihe agent used, since mnng induced clones had the m g e s t immunogeniciues and uv-b ihe weakest the smng neoantigens expressed by mnng induced imm+ were varunt-sppifc. while uv and s -d d r induced clones displayed significant cross-reactivilies not attributable to the parental t u n a antigen. increased or inappropriate expression of class-l mhc antigens did not correlate with the imm+ phenotype. we investigated the phenotypes of the spleen cells medrating tumor rejccuon using the local adopllve lransfer a s a y (lata). variant-specific immunity w mnng, -azacdr and uv induced imm+ were all m d a t c d by thy . +. l t +. lyc . -t cells. afrw immunization with high numbers of imm+ w engender both anti-lmm+ and anu-parental immunity. both cd + and cdw effectors rejecled the imm+ in lata, while only ihe c w + t cells could wnsfer resistance w the parent immunity the parenlal tumor anugen engendered by the imm+ suggeslcd associative recognition of the parental and neoantigem wgelher on ihe cell surface. this hypochfsis was supported by failure of lmm+ u) pmlect againu an antigenically disunct tumor (mca-d) admixed with it, either at the lime of immunization or at challenge. fusion of ihe h m * vanant with mca-d yielded a unique, hybrid parental umor antigen that was associatively recognized with rhc original imm+ neoanugen, demomirating the importance of antigen cocxpression. grant rr- - . w e have recently demonstrated and reported that substitution of anionic side chain carboxylic groups with aminoethylamide groups on protein antigens exhibits a pattern of enhanced immunogenicity both in vivo and in vitro. this enhanced immunogenicity was also observed in low responder strains of mice and we investigated the mechanism by which it is achieved. we examined antigen processing and presentation of native (nbsa) and modified bsa (mbsa) to t helper c e h isolated from c /bl low responder mice. a greatly reduced amount of mbsa than nbsa was required to activate both nbsa and mbsa primed th. proliferation of nbsa and mbsa primed t cells increased in proportion to the amount of time of exposure of the antigen presenting cells (apc) to nbsa, peaking at h. conversely, apc required less than min exposure to mbsa to achieve optimal activation, indicating rapid uptake of mbsa. paraformaldehyde fixed apc recognized mbsa without a lag phase processing, indicating that this event also occurred quite rapidly. apc processed nbsa w a s presented to primed t cells more effectively than the soluble antigen m shown by the increased rate of t cell proliferation. in contrast, mbsa was equally well presented to th cells by apc m in soluble unprocessed form. our data demonstrate that the reduced response in low responders is greatly enhanced by a modified antigen which is rapidly taken up and processed by apc. b cells which bear surface innunoglobulin (sig) receptors specific for a particular antigen are abile t o present fragments of that antigen very efficiently t o t cells. this i s due. in part, t o the high affinity of the receptor, which facilitates antigen binding at low concentrations. using tnp-abc and specific antigen, we have demonstrated that the tnp-abc process antigen very effectively. w e have compared specific antigen with i t s polyclonal analog, anti-ig, and demonstrated differences in the kinetics of degradation of anti-ig and tnp-antigens by tnp-aex. both antigen and anti-ig bound by tnp-abc are degraded into small fragments which are released into the supernatant. however, the following differences have been found: ) the rate of release of small fragments of tnp-antigen parallels the rate at which these cells become able to directly conjugate with t cells (a lneasure of antigen presentation), reaching a plateau between and hours. in contrast, the degradation of anti-ig and release of fragnents continues for hours. ) analysis of initial kinetics demonstrated that release of fragments of tnp-antigen begins minutes after binding; there i s no significant release of anti-ig fragrnents u n t i l about minutes. ) in contrast to anti-ig where there i s significant accumulation of degradation intermediates within the cells, there i s very l i t t l e intracellular accumulation of intennediate-size fragments of tnp-antigen. thus, we propose that the processing of antigen bound via specific sig may involve a specific intracellular pathway and that intracellular routing may be determined either by the degree of cross-linking of sig induced by antigen vs anti-ig or the mode of interaction of the various ligands with sig. alt*, departments of biochemistry (*) and medicine (+), college of physicians and surgeons of columbia univerity, new york, new york . we have recently analysed the structure of the / t cell receptor (tcr) expressed by the normal human thymocyte clone cii. cii expresses a c ' constant region that is a polymorphic form lacking a copy of an izternal exon; the sequence of this constant region accounts for the size of the chain and noncovalent linkage of and chains in the cii tcr. in order to elucidate its role, this / tcr will be reconstituted in immortalized t-cell lines. in addition, the productively rearranged human / receptor will be transgenically introduced into mice in order to assess the effect of the complete receptor on the development of t cells. the humoral immune response to human immunodeficiency virus has been shown to contain antibodies which act to mediate the uptake of virus through fc receptor mediated mechanisms. it is therefore possible that vaccination with the entire envelope polypeptide may present immunologic determinants that enhance infection. one means by which to generate an immune response to hiv that shall possess neutralizing activity in the absence of infection enhancing activity is to generate anti-idiotypic abs that bear the internal image of neutralizing human antibodies directed against hiv. we affinity purified human antibodies from hiv+ patients on a viral lysate column. we have produced monoclonal anti-idiotypic antibodies directed against these abi's. two of these monoclonals were shown to he ag inhibitable by their ability to inhibit the binding of p o l y e l o n a l human antisera to hiv viral lysate on ortho hiv ab t,est, wells. one monoclonal, b , when coupled t.o klh and used to immunize mice, produced an abs that. bound to viral lysate in an elisa assay. an affinity column containing rbs was used to purify an abi that was shown to bind to p and p hy western blot analysis. these data suggest that br may be a potential vaccine candidate. we have recently described a transgenic mouse model which co-expresses the tcr u and fl chains from the c cell line (recognized by the b anti-clonotype). t celh bearing the transgenic clonotype are positively selected by elements of the h-zb mhc for expression on cd ' cells. thus in the periphery of h-zb animals - % of the t cells are bz /cd *. the same peripheral expression is observed when the transgenes are expressed in f animals bearing a "neutral" mhc haplotype (eg. h-zb'*). however, when the transgene hi expressed in f animals which also express the h- ld gene product, negative selection occurs by clonal deletion. however, this deletion is functional rather than structural as the b clonotype is present on - % of peripheral t cells. these cells are unusual in that they express neither of the characteristic peripheral molecules cd or cd . the absence of cd expression on the b * cells appears to allow these potentially self-reactive clones to exist without evidence of autoimmunity. the original clone as well as b +/cd + cells from h- b animals are strongly inhibited by anti-cd reagents. in an effort to understand the process of negative selection and self-tolerance we have examined the capacity of these cells to be activated directly by the anti-clonotype rather than antigen (h- ld). the results demonstrate that the clonotype is fully functional on these double negative cells, indicating a normal maturation in the thymus. further examination of their surface phenotype also supports the conclusion that these are fully mature cells which are phenotypically distinct from double negative cells which exist in the thymus of h- b animals. of imunohematology. azl, leiden, the netherlands; praxis biologics, rochester, new york, usa and 'university of southampton, uk. immunity to disease caused by neisseria menineitidis is associated with the presence of bactericidal and opsonic antibodies to the capsular polysaccharide (cps), lipopolysaccharide and to outer membrane proteins (omps). the cps of group a and c meningococci are proven efficacious vaccines, although the immunogenicity in infants is poor and the immunity is of short duration. the combination with t-helper epitopes will certainly improve the immunogenic properties of these t-independent (ti,) antigens. the group b cps is poorly immunogenic in humans probably because of tolerance due to structural similarity to host glycopeptides and/or glycolipids. we have focused our research onto the class omps which show limited heterogeneity amongst meningococci. murine monoclonal antibodies to these proteins are highly bactericidal in vitro and will be used to map b-cell epitopes. t-epitopes have been identified by theoretical prediction of immunodominant sites by analysis of the amino acid sequence of the omp followed by their solid phase synthesis and subsequent testing for polyclonal activation of t-lymphocytes obtained from hl -typed volunteers immunized with the omp. in addition human t-cell clones are generated with omps and maintained with antigen, ebv-transformed b-cells, fresh feeders and ril . the clones are tested for antigen specificity, io vitro helper function, mhc restriction element, expression of surface markers and recognition of common meningococcal t-cell epitopes. c demonstration o f p-azobenzene-arsonate-l-tyrosine (aba-tyr) speclfic t cells in low responder h- mice by il- supported t cell proliferatlon previous studies have shown that h-zb mice immunized with aba-tyr fail to produce aba specific delayed-type hypersensitivity and show little or no t cell proliferation in vitro to aba-tyr. these observations suggest that h-zb mice are deficient in th cells that respond to aba-tyr. by contrast, immunization of h- b mice with tnp conjugates of aba-tyr revealed good cognate help, suggesting that these mice do possess aba-tyr specific th cells and that such cells are not revealed in conventional lymphoproliferative assays. because such assays are widely used to evaluate ir gene control and to map t cell epitopes, the databases generated from such studies may seriously under represent the total number of responder phenotypes and t cell epitopes. because of this concern, we established culture conditions that wlii support aba-tyr specific t cell proliferation in h- b mice. in these studies, c bu .l mice were immunized s.c. with aba-tyr and to days later the draining lymph nodes were cultured with varying doses of aba-tyr or with varying doses of aba-tyr and varying doses of recombinant il- alpha (rll-la), a known costimulator of th cells. culture with aba-tyr alone produced no proliferation. by contrast, culture with aba-tyr and rll-la revealed t cell proliferation that titrated with the dose of aba-tyr and the dose of rll-la specific for conalbumin presented on ryngeneic antigen presenting cells and dependent on il- for its proliferation, was used a s an indicator cell for the ability of neonatal murine spleen cells to present antigen and produce il- and il- .the antigen presenting capacity of neonatal spleen cells is low. during antigen presentation there is an augmentation of il- and il- production by the antigen presenting spleen cell population. however, neonatal spleen cells do not respond as well a s adult cells. the low levels o f il- can not be attributed t o a low potential for producing il- since neonatal cells produce high levels of il- after induction by a crude il- inducer factor (il- -if).the this impairment leads t o a decreased stimulus of the -helper cell to produce inducer factors which leads t o low levels of il- and il- production by the neonatal cells during antigen presentation. no suppressor mechanisms responsible for the l o w interleukin production were detected. human or murine class i genomic dna was transfected into a b-lymphohlastoid x t-lymphoblastoid hybrid cell line. this fusion hybrid has lost both t cell derived copies of chromosome six and contains deletions spanning the class i region on both copies of chromosome six derived from the b-cell parent. previous data have described a transacting factor within this region that is responsible for class i antigen expression. hla-bw and b glycoproteins, although synthesized, were not transported to the plasma membrane in the hybrid. were surface expressed. these data suggest a fundamental difference between human and mouse histocompatibility antigens in their requirements for intracellular transport. the role of glycans in this transport dicotomy is currently under investigation. in addition, hybrid human-murine genes are being used to identify regions of the class i molecules involved in this transport phenomenon. we probed t h e means by which t h e a n t l g e n p r e s e n t l n g c e l l (apc) handles t h e a n t i g e n produce p e p t i d e s t h a t bind t o mhc-molecules. we propose t h e e x i s t a n c e o f a new type o f i n t e r n a l image i n which immunoglobulin v-region peptides. formed by processing, imitate peptides from conventional a n t i g e n s . w e r e f e r t o such denatured i n t e r n a l images as r e s i d u e internal images, since t h e y a r e a s s o c i a t e d w i t h t h e r e s i d u e of p e p t i d e s remaining a f t e r processing. in some cases, r e s i d u e internal images may be actual sequence images, i . e . , the v-region sequence m y be i d e n t i c a l t o the conventional a n t i g e n sequence.to be class i h- ka-restricted cytolytic t lymphocytes (ctl) are directed against two immunodominant sites on the a/jap/ / influenza hemagglutinin (ha) that can be mimicked by synthetic oligopeptides spanning residues - in the ha and - in the hydrophobic, transmembrane region. analysis of the fine specificity of hal-specific ctl clones demonstrated that these ctl clones can be subdivided into at least two group based on their patterns of recognition of closely related influenza h n field strains and a monoclonal antibody derived variant of a/guiyang/ / . using a series of nested synthetic peptides spanning the - region, the minimal amino acid residues necessary for recognition by the two groups of ctl clones were defined and found to consist of two separate but overlapping sites. sequence comparison of the ha of the a/jap/ / , the influenza field isolates and the monoclonal antibody derived variant has identified two amino acids, asn at position and gly at position , that are critical for t cell recognition. thus, animo acid substitutions induced either by antigenic drift or by monoclonal antibody selection can affect class i ctl recognition. pretreatment with a n t m e s reactive with class mhc anti-has previously been reported to be successful in exov~~kj a n t i g e n -w i r q dendritic cells (m) fran rodent tissue grafts. we have extended these exper-to inta? whole organ grafts. ilia pnmxses also demcnstrated a prolonged survival ( f days) ccapared to controls ( t days) (p < . ) tihen v l a n t e d into streprozatocin treat& da recipients. antigenic variation in the haemagglutinin (ha) of influenza a viruses frequently introduces new oligosacchekide attachment sites ( aan-x-serlthreo) and carbohydrate addition prevents antibody recognition by steric hindrance. acid substitution in mutant viruses of the h n subtype (ha asp+asn), that introduces an n-glycosylation site (hn gcys &thr ), abrogates antibody and cd + t recognition. infected with x virus recognise a synthetic peptide corresponding to antigenic site e, ha - , and are sensitive to a single substitution (ha asp-basn) in mutant viruses. virus infected target cells, thereby confirming that carbohydrate addition prevents cd ' t cell recognition. here ve show that an amino cell i-ad restricted, ha specific t cell clones f r m balblc mice-reviously recognition of mutant viruses is restored however by tunicamycin-treatment of key: cord- -joyan ij authors: sewell, andrew k. title: why must t cells be cross-reactive? date: - - journal: nat rev immunol doi: . /nri sha: doc_id: cord_uid: joyan ij clonal selection theory proposed that individual t cells are specific for a single peptide–mhc antigen. however, the repertoire of αβ t cell receptors (tcrs) is dwarfed by the vast array of potential foreign peptide–mhc complexes, and a comprehensive system requires each t cell to recognize numerous peptides and thus be cross-reactive. this compromise on specificity has profound implications because the chance of any natural peptide–mhc ligand being an optimal fit for its cognate tcr is small, as there will almost always be more-potent agonists. furthermore, any tcr raised against a specific peptide–mhc complex in vivo can only be the best available solution from the naive t cell pool and is unlikely to be the best possible solution from the substantially greater number of tcrs that could theoretically be produced. this 'systems view' of tcr recognition provides a plausible cause for autoimmune disease and substantial scope for multiple therapeutic interventions. supplementary information: the online version of this article (doi: . /nri ) contains supplementary material, which is available to authorized users. jonathan kipnis's homepage: http://www.medicine.virginia. edu/basic-science/departments/neurosci/faculty/kipnis t cells recognize peptides bound to mhc class i and class ii molecules at the cell surface . the specificity of this recognition is conferred by the clonotypic αβ t cell receptor (tcr), which is made from two separate chains manufactured from variable (v), diversity (d), joining (j) and constant (c) gene fragments through a process of somatic gene rearrangement. this process involves nucleotide insertions and deletions at v(d)j junctions in each chain. the 'randomization' of v(d)j junctions and the fact that the tcr is a heterodimer of two separately rearranged chains results in a theoretical repertoire of > unique αβ tcrs in the mouse , . the theoretical number of possible tcrs in humans is likely to be orders of magnitude larger, as humans possess tcrβ variable genes as compared with the genes in mice, with all other variables being comparable . why must t cells be cross-reactive? abstract | clonal selection theory proposed that individual t cells are specific for a single peptide-mhc antigen. however, the repertoire of αβ t cell receptors (tcrs) is dwarfed by the vast array of potential foreign peptide-mhc complexes, and a comprehensive system requires each t cell to recognize numerous peptides and thus be cross-reactive. this compromise on specificity has profound implications because the chance of any natural peptide-mhc ligand being an optimal fit for its cognate tcr is small, as there will almost always be more-potent agonists. furthermore, any tcr raised against a specific peptide-mhc complex in vivo can only be the best available solution from the naive t cell pool and is unlikely to be the best possible solution from the substantially greater number of tcrs that could theoretically be produced. this 'systems view' of tcr recognition provides a plausible cause for autoimmune disease and substantial scope for multiple therapeutic interventions. the diversity of tcrs is based on the six complementarity-determining regions (cdrs), which engage both the peptide and the mhc molecule (fig. ) . typically, mhc class i and class ii molecules present peptides from endogenous and exogenous antigens, respectively. the mhc class i molecule has a closed-ended peptide-binding groove and binds peptides of - amino acids in length. longer peptides become increasingly distorted in the central region of the mhc class i molecule as the peptide length increases, resulting in peptide 'bulging' , . by contrast, the ends of the mhc class ii peptide-binding cleft are open, allowing even longer peptides to extend beyond this groove without bulging (fig. b,c) . the clonal selection theory , proposed that individual lymphocytes are specific for a single antigen and that the recognition of alternative ligands is unlikely. for many years the concept of huge numbers of tcrs successfully providing immunity to all foreign peptides in a 'one-clonotype-one-specificity' paradigm was accepted. however, several workers questioned this concept [ ] [ ] [ ] [ ] . most notably, don mason called for the abandonment of such a notion in his seminal thesis on the topic (see ref. ). many of the reasons for this paradigm shift were based on the simple arithmetic of effective immunity requiring the recognition of > potential foreign peptides. indeed, put in the context of t cells weighing > kilograms, the notion of immune coverage by a naive pool of monospecific tcrs as suggested by the clonal selection theory is clearly absurd . there are only t cells in a human, and more recent studies have estimated that there are < distinct tcrs in the human naive t cell pool . in humans, mhc molecules are encoded within the hla locus. the hla locus is the most polymorphic region of the human genome and is known to encode more than , allelic variants across the population, with a large number of these variants present at appreciable frequencies . some hla loci are among the fastest evolving coding regions in the human genome . each individual expresses six different classical peptide-presenting hla class i molecules (two hla-a, two hla-b and two hla-c) and six hla class ii molecules (two hla-dr, two hla-dq and two hla-dp). the expression of a wide variety of hla molecules ensures that individuals across the population present different antigenic peptides and provides the greatest chance that some individuals may survive any emerging infection. it is extremely difficult to link hla diversity to past pandemics, but evidence of the importance of infectious diseases in driving hla selection can be seen with current emerging infectious diseases. for example, homozygosity at hla class i alleles results in faster disease progression during hiv infection , and some hla class i alleles are associated with lower viral loads and protection from disease . various factors in addition to t cell immunity are thought to contribute to the maintenance of hla diversity, including natural killer cell recognition , mate selection , and transmissible tumours . overall, the fact that mutations that alter the amino the closed ends of the mhc class i binding groove cause long peptides to 'bulge' out of the binding groove, and this bulging increases with each additional amino acid in the peptide. by contrast, the ends of the mhc class ii binding cleft are open, which allows the accommodation of much longer peptides without the need for peptide kinking. d,e | the images show hla-a* (in grey) presenting the immunodominant glctlvaml peptide (stick model) from epstein-barr virus and hla-dr (in grey) presenting a peptide from myelin basic protein (mbp). tcrs dock on a peptide-mhc complex in a diagonal mode that is conserved for binding to mhc class i and class ii molecules. the colours indicate the docking footprints of the as tcr and msc- c tcr on their cognate peptide-mhc complexes and show the 'footprints' on the mhc complex of the six cdr loops. in general, the germline-encoded cdr and cdr loops interact mainly with the mhc molecule itself, whereas the hypervariable cdr loops sit over the peptide. however, the small structural database that has been compiled to date already contains examples in which cdr and cdr make substantial interactions with the peptide and in which cdr has an important role in contacting the mhc molecule , . tcr binding degeneracy and structure the recognition by tcrs of all hla molecules and a roughly conserved diagonal mode of binding on peptide-mhc complexes suggest that tcr interactions conform to some 'rules of engagement' (fig. ) . such rules have been proffered in the form of a tcr 'interaction codon' that interacts with mhc class ii molecules, and in the form of a 'restriction triad' that consists of three largely conserved residues in mhc class i molecules that interact with tcrs. these rules fit the generally observed arrangement of tcr-peptide-mhc interactions, in which the germline-encoded (that is, non-rearranged) cdr α, cdr β, cdr α and cdr β elements of the tcr contact the germline element of the mhc molecule, whereas the non-germline (that is, somatically rearranged) cdr α and cdr β loops contact the 'random' peptide element (fig. ) . however, these convenient rules fail to match all the structures of tcr-peptide-mhc complexes that have been generated to date , and mhc mutational studies show that the dependency on fixed pairwise interactions between a tcr and a peptide-mhc complex varies widely between individual tcrs . the peptide-mhc complex itself can also change its confirmation following tcr binding [ ] [ ] [ ] . thus, it is clear that tcr-peptide-mhc interactions are not rigidly conserved but rather allow for considerable flexibility within the confines of some general orientation and binding rules. the tumour-specific dmf tcr provides an excellent example of how large changes in tcr orientation can increase t cell cross-reactivity. the dmf tcr engages the nine-amino-acid ( -mer) peptide aagigiltv and the -mer peptide elagigiltv (which have overlapping sequences) in the context of hla-a* by adopting a different orientation for the two peptide-mhc complexes . tcr-binding plasticity can extend beyond different peptide binding registers or different peptide binding angles on peptide-mhc complexes because the cdr loops can be extremely flexible , . the mouse c tcr structure has been solved in complex with eqykfysv-h -k b (ref. ), eqykfysv-h -k bm (ref. ), siyryygl-h -k b (ref. ) and box | extensive t cell cross-reactivity and apparent specificity are not incongruous from the proteinogenic amino acids, it is possible to generate vast numbers of peptides of a length that can be presented by mhc molecules (see the table). t cells are specific because any given t cell can recognize only a tiny fraction of the 'universe' of peptides that can be presented by any given mhc molecule, but they are multispecific because the peptide universe is so large. by way of example, a t cell that recognizes million -mer ( -amino-acid) peptides will have less than a in million chance of recognizing any -mer peptide chosen at random from the entire peptide universe. these numbers indicate that if a t cell that recognizes million different -mer peptides was tested for recognition of random -mer peptides at a rate of every minute then on average it would take over years before a cross-reaction was seen! even the total number of overlapping peptides that can be made from the entire human proteome is an extremely small fraction of all possible peptides (for example, fewer than of the total possible number of -mer peptides (> ) can be made from the human proteome). in the environment in which t cells function, the important number is the frequency of functional recognition of unrelated peptides that can be processed and presented by mhc molecules. assuming that just % of possible peptides are presented by an mhc molecule, then the functional recognition of -mer peptides by a single tcr translates into a frequency of cross-reactivity of in , , which is in good accord with an experimental attempt to directly measure this parameter . thus, the sheer size of the possible peptide universe allows t cells to be enormously cross-reactive while appearing to be very specific within the environment in which they are required to operate. acid sequence of hla class i and class ii molecules are clustered around the peptidebinding cleft and often alter the peptide sequence that is preferentially bound by the hla molecule [ ] [ ] [ ] strongly suggests that hla diversity is upheld to increase the variety of peptides displayed. the tcr recognizes peptide antigens presented by all hla variants. unlike the b cell receptor, the protein sequence of the tcr is fixed, and the tcr never undergoes affinity maturation. thus, tcrs expressed by naive t cells are required to respond to all foreign antigens despite never having encountered them before and being unable to adapt to them at the protein sequence level. if the tcr repertoire was unable to recognize virtually all foreign peptides bound to self mhc molecules, then pathogens -which usually evolve many millions of times faster than their vertebrate hosts -would be expected to rapidly evolve to exploit these t cell 'blind spots' and overwhelm the host. it is difficult to conceive of any obvious universal mechanism that might transmit knowledge of 'presentable' epitopes from previous infections between generations within the tcr cdr loops . in the absence of 'prior knowledge' of the epitopes that might be encountered, t cell immunity must provide immune cover for all possible foreign peptides that contain appropriate anchors for binding to self mhc molecules . this universal cover represents a major challenge to the immune system, as the possible array of peptides that can be manufactured from the proteinogenic amino acids of a length that can bind to self mhc molecules is vast (> ) (box ). in fact, the theoretical number of possible peptides that t cells might provide immunity to is even greater, as it is possible to raise specific t cell responses to peptides that contain amino acids with post-translational modifications, such as glycosylation , citrullination , phosphorylation , , cysteinylation and dimerization , . thus, the number of potential foreign peptide-mhc complexes that t cells might encounter dwarfs the number of tcrs available. here, i consider how the challenge of this disparity has been met by compromising on antigen specificity so that individual t cells are capable of responding to enormous numbers of different peptide-mhc complexes. this inevitable, extensive t cell cross-reactivity has some profound consequences, including providing a plausible cause for autoimmune disease. i also discuss how the consequences of tcr binding degeneracy offer substantial scope for multiple therapeutic interventions. the recently described e tcr -which was isolated from a patient with type diabetes and which recognizes residues - of the preproinsulin molecule (ppi [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] presented in the context of hla-a* (ref. ) -does not undergo structural rearrangements following ligand binding but is still hugely cross-reactive. despite a rigid 'lock and key' binding mode, t cells expressing the e tcr respond to over . million -mer peptides at least as strongly as they respond to the ppi - peptide , . peptides were identified that were > -fold more potent than ppi - at activating e tcr-expressing t cells but that differed from ppi - at seven of the ten amino acid positions . this promiscuity is explained by the structure of the e tcr-ppi - -hla-a complex, in which the tcr exhibits peptide-centric binding that is focused on just two amino acids in the peptide . this residue-focused mode of binding presumably allows for substitutions at other positions that, in some cases, must considerably stabilize the interaction. in another example of such peptide-centric binding, a single amino acid interchange within two hiv envelope epitopes was shown to reciprocally swap the specificities of two cd + t cell clones , suggesting that a dominant focus on a single amino acid residue in the peptide within a peptide-mhc complex might be reasonably common. indeed, the tcr-peptide-mhc structures that have been described to date show that usually only a few upward-facing residues from the peptide contribute to the inter action of the tcr with the peptide-mhc complex. thus, data from the limited number of tcr structures available indicate that tcrs can exhibit substantial binding degeneracy by being extremely flexible and/or through a focused interaction that is dominated by a few peptide residues (fig. ) . together, this binding promiscuity at the tcr interface and the flexible mhcbinding 'motifs' that often allow the accommodation of several amino acids at primary mhc anchor positions enable a substantial number of peptides to act as agonists for any given tcr. it is possible to generate vast numbers of peptides of the length recognized by t cells from the proteinogenic amino acids . even conservative estimates predict that substantially more than % of these peptides will possess anchors that allow them to bind to any single mhc molecule. taking -mer peptides as an example, it is possible to generate > different peptides of a | macro-level changes enable the t cell receptor (tcr) to bind to peptide-mhc complexes with an altered peptide binding angle (red dotted line) and/or peptide binding register (black dotted line) within a roughly diagonal binding mode . the cartoon shows 'footprints' of the tcr complementaritydetermining region (cdr) loops projected down onto the peptide-mhc platform. b | micro-level cdr loop flexibility enables the accommodation of different peptide-mhc 'landscapes'. the cartoon shows a side view of a tcr engaging a peptide-mhc complex. c | structural studies show that most tcrs focus on two to four upward-facing peptide residues. in this example, the tcr is focused on the two peptide residues shown in red. such residue-focused interaction allows the tcr to tolerate multiple amino acid substitutions at other positions in the peptide (indicated by different colours). the above examples are not mutually exclusive and represent only some of the possibilities. mhc-binding motifs often allow for different residues at primary mhc anchors . it should also be noted that tcrs can change the conformation of the peptide-mhc complex following engagement - . amino acids in length from the amino acids. assuming that at least % (> ) of these peptides can bind to a given self mhc molecule, a heterozygous human antigenpresenting cell could theoretically present more than × different -mer peptides on its six mhc class i molecules and six mhc class ii molecules. furthermore, as mhc class ii molecules can present longer peptides that can 'frame-shift' within the open-ended binding groove (fig. ) , mason calculated that each mhc class ii molecule could theoretically present almost different -mer peptides, assuming that % of all peptides associate with mhc class ii molecules , and this is without even considering the possibility of post-translational modifications. in summary, the number of potential peptide antigens exceeds the number of tcrs available to respond to them by many orders of magnitude, so t cells can only provide comprehensive immune cover if each one is capable of recognizing many peptides. the theoretical arguments of mason suggesting that t cells must each recognize on average at least million individual peptides have recently gained traction as a result of data that demonstrate this level of cross-reactivity and provide plausible structural mechanisms for its occurrence. all t cells are 'auditioned' in the thymus and only those that react weakly with a self peptide-mhc ligand are positively selected . t cells bearing tcrs that react strongly to self antigens are 'culled' at this stage. extensive tcr binding degeneracy and cross-recognition of peptide-mhc molecules by thymocytes has been elegantly demonstrated by studies showing that a remarkably comprehensive t cell repertoire can be selected by a single peptide and that the resulting t cells can be activated by peptides that are unrelated in sequence to the peptide that they were selected on . further compelling evidence that t cells can exhibit extensive cross-reactivity comes from studies with combinatorial peptide libraries that comprise almost all possible peptides of a particular length , , [ ] [ ] [ ] [ ] . these libraries are usually used as a series of sub-libraries laid out in positional-scanning format such that there is a sub-library with each amino acid fixed in each position and with all other positions made up of an equimolar mix of the remaining amino acids (of note, cysteine is generally excluded from the 'random' positions to avoid problems of oxidation) (see supplementary information s (figure)). studies with these libraries in t cell activation assays indicate that agonist ligands can contain several different amino acids at many positions. several studies have gone on to use this approach to prove the 'mason hypothesis' and show that individual t cell clones really can recognize over a million different individual peptides in the context of a single mhc molecule , , . the antigen sensitivity of a t cell and its ability to respond to weaker tcr ligands are inexorably linked. t cell sensitivity to an antigen is not a fixed parameter. memory t cells can recognize concentrations of a peptide antigen that are > -fold lower than those recognized by naive t cells , , and individual t cell clones can generate progeny with both high and low antigen sensitivities . antigen sensitivity can be regulated by changes in tcr expression levels or clustering on the cell surface, by changes in the expression or function of co-stimulatory molecules, by differential control of phosphatase pathways that dampen t cell signalling or by alterations in the glycosylation status of the tcr or other cell-surface molecules (reviewed in ref. ). although these mechanisms may regulate the antigen sensitivity of t cells, and thus the ability of t cells to cross-recognize weak tcr ligands, it is difficult to conceive how they might be used to tune the biophysics of tcr engagement with a specific ligand. by contrast, the cd and cd glycoproteins have a unique role in 'co-receiving' peptide-mhc molecules by binding to largely invariant sites on mhc class ii and mhc class i molecules, respectively . thus, these coreceptors might possess an ability to differentially regulate the responsiveness of the tcr to the ligand and thereby modulate tcr specificity . indeed, cd is known to affect both the on-rate , and off-rate , of tcr-peptide-mhc class i engagement and therefore can modulate the kinetics of tcr binding by different peptide-mhc ligands. we have demonstrated how the strength of the peptide-mhc class i-cd interaction can have substantial effects on t cell cross-reactivity . it is important to realize that, although the tcr sequence is invariant, tcr sensitivity to agonist ligands (and therefore t cell cross-reactivity) is not fixed and can be varied throughout development by a number of parameters . the idea that immune cover is provided by limited numbers of highly cross-reactive t cells has both positive and negative implications. the presence of pools of cross-reactive t cells that each recognize large numbers glossary altered peptide ligands (apls) . peptide analogues that are derived from an original antigenic peptide. they commonly have amino acid substitutions at residues that contact the t cell receptor (tcr) and alter tcr engagement, resulting in different activation consequences than those induced by the wild-type ('index') antigenic peptide. a measure of how sensitive t cells are to the density of cognate antigen on the antigen-presenting cell surface. t cell receptor (tcr) affinity for a peptide-mhc complex has a large role in antigen sensitivity, but the parameter is also affected by the expression of other molecules that influence cell-cell contact or the downstream signal transduction that results from tcr-peptide-mhc engagement. a theory proffered by niels jerne which states that there is already a vast array of lymphocytes in the body before any infection. any challenge with antigen selects, and clonally expands, a single corresponding lymphocyte (b cell or t cell) from the pre-existing lymphocyte pool of differing specificities, and this clonal lymphocyte population then eliminates the antigen. (cdrs). the regions within antigen receptors that complement the shape of an antigen. the cdrs are the most variable part of the antigen receptor and are largely responsible for the diversity in these molecules. the cdrs allow antibodies and t cell receptors to recognize a vast repertoire of antigens. the term used to describe how an immune response to a pathogen can provide immunity to a non-identical pathogen. heterologous immunity can be mediated by cross-reactive t cells or antibodies. resemblance between epitopes contained in microbial and host proteins, leading to cross-reactivity of t cells in the host. a 'footprint' of immune responses is established during the first exposure to a pathogen. these specific memory t cell populations are preferentially re-expanded when re-exposed to the same antigen or one that is similar, thereby limiting the clonal expansion of new antigenspecific t cells. a similar mechanism has been proposed for b cell responses. the reaction of t cells to more than one distinct peptide-mhc ligand. refers to the promiscuity of t cell receptor (tcr) engagement that allows a single tcr to bind to different peptide-mhc complexes. pathogen-derived peptide self peptide t cell priming cross-recognition autoimmune attack tcr tissue cell of peptides but that do not respond to self peptides in the periphery has a number of positive consequences. first, a cross-reactive t cell repertoire generates a near perfect solution to the huge challenge of providing effective immune cover by allowing a limited number of t cells to provide immunity against virtually all foreign peptides that can bind to self mhc molecules. second, a system with a limited number of hugely cross-reactive t cells is both temporally and spatially favourable, as far fewer t cells are needed to scan any infected cell than if the clonal selection theory was rigidly upheld. third, the corollary of extensive t cell crossreactivity is that several tcrs are likely to recognize any one peptide (and thus that t cell responses are polyclonal). polyclonal recognition of peptide-mhc molecules makes it substantially more difficult for pathogens to escape immune recognition, as a mutation that escapes recognition by one tcr might be recognized by another. fourth, extensive t cell cross-reactivity also provides excellent conservation of resources by generating 'one weapon with several triggers' . several documented examples show that an individual t cell clone can target more than one infection through different peptides, a phenomenon known as heterologous immunity . heterologous immunity between related pathogens is common. it is well known that immunity to cowpox provides cover for smallpox , and the tuberculosis vaccine bacterium mycobacterium bovis bacillus calmette-guérin (bcg) can provide some protection against leprosy . but, the existence of extensive t cell crossreactivity means that heterologous immunity can extend beyond the cross-recognition of pathogens with high sequence similarity to allow, for example, bcg-induced t cells to also provide immunity against poxviruses . similarly, cd + t cells specific for the human papillomavirus hla-a -restricted ymldlqpet peptide also recognize the hla-a -restricted tmldiqped peptide from coronavirus . indeed, cd + t cellmediated heterologous immunity can extend to very dissimilar antigens. for example, cells that are specific for the immunodominant gilgfvftl peptide from influenza virus can often recognize the epstein-barr virus epitope glctlvaml or the immuno dominant hiv-derived slyntvatl antigen (all of which are hla-a restricted). the extent of heterologous immunity and its importance to human immunity is not yet fully known. the potential positive outcomes of this phenomenon are clear, but heterologous immunity could also have deleterious effects. documented negative consequences of heterologous immunity include influenza-specific cd + t cells contributing to lymphoproliferation in epstein-barr virus-associated mononucleosis or cross-recognizing a peptide derived from hepatitis c virus (hcv) , which increases the severity of hcv-associated liver pathology . it is also possible that heterologous immunity via t cell cross-reactivity could encourage a suboptimal response to the second pathogen owing to 'original antigenic sin' . this antigenic sin could extend beyond the simple case of suboptimal sensitivity to the second antigen to a situation in which the original antigen has established a t helper (t h )-t h -or t h -type response bias that is inappropriate for the second pathogen. however, the most obvious and detrimental consequence of t cell cross-reactivity to vast numbers of individual peptides is the potential such a system has for causing autoimmunity (fig. ) . although strongly selfreactive t cells are deleted in the thymus , weakly cross-reactive t cells may survive and become activated in the periphery through the cross-recognition of peptides from infectious agents, a phenomenon known as molecular mimicry [ ] [ ] [ ] [ ] . memory t cells can be stimulated by peptide concentrations more than -fold lower than those required to stimulate naive t cells , . it is therefore likely that a memory t cell could be stimulated by a cross-reactive peptide with an affinity for the tcr that is far lower than that of the original pathogen-derived peptide. in such a situation, pathogen-mediated priming would be obligatory before functional crossrecognition of a self peptide, a notion that is consistent with the observation that infection can precipitate autoimmune diseases , . the compromise imposed by t cells being hugely cross-reactive in order to provide complete immune cover dictates that an individual tcr-peptide-mhc pairing is highly likely to be suboptimal. thus, it should be possible to improve the binding of any given tcr to its cognate antigen by enhancing the specific molecular matching. indeed, yeast display , phage display and computational design , have been used to produce tcrs that bind to peptide-mhc complexes with extremely high affinities (k d < pm) and half-lives of many hours. the mhc class i pathway is predicted to present at least one peptide at the cell surface from every internally produced protein . this allows tcrs to potentially target any cell based on its expression of any protein (fig. a) . consequently, tcrs might have considerable advantages over regular antibody-based therapies, as they can target a substantially greater number of cellular proteins. furthermore, there is now substantial evidence that it is possible to improve the affinity of almost any peptide antigen for a given natural tcr. thus, there is ample scope for the rational design of therapeutic interventions that exploit the fact that most natural tcr-peptide-mhc interactions can be improved upon. enhanced tcrs in tcr gene transfer therapy. the rigours of thymic selection ensure that natural tcrs bind to ubiquitous self or tumour-associated antigens with substantially lower affinities than they bind to pathogen-derived antigens . natural tcr-peptide-mhc interactions have affinities (measured in terms of k d ) in the , . within this range of tcr binding affinities, the affinity and/ or half-life correlates with antigen sensitivity , , placing natural antitumour t cells at a distinct disadvantage compared with their pathogen-reactive counterparts. the transfer of tcr genes into recipient host t cells followed by the adoptive transfer of the t cells to patients allows the passive transfer of immunity and can provide a useful mechanism for breaking tolerance to tumour antigens . this strategy has already shown some promise in patients with malignant melanoma , but there is room for improvement. the transfer of genes encoding tcrs that have been affinity matured to bind to tumourassociated peptide-mhc complexes with affinities as high as those of the best antiviral t cells (k d = nm) enhanced tcrs as soluble therapies. highaffinity soluble tcrs provide an efficient means for the cellular targeting of intracellular antigens that are presented by mhc molecules in vivo (fig. a) . soluble tcrs can be linked to other molecules, such as antibody fab fragments, and can deliver these molecules to sites of antigen expression in vivo . despite the low copy number of most peptide-mhc molecules (< copies per cell), we have recently used a soluble tcr fused to a cd -specific fab fragment to induce tumour regression in vivo . these bispecific t cell-engaging tcrs function by recruiting polyclonal t cells via the cd specific fab component but do not by themselves crosslink tcrs or induce t cell activation. once these molecules are bound to a target cell surface, they become potent activators of antigen-experienced cd + t cells and promote the lysis of targets expressing as few as ten cognate peptide-mhc complexes (fig. b) . a similar approach could be used to dampen autoimmunity by crosslinking inhibitory receptors such as cytotoxic t lymphocyte antigen (ctla ). the fact that any tcr will be capable of recognizing enormous numbers of ligands paves the way for therapies based on altered peptide ligands (apls). apls can have advantages over natural ligands, as they can bind strongly to tcrs and can break tolerance to self ligands (including tumour-derived ligands). previous assumptions about apls, such as the suggestion that altering a buried anchor residue will not substantially alter tcr binding, have proved to be incorrect . nevertheless, combinatorial screening of peptide (or non-peptide) ligands can be used to determine the preferred binding 'landscape' of any tcr and circumvent the requirement for any assumptions. the nature of the system makes it highly likely that each tcr has a different preferred binding landscape. this then enables relatively precise targeting of specific tcrs within populations of antigen-specific t cells through a process termed tcr-optimized peptide skewing of the repertoire of t cells (topsort), which can be used to sort the most effective clonotypes (fig. ) . the widespread applicability of figure | enhanced tcrs as soluble therapies. a | the mhc class i presentation pathway presents peptides at the cell surface from intracellular proteins. this potentially allows soluble high-affinity 'monoclonal' t cell receptors (tcrs) to target any cell based on its expression of any protein. 'monoclonal' tcrs are able to use the mhc class i presentation pathway to 'see inside' cells and scan them for internal anomalies. this 'x-ray vision' opens up access to a far greater range of disease-relevant antigens than are available for monoclonal antibodies. tcrs can be engineered to deliver a variety of molecules that stimulate or suppress the immune system. potential 'payloads' include antibody fab fragments that then deliver a signal to immune cells. as mhc-bound peptides are often present at low copy numbers (< copies per cell), the payloads delivered by tcrs must act at very low concentrations. b | high-affinity tumour-specific tcrs that are manufactured as bispecific t cell-engaging molecules by linking them to cd -specific fab fragments can direct the lysis of tumour cells by cd + t cells and thereby induce the regression of established tumours . these molecules do not activate t cells as monomers at the concentrations used. t cell-engaging tcrs bind to the cognate antigen on the tumour cell surface with long half-lives and 'present' the linked cd -specific fab fragments. these fab fragments then crosslink tcrs on the surface of antigen-experienced cd + t cells, resulting in cellular activation and elimination of the target cell . the delivery of toxins with soluble tcrs is not recommended, as the soluble tcr constructs are taken up by scavenging cells such as macrophages. thus, molecules that deliver a particular signal to a specific effector cell are preferable. for example high-affinity tcrs could be used to downregulate immune responses by signalling through inhibitory receptors such as cytotoxic t lymphocyte antigen (ctla ) (not shown). this approach is dependent on the effective clonotype being 'public' (that is, occurring in all individuals with the restricting hla molecule) or having a public motif that is shared by all individuals with the relevant hla molecule. our own preliminary studies using ex vivo peripheral blood mononuclear cells show that this approach can be used to skew the clonotypes that respond to a tumour antigen (j. ekeruche-makinde et al., unpublished observations). a similar approach could be used to skew the clonotypes induced by a vaccination against hiv towards those that are known to be more difficult for hiv to escape from. accumulating evidence, including direct estimates of the total number of tcrs in a human, supports mason's notion that we should abandon the 'one-clonotypeone-specificity' paradigm suggested by clonal selection theory in favour of a 'one-clonotype-millions-of-specificities' reality. the simple arithmetic of t cell immunity allows t cells to be highly cross-reactive while appearing to be exquisitely specific in the environment in which they are expected to function . however, the realities of t cell immunity dictate that tcrs are very rarely an optimal fit for a real antigen and that real mhc-presented peptide antigens are rarely the optimal agonists for a given tcr. this compromise provides multiple opportunities for rational therapeutic interventions based on the directed manipulation of t cell immunity. clonotypic t cell receptors (tcrs) that recognize the same antigen are not all equal, and one tcr may provide the most effective immunity. in the case of hiv for example, one tcr may be more difficult for the virus to escape from than other tcrs. if the required tcr is public (that is, it occurs in all individuals with the restricting hla molecule) or has a public-type motif, then a tcr-optimized peptide for this clonotype could be used to skew the response towards the most effective clonotype(s). there are no known rules that enable the prediction of which tcrs a particular ligand will stimulate. thus, this process requires pre-testing using in vitro priming assays to ensure that it induces the required clonotype(s) while minimizing the induction of suboptimal clonotypes. ligand recognition by αβ t cell receptors t cell receptor gene diversity and selection t-cell antigen receptor genes and t-cell recognition imgt, the international immunogenetics information system how tcrs bind mhcs, peptides, and coreceptors conformational restraints and flexibility of -meric peptides in complex with hla-b* t cell receptor recognition of a 'super-bulged' major histocompatibility complex class i-bound peptide the natural-selection theory of antibody formation the somatic generation of immune recognition a very high level of crossreactivity is an essential feature of the t-cell receptor specificity and degeneracy of t cells polyspecificity of t cell and b cell receptor recognition structural basis for t cell recognition of altered peptide ligands: a single t cell receptor can productively recognize a large continuum of related ligands a direct estimate of the human αβ t cell receptor diversity imgt/hla and imgt/mhc: sequence databases for the study of the major histocompatibility complex a uniquely high level of recombination at the hla-b locus hla and hiv- : heterozygote advantage and b* -cw* disadvantage impact of mhc class i diversity on immune control of immunodeficiency virus replication in search of the 'missing self': mhc molecules and nk cell recognition discrimination of mhc-derived odors by untrained mice is consistent with divergence in peptide-binding region residues control of mating preferences in mice by genes in the major histocompatibility complex transmission of a fatal clonal tumor by biting occurs due to depleted mhc diversity in a threatened carnivorous marsupial positive darwinian selection promotes charge profile diversity in the antigen-binding cleft of class i majorhistocompatibility-complex molecules population biology of antigen presentation by mhc class i molecules hla supertypes and supermotifs: a functional perspective on hla polymorphism mammalian n-glycan branching protects against innate immune self-recognition and inflammation in autoimmune disease pathogenesis arthritis induced by posttranslationally modified (citrullinated) fibrinogen in dr -ie transgenic mice phosphorylation-dependent interaction between antigenic peptides and mhc class i: a molecular basis for the presentation of transformed self phosphorylated self-peptides alter human leukocyte antigen class i-restricted antigen presentation and generate tumor-specific epitopes modification of cysteine residues in vitro and in vivo affects the immunogenicity and antigenicity of major histocompatibility complex class i-restricted viral determinants the hla-a* -restricted h-y antigen contains a posttranslationally modified cysteine that significantly affects t cell recognition structural evidence for a germline-encoded t cell receptor-major histocompatibility complex interaction 'codon' hard wiring of t cell receptor specificity for the major histocompatibility complex is underpinned by tcr adaptability t cell receptor cross-reactivity directed by antigen-dependent tuning of peptide-mhc molecular flexibility structure of the complex between human t-cell receptor, viral peptide and hla-a a t cell receptor flattens a bulged antigenic peptide presented by a major histocompatibility complex class i molecule tcrs used in cancer gene therapy cross-react with mart- /melan-a tumor antigens via distinct mechanisms conformational changes and flexibility in t-cell receptor recognition of peptide-mhc complexes disparate degrees of hypervariable loop flexibility control t-cell receptor cross-reactivity, specificity, and binding mechanism structural basis of plasticity in t cell receptor recognition of a self peptide-mhc antigen structural comparison of allogeneic and syngeneic t cell receptor-peptide-major histocompatibility complex complexes: a buried alloreactive mutation subtly alters peptide presentation substantially increasing vβ interactions a functional hot spot for antigen recognition in a superagonist tcr/mhc complex how a single t cell receptor recognizes both self and foreign mhc a single t cell receptor bound to major histocompatibility complex class i and class ii glycoproteins reveals switchable tcr conformers ctls are targeted to kill β cells in patients with type diabetes through recognition of a glucose-regulated preproinsulin epitope structural basis for the killing of human β cells by cd + t cells in type diabetes a single autoimmune t cell receptor recognizes more than a million different peptides a single amino acid interchange yields reciprocal ctl specificities for hiv- gp allele-specific motifs revealed by sequencing of self-peptides eluted from mhc molecules positive and negative selection of t cells the repertoire of t cells shaped by a single mhc/peptide ligand t cells can be activated by peptides that are unrelated in sequence to their selecting peptide cd controls t cell crossreactivity antigen arrays in t cell immunology exploring immunological specificity using synthetic peptide combinatorial libraries structure of an autoimmune t cell receptor complexed with class ii peptide-mhc: insights into mhc bias and antigen specificity quantitative determination of tcr cross-reactivity using peptide libraries and protein databases cd + memory t cells (cd high , ly- c + ) are more sensitive than naive cells (cd low , ly- c -) to tcr/cd signaling in response to antigen response of naive and memory cd + t cells to antigen stimulation in vivo cutting edge: cd + t cell clones possess the potential to differentiate into both high-and low-avidity effector cells tricks with tetramers: how to get the most from multimeric peptide-mhc the t cell receptor as a multicomponent signalling machine: cd /cd coreceptors and cd in t cell activation coreceptor cd -driven modulation of t cell antigen receptor specificity cd kinetically promotes ligand binding to the t-cell antigen receptor different t cell receptor affinity thresholds and cd coreceptor dependence govern cytotoxic t lymphocyte activation and tetramer binding properties interaction between the cd coreceptor and major histocompatibility complex class i stabilizes t cell receptor-antigen complexes at the cell surface cd modulation of t-cell antigen receptor-ligand interactions on living cytotoxic t lymphocytes no one is naive: the significance of heterologous t-cell immunity the history of the smallpox vaccine the role of bcg in prevention of leprosy: a metaanalysis cd t-cell-mediated heterologous immunity between mycobacteria and poxviruses human papillomavirus type e peptide-directed cd + t cells from patients with cervical cancer are cross-reactive with the coronavirus ns protein broad cross-reactive tcr repertoires recognizing dissimilar epstein-barr and influenza a virus epitopes cross-reactivity between hla-a -restricted flu-m : - and hiv p gag: - epitopes in hiv-infected and uninfected individuals cross-reactive influenza virus-specific cd + t cells contribute to lymphoproliferation in epstein-barr virus-associated infectious mononucleosis cross-reactivity between hepatitis c virus and influenza a virus determinantspecific cytotoxic t cells heterologous t cell immunity in severe hepatitis c virus infection molecular mimicry in t cell-mediated autoimmunity: viral peptides activate human t cell clones specific for myelin basic protein molecular mimicry and immunemediated diseases molecular mimicry by herpes simplex virustype : autoimmune disease after viral infection molecular mimicry and autoimmunity infection, mimics, and autoimmune disease selection of functional t cell receptor mutants from a yeast surface-display library directed evolution of human t-cell receptors with picomolar affinities by phage display cutting edge: evidence for a dynamically driven t cell signaling mechanism interplay between t cell receptor binding kinetics and the level of cognate peptide presented by major histocompatibility complexes governs cd + t cell responsiveness human tcr-binding affinity is governed by mhc class restriction control of hiv- immune escape by cd t cells expressing enhanced t-cell receptor kinetic proofreading in t-cell receptor signal transduction adoptive immunotherapy for cancer: harnessing the t cell response cancer regression in patients after transfer of genetically engineered lymphocytes monoclonal tcr-redirected tumor cell killing modification of mhc anchor residues generates heteroclitic peptides that alter tcr binding and t cell recognition bias in the αβ t-cell repertoire: implications for disease pathogenesis and vaccination quantitating t cell cross-reactivity for unrelated peptide antigens genetic and structural basis for selection of a ubiquitous t cell receptor deployed in epstein-barr virus infection structure of a tcr with high affinity for selfantigen reveals basis for escape from negative selection germ line-governed recognition of a cancer epitope by an immunodominant human t-cell receptor the shaping of t cell receptor recognition by self-tolerance we thank s. smith and n. watson for editing the manuscript. we thank the members of the kipnis laboratory for their valuable comments during multiple discussions of this work. n.c.d. is the recipient of a hartwell foundation postdoctoral fellowship. this work was primarily supported by a grant from the us national institute on aging, national institutes of health (award ag to j.k.). heath park, cardiff, uk. e-mail: sewellak@cardiff.ac.uk my studies in this area were made possible by the generous support of the uk biotechnology and biological sciences research council to my colleagues and myself (grant bb/ h / ). i thank d. cole and b. baker for helpful discussions. the authors declare no competing financial interests. the author declares no competing financial interests. andrew k. sewell's homepage: http://www.tcells.org see online article: s (figure) key: cord- -ss g jkg authors: jakhar, renu; gakhar, s.k title: an immunoinformatics study to predict epitopes in the envelope protein of sars-cov- date: - - journal: biorxiv doi: . / . . . sha: doc_id: cord_uid: ss g jkg covid- is a new viral emergent human disease caused by a novel strain of coronavirus. this virus has caused a huge problem in the world as millions of the people are affected with this disease in the entire world. we aimed to design a peptide vaccine for covid- particularly for the envelope protein using computational methods to predict epitopes inducing the immune system and can be used later to create a new peptide vaccine that could replace conventional vaccines. a total of available sequences of sars-cov- were retrieved from ncbi for bioinformatics analysis using immune epitope data base (iedb) to predict b and t cells epitopes. then we docked the best predicted ctl epitopes with hla alleles. ctl cell epitopes namely interacted with mhc class i alleles and we suggested them to become universal peptides based vaccine against covid- . potentially continuous b cell epitopes were predicted using tools from iedb. the allergenicity of predicted epitopes was analyzed by allertop tool and the coverage was determined throughout the worlds. we found these ctl epitopes to be t helper epitopes also. the b cell epitope, srvknl and t cell epitope, flafvvfll were suggested to become a universal candidate for peptide-based vaccine against covid- . we hope to confirm our findings by adding complementary steps of both in vitro and in vivo studies to support this new universal predicted candidate. as we all know the corona virus has stopped the movements of the entire world. this virus is so deadly that it is taking lives of the more than thousands of people every day and affecting millions of people on the globe. although the disease was first reported in the wuhan city of china, where the virus was isolated from a patient with respiratory symptom in dec , [ , ] later identified it by the name of covid- [ ] . world health organization (who) announced this disease as pandemic disease that spread from china to more than a hundred countries in the world. by may , , the disease had already struck more than million persons of whom thousands of peoples died from covid- infection majority of them were reported from china, italy, united state of america, britain and spain. corona viruses are the large group of viruses belonging to the family coronaviridae and the order nidovirales that are common among animals [ ] . the coronaviridae family is divided into four genera based on their genetic properties, including alpha, beta, gamma and delta corona virus genus [ ] . the -ncov is enveloped positive-sense rna, beta corona virus with a genome of . kb [ ] . they are zoonotic, transmitted from animals to humans [ ] . covid- affects the respiratory system (lungs and breathing tubes). most covid- patients developed severe acute respiratory illness with symptoms of fever, cough, and shortness of breath. maximum reported cases of covid- have been linked through travel to or residence in countries in this region [ , ] . presently there are no clinically approved vaccines available in the world for this disease. the development of new vaccine for this new emergent strain by using therapeutic and preventive approach can be readily applied to save human lives. the use of peptides or epitopes as therapeutics is a good strategy as it has advances in design, stability, and delivery [ , ] . moreover, there is a growing importance on the use of peptides in vaccine design by predicting immunogenic ctl, htl and b cell epitopes from tissue-specific proteins of organisms [ , ] . among the structural proteins of sars-cov- , the cov envelope (e) protein is a small integral membrane protein involved in life cycle of virus. it involves in envelope formation, and some other aspects like assembly formation, budding, and pathogenesis. thus, it is considered to be a promising target for effective covid- vaccine design [ ] . more importantly, t-cell-based cellular immunity is essential for cleaning sars-cov- infection because it is memory based [ , ] . also, the low mutation rate of the e protein or it is a highly conserved protein that can elicits both cellular immunity, and neutralizing antibody against covid- is necessary for an efficient vaccine development [ , ] . therefore, in this study, an immunoinformatics based approach was adopted to identify a candidate epitopes against envelope protein of sars-cov- that could be appropriately activate a significant cellular, and humoral immune response [ , ] . the aim of this study is to analyze envelope protein strains using in silico approaches looking for the conservancy, which is further studied to predict all potential epitopes that can be used after in vitro and in vivo confirmation as a therapeutic peptide vaccine [ , , ] . the protein sequence of envelope protein from severe acute respiratory syndrome coronavirus isolate indian strain (sars-cov- / /human/ /ind) with accession no. qia . was retrieved from the ncbi. the antigenicity of this sequence was predicted by the vaxijen v . server [ ] with default parameter. in the present study, envelope protein was found to be a potential antigenic protein with good antigenicity score. a total of envelope protein sequences were retrieved from the ncbi database till april . these sequences retrieved were collected from different parts of the world; retrieved sequences and their accession numbers are listed in the supplementary file. further, the multiple sequence alignment of envelope protein sequences was carried out through clustal w. envelope protein d structure was obtained by swissmodeller which uses homology detection methods to build d models [ ] . ucsf chimera was used to visualize and minimize the d structures [ ] , and structure validation was carried out with saves [ ] . homology modelling was achieved to establish conformational b cell epitope prediction and for further verification of the surface accessibility and hydrophilicity of b lymphocyte epitopes predicted, as well as to visualize all predicted t cell epitopes in the structural level. b cell epitope is the portion of an immunogen, which interacts with b-lymphocytes. as a result, the b-lymphocyte is differentiated into an antibody-secreting plasma cell and the memory cell. thus, the iedb resource was used for analysis. envelope protein was subjected to bepipred linear epitope prediction [ ] , emini surface accessibility [ ] , kolaskar and tongaonkar antigenicity [ ] , parker hydrophilicity [ ] , chou and fasman beta turn [ ] and karplus & schulz flexibility prediction [ ] prediction methods in iedb, that predict the probability of specific regions in the protein to bind to b cell receptor, being in the surface, being immunogenic, being in a hydrophilic region and being in a beta turn region, respectively. potentially continuous b cell epitope was predicted using tool ellipro from iedb resource [ ] . the allergenicity of predicted epitopes was analyzed by allertop tool [ ] . toxinpred server was used to predict toxicity assessment of epitopes [ ] . t-cell epitopes were predicted by the netctl server [ ] . the parameter was set at to have the highest specificity and sensitivity of . and . , respectively and all the supertypes were taken during the submission of a protein sequence. a combined algorithm of major histocompatibility complex (mhc)- binding, transporter of antigenic peptide (tap) transport efficiency and proteasomal cleavage efficiency were used to predict the overall scores [ ] . on the basis of the combined score first, five best epitopes were selected for further testing as putative epitope vaccine candidates. mhc- binding t cell epitope was predicted by iedb by using the stabilized matrix method (smm) for each peptide [ ] . prior to prediction, all epitope lengths were set as mers, conserved epitopes that bind to many hla alleles at score equal or less than . percentile rank were selected. for further analysis, alleles having ic less than nm were selected. overall, the higher immunogenicity of peptides shows more expected to be ctl epitopes than those having lower immunogenicity. therefore, the iedb immunogenicity prediction tool was used for the prediction of the immunogenicity of the candidate epitopes [ ] . analysis of peptide binding to mhc class ii molecules was assessed by the iedb mhc ii prediction tool, where smm based netmhciipan . server was used [ ] . it covers all hla class ii alleles including hla-dr, hla-dq, and hla-dp [ ] . ic below nm show maximum interaction potentials of htl epitope and mhc ii allele [ ] . accordingly, five top epitopes were selected. the predicted htl epitopes were submitted to the ifn epitope server to check whether the mhcii binding epitopes had the ability to induce ifn-γ [ ] . all potential mhc i and mhc ii binders from envelope protein were assessed for population coverage against the whole world population that had been reported covid- cases. calculations achieved using the selected mhc-i and mhc-ii interacted alleles by the iedb population coverage calculation tool [ ] . epitopes of mhc i alleles that predicted to bind with percentile rank below . were selected as the ligands, which are modeled using pep-fold online peptide modeling tool [ ] . the receptor mhc i allele d structure was obtained from the pdb server [ ] . patchdock program was used for all dockings [ ] . pymol and chimera were used for visualization and determination of binding affinity and to show the suitable epitopes binding with the lowest energy. the protein sequence of envelope protein from severe acute respiratory syndrome coronavirus isolate indian strain retrieved in fasta format was screened using the vaxijen server to predict the immunogenicity. in the present study, the qia . ) was predicted to be antigenic protein based on the overall score by the vaxijen server and this has been indicated as an immunogenic protein. a total of envelope protein sequences retrieved from the ncbi database were aligned, to see the conservation of predicted epitopes. by means of iedb analysis resource b and t cell epitopes were predicted and population coverage was calculated. three-dimensional structure of envelope protein of the sars-cov- was modelled using the homology structure modelling tool swissmodeller (fig. ) . this protein showed a good model with swissmodeller by using pdb id: x respectively as a template has more than % identity and % similarity with the query structure. these models were energy minimized by using chimera. the ramachandran plot and prosa z-score validation (fig. ) indicated that > % residues in the favoured region for the modelled envelope protein. the conformational b-cell epitopes were also obtained in five chains of envelope protein by using ellipro. ellipro gives the score to each output epitope, which is protrusion index (pi) value averaged over each epitope residue [ ] . some ellipsoids approximated the tertiary structure of the protein. the highest probability of a conformational epitope was calculated at % (pi score: . ). residues involved in conformational epitopes, their number, location and scores are shown in table , srvknl residues were found have highest pi score. this epitope is antigenic, nonallergic, nontoxin, and conserved in sars-cov- . also, their positions on d structures are shown in envelope protein from the sars-cov- was analyzed using the iedb mhc- binding prediction tool to predict the t cell epitope suggested interacting with different types of mhc class i alleles. based on netctl and smm-based iedb mhc-i binding prediction tools with higher affinity (ic less than ) were predicted to interact with different mhc- alleles. the predicted total score of proteasome score, tap score, mhc score, processing score, and mhc-i binding are summarized as a total score in table table . among these t-cell epitopes, -mer epitope, flafvvfll was found to have the highest immunogenicity which was maximum than above said epitope and found to have more number of allelic interactions with good population coverage than other epitopes. by the same way in iedb mhc- binding prediction tool, t-cell epitopes from the sars-cov- were analyzed using the mhc-ii binding prediction method; based on smm based netmhciipan with ic less than . there were top predicted epitopes found to be nonallergic and antigenic interact with mhc-ii alleles for which the peptide (core) (table- and ) . epitopes that are suggested interacting with mhc-i and ii alleles (especially high affinity binding epitopes and that can bind to a different set of alleles) were selected for population coverage analysis. the results of population coverage of all epitopes are listed in table and . flafvvfll epitope that interacts with most frequent mhc class i and ii alleles gave a high percentage against the whole world population by the iedb population coverage tool. the maximum class i and ii combined population coverage ( . %) for this proposed epitope was found in north america (table- ), while the higher population coverage in europe ( . %) and east asia ( . %) followed by south asia ( . %) and north africa ( . %) then northeast asia ( . %) and southeast asia ( . %). table represents the populations for which the mhc i and ii class combined coverage of other areas. proposed t-cell epitopes, flafvvfl (green) and b cell epitopes, srvknl (yellow) in pantamer structure of e protein of sars-cov- . the predicted t cell epitope flafvvfll that interacted with selected human's mhc- and ii alleles were used as ligands (fig. ) to detect their interaction with alleles /receptors, by docking techniques using on-line software patchdock. after successful docking by patchdock, the refinement and re-scoring of the docking results were carried out by the firedock server. after refinement of the docking scores, the firedock server generates global energies/ binding energies for the best solutions. chimera was used to visualize the best results. the d structure of epitopes was predicted using pep-fold and energy minimization was carried out by using chimera. based on the binding energy in kcal/mol unit, the lowest binding energy (kcal/mol) was selected to obtain a best binding (pose) and to predict real ctl and htl epitope as possible. the receptors used for docking studies included reported hlas, hla-c* : (pdb id: efx) for class i and hla-drb * : (pdb id: aqd) for class ii. hla-c* : and hla-drb * : was observed to have the interaction with the flafvvfll epitope with lower binding energy, - . kcal/mol and - . kcal/mol respectively ( fig. and ) . the predicted peptide showed significant binding affinities with all hlas. also, the binding energy of the predicted epitopes were compared with the binding energy of the already experimentally verified peptides and found to be negative [ ] . in this study, we aimed to determine the highly potential immunogenic epitopes for b and t since the immune response of t cell is long lasting response comparing with b cell, where the antigen can easily escape the antibody memory response [ ] additionally, cd + t and cd + t cell responses play a major role in antiviral immunity [ ] , designing of a vaccine against t cell epitope is much more promising. flafvvfll epitope could be used as a potential candidate because it had a maximum combined score and immunogenic score. moreover, it possessed the maximum number of hla binding alleles amongst other ctl and htl. this epitope was found to be antigenic, non-toxin and nonallergic. an ideal epitope should be highly conserved. the conservancy analysis of this epitopes indicated that this epitope was found to have been conserved in all sequences of the sars-cov- consider in this study. we found these ctl epitopes to be htl epitopes. the overlapping between mhc class i and ii t cell epitopes suggested the possibility of antigen presentation to immune cells via both mhc class i and ii pathways especially the overlapping sequences. to conclude, by using e protein one epitope, srvknl was proposed for an international therapeutic peptide vaccine for b cell. regarding t cell, the flafvvfll epitope was highly recommended as a therapeutic peptide vaccine to interact with both mhc class i and ii. we recommend in vitro and in vivo validation for the efficacy and efficiency of these predicted candidate epitopes as a vaccine as well as to be used as a diagnostic screening test. author contribution: renu jakhar conducted the study, performed in silico analysis and wrote the manuscript. s.k. gakhar plans the study and revises the manuscript. outbreak of pneumonia of unknown etiology in wuhan china: the mystery and the miracle a new coronavirus associated with human respiratory disease in china the -new coronavirus epidemic: evidence for virus evolution emerging coronaviruses: genome structure, replication, and pathogenesis a familial cluster of pneumonia associated with the novel coronavirus indicating person-to-person transmission: a study of a 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use of peptides in vaccine design vaxijen: a server for prediction of protective antigens, tumour antigens and subunit vaccines swiss-model: homology modelling of protein structures and complexes ucsf chimera, a visualization system for exploratory research and analysis stereochemistry of polypeptide chain configurations prediction of residues in discontinuous b-cell epitopes using protein d structures induction of hepatitis a virus-neutralizing antibody by a virus-specific synthetic peptide a semi-empirical method for prediction of antigenic determinants on protein antigens new hydrophilicity scale derived from highperformance liquid chromatography peptide retention data: correlation of predicted surface residues with antigenicity and x ray-derived accessible sites prediction of the secondary structure of proteins from their amino acid sequence prediction of chain flexibility in proteins protection from ebola virus mediated by cytotoxic t lymphocytes specific for the viral nucleoprotein allertop -a server for in silico prediction of allergens open source drug discovery consortium. in silico approach for predicting toxicity of peptides and proteins large-scale validation of methods for cytotoxic t-lymphocyte epitope prediction sensitive quantitative predictions of peptide-mhc binding by a 'query by committee'artificial neural network approach the immune epitope database (iedb) . properties of mhc class i presented peptides that enhance immunogenicity netmhciipan- . , a common panspecific mhc class ii prediction method including all three human mhc class ii isotypes protection from ebola virus mediated by cytotoxic t lymphocytes specific for the viral nucleoprotein toward more accurate pan-specific mhcpeptide binding prediction: a review of current methods and tools novel immunoinformatics approaches to design multi-epitope subunit vaccine for malaria by investigating anopheles salivary protein predicting population coverage of t-cell epitope-based diagnostics and vaccines pep-fold: an updated de novo structure prediction server for both linear and disulfide bonded cyclic peptides the rcsb protein data bank: a redesigned query system and relational database based on the mmcif schema patchdock and symmdock: servers for rigid and symmetric docking a comprehensive analysis of aminopeptidase n protein (apn) from anopheles culicifacies for epitope design using immuno-informatics models key: cord- -z vmhopa authors: ji, wei; niu, ling; peng, weiyu; zhang, yongli; cheng, hao; gao, feng; shi, yi; qi, jianxun; gao, george f.; liu, william j. title: salt bridge-forming residues positioned over viral peptides presented by mhc class i impacts t-cell recognition in a binding-dependent manner date: - - journal: mol immunol doi: . /j.molimm. . . sha: doc_id: cord_uid: z vmhopa the viral peptides presentation by major histocompatibility complex class i (mhc i) molecules play a pivotal role in t-cell recognition and the subsequent virus clearance. this process is delicately adjusted by the variant residues of mhc i, especially the residues in the peptide binding groove (pbg). in a series of mhc i molecules, a salt bridge is formed above the n-terminus of the peptides. however, the potential impact of the salt bridge on peptide binding and t-cell receptor (tcr) recognition of mhc i, as well as the corresponding molecular basis, are still largely unknown. herein, we determined the structures of hla-b* and h- k(d) in which two different types of salt bridges (arg -glu or arg -glu ) across the pbg were observed. although the two salt bridges led to different conformation shifts of both the mhc i α helix and the peptides, binding of the peptides with the salt bridge residues was relatively conserved. furthermore, through a series of in vitro and in vivo investigations, we found that mhc i mutations that disrupt the salt bridge alleviate peptide binding and can weaken the tcr recognition of mhc i-peptide complexes. our study may provide key references for understanding mhc i-restricted peptide recognition by t-cells. the presentation of viral peptides to host t lymphocytes is crucial for adaptive cellular immunity to clear viruses during the infection. in the structures of mhc i presenting these short peptides, the α and α domains of mhc i form a bed-like groove to comfortably accommodate the peptides: two α-helices as the bedrails and the β-sheets beneath as the mattress (bjorkman et al., ; niu et al., ) . the short peptides interact with the amino acids within the peptide binding groove (pbg) through different binding modes. first, the featured residues at the second position (p ) from the n-terminus and the last residue (pΩ) at the c-terminus of the peptides, termed primary anchor residues, protrude into the specific pockets in the pbg of the mhc i. though the positions of the primary anchors on the peptides are relatively conserved, the binding modes of the anchoring residues of the peptides to different mhc i molecules are diversified due to polymorphisms in the pocket-forming residues. second, the peptides also interact with the pbg via contacts between the backbone atoms of the peptides and the pbg residues (mitaksov and fremont, ) . third, a hydrogen bond network is formed between the two termini of the peptides and the conserved residues in the pbg. the latter two interaction modes are relatively conserved between different mhc i molecules (fremont et al., ; madden et al., ) . generally, mhc i binding peptides are located in the bottom of a ringent pbg, which leaves a solvent-exposed top surface of the entire peptide from the n-to the c-terminus, ready for the recognition of the t-cell receptors (tcr) (bjorkman et al., ) . however, based on the structures of a series of mhc i molecules, such as human hla-b* (madden et al., ) , rhesus macaque mamu-a* , and mouse h- k d (mitaksov and fremont, ; zhou et al., ) , there is a salt bridge positioned over the peptides formed by opposite charged residues from the α and α helices of mhc i, respectively. in humans and macaques, the salt bridge is formed by the positively charged arg on the α helix and the negatively charged glu on the α helix. meanwhile, in mice, the involved residues are arg on α helix and glu on α helix. both of these types of salt bridges overhang the n-terminal portion of the t-cell epitopes in the groove. obviously, the salt bridge forms a ligature to tightly fix the peptides into the pbg. however, thus far, it is still largely unknown how the formation of these salt bridges in mhc i molecules impacts peptide binding. further, considering that these salt bridges are also solvent-exposed, they are in the position to be recognized by tcrs. generally, t-cell activation by the mhc-peptide complex may occur in three correlated ways: ) direct interaction of the tcr with the mhc, termed mhc restriction; ) direct interaction of the tcr with the solvent-accessible peptide main chain and side chains; and ) interaction of the peptide with the tcr mediated by conformational perturbations in the mhcpeptide complex (baker et al., ; gras et al., ) . crystallographic studies clearly demonstrate that the polymorphic amino acids in the grooves of mhc i molecules from different mammals may influence the peptide conformation, subsequently impacting t-cell recognition (borbulevych et al., ; hulsmeyer et al., ; insaidoo et al., ) . nevertheless, to our knowledge, it has not been determined if the salt bridges in some mhc i molecules impact tcr recognition through a direct interaction with the tcr or indirectly through modulation of the peptide conformation. herein, by determining the crystal structures of human mhc i hla-b* complexed with a severe acute respiratory syndrome coronavirus (sars-cov) nucleocapsid (n)-derived t-cell epitope (oh et al., ) and mouse mhc i h- k d bound to an immunodominant t-cell epitope from human hepatitis b virus (hbv) core antigen (hbc) (li et al., ) , we clearly demonstrated the molecular features of mhc i molecules with two different salt bridges formed by the residues pairs arg -glu and arg -glu , respectively. we further investigated the impacts of the salt bridges on peptide binding and t-cell recognition by constructing a series of mhc i mutants in vitro and in vivo. our results elucidated the key features of mammalian mhc i molecules with salt bridges in the pbg and will benefit t-cell based diagnosis and vaccine development. the human hbv hbc protein-derived peptide hbc - (syvnt-nmgl) (li et al., ) and sars-cov n protein-derived peptide n - (getalallll) (oh et al., ) were synthesized with % purity by reverse-phase high performance liquid chromatography (scilight biotechnology, beijing, china). the peptides were stored at − °c as freeze-dried powders and were dissolved in dimethyl sulfoxide before use. the expression plasmid for mouse mhc i h- k d was constructed previously in our lab (zhou et al., ) . we also constructed three h- k d salt bridge mutants (r a, e a, and r a&e a) and three control mutants (s a, r a, a g) based on the wt plasmid (genewiz). for the hla-b* expression construct, the gene (genbank q ) was synthesized and ligated into the pet a vector (shanghai generay). the three hla-b* salt bridge mutants (r a, e a, r a&e a) and three control mutants (i a, r a, a g) were also constructed on the basis of the wt construct. six-to eight-week-old female balb/c mice were purchased from vital river laboratory animal technology company (beijing, china) and raised under specific pathogen-free conditions. all experiments were performed in strict compliance with the guide for the care and use of laboratory animals of the people's republic of china and approved by the committee on the ethics of animal experiments of national institute for viral disease control and prevention, chinese center for disease control and prevention. mice in the experimental group were subcutaneously injected at multiple sites with hbv peptide hbc - and the n-terminal fragment n ( - aa) of murine gp emulsified in complete freund's adjuvant for the first dose. mice were administered the mixture of peptide, gp fragment, and incomplete freund's adjuvant twice in -week intervals for the next two doses. mice were sacrificed to harvest splenocytes on day after the final immunization. mice in the control group were immunized with a mixture of phosphate buffered saline (pbs), gp fragment, and freund's adjuvant. antigen-specific t lymphocyte responses were detected with an ifnγ-secreting elispot assay by using splenocytes as previously described (tan et al., ) . briefly, -well plates were coated with μl/well of mg/ml anti-mouse ifn-γ antibody (bd pharmingen, san diego, ca) overnight at °c. after washing with pbs twice, the plates were blocked with culture medium for h at room temperature. mouse spleens were ground and filtered through cell strainers. cells were processed with red blood cell lysis buffer ( . m nh cl and μm tris, ph . ). we washed cells with pbs twice and centrifuged them to harvest lymphocytes. fresh mouse splenocytes ( . × ) in μl roswell park memorial institute (rpmi) medium with % fetal bovine serum (fbs) were seeded in each well. then, the peptides ( μg/ml) were added to the wells and incubated at °c in % co for h. phytohaemagglutinin (pha) was added as the positive control for nonspecific stimulation. cells incubated with medium alone were employed as a negative control that produced less than five spots in % of the experiments. finally, the cells were removed, and the plates were processed according to the manufacturer's instructions (bd). the colored spots, which represent epitope-specific t-cells, were counted and analyzed using an automatic elispot reader (ctl corp.). murine mhc class i h- k d and β m or human mhc class i hla-b* heavy chain and human β m were overexpressed in escherichia coli as inclusion bodies and subsequently refolded in vitro in the presence of a high concentration of peptide, as described previously (xiao et al., ) . briefly, the dissolved mhc i heavy chain and β m inclusion body and peptides were diluted at a molar ratio of : : , respectively, in refolding buffer ( mm tris−hcl, mm l-arginine, mm edta-na, mm glutathione [gsh], and . mm l-glutathione oxidized [gssg]). after h of slow stirring at °c, the mhc i/peptide complex was then concentrated and purified via superdex / g l (ge healthcare) chromatography. wt h- k d and the three h- k d mutant-restricted tetramers of hbvpeptide were prepared as previously described (liu et al., a; zhang et al., ; zhou et al., ) . briefly, recombinant h- k d /peptide complexes were purified and then biotinylated by incubation with dbiotin, atp, and the biotin protein ligase bira (avidity) at °c for h. the biotinylated h- k d was further purified by gel filtration to remove free biotin, and then the multimers were produced by using pe-streptavidin (sigma). cells from the subjects were stained with pe-tetramer and fitc-conjugated anti-cd antibody. all samples were analyzed with a facscalibur flow cytometer (bd biosciences) after staining. the conditions for the protein purification and the crystal growth of the complex formed by h- k d with hbv peptide hbc - was described previously (zhou et al., ) . human β m was used to generate the h- k d /hbc - complex. the renatured hla-b* /n - complex was further purified by resource q anion-exchange chromatography before crystallization. crystallization was performed using the hanging drop vapor diffusion technique. the concentration of protein was mg/ml, and the crystals grew in . m sodium citrate tribasic dehydrate (ph . ) and % (w/v) polyethylene glycol at °c. for cryoprotection, crystals were transferred to reservoir solutions containing % glycerol, flash-cooled, and maintained at k in a stream. x-ray diffraction data were collected at k at the ssrf beamline bl u (shanghai, china) at a wavelength of . Å ( table ). the structure of h- k d /hbc - and hla-b* /n - were resolved through the molecular replacement method using the mhc i structures with pdb codes fg and f m, respectively (liu et al., b) as the model in the crystallography and nmr system (cns) program. detailed model building was performed by hand using coot, and restrained refinement was performed using refmac . the stereochemical quality of the final model was assessed with the program procheck. structure-related figures were processed by pymol. to evaluate the thermostability of different mhc i complexes and also their mutants, we used cd spectroscopy as previously described (n. zhang et al., ) . we repeated at least three times for each protein. all complexes were prepared as described above and diluted to . mg/ ml in mm tris−hcl (ph . ) and mm nacl. thermal denaturation curves were determined by monitoring the cd value at nm using a -mm optical path-length cell as the temperature was raised from to °c at a rate of °c/min. the temperature of the sample solution was directly measured with a thermistor. the fraction of unfolded protein was calculated from the mean residue ellipticity (θ) by the standard method. the unfolded fraction (%) is expressed as (θ -θ n ) / (θ u -θ n ), where θ n and θ u are the mean residue ellipticity values in the fully folded and fully unfolded states, respectively. the midpoint transition temperature (t m ) was determined by fitting the data to the denaturation curves using the origin . program (originlab). for comparisons between multiple groups, two-way anova with bonferroni post-tests was performed. one-way anova analysis with bonferroni post-tests was used for comparison between multiple columns. statistical significance of differences between two columns was determined by student's t-test all tests were two-tailed with a significance level of . . all data analyses were performed with graphpad prism. the coordinates and structure factors of sars-cov peptide n - complexed to hla-b* and hbv peptide hbc - complexed to h- k d have been deposited in the pdb under accession numbers iex and vgk, respectively. the retrievement of the mhc i protein sequences available in the ipd-mhc database (https://www.ebi.ac.uk/ipd/mhc/) showed that %- % of the mhc i alleles available possess the paired residues for the potential salt bridge formation ( table ). the type salt bridge is formed by r on the α -helix and e on the α -helix, such as in h- k d , while the type salt bridge is constructed by r and e on the α -and α -helices, respectively, such as in hla-b* . moreover, we retrieved the allele frequency of mhc i molecules which are confirmed to possess salt bridge from the crystal structures in . the allele h- k d with type salt bridge is also common in mouse mhc alleles. thus, the impacts of the salt bridge formation overhead the pbg on the peptide binding and recognition by tcr would be a substantial phenomenon for different vertebrates, which has emerged in fish. to determine the potential role of the mhc i salt bridge in peptide binding, we utilized the peptide hbc - to facilitate the in vitro renaturation of h- k d and its salt bridge mutants h- kd-m (mutant r a), h- kd-m (mutant e a), and h- kd-m (mutant r a& e a) followed by size exclusion chromatography (gel filtration) analyses. the control mutants h- kd-c (mutant r a), h- kd-c (mutant a g), h- kd-c (mutant s a), which preserve the salt bridge were proceeded the same experiment as control mutants. compared to wild type (wt) of h- k d , all the three salt bridge mutants generated relatively lower yields of refolded products at the size expected for an mhc i monomer. however, the control mutants showed similar yields of renatured products as wt h- k d (fig. a and table s ). the binding stabilities of the peptide hbc - with h- k d and all the mutants were further analyzed by circular dichroism (cd) spectroscopy (fig. b) , with the t m s determined from melting curves. wt h- k d complexed with the hbc - peptide was stable, with a t m of . ± . °c. as expected, the h- k d salt bridge mutants h- kd-m , h- kd-m , and h- kd-m displayed significantly decreased stability with lower t m s ( . ± . °c for h- kd-m , . ± . °c for h- kd-m , and . ± . °c for h- kd-m ). moreover, the t m s of the control mutants have no statistically differences with wt h- k d ( . ± . °c for h- kd-c , . ± . °c for h- kd-c , . ± . °c for h- kd-c ). we also determined the binding capacity of the sars-cov-derived peptide n - with the human mhc i hla-b* , and its salt bridge mutants b -m (mutant r a), b -m (mutant e a), and b -m (mutant r a&e a) and its control mutants b -c (mutant a g), b -c (mutant r a), b -c (mutant i a). we found that three salt bridge mutants b -m ( . ± . °c), b -m ( . ± . °c), and b -m ( . ± . °c) displayed significantly lower binding capacity for the peptide compared to wt hla-b* ( . ± . °c) ( fig. c and d ), while the control mutants showed the identical binding capacity with the peptides as wt hla-b* ( fig. c and d) . thus, the salt bridge formation in both pbgs of h- k d and hla-b* contributed to the binding of the peptides, though breaking the salt bridge did not abolish peptide binding. to further investigate the role of the mhc i salt bridge above the pbg in t-cell recognition, we established a mouse model via three rounds of fortnightly in vivo injection of the hbv-derived peptide hbc - . elispot assays were then performed using freshly isolated splenocytes to assess the t-cell immune responses induced by the peptide. no specific reactivity of ifn-γ secretion could be detected in splenocytes separated from placebo-immunized mice (< sfcs/ splenocytes). in contrast, cd + t-cells from splenocytes of the mice immunized with peptide hbc - presented strong ifn-γ production ( fig. a) . the splenocytes from the peptide-inoculated mice were also stained with the h- k d tetramers prepared in the presence of peptide hbc - . the splenocytes from hbc - -inoculated mice contained . ± . % of peptide-specific cd + t-cells ( fig. b and c) . no peptide-specific cd + t-cells were identified from splenocytes of mice inoculated with placebo, as judged by hbc - tetramer staining. we also constructed tetramers based on a series of mutated h- k d mhc i heavy chains, including h- kd-m , h- kd-m , and the dual mutant h- kd-m . when compared to the wt tetramer, the three mutant tetramers stained a lower proportion of hbc - -specific cd + t-cells within splenocytes from mice inoculated with peptide hbc - ( . ± . %, . ± . %, and . ± . %, respectively). these results indicated that mutations of the residues forming the salt bridge positioned over the peptide may impact specific-t-cell recognition. though both h- k d and hla-b* contain the conserved negatively charged residue e , the structures of these two mhc i molecules clearly display two different types of salt bridges over the n-terminus of their complexed peptides (fig. ) . compared to h- k d , the salt bridge of hla-b* is located closer to the n-terminus of the pbg, and an angle of˜ °can be observed between the positions of the two salt bridges. this is due to the different positively charged residues in the salt bridges of the two mhc i molecules. however, in the structures of some mhc i available online thus far (e.g., h- l d , h- d b , and h- d d ), though they possess the same salt bridge-forming residues (r and e ) as hla-b* , no salt bridge is formed (pdb codes: ld , ce , bii) (supplemental fig. s ). the "broken bridge" is also found in several hla-b* structures (e.g. pbd codes: bst and jge) (supplemental fig. s ). particularly, h- d d possesses both r and r in its α helix, but no salt bridge is formed the balb/c mice were immunized with hbc - peptide (peptide group) or pbs (adjuvant group) together with adjuvants. elispot assays were performed using freshly isolated mouse splenocytes. the non-specific stimulant pha was used as a positive control, and mock indicates the negative control without any stimulant. (b) peptide-specific cd + t-cells stained by tetramers of h- k d and mutants. the hbc - peptide-specific cd + t-cells in the freshly isolated splenocytes from vaccinated mice were stained by h- k d tetramer, kd-m (mutant r a) tetramer, kd-m (mutant e a) tetramer, and kd-m (mutant r a&e a)tetramer, respectively. the hollow dots represent the peptide-immunized group, and the black dots represent the adjuvant group. the control was staining with unrelated tetramer (hla-a /influenza peptide gl ). error bars represent means ± sd. n = mice for per peptide group, n = mice for per adjuvant group. (c) the representative peptide-specific cd + t-cells stained by tetramers of h- k d and mutants. the statistical analysis between two groups used two-way anova with bonferroni post-tests. ** p < . , *** p < . . between e and either of these positively charged residues. next, we analyzed the hydrogen bonds within the two residues forming the salt bridge and the adjacent residues of both mhc i and the peptides (fig. ) . three different interactions contribute to the formation of the salt bridges: ) the electrostatic interaction between r or r in the α helix and the e in the α helix. the hydrogen bonds constructed between residues r and e in h- k d and between r and e in hla-b* are . Å and . Å, respectively. ) r or r in the salt bridge can form a hydrogen bond with the carbonyl group of the p residue of the bound peptide in the pbg (as in both the h- k d and hla-b* structures, and through a water molecule in mamu-a* ). ) for the mhc i-binding peptides with a hydrophilic residue at the p position, hydrogen bonds can be found between e and the side chains of the p residue of the peptide (as in the h- k d structure but not hla-b* ) (fig. ) . in hla-b * and hla-b* , a water molecule links the interaction between r and e (fig. ) . further analysis indicated that this water molecule is conserved in a series of hla-b* structures ( uxs, a , bsr, b s, bp , of , uxw, and w w) (supplemental fig. s ) . we further analyzed the structures of h- l d , h- d b , and h- d d , which contain the same residues for salt bridge forming (r and e ) as hla-b* but do not contain salt bridges. in the mhc i h- l d , h- d b , and h- d d structures, r is pulled away by e on the α helix, and r or k forms hydrogen bonds with e of the α helix. thus, no salt bridge is formed in these three mhc i structures despite the fact that they contain the corresponding residues (supplemental fig. s ). to investigate the potential influence of salt bridge formation on the structural conformation of the mhc/peptide complex, we superimposed the structures of the mhc i molecules with the two different salt bridges: r -e (h- k d , mamu-b* , and mamu-b* ) and r -e (hla-b* , mamu-a* , rt -a c, hla-b* , hla-b* , and hla-b* ) (fig. a) . compared to the structure of mhc i with the r -e salt bridge, the α -helix of mhc i molecules with the r -e salt bridge has an conformational shift (approximately . Å between h- k d and hla-b* ), using the α -helix of mhc i as the benchmark during the alignment (fig. b) . the peptides in the structures of mhc i complexes with r -e salt bridges also display conformational differences for the two residues at the p and p positions compared to the peptides under the r -e salt bridges (fig. c ). the cα of the p residues in the h- k d -binding peptides are closer to the c-terminus of the pbg and located lower and closer to the bottom of the groove (fig. d and e ). the r -e salt bridge acts as a "seatbelt" for the peptide, pushing both the conformational shift of the peptides and the α -helices. herein, we determined the crystal structures of hla-b* and h- k d molecules with two representative different salt bridges spanning the presented peptides. compared to wt, the peptide binding capacity of the salt bridge mutants was significantly lower. meanwhile, the control mutants showed no statistical difference. though the two salt bridges had distinct impacts on the conformational changes of the mhc i structures, both enhanced peptide binding and potentially contributed to tcr recognition. though the mhc i salt bridge-breaking mutants still bound to the peptides, their binding capacity were lower than wt. the impact on the thermostability of hla-b* by the mutants was not as much as that observed in the h- k d mutants. this is may be due to the intrinsic features of different salt bridges in the two molecules. using structural analysis, we found that the enhancement effect of peptide binding to mhc i was not only contributed by the direct formation of the salt bridge above the peptide main chain but also contributed by the conserved hydrogen bonds between the salt bridge-forming residues with the atoms on the main chain and the p residues of the peptides. a previous study indicates that a salt bridge (also called an ion pair in the study) formed by argα and aspβ in mhc class ii heterodimers is also critical for surface expression and peptide presentation (nalefski et al., ) . however, unlike the salt bridge overhanging the mhc i peptide, this argα -aspβ salt bridge is located under the main chain . the interactions between the two residues forming the salt bridge and the adjacent residues of both mhc i and the peptides. the interactions between r /r and e that form the salt bridge are denoted as red arrows. the cyan arrows represent the hydrogen bond between e and the side chains of the p residues of the peptides. the dark blue arrow marks the hydrogen bond between r or r and the carbonyl group of the p residue of the bound peptide in the pbg. in hla-b* , hla-b* and mamu-a* , the cyan dots represent water molecules. the black dashed lines in the structures represent the hydrogen bonds. (for interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article). of the mhc ii-presented peptide and may contribute to p binding and the c-terminal hanging conformation of the mhc ii-bound peptides. the salt bridge above the peptide both enhances mhc i-peptide binding and contributes to tcr recognition of mhc i-peptide complexes. birkinshaw et al. studied that a prototypical autoreactive bk tcr bound cd a when the presented ligands were permissive. tcr docked over the a' roof of cd a and a central cd a salt-bridge network (arg -glu salt-bridge) prevented the interaction between bk tcr and permissive ligands, and its autoreactivity was determined by contacts with cd a. nonpermissive ligands disrupted salt bridge interaction of cd a, disrupt the properties of a' roof and could hinder engagement of hydrophobic cdr β loop of the bk tcr with cd a (birkinshaw et al., ) . nurzia et al. indicate that the positive charging of arg is preferred for the tcr recognition of ankylosing spondylitis-associated b* complexes, as revealed by the r k and r a mutants with an overall reduced capability to present peptides to cd + t-cells (nurzia et al., ) . our data also demonstrate that tcell recognition was significantly alleviated when we broke the mhc i salt bridge with r a and e a mutations. previously determined complex structure between hla-b* : and kfj tcr showed that both the r and e of hla-b* : can form hydrogen bonds with the residues in the cdr α and cdr α loops of the tcr, respectively (chan et al., ) . this indicated that the residues in the salt bridge can be directly-recognized by the tcr (tynan et al., ) . meanwhile, our analysis also demonstrated that the salt bridge can lead to a conformational shift of the presented peptide and the relative locations of the α or α helices, which may subsequently and indirectly impact tcr recognition. further structural studies are required to investigate the accurate and detailed recognition of mhc i/peptide complex containing the salt bridge by the tcr. nevertheless, our current study still has limitations. the mutant mhc i tetramers used in this study including the h- k d mutants at r and/or e a can dismantle the salt breakage and then may indirectly alter t cell response. however, these residues can also be recognized by tcr, thus, the mutation of these residues may also directly influence the tcr response. we cannot exclude the possibility that the altered t cell response was due to altered contacts with the mutated residues, rather than the loss of the salt bridge interaction. in summary, our study indicates that the two different salt bridges in mammalian mhc i molecules, represented by hla-b* and h- k d , respectively, have different structural characteristics. and the mutations of the salt bridge-forming residues may impact both peptide binding and tcr recognition, directly or indirectly. our data is helpful for the understanding of the cd + t-cell recognition of mhc i-presented peptides. structural and dynamic control of t-cell receptor specificity, cross-reactivity, and binding mechanism alphabeta t cell antigen receptor recognition of cd a presenting self lipid ligands structure of the human class i histocompatibility antigen, hla-a t cell receptor cross-reactivity directed by antigen-dependent tuning of peptide-mhc molecular flexibility divergent t-cell receptor recognition modes of a hla-i restricted extended tumourassociated peptide crystal structures of two viral peptides in complex with murine mhc class i h- kb a structural voyage toward an understanding of the mhc-i-restricted immune response: lessons learned and much to be learned loss of t cell antigen recognition arising from changes in peptide and major histocompatibility complex protein flexibility: implications for vaccine design generation of murine ctl by a hepatitis b virus-specific peptide and evaluation of the adjuvant effect of heat shock protein glycoprotein and its terminal fragments diverse peptide presentation of rhesus macaque major histocompatibility complex class i mamu-a revealed by two peptide complex structures and insights into immune escape of simian immunodeficiency virus crystal structure of cell adhesion molecule nectin- /cd and its binding to immune receptor dnam- / cd cross-allele cytotoxic t lymphocyte responses against pandemic h n influenza a virus among hla-a and hla-a supertype-positive individuals the antigenic identity of peptide-mhc complexes: a comparison of the conformations of five viral peptides presented by hla-a the structure of hla-b reveals nonamer self-peptides bound in an extended conformation structural definition of the h- kd peptide-binding motif an ion pair in class ii major histocompatibility complex heterodimers critical for surface expression and peptide presentation structural basis for the differential classification of hla-a* and hla-a* into the a and a supertypes interaction pattern of arg in the a-pocket of differentially disease-associated hla-b subtypes suggests distinct tcr binding modes engineering t cells specific for a dominant severe acute respiratory syndrome coronavirus cd t cell epitope hemagglutininspecific cd (+) t-cell responses following -ph n inactivated split-vaccine inoculation in humans a t cell receptor flattens a bulged antigenic peptide presented by a major histocompatibility complex class i molecule diversified anchoring features the peptide presentation of dla- * : first structural insight into domestic dog mhc class i crystal structure of swine major histocompatibility complex class i sla- and identification of pandemic swine-origin influenza a h n virus cytotoxic t lymphocyte epitope peptides evaluation of zika virus-specific t-cell responses in immunoprivileged organs of infected ifnar -/-mice screening and identification of severe acute respiratory syndrome-associated coronavirus-specific ctl epitopes complex assembly, crystallization and preliminary x-ray crystallographic studies of mhc h- kd complexed with an hbv-core nonapeptide we thank prof. jianfang zhou (national institute for viral disease control and prevention, chinese center for disease control and prevention) for excellent assistance with flow cytometry. we thank dr. minghai zhou (the scripps research institute, scripps florida, usa) for his excellent work in crystallization of the proteins. we also thank dr. jianhui li (institute of biophysics, chinese academy of sciences) for instruction in circular dichroism spectroscopy. supplementary material related to this article can be found, in the online version, at doi:https://doi.org/ . /j.molimm. . . . key: cord- - uwhxs authors: plaisted, warren c.; weinger, jason g.; walsh, craig m.; lane, thomas e. title: t cell mediated suppression of neurotropic coronavirus replication in neural precursor cells date: - - journal: virology doi: . /j.virol. . . sha: doc_id: cord_uid: uwhxs neural precursor cells (npcs) are the subject of intense investigation for their potential to treat neurodegenerative disorders, yet the consequences of neuroinvasive virus infection of npcs remain unclear. this study demonstrates that npcs support replication following infection by the neurotropic jhm strain of mouse hepatitis virus (jhmv). jhmv infection leads to increased cell death and dampens ifn-γ-induced mhc class ii expression. importantly, cytokines secreted by cd + t cells inhibit jhmv replication in npcs, and cd + t cells specifically target viral peptide-pulsed npcs for lysis. furthermore, treatment with ifn-γ inhibits jhmv replication in a dose-dependent manner. together, these findings suggest that t cells play a critical role in controlling replication of a neurotropic virus in npcs, a finding which has important implications when considering immune modulation for npc-based therapies for treatment of human neurologic diseases. transplantation of multipotent neural precursor cells (npcs) is emerging as a feasible therapeutic strategy for the treatment of a variety of neurological disorders. recent studies have demonstrated both short and long-term clinical benefits following npc engraftment within the context of rodent models of alzheimer's disease, parkinson's disease, huntington's disease, and acute spinal cord injury (blurton-jones et al., ; mcbride et al., ; van gorp et al., ; yasuhara et al., ) . furthermore, in murine and non-human primate models of the neuroinflammatory disease multiple sclerosis (ms) the ability of human npcs to function as modulators of the immune system in addition to replacing lost or damaged neural cell populations has been suggested (aharonowiz et al., ; pluchino et al., pluchino et al., , . however, despite the clinical and histological benefits of npc transplantation in pre-clinical animal models of neurologic disease, there is limited evidence addressing the capacity of neural grafts to act as reservoirs for viral replication. studies using the non-polio enterovirus coxsackievirus b (cvb) demonstrate the ability of cvb to preferentially replicate in murine npcs (ruller et al., ) . the ensuing carrier-state infection results in increased cell death and impaired differentiation potential in vitro, as well as inflammation, microgliosis, and a variety of cns developmental defects in vivo (ruller et al., ; tsueng et al., ) . intracerebral infection of neonates with murine cytomegalovirus (mcmv) results in the loss of neural stem cells and their neuronal progeny, as well as a decrease in the production of neurotrophins imperative to normal brain development (mutnal et al., ) . borna disease virus (bdv) infection of human fetal human npcs results in cell death upon differentiation and impaired neurogenesis (brnic et al., ) . thus, the role of neural stem and progenitors as targets for a variety of neuroinvasive viruses is evident, while the consequences of infection within the context of cellular therapy remain to be elucidated. complicating npc-based therapies is the controversial issue of antigenicity of transplanted cells and immune-mediated recognition. a growing body of evidence suggests npcs are not immunoprivileged, as has previously been reported (hori et al., ) . indeed, we have shown that npcs derived from post-natal c bl/ brains express the co-stimulatory molecules cd and cd and up-regulate major histocompatibility complex (mhc) molecules in response to the pro-inflammatory cytokine interferon gamma (ifn-γ) (weinger et al., ) . furthermore, allogeneic npcs are rapidly rejected via a t cell mediated mechanism following intraspinal transplantation into mhc-mismatched recipients (weinger et al., ) . similarly, human npcs have the capacity to express mhcs i and ii and induce t cell proliferation (goya et al., ) . the apparent antigenicity of npcs suggests successful engraftment may require the use of immunomodulatory agents and lifelong suppression of the immune system, as with solid organ transplants. however, an unintended consequence of immune suppression is the potential for latent viruses to become activated, or for uncontrolled viral replication to occur following opportunistic infection (crough et al., ; jordan et al., ; wynn et al., ; young et al., ) . therefore, it is imperative to understand the consequences of neurotropic virus infection of npcs as cellreplacement therapies continue to move into the clinic (gupta et al., ; riley et al., ) . in this study, we demonstrate that cultured murine npcs are infected by the neurotropic jhm strain of mouse hepatitis virus (jhmv), which induces acute encephalomyelitis and chronic demyelination when injected intracranially into immunocompetent mice. jhmv-infected npcs support replication that ultimately results in increased cell death over time. importantly, cd þ t cells kill npcs pulsed with viral-peptides, and jhmv replication in npcs was suppressed, in part, by ifn-γ secreted from virus-specific cd þ t cells. npcs express the mhv receptor ceacam a and are infected by jhmv jhmv is a neurotropic coronavirus with relatively restricted tropism for glial cells through recognition and binding to the receptor carcinoembryonic antigen-cell adhesion molecule a (ceacam a) (hirai et al., ; thorp and gallagher, ) . ceacam a expression in mouse tissues is widespread and can be detected on the surface of a variety of epithelial cells in the gastrointestinal, respiratory, and reproductive tracts, as well as on small vascular endothelia and hematopoietic cells (hemmila et al., ) . however, ceacam a expression is not ubiquitous, and although it is known to be located at the surface of resident cells of the cns including glia, expression by neural stem or progenitor cells has not been evaluated. to determine if npcs derived from c bl/ transgenic mice engineered to express gfp (gfp-npcs) express ceacam a, mrna was isolated from cultured npcs and receptor expression was evaluated by pcr. using ceacam a-specific primers, pcr amplicons were detected in npcs, as well as mixed splenocytes from c bl/ mice acting as controls (fig. a) , and nucleotide sequencing confirmed homology with the specified region of the gene (data not shown). furthermore, cell surface expression of ceacam a was confirmed with more than % of npcs expressing the receptor as determined via flow cytometric analysis (fig. b) . we next infected sox þ gfp-expressing npcs with jhmv to assess susceptibility to infection. infected npc cultures were fixed h post-infection (p.i.) and stained with an antibody specific for the carboxyl terminus of the jhmv nucleocapsid (n) protein and subsequently imaged using fluorescence microscopy. compared to non-infected npcs that form a confluent monolayer when grown in tissue culture-treated, matrigel-coated vessels, sox þ npcs infected at a multiplicity of infection (m.o.i.) of . displayed jhmv-specific syncytia formation by h post-infection ( fig. a) . correspondingly, increasing viral titers were detected when plaque forming unit (pfu) assays were performed on supernatants harvested from jhmv-infected npc cultures at , , and h p.i. (fig. b) . furthermore, determination of lactate dehydrogenase (ldh) released into the supernatants of infected cultures at defined time p.i. revealed increased npc death over time, ranging from . . % at h p.i., increasing to . . % at h p.i., and peaking at . . % by h (fig. c ). as jhmv replication has been reported to occur via ceacam a-dependent and independent mechanisms (nakagaki and taguchi, ) , we performed a monoclonal antibody blockade to determine the role of ceacam a in the spread of jhmv infection in cultured npcs (fig. d ). by h p.i., significant (p o . ) inhibition of viral replication was observed in anti-ceacam a-treated cells ( .  .  pfu/ml, n¼ ) when compared to non-treated, jhmvinfected npcs ( .  .  pfu/ml). under normal culture conditions, expression of mhc classes i and ii is undetectable on npcs, yet mhc expression can be induced by treatment with ifn-γ (chen et al., ; weinger et al., ) . to investigate if jhmv infection alters mhc class i and/or ii expression on npcs, we compared surface expression levels of these molecules on non-infected and infected cells in the absence or presence of u/ml ifn-γ. our findings indicated r % of npcs were found to be positive for mhc class i ( fig. a fig. a and c). however, mhc class ii was detected on a significantly (p o . ) lower fraction ( . . %, n ¼ ) of infected, ifn-γ-treated npcs compared to non-infected, ifn-γ-treated npcs ( . . %, n¼ ) (fig. c) . furthermore, mhc class ii could not be detected on the majority of jhmv-infected npcs as determined by dual staining for viral antigen and mhc class ii (fig. d ). cd þ and cd þ t cells are pivotal in controlling jhmv replication within the infected cns (sussman et al., ; williamson and stohlman, ). virus-specific effector cd þ t cells help control replication in infected astrocytes and microglia through cytolytic activity (bergmann et al., ) . in addition to secreting ifn-γ that limits viral replication in oligodendrocytes, cd þ t cells carry out perforin-dependent cytolysis of astrocytes and microglia (bergmann et al., ; williamson and stohlman, ) . we co-cultured virus-specific ctls at diminishing effectorto-target (e:t) ratios with npcs pulsed with the immunodominant cd peptide specific for jhmv spike (s) glycoprotein spanning amino acids - (s - ), and treated with ifn-γ to induce mhc class i expression. subsequently, ldh released in the supernatants was evaluated to quantify ctl-mediated npc lysis; rma-s cells, a murine lymphoma cell line that presents viral peptides to ctls in an mhc class i dependent manner, were used as positive control (debruijn et al., ) . npcs pulsed with s - peptide were specifically lysed by virus-specific ctls at an e:t ratio of - (p o . , n ¼ ), indicating that virus-specific cd þ t cells are capable of recognizing and directly killing jhmv-infected npcs in vitro (fig. ) . importantly, this cytolytic effect waned as the e:t to target ratio declined. cd þ t cells have both indirect and direct antiviral roles during acute jhmv-induced encephalomyelitis, which include inducing the effector functions of virus-specific ctls, along with ifn-γ secretion (savarin et al., ; stohlman et al., fig. . virus-specific cd þ t cells target s - pulsed npcs for lysis. ctls were harvested from mice immunized with the dm variant of jhmv and co-cultured at varying effector:target ratios with s - pulsed, ifn-γ-treated npcs for h, and lactate dehydrogenase released into the supernatant was subsequently measured. non-ifn-γtreated rma/s cells pulsed with μm s - were used as a positive lysis control. negative selection was performed to purify the respective t cell populations. npc media was conditioned with cd þ t cell cytokines for h and then added to jhmv-infected npcs. supernatants from either naïve or virus-specific cd þ t cells suppressed viral replication in npcs at and h post-infection, with the most significant inhibitory effects observed in groups treated with media enriched with virus-specific cd þ t cell cytokines (fig. a) . however, while the suppressive effects of naïve t cell media appeared to wane by h p.i. ( .   pfu/ml), supernatants from npcs treated with virusspecific cd þ t cell conditioned media maintained low viral titers ( .  .  pfu/ml) in comparison to non-treated controls ( .  .  pfu/ml; fig. a ). t cell derived ifn-γ is critical in controlling jhmv replication in the cns (bergmann et al., ; smith et al., ) . furthermore, treatment with ifn-γ specifically inhibits jhmv replication in oligodendrocyte progenitors (opcs) derived from c bl/ npcs, and inhibition of ifn-γ signaling in oligodendrocytes is associated with increased viral loads and mortality (parra et al., ; whitman et al., ) . we evaluated levels of ifn-γ in naïve-versus-dm specific cd þ t cell conditioned media by enzyme-linked immunosorbent assay (elisa); absorbance values from media conditioned with dm-cd þ t cells were increased $ -fold when compared to naïve t cell conditioned media (p o . ; fig. b ). we subsequently treated jhmv-infected npcs with varying amounts of mouse recombinant ifn-γ for h and determined its effects on viral titers. npcs treated with or u/ml ifn-γ maintained high jhmv titers ( .  .  pfu/ml and .  .  pfu/ml, respectively) in relation to non-treated groups ( .  .  pfu/ml; fig. c ). however, jhmv replication in npcs was reduced in cultures treated with or u/ml ifnγ ( .   pfu/ml and .  .  pfu/ml, respectively; fig. c ). we next performed a -h time course to further probe the effects of ifn-γ ( u/ml) on jhmv-infected npcs. a reduction from .  .  pfu/ml to .  .  pfu/ml was observed in ifn-γ-treated cultures by h post-treatment when compared to non-treated groups (p o . , n ¼ ), and jhmv levels were reduced to .  .  in ifn-γ treated cultures, versus .  .  in non-treated cultures, by h post-treatment (po . ) (fig. d ). we previously showed that multiple pro-inflammatory cytokines secreted by dmspecific t cells have synergistic effects with ifn-γ (weinger et al., ) . to confirm the role of ifn-γ as the major cytokine contributing to suppression of jhmv replication in infected npc cultures, monoclonal antibody blockade against the ifn-γ receptor was performed on npcs before and during treatment with virusspecific cd þ t cell enriched media. as expected, by h p.t. jhmv levels were significantly (po . ) reduced in conditioned media treated cultures compared to npcs grown in non-conditioned media ( .  .  and .  .  pfu/ml, respectively; fig. e ). however, treatment with anti-ifn-γ receptor resulted in higher (po . ) viral titers ( .  .  ) compared to cd þ t cell media treated cultures, thereby confirming the pivotal role of ifn-γ in cd þ t cell mediated suppression of jhmv in npcs. we have previously shown that ifn-γ treatment of jhmvinfected opcs increases ifn-α/β secretion, and treatment with ifn-β suppresses jhmv replication (whitman et al., ) . type i interferon (ifn-β) levels in jhmv-infected, ifn-γ treated npc supernatants were assessed by elisa and ifn-β was not detected above background levels (data not shown). we evaluated the expression of the jhmv receptor ceacam a on npcs following treatment with u/ml ifn-γ and did not observe a change in the frequency of cecam aþ npcs between treated and non-treated groups at h p.t. (fig. a and b) (matthews et al., ) . to determine if m transcripts were decreased following ifn-γ treatment, gene-specific quantitative pcr (qpcr) was performed on total rna extracts from jhmv-infected npcs and m transcript levels were normalized to β-actin. m expression was significantly reduced in ifn-γ treated npcs compared to non-treated npcs at and h p.t. (po . ; fig. c ). these findings suggest that the ifn-γinduced inhibitory effect on jhmv replication within npcs is related to both muted expression of ceacam a and inhibition of viral rna synthesis. this study demonstrates that npcs derived from the brains of post-natal c bl/ -gfp mice express the jhmv receptor, cea-cam a, and support viral replication following ceacam adependent infection. additionally, jhmv infection of cultured npcs induces cytopathic effects over time as evidenced by syncytia formation and elevated ldh levels. within the context of jhmv infection of the cns, these findings demonstrate that resident npcs present within defined anatomical niches may be susceptible to viral infection. moreover, we have previously shown that intraspinal transplantation of npcs into mice persistently infected with jhmv results in clinical recovery associated with remyelination (carbajal et al., ; totoiu et al., ) . data presented within this report argues that transplanted npcs may be susceptible to jhmv infection, a finding that highlights important clinical implications for emerging therapies utilizing npcs to treat human neurologic disease as engrafted cells may be susceptible to infection by persistent neurotropic viruses. jhmv infection has previously been shown to inhibit constitutive expression of mhc class i in mouse primary astrocyte cultures and to block ifn-γ-induced mhc class ii expression on murine cerebral endothelial cells (correale et al., ; joseph et al., ) . here, we show that jhmv does not significantly affect mhc class i or ii expression following infection of cultured npcs in the absence of ifn-γ. however, ifn-γ-induced expression of mhc class ii was reduced following jhmv infection. mhc expression plays an important role in immune surveillance during viral infection, and control of jhmv replication within the cns requires antigen recognition by mhc class i and mhc class ii restricted cd þ and cd þ t cells (bergmann et al., ; sussman et al., ; williamson and stohlman, ) . impaired expression of mhc class ii following ifn-γ-treatment of infected npcs may be a mechanism employed to subvert detection by infiltrating virus-specific cd þ t cells. nonetheless, conditioned medium from virus-specific cd þ t cells was able to suppress jhmv replication within npcs, likely due to the effects of ifn-γ. supporting this notion, treatment of infected npcs with recombinant mouse ifn-γ had a dose-dependent inhibitory effect on virus replication, and blocking ifn-γ receptor abrogated the observed suppressive effects. ifn-γ treatment resulted in fewer ceacam a-expressing npcs with a concomitant decrease in jhmv membrane glycoprotein transcripts, suggestive of viral entry inhibition and reduced virion assembly. we also observed that npcs pulsed with the cd -specific viral peptide s - were detected and killed by virus-specific cd þ t cells, indicating that virallyinfected npcs may be targeted for lysis by ctls infiltrating into the cns in response to infection. collectively, our findings argue that t cells are important for controlling viral replication within npcs through both cytolytic activity and ifn-γ secretion. lineage fate mapping of neural stem/precursor cells residing within the subventricular zone of lateral ventricles and subgranular zone of the hippocampus demonstrates the ability of these cells to differentiate into neurons and glia throughout development (doetsch, ; gage, ) . furthermore, endogenous npcs have been shown to proliferate, migrate, and differentiate in response to acute cns inflammatory events, such as with spinal cord injury, stroke, and experimental models of chronic inflammatory demyelinating disorders (picard-riera et al., ; yagita et al., ; zhang et al., ) . though viewed as a glial tropic virus, this study highlights the potential for npcs to serve as a reservoir for jhmv infection and replication. ctl-mediated lysis of jhmv-infected npcs may be detrimental to npc-mediated repair during cns inflammation, and a loss of npcs destined to become oligodendrocytes could contribute to limited remyelination observed in the jhmv-infected cns. additionally, our findings have clinical relevance, as npcs are currently being employed in clinical trials for spinal cord injury as well as for treating the pelizaeus-merzbacher disease, a genetic disorder that affects the growth of the myelin sheath (gupta et al., ; mayor, ) . as npcs used for clinical trials are unlikely to be "self-derived", they would be subject to immune recognition and potential destruction by both innate and adaptive immune responses, necessitating long-term immune suppression to prevent graft rejection (chen et al., ; swijnenburg et al., ; weinger et al., ) . several classes of immunosuppressive drugs used during transplantation, including calcineurin inhibitors i.e. cyclosporine and fk , inhibit the activation and/or proliferation of t cells. such immunosuppressive drugs would foster an environment whereby opportunistic infection or reactivation of latent virus might occur. this raises the possibility that transplanted npcs may be subject to infection, and in the absence of adequate immune surveillance of the cns, could lead to damage/ death of engrafted cells. with this in mind, careful consideration should be given to potential viral infection when contemplating npc grafting for treating neurological disease. the jhm strain of mouse hepatitis virus (j . v- ) was added to npc cultures at a multiplicity of infection (moi) of . pfu/cell. virus was allowed to absorb overnight ( - h) before media were replaced. supernatants of infected cultures were collected at defined time p.i. and viral titers were determined using the dbt astrocytoma cell line as previously described (hirano et al., ) . npcs derived from the striatum of post-natal day transgenic c bl/ mice expressing enhanced green fluorescent protein (gfp) were cultured as previously described (carbajal et al., ) . npc media consisted of dmem/f with glutamax (gibco), n supplement ( x, gibco), ciprofloxacin hydrochloride ( μg/ ml, cellgro), gentamicin ( μg/ml, sigma-aldrich), fungizone ( . μg/ml, gibco), penicillin/streptomycin ( u/ml, gibco), and human epidermal growth factor ( ng/ml, sigma-aldrich). recombinant mouse ifn-γ was purchased from cell sciences. for studies involving blockade of ceacam- a, npcs were infected overnight and monoclonal antibody cc (ebiosciences) was subsequently added at a concentration of μg/ml. media were harvested h p.i. and plaque assay performed to determine viral titers. experimental blockade of ifn-γ receptor was performed using jhmv-infected npcs incubated with nm (final) antimouse cd (ifn gamma receptor ; ebiosciences) or nm purified rabbit igg (control; bd pharmigen) for h before media were replaced with non-conditioned or cd þ t cell conditioned media þ/ À anti-mouse cd or rabbit igg. supernatants were harvested h post-treatment and viral titers determined. cultured npcs were dissociated using . % trypsin-edta and suspended in pbs containing . % bsa and mm edta (invitrogen). cells were subsequently treated with blocking antibody (purified rat igg b anti-mouse cd /cd monoclonal antibody, : ; bd biosciences) for min at c before being incubated with antibodies specific for ceacam a (apc-conjugated, . μg/ test, ebioscience), mhc class i (pe-conjugated, : , ebioscience), or mhc class ii (pe-conjugated, : , bd biosciences), for - min. in experiments where facs analysis of jhmv was performed, npcs were fixed with % paraformaldhyde for min before being permeabilized using bd perm/wash buffer (bd biosciences). the anti-jhmv mab j. . specific for the carboxyl terminus of the viral nucleocapsid (n) protein was conjugated to alexa fluor using the apex labeling system (life technologies) and used at a final concentration of . ng/ml. detection of fluorescence was performed using a lsr ii flow cytometer (bd biosciences) and analysis of facs data was performed with flowjo software (tree star). total rna was isolated from c bl/ splenocytes and npcs using trizol reagent (invitrogen) and purified by phenol-chloroform extraction. cdna was reverse transcribed from rna according to manufacturer's instructions using the superscript iii first-strand synthesis system (invitrogen) and random hexamers. standard pcr for ceacam a expression was performed with an eppendorf mastercycler using the platinum taq dna polymerase kit (invitrogen) and the following primers purchased from integrated dna technologies: ttccctggggaggactactg (forward primer) and tgtatgcttgcc ccgtgaaat (reverse primer). gene products were run alongside a kb plus dna ladder (invitrogen) on a % agarose gel containing ethidium bromide before being imaged using the bio-rad geldoc system. for quantitative rt-pcr experiments, primers specific for the jhmv membrane protein (forward: cgagccgtagcatgtttatcta; reverse: cgcatacacgcaattgaa-cata) were designed using primerquest software (integrated dna technologies, inc.). sybr green real-time pcr master mix (life technologies) was used according to manufacturer's specifications and rt-pcr was performed using the applied biosystem viia real-time pcr system. c t values of m protein transcripts were normalized to β-actin c t values (forward: ggcccagagcaa-gagaggtatcc; reverse: acgcacgatttccctctcagc) and compared using the ΔΔc t method. to evaluate jhmv infection of cultured npcs, cells were dissociated and plated on slides or cover slips coated with reduced growth factor matrigel (bd biosciences). npcs were infected with jhmv overnight and fixed h p.i. with % paraformaldehyde for min at room temperature. immunofluorescence staining was performed as previously described (whitman et al., ) using the anti-jhmv mab j. . ( : dilution) specific for the carboxyl terminus of the viral nucleocapsid (n) protein and the alexa fluor goat anti-mouse igg secondary antibody (life technologies), as well as rabbit monoclonal anti-sox (epitomics) and alexa fluor goat anti-rabbit igg secondary antibody (life technologies). slides were imaged using a nikon eclipse ti inverted microscope. npc death due to jhmv infection was evaluated at , , and h p.i. by measuring lactate dehydrogenase released by lysed cells according to manufacturer's recommendations using the cytotox non-radioactive cytotoxicity assay (promega). briefly, spontaneous and virus-induced ldh levels were determined using the following formula: % lysis¼ (experimental ldh release)/(maximum ldh release). ldh levels from jhmv-infected cultures were then normalized to spontaneously released ldh and expressed as cell death due to infection (%). cd þ t cell isolation for npc media conditioning c bl/ mice were infected with an i.p. injection of .  pfu of a demyelinating (dm) variant of jhmv. on day p.i., cd þ t cells were isolated from spleens by negative selection according to manufacturer's specifications using the easysep mouse cd þ t cell isolation kit (stemcell technologies). briefly, red blood cell depleted splenocytes were suspended at a concentration of  cells/ml in pbsþ % fbs with mm edta. normal rat serum was added at the appropriate concentration and cells were incubated with a cocktail containing a combination of biotinylated monoclonal antibodies directed against cd a, cd b, cd c, cd , cd r/b , cd b, tcrγ/δ and ter , for min. subsequently, a suspension of streptavidin-coated magnetic particles in pbs was added and incubated with the cells for . min; buffer was added to the appropriate volume, and cells were incubated in the easysep magnet for . min to foster binding of magnetically-labeled unwanted cells to the tube walls before cd þ t cells were poured off. to generate cd þ t cell conditioned npc media, the magnetically-labeled fraction following depletion of total t cells was collected using the easysep mouse t cell isolation kit (stemcell technologies). this enriched fraction was treated with μg/ml mitomycin-c (ag scientific), and  cells were co-cultured with  cd þ t cells in ml npc media containing μm cd -specific membrane (m) glycoprotein spanning amino acid residues - (m - , bio-synthesis) for h. cd þ t cell conditioned media were administered to jhmvinfected npcs and supernatants were harvested , , and h p.i. for determination of viral titers. levels of ifn-γ in cd þ t cell conditioned media were determined by elisa using the mouse ifn-γ duoset according to manufacturer's recommendations (r&d systems). interferon-β levels in jhmv-infected npc cultures were evaluated using the verikine mouse interferon beta elisa kit (pbl assay science). the animal protocols and procedures used for these studies were reviewed and approved by the institutional animal care and use committee of the university of california, irvine. npcs were seeded at a density of , cells/well in a flatbottom -well format tissue culture plate (corning life sciences) and pulsed overnight with μm of the immunodominant cd peptide specific for mhv spike (s) glycoprotein spanning amino acids - (s - , bio-synthesis). npcs were simultaneously treated overnight with u/ml ifn-γ to induce mhc class i expression for the presentation of s - . cd þ t cells isolated from dm-infected c bl/ mouse splenocytes (as mentioned for cd þ t cells) using the easysep mouse cd þ t cell isolation kit (stemcell technologies) were then plated with npcs at effector-to-target (e:t) ratios ranging from : to . : . cocultures were incubated for h at c in % co at a final volume of μl/well. the amounts of lactate dehydrogenase released from lysed cells were determined using a cytotox non-radioactive cytotoxicity assay (promega). the percentage of ctlmediated lysis was determined as specified by the manufacturer's protocols. rma-s 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endogenous retroviruses in antibody-deficient mice stroke transiently increases subventricular zone cell division from asymmetric to symmetric and increases neuronal differentiation in the adult rat this work was supported by the national institutes of health (nih) grant r ns to t.e.l. c.m.w. is supported by the california institute for regenerative medicine (cirm) grants rm - and tr - , the national multiple sclerosis society (nmss) collaborative center research award ca -a- , and the gleis family foundation. w.c.p. is supported by nih predoctoral training grant t ns - and j.g.w. is supported by nmss post-doctoral fellowship fg -a- . key: cord- -y wf f authors: zhang, guang lan; srinivasan, kellathur n.; veeramani, anitha; august, j. thomas; brusic, vladimir title: pred(balb/c): a system for the prediction of peptide binding to h (d) molecules, a haplotype of the balb/c mouse date: - - journal: nucleic acids res doi: . /nar/gki sha: doc_id: cord_uid: y wf f pred(balb/c) is a computational system that predicts peptides binding to the major histocompatibility complex- (h (d)) of the balb/c mouse, an important laboratory model organism. the predictions include the complete set of h (d) class i (h -k(d), h -l(d) and h -d(d)) and class ii (i-e(d) and i-a(d)) molecules. the prediction system utilizes quantitative matrices, which were rigorously validated using experimentally determined binders and non-binders and also by in vivo studies using viral proteins. the prediction performance of pred(balb/c) is of very high accuracy. to our knowledge, this is the first online server for the prediction of peptides binding to a complete set of major histocompatibility complex molecules in a model organism (h (d) haplotype). pred(balb/c) is available at . the t cells of the immune system recognize antigens as short peptide fragments (t-cell epitopes) derived from self or foreign proteins. self proteins include all proteins produced by the cells of the host. foreign peptides are derived from pathogens, environmental antigens, tumor cells and transplanted tissue. immune recognition of both self and foreign antigens involves proteolytic processing of antigens, binding of the peptide epitopes by major histocompatibility complex (mhc) molecules and presentation of selected peptide epitopes on the cell surface to activate of t cells ( ) ( ) ( ) ( ) . cytotoxic t cells (cd + ) recognize peptides bound to mhc class i molecules and helper t cells (cd + ) recognize antigen in the context of mhc class ii molecules. mhc class i molecules are present in all cells and bind mainly endogenous peptides (those produced within the presenting cell), whereas class ii molecules are present mainly in cells that recognize foreign proteins, such as macrophages and dendritic cells. t-cell epitopes are critical for the immune response to infectious, autoimmune, allergic and neoplastic disease. they have been studied for the development of peptide-based vaccines ( ) and may also be important in the diagnosis of pathogens. it is estimated that between and % of all peptides can bind a particular mhc molecule ( ) . traditional approaches to the identification of t-cell epitopes that involve various biochemical and functional assays of overlapping peptides derived from proteins of interest are costly and not applicable to large-scale studies. accurate predictions using computer models help speed up the identification of t-cell epitopes ( ), minimize the number of experiments necessary and enable systematic scanning for candidate t-cell epitopes from larger sets of protein antigens, such as those encoded by complete viral genomes ( ) . the balb/c inbred laboratory mouse strain is one of the most commonly used animal models in immunological studies and has been used extensively in vaccine research ( , ) . balb/c mice express three class i (h -k d , h -l d and h -d d ) and two class ii (i-a d and i-e d ) molecules. several publicly available prediction systems for mhc class i and class ii binding peptides provide the prediction models for (histocompatibility complex- ) h d alleles. syfpeithi ( ) has h -k d and h -l d models, bimas ( ) has h -k d , h -d d and h -l d models, and rankpep ( ) has models for *to whom correspondence should be addressed: tel: if an article is subsequently reproduced or disseminated not in its entirety but only in part or as a derivative work this must be clearly indicated. for commercial re-use, please contact journals.permissions@oupjournals.org all h d molecules. syfpeithi uses binding motifs, whereas bimas and rankpep use binding matrices. these are general servers that contain prediction models for a range of mhc molecules in human and mouse, and several other mammalian organisms, but the accuracies of individual models have not been determined. on the other hand, quantitative matrices for h -k b , h -d b , h -l d and h -k k ( , ) have been developed and validated. pred balb/c is a computational system for the prediction of peptides binding to all five mhc molecules in balb/c mice (h d ) class i (h -k d , h -l d and h -d d ) and class ii (i-a d and i-e d ) that allows analysis of proteins for the presence of binding motifs to all five h d molecules in parallel. we derived the initial quantitative matrices for pred balb/c using logarithmic equations based on the frequency of amino acids at specific positions within the training set of mer peptides as described previously ( ) . the initial matrices were refined by including information on the consensus ( ) and other binding motifs, for example, h -k d binding peptides that have i, l or m at major anchor position p ( ) . the anchor positions (e.g. positions and in k d binding peptides) were assigned higher weights than other positions. in addition, the prediction scores were inspected for all permissible amino acids at each of the anchor positions. all amino acids at the anchor positions other than the permissible ones were assigned low scores to exclude peptides with non-permissible amino acids from the list of predicted binders. the final binding scores were normalized to a scale of - and the final models were tested and validated rigorously. to our knowledge, pred balb/c is the first online server for the prediction of peptides binding to a complete set of mhc molecules in a model organism (h d haplotype). the training data containing binding and non-binding peptides were extracted from mhcpep ( ), mhcbn ( ) , syfpeithi ( ) , jenpep ( ) and a set of non-binders (v. brusic, unpublished data). the mer peptides were used for deriving h d class i matrices because the majority of peptides that bind these molecules are amino acids long ( ) . although the majority of h d class ii binding peptides are - amino acids long, their binding cores are amino acids long ( , ) . an iterative elimination method starting from i-a d and i-e d motifs in syfpeithi ( ) was used to identify the core mer regions from long peptides (k.n. srinivasan, g.l. zhang, a. veeramani, j.t. august and v. brusic, manuscript in preparation). the number of peptides in the training sets is shown in table . no one method of predicting peptide-mhc binding consistently outperforms the rest and the most appropriate predictive model depends on the amount of data available in ref. ( ) . in our previous work, an artificial neural network method and hidden markov models were applied to the prediction of human leukocyte antigens (hlas) binding peptides ( , ) , where more training data were available. because relatively small training data sets are available for h d , we adopted matrix models as the prediction method. five · matrices were built, one for each of the five h d alleles, and -fold cross-validations were performed to test the accuracy of the prediction models. the results show that pred balb/c predicts peptides binding to i-e d , i-a d and h -k d with excellent accuracy [area under the receiver operating characteristic (roc) curve, a roc > . ], and to h -d d and h -l d with good accuracy (a roc > . ). the models were also rigorously tested using experimentally known peptides from viral, prokaryotic and eukaryotic origins ( , ( ) ( ) ( ) ( ) and validated by in vivo studies using severe acute respiratory syndrome (sars) nucleocapsid and hiv gag proteins. the h d models accurately predicted out of elispot positive regions from balb/c mice splenocytes immunized with sars nucleocapsid dna vaccines (data not shown). the web interface of pred balb/c uses a set of graphical user interface forms. the interface was built using a combination of perl, cgi and c programs. pred balb/c has been implemented in a sunos . unix environment. users have the option to predict peptides binding to all h d molecules, h d class i molecules, h d class ii molecules or a single h d molecule. the default selection on the webpage is 'all h d ' molecules. to perform predictions using pred balb/c , the user must paste a protein sequence into a textbox and assign a name to the sequence. the sequence must contain between and amino acids. if the prediction is run with an input sequence containing symbols other than the amino acid codes (spaces and carriage returns are allowed) or the total sequence length is outside the - amino acids range, an error message will be displayed. the input can be either a contiguous protein sequence or a list of peptides, one per line. the default selection on the webpage is 'protein sequence' (figure a) , which means the input sequence is treated as a contiguous protein sequence (carriage returns and line breaks will be ignored). the pred balb/c input processing program decomposes protein sequence (or the list of peptides) into a series of mer peptides overlapping by eight amino acids. individual mer peptides are then submitted for prediction. predicted binding scores for all mers are displayed in the result tables ( figure b) . the mer binding scores are within the range - ; the higher the score, the higher the probability of the peptide being a binder. pre-d balb/c has the option to plot the binding scores of all the overlapping mer peptides as a graph, in which the x-axis represents the start position of a mer peptide and the y-axis represents the binding score of the mer peptide ( figure c ). the user can sort the peptides by their binding scores and choose to view only predicted binders with binding scores above a certain threshold. to assess prediction accuracy, we used measures of sensitivity se = tp/(tp + fn) and specificity sp = tn/(tn + fp) (tp: true positives; tn: true negatives; fp: false positives; fn: false negatives). the higher the value of sp, the lower is the value of se, which results in lower number of both tps and fps. the lower the value of sp, the higher is the value of se, which results in higher number of both tps and fps. raw binding scores are mapped to a linear scale that corresponds to sp values, and therefore the prediction thresholds across different models have similar meaning. for example, when a user sets the threshold to , the specificity of the predictions to all five alleles is . . the corresponding sensitivities of each model can be viewed at http://antigen.i r. a-star.edu.sg/predbalbc/html/specificity.html. when users select the input sequence type to be 'a list of peptide sequences', the input sequences separated by carriage returns or line breaks are treated as different peptides (figure a ). all overlapping mers in each peptide are submitted for prediction. in the result tables, predicted binding scores are represented by the highest individual mer binding score within the input peptide. the predicted binding scores of individual mers in each peptide in the list are not shown ( figure b ). to display the top-scoring mer peptides from each input peptide, the user can use the function 'view binding peptides at threshold ' (figure b ). in the result page ( figure c ), the mers with binding scores equal to or above the threshold of are aligned with the input peptides. the predicted mers are displayed with the names of the h d alleles to which the mer binding scores are above the threshold. for example, the first input peptide, ypilpeylqcvk, has binding scores . , . , . , . and . to h -d d , h -k d , h -l d , i-a d and i-e d , respectively ( figure b ). the alignment view of the predicted binding peptides at threshold , which indicates that the specificity of the prediction is . . at threshold (sp level . ), there are no mer binders to h d class ii alleles and the mer ilpeylqcv has the highest binding score to h -d d , . . thus, in figure c , this mer is aligned with the input peptide and followed by 'd d '. conclusion pred balb/c marks a new direction in predictive modeling of mhc-binding peptides and t-cell epitopes. the main advantage is that pred balb/c focuses on a complete organism and its predictions represent a complete set of predicted targets of t-cell immune responses. the focus on the complete set of mhc alleles is closer to studies involving laboratory animals. this approach provides a more complete view of the immune responses of an organism. the balb/c mouse is an important laboratory model and pred balb/c is, therefore, useful for the analysis of immunization regimens and deciphering responses to infections. further development of pred balb/c will include addition of matrices for prediction of mer and mer binders to h d class i molecules and further improvement of prediction matrices by cyclical refinement-using newly defined binders and non-binders from experiments. mechanisms of mhc class i-restricted antigen processing proteases involved in mhc class ii antigen presentation cut and trim: generating mhc class i peptide ligands class ii mhc peptide loading by the professionals genome-wide characterization of a viral cytotoxic t lymphocyte epitope repertoire computational binding assays of antigenic peptides computational methods for prediction of t-cell epitopes-a framework for modelling, testing, and applications rapid determination of hla b * ligands from the west nile virus ny genome a peptide mimotope of type pneumococcal capsular polysaccharide induces a protective immune response in mice vaccination by genetically modified dendritic cells expressing a truncated neu oncogene prevents development of breast cancer in transgenic mice syfpeithi: database for mhc ligands and peptide motifs scheme for ranking potential hla-a binding peptides based on independent binding of individual peptide side-chains prediction of mhc class i binding peptides using profile motifs an automated prediction of mhc class i-binding peptides based on positional scanning with peptide libraries new horizons in mouse immunoinformatics: reliable in silico prediction of mouse class i histocompatibility major complex peptide binding affinity methods for prediction of peptide binding to mhc molecules: a comparative study efficient binding to the mhc class i k(d) molecule of synthetic peptides in which the anchoring position does not fit the consensus motif mhcpep, a database of mhc-binding peptides mhcbn: a comprehensive database of mhc binding and non-binding peptides jenpep: a novel computational information resource for immunobiology and vaccinology peptides naturally presented by mhc class i molecules chemistry of peptides associated with mhc class i and class ii molecules crystal structures of two i-a d -peptide complexes reveal that high affinity can be achieved without large anchor residues neural models for predicting viral vaccine targets multipred: a computational system for prediction of promiscuous hla binding peptides prediction of major histocompatibility complex binding regions of protein antigens by sequence pattern analysis the mhc class i-restricted immune response to hiv-gag in balb/c mice selects a single epitope that does not have a predictable mhc-binding motif and binds to k d through interactions between a glutamine at p and pocket d influenza a virus-specific h- d restricted cross-reactive cytotoxic t lymphocyte epitope(s) detected in the hemagglutinin ha subunit of a/udorn/ identification of murine cytotoxic t-lymphocyte epitopes of bovine herpesvirus this project has been funded in part by us federal funds from the national institute of allergy and infectious diseases, national institutes of health, department of health and human services, under grant no. u ai and contract no. hhsn c. funding to pay the open access publication charges for this article was provided by the institute for infocomm research.conflict of interest statement. none declared. supplementary material is available at nar online. key: cord- -tpqf q authors: thanthrige-don, niroshan; abdul-careem, mohamed f.; shack, l. allen; burgess, shane c.; sharif, shayan title: analyses of the spleen proteome of chickens infected with marek's disease virus date: - - journal: virology doi: . /j.virol. . . sha: doc_id: cord_uid: tpqf q marek's disease virus (mdv), which causes a lymphoproliferative disease in chickens, is known to induce host responses leading to protection against disease in a manner dependent on genetic background of chickens and virulence of the virus. in the present study, changes in the spleen proteome at , and days post-infection in response to mdv infection were studied using two-dimensional polyacrylamide gel electrophoresis. differentially expressed proteins were identified using one-dimensional liquid chromatography electrospray ionization tandem mass spectrometry ( d lc esi ms/ms). comparative analysis of multiple gels revealed that the majority of changes had occurred at early stages of the disease. in total, protein spots representing host proteins were detected as either quantitatively (false discovery rate (fdr) ≤ . and fold change ≥ ) or qualitatively differentially expressed at least once during different sampling points. overall, the proteins identified in the present study are involved in a variety of cellular processes such as the antigen processing and presentation, ubiquitin–proteasome protein degradation (upp), formation of the cytoskeleton, cellular metabolism, signal transduction and regulation of translation. notably, early stages of the disease were characterized by changes in the upp, and antigen presentation. furthermore, changes indicative of active cell proliferation as well as apoptosis together with significant changes in cytoskeletal components that were observed throughout the experimental period suggested the complexity of the pathogenesis. the present findings provide a basis for further studies aimed at elucidation of the role of these proteins in mdv interactions with its host. marek's disease (md) in chickens is caused by gallid herpesvirus (gahv- ) or marek's disease virus (mdv). hereafter, this virus is referred to as mdv. mdv is a highly prevalent alpha-herpesvirus and the disease it causes is characterized by transient neurological signs and immunosuppression at early stages that could subsequently be followed by lymphoma formation in various visceral organs in susceptible birds. upon infection via inhalation, in all infected birds, mdv is taken to various lymphoid organs, such as spleen, thymus and bursa of fabricius where early cytolytic infection in b cells that proceeds to latent infection of t cells occurs. while a lifelong latent phase could occur in genetically md-resistant birds and in those protected by vaccination, late reactivation of latent virus in susceptible birds could cause transformation of mainly cd + t cells, leading to lymphoma formation. meanwhile, active replication of mdv occurs in feather follicles of infected birds regardless of their genetic susceptibility rendering them a continuous source of infectious viruses (baigent and davison, ) . although md is currently controlled by vaccination, there have been periodical md outbreaks caused by new strains of mdv with increased virulence (witter, ) . the potential of mdv to evolve and overcome vaccinal immunity is considered as a major threat for a sustainable md control strategy. however, many aspects of mdv-host interactions are still being elucidated (baaten et al., ) . to date, several studies have been conducted to examine host gene expression in response to mdv infection on a relatively large scale using various genomic techniques, such as microarrays. changes in gene expression of chicken embryo fibroblasts (cef) infected with rb b, a very virulent strain of mdv, were studied by morgan et al. ( ) using a microarray containing expressed sequence tags. these authors reported the differential expression of a number of host genes including those associated with inflammation, antigen presentation and cell growth. an in vivo study by our group using the same virus strain and a small-scale microarray has revealed significant changes in the expression of genes encoding cell surface molecules, transcription and signal transduction molecules as well as cytokines (sarson et al., ) . while studies of this nature, in the context of md in particular and various other viruses in general (reviewed in piersanti et al., ) would certainly enhance our understanding of hostpathogen interactions, further expansion of this knowledge with proteomic studies is still important (reviewed in burgess, ; zhang et al., ) . this is partly because of the possible inconsistency between the expression of genes at the transcript and protein levels (gygi et al., ) . furthermore, viruses can induce post-translational modifications in host proteins without affecting the mrna expression (liu et al., ) . fig. . representative d gel images of mdv-infected and uninfected control spleen proteomes with their respective sampling points. arrows with accompanying spot numbers show successfully identified protein spots that were uniquely expressed (qualitative differences) in each group at corresponding time point. please refer to table for identities of corresponding spot numbers and to fig. for the map of quantitatively differentially expressed spots. in a recent in vitro proteomic study, ramaroson et al. ( ) inventoried and proteins expressed in mdv-infected and mock-infected cef, respectively. several other proteomic studies in the context of md have been conducted to model the proteome of mdvtransformed cd + t lymphocytes in vitro. notably, proteomic modeling of mdv-transformed cd hi cd + t lymphocytes has suggested that these cells exhibit a regulatory t cell phenotype (buza and burgess, ; shack et al., ) . further, such studies have been able to describe the fundamental differences between mdv-transformed t cells and their non-transformed healthy counterparts with regard to activated signalling pathways . with respect to viral protein expression, liu et al. ( ) identified a number of unique proteins expressed during the lytic phase of mdvinfected cef using a mass spectrometry-based proteomic approach. these studies highlight the potential of various proteomic tools in understanding the dynamics of host-pathogen interactions during various phases of md. the current study was intended to investigate the dynamics of host protein expression across the various phases of mdv life cycle in mdvinfected chickens. while we were able to detect more than separate protein spots for each sample, here we report more than significantly differentially expressed proteins identified using two-dimensional gel electrophoresis ( de) and mass spectrometry. putative importance of some of these proteins in the context of md is discussed. proteins from spleens of mdv-infected and uninfected control chickens at , and days post-infection (dpi) were extracted and analyzed by de in order to compare the protein expression profiles between each group. on average, ± distinct protein spots could be resolved by de using ph - nl ipg strips loaded with fig. . a representative gel image showing d gel electrophoresis map of the relative locations of spots that displayed significant quantitative differential expression (fdr ≤ . and fold change ≥ ) at least once during different sampling times. this image represents the proteome of dpi mdv-infected spleen. because of the absence of certain spots in this gel, arrows for spot , , and represent the relative positions only. please refer to table for identities of corresponding spot numbers and to fig. for maps of qualitatively differentially expressed spots. fig. . comparison of total numbers of significantly differentially expressed protein spots in mdv-infected spleens at various sampling time points. in calculation of total number of spots in each category, newly induced protein spots were considered as upregulation and the absence of spots compared to uninfected controls was considered as down-regulation. μg of total proteins. the molecular weights of spots ranged from to kda. differences in spot intensity were identified as either qualitative or quantitative changes (figs. and ). on average there were over spots on each gel, however only , and spots from each group at consecutive sampling points were considered for statistical comparison. because, according to our selection criteria, only those spots which were present in at least of gels in both infected and control groups at a given sampling point were considered for quantitative comparisons. the highest number of qualitative differences in spots was detected at dpi (n = ). this number decreased in the subsequent sampling time point at dpi (n = ) and the lowest number was at dpi (n = ). although a similar pattern was seen in the total number of spot differences, the highest number of quantitatively significant differences (false discovery rate or fdr ≤ . and fold change ≥ ) in spot expression was detected at dpi (n = ), which was followed by dpi (n = ) and dpi (n = ). among these spots, there were , and spots identified as significantly up-regulated at , and dpi, respectively. the rest were significantly down-regulated (fig. ) . taken together, protein spots were detected as either quantitatively or qualitatively differentially expressed at least once during different sampling points. in order to obtain the identities of the differentially expressed spots, the spots were manually excised from preparative gels prepared by loading mg of total proteins and staining with coomassie blue. subsequently, trypsin-digested spots were identified by d lc esi ms/ ms. peptide identities with fdr b . were considered significant and, in total, the identity of proteins in different spots representing different proteins was determined (table ) . moreover, several spots contained peptides generated from multiple proteins. while we have used the protein with the highest relative abundance under corresponding spot number throughout our discussion, the complete list of spots with multiple identities has been provided as supplementary table . several proteins were differentially expressed at more than one time point, e.g. cathepsin d (cathd), natural killer cell enhancing factor isoform (nkef), eukaryotic elongation factor (eef ), aldolase b (aldob), membrane associated guanylate kinase (magi ), a predicted hypothetical protein (hp ) and beta actin (actb) at all three times. in total, proteins were differentially expressed exclusively at dpi. notably, several proteins involved in antigen presentation pathways, the ubiquitin-proteasome protein degradation system and a number of spots representing several cellular structural proteins were among those that were differentially fig. . venn diagram summarizing the spots that were significantly differentially expressed in the spleen tissues of mdv-infected chickens according to their corresponding time of sampling. these identities include both quantitatively and qualitatively differentially expressed spots. the identities of spots which were commonly expressed were placed in overlapping areas accordingly. corresponding spot numbers are in parentheses. refer to table for the respective protein names. expressed exclusively at dpi. there was only one spot specific to each of and dpi. these spots were identified as glutathione-stransferase theta ( dpi) and proliferating cell nuclear antigen ( dpi). the significant changes detected with the rest of the spots overlapped between each time point in varying numbers. interestingly, some of the spots representing a particular protein showed opposite directions of regulation even within the same group. for example, three spots with different molecular weights and pi were identified as cathd (table ) . of these three spots, two spots with molecular weights of . and . kda were found to be up-regulated in infected birds at more than one time point. the summarized distribution of the identities of spots identified at each time point is presented in fig. . forty-percent of the identified proteins were associated with the cytoplasm and the plasma membrane (terms go: and go: respectively). furthermore, there were % nuclear and nuclear envelope proteins (go: and go: ), % cytoskeleton-associated proteins (go: ) and % extracellular proteins (go: ). nine-percent of proteins did not have any go annotation with respect to their cellular compartment, hence, were categorized very broadly as being associated as a "cellular component" (go: ). for biological processes, the highest associations ( %) were with metabolic processes (go: ). another % associations were with nucleic acid metabolism (go: ), while % and % were associations with transport (go: ) and cell communication (go: ), respectively. among the remaining associations, % were associated with cell death (go: ) (fig. ) . virus genome copy numbers in infected spleens were determined using quantitative real-time pcr (qpcr). using conventional pcr screening, mdv-meq gene could be amplified from dna of all infected spleens but not from any of the uninfected controls (data not shown). results of the subsequent qpcr analysis of infected samples are presented in fig. . the average mdv-meq copy numbers were . × ± . × (n = ), . × ± . × (n = ) and . × ± . × (n = ) per ng of spleen dna at , and dpi, respectively (fig. ). there was a statistically significant difference between genome copy numbers at dpi compared to other time points. in the present study, we have profiled the global protein expression changes in the chicken spleen in response to mdv infection at various time points representing the different phases of mdv life cycle. furthermore, we have determined the identity of the spots that were differentially expressed between infected and uninfected birds using mass spectrometry. among the proteins that were differentially expressed, there was considerable number of proteins that had multiple spots in d gels. in addition, there was some degree of disagreement between the expected and experimental molecular weights in case of some of the proteins. this could be due to several reasons. first, some proteins exist as different isoforms (therefore different pi) as well as different intermediate stages between translation and functionally mature form (i.e. pre-pro-and pro-forms of protein). this could change the molecular weight and/or the isoelectric point (for example, please see the discussion regarding different spots of cathd below). another reason is possible protein degradation between sample collection and processing. interestingly, no protein with multiple matches showed any specific migration pattern in our d gels (such as trains of spots). while current data are not enough to determine the exact reason for such discrepancies, it should be noted that several previous studies using d gels have also reported the presence of similar patterns of proteins with multiple spots (dupont et al., ; liu et al., ; saldanha et al., ; zheng et al., ) . analysis of viral load in spleen tissue revealed a significant increase over time. although viral genome load may not be a direct indicator for the degree of infection, there is a correlation between mdv genome load and the number of infected cells (bumstead et al., ) as well as with subsequent md incidence (islam et al., ) or protection conferred by vaccines against md (abdul-careem et al., ) . based on our previous observations, virus genome load of around × copies/ ng of spleen dna is correlated with development of tumors in infected birds (abdul-careem et al., ) . therefore, it is conceivable that the virus copy numbers observed in the present study would be an indicative of a high level of infection. the proteins identified in the present study are involved in a variety of cellular processes, notably the antigen processing and presentation, ubiquitin-proteasome protein degradation, formation of the cytoskeleton, cellular metabolism, signal transduction and regulation of translation (table ). the significance of these processes in chicken-mdv interaction is discussed below. although this was not determined in the present study, temporal changes in the cellular composition of the spleen may have, at least in part, influenced the whole spleen organ proteome. however, the advantage of our method is that the proteins that are differentially expressed could be used to point to cell subsets and/or mechanisms that have a potential involvement in mdv-host interactions and could be targeted for future studies. among the differentially expressed spots, several proteins were identified that are either directly or indirectly involved in the ubiquitin-proteasome (upp) and antigen presentation pathways (table ). in our study, we identified three differentially regulated proteins, namely ubiquitin c-terminal hydrolase l (uchl ), proteasome (prosome, macropain) subunit beta type (psmb ) and predicted protein similar to mouse proteasome s subunit atpase (psmc ) (also known as msug ), which are involved in upp (table ) . among them, chicken uchl (alias uch- ) has % amino acid similarity with its human counterpart (baek et al., ) , which is a deubiqutinating enzyme (dub) that functions as a negative regulator of ubiquitination of proteins as well as facilitates recycling of ubiquitin. increasing evidence suggests that dubs are important regulators of many cellular processes such as endocytosis, apoptosis and various signalling pathways (wing, ) . several virus-encoded proteins such as epstein-barr virus (ebv)-encoded epstein-barr nuclear antigen and herpes simplex virus (hsv- ) regulatory protein icp have been shown to interact with dubs for viral survival in infected cells (reviewed in lindner, ) . although there is a high amino acid identity between human uch-l and chicken uch- , they differ with respect to their substrate specificity and tissue distribution (baek et al., ) . therefore, the chicken ortholog of uch-l may not have a similar role in viral infections. more functional studies are needed to understand the significance of the down-regulation of uchl in mdv infection. down-regulation of psmb at dpi probably indicates the modification of proteasome into immunoproteasome under the influence of interferon (ifn)-γ to enhance the generation of peptides for binding to major histocompatibility complex (mhc) class i molecules. in this process, three constitutive beta subunits of the s proteasome, namely delta, x and z (psmb ), are replaced by ifn-γ inducible low molecular mass peptide (lmp)- , lmp- and multicatalytic endopeptidase complex-like (mecl) catalytic subunits, respectively (griffin et al., ) . although we could not detect upregulation of ifn-γ inducible subunits, given the reciprocal regulation between constitutive and ifn-γ inducible subunits (hisamatsu et al., ) , down-regulation of psmb may indicate the effect of induced ifn-γ. in line with that, mdv is known to induce ifn-γ in chickens as early as dpi and remains up-regulated until at least dpi (xing and schat, ) . moreover, although mdv is capable of down-regulating mhc-i expression in vitro, elevated ifn-γ has been shown to reverse the effect of mdv on mhc-i (levy et al., ) . our present observations do not provide evidence for differential regulation of mhc-i expression. however, given the possible enhancement of immunoproteasome activity, it is conceivable that there is an enhanced mhc-i-mediated antigen presentation. the s proteasome subunit sug up-regulates mhc-ii expression by interaction with class ii transactivator (ciita) gene and mhc-ii proximal promoter in human. mhc-ii expression is reduced in the absence of the expression of sug (bhat et al., ) . in agreement with this, the observed absence of predicted protein, msug in infected birds is associated with a significant decrease in the expression of the mhc class ii alpha chain (b-la) in infected spleens at dpi. reduction of the mhc-ii expression is known to be a predominant way of evading the host immune response by a number of viruses including herpesviruses (hegde et al., ) . in agreement with our present observation of down-regulation of mhc-ii expression, previous work from our laboratory showed that mdv infection causes significant downregulation of invariant (ii) chain gene expression in the chicken spleen tissue (sarson et al., ) . in contrast to this observation, niikura et al. ( ) have shown an up-regulation of mhc-ii in bursa cells of chickens infected with md , another very virulent strain of mdv. however, in contrast to our present infection model which used outbred spf chickens, they have used chickens from a cross between two inbred lines, i and , both of which are susceptible to md (bacon et al., ) . therefore, it is possible that the difference in genetic background of infected birds may have, at least in part, contributed to these contradicting observations. taken together, our observations suggest that there is enhanced antigen processing, presumably mediated by elevated ifn-γ for mhc class i pathway, while there is a down-regulation of mhc class iimediated antigen presentation at least in our experimental model at early stages of the disease. in the current study, altered spot profiles were observed for a number of cytoskeleton-associated proteins representing all three main categories of cytoskeleton proteins, namely microfilaments, intermediate filaments and microtubules. among the microfilament proteins, all detected actin spots, except for alpha- actin (acta ) and a predicted protein similar to coactosin-like (cotl ) at dpi, were either newly induced or significantly up-regulated across the experimental period. intermediate filament, lamin b (lmnb ) spots also were either up-regulated or newly induced in infected birds at all times. however, microtubule protein, tubulin β b (tubb b) was significantly down-regulated in infected birds at dpi (table ) . changes in cytoskeleton proteins have been previously described in several other viral infections, such as ibdv (zheng et al., ) , severe acute respiratory syndrome (sars)-associated coronavirus (jiang et al., ) and human papillomavirus type (akgül et al., ) . herpesviruses are known to interact with the actin filament system and its regulatory protein, rho gtpase, at various stages of infection (favoreel et al., ) . in addition to actins, here we have identified a rho gtpase regulatory protein, d -gdi, as a protein that was differentially expressed in spleen of infected chickens (discussed in detail elsewhere in the discussion). this may indicate a putative role of the same system in the context of mdv infection. schumacher et al. ( ) showed that mdv-encoded us ortholog protein causes depolymerization of the actin stress fibers. however, its role in mdv replication and/or spreading is still unclear. stathmin (stmn ), which also plays a role in the regulation of the microtubule system, has been shown to be up-regulated in ebv-infected b lymphocytes in human as early as dpi (baik et al., ) . in the present study, we could not detect changes in stmn in spleen at dpi. however, the expression of this protein was induced by dpi. as a possible result of regulation by stmn , tubb b, which is a component of the microtubule filament system, also showed a similar pattern of expression in mdv-infected spleen tissues. however, if that change has any direct relationship with the stmn is not known. apart from its interaction with the microtubule system, stmn has been shown to interact with heat shock protein (hsp ) proteins, particularly with heat shock cognate (hsc ) (manceau et al., ) . in line with that, we have also identified significant changes in the expression of hsp proteins, including hsc . another cellular structural protein, lmnb , which is associated with the nuclear membrane, was up-regulated in infected birds throughout the experimental period as a probable result of the egress process of virus nucleocapsids from the infected nuclei. the nucleocapsids of herpesviruses acquire a temporary envelope from the inner nuclear membrane before egress by budding off from the infected cell nuclei (granzow et al., ) . in line with that, camozzi et al. ( ) have reported various biochemical and structural modulations, including mislocalization of lamin proteins in the nuclear envelope of cells infected with human cytomegalovirus. although camozzi et al. ( ) did not observe any quantitative changes in the expression of lamin proteins, qualitative changes in the expression of the same protein have been described in infections with several other viruses such as ibdv (zheng et al., ) , enterovirus (leong and chow, ) and sars-associated coronavirus (jiang et al., ) . similar to the other members of the family, mdv particles egress from the nucleus of infected cells through budding (baigent and davison, ) . therefore, it is conceivable that the up-regulation of lmnb protein observed in the present experiment is, at least partly, the result of such interaction. proliferating cell nuclear antigen (pcna), which is one of the critical proteins in cell survival, was significantly up-regulated in infected spleens at dpi. pcna is an essential component in dna synthesis process in the cell. therefore, it is likely that induction of pcna represents highly replicating cell populations in the spleen at dpi. while it is possible that elevated pcna expression represents a transformed cell population, possible interactions between pcna and viral proteins might be also occurring. for example, hsv- encoded protein, icp . , interacts with pcna of infected cells, presumably preventing virus-induced translational arrest (brown et al., ; harland et al., ) . the possibility of a yet unidentified mdv protein interacting with pcna remains to be investigated. icp . has also been shown to interact with phosphatase , preventing the inactivation of eukaryotic elongation factors (eef), a group of proteins which play an important role in protein biosyntheses, hence being important in various cell processes including proliferation (thompson and sarnow, ) . eef was among the proteins that were up-regulated at both and dpi. increased levels of eef have been shown in response to human immunodeficiency virus (hiv) protein, vpr, and have been shown to have the potential of preventing vpr-mediated apoptosis in cd + t cells (zelivianski et al., ) . d -gdp-dissociation inhibitor (d -gdi) (also known as ly-gdi) was differentially regulated in infected birds compared to uninfected controls. as mentioned above, ly-gdi represents a group of proteins i.e. gdi, which are involved in the regulation of another group of proteins, rho family gtpases. apart from its involvement in the organization of the cytoskeleton, rho-gtpases are also involved in cell signalling and proliferation. gdis are involved in the regulation of shifting of rho gtpase between the active gtp-bound form and the inactive gdp-bound form. our present observations showed a more than -fold increase of an . kda d -gdi spot while there was a significant decrease of the presumably intact protein of kda. while activation as well as apoptosis of different types of cells in response to viral infections has been well documented, our present observations may highlight some of the molecules involved in these processes in the context of mdv infection. cathepsin d (cathd), a lysosomal aspartic proteinase, was among the proteins that were differentially expressed in infected spleens at all three time points. there were two spots with molecular weight of approximately and kda which were up-regulated, while another one ( . kda) was down-regulated. cathd is synthesized as a single chain pre-pro-enzyme and after being cleaved into several successive intermediates, it forms the mature + kda form consisting of a heavy and a light chain in the lysosome (laurent-matha et al., ) . therefore, processing of the . kda intermediate into the mature enzyme appears to be induced by mdv infection. cathd has been shown to be an important mediator of apoptosis induced through the lysosomal pathway (guicciardi et al., ) . while cathd appears to be involved in the intrinsic pathway of the induction of apoptosis in activated t lymphocytes in human (bidere et al., ) , it may also play a significant role in the resolution of inflammation by inducing apoptosis in neutrophils (conus et al., ) . apart from its role in the induction of apoptosis, increasing evidence suggests that cathd plays a significant role in cancer progression and metastasis. in the context of md, several studies have shown the occurrence of cell death in mdvinfected organs such as the bursa of fabricius (st hill and sharma, ) , thymus (morimura et al., ) and in peripheral blood mononuclear cells (morimura et al., ) . while it is conceivable that up-regulated cathd may play a role in apoptosis during the early stages of md, it may also have a role in t cell transformation in the later stages of the disease; however, this needs to be further studied. transglutaminase (tgm ) is among the proteins with the highest fold increase in spleens of infected chickens. generally, transglutaminases are involved in post-translational modification of proteins (beninati and piacentini, ) . tgm is highly expressed in the prostate gland of humans but little information is available about the function of this protein in chickens. tgm , which is a ubiquitously expressed protein in human (also known as tissue tgm, ttgm), has been shown to play a significant role in stress response (ientile et al., ) notably in apoptotic cell death. while tgm is up-regulated in apoptotic cells, it acts as a molecular glue and appears to stabilize the dying cells to prevent release of intracellular molecules prior to clearance by phagocytosis (fesus and szondy, ) thereby preventing adverse effects, such as excessive inflammatory reactions. assuming chicken tgm has a similar role as that of human tgm , above observations may explain the putative role of the former in the context of host-mdv interactions in the spleen to prevent collateral damage to the neighbouring cells. according to go classification based on the biological process, the highest association of identified proteins in the current study was with metabolic processes ( %) (fig. ) . notably, several metabolic enzymes associated with glycolysis have been found differentially regulated. among them, a . kda spot representing aldolase b fructose-bisphosphate (aldob) was constantly up-regulated in infected spleens at all time points. interestingly, another spot with similar identity and molecular weight but slightly different pi was significantly down-regulated only at dpi. while different pis for the same protein is possible with structural changes such as phosphorylation, functional relevance in this context needs to be elucidated. another two newly induced glycolytic enzymes, each at and dpi, were identified as triosephosphate isomerase (tpi ) and phosphoglycerate mutase (pgam ), respectively. viruses utilize host cell metabolic process for their replication process. in agreement with the present observations, significantly elevated levels of several serum enzymes, including aldolase b, in response to mdv infection in vivo has been previously described (ivanov et al., ) . further, mdvencoded protein pp has been shown to up-regulate cellular metabolic activities in vitro as determined by the enhanced activity of mitochondrial dehydrogenases (li et al., ) . similarly, human cytomegalovirus infection in fibroblasts has also shown overall upregulation of a number of glycolytic enzymes (munger et al., ) . in conclusion, findings of the present study highlight some of the mechanisms involved in the host response in the spleen to mdv infection during various time points representing different stages of mdv pathogenesis. although the functions of the proteins, which were identified here, were not studied, it is likely that all or some of them are involved in host-virus interactions. one of the limitations of the tools used in this study is the inefficiency of detecting low abundance proteins or those with low molecular weights, such as cytokines and chemokines. therefore, a more comprehensive study is needed to elaborate on our present observations and to further explore other proteins that may play a role in pathogenesis of the virus as well as host responses to this virus. all the chickens used in this experiment were one-day old specific pathogen free (spf) chickens obtained from the animal disease research institute, canadian food inspection agency (ottawa, ontario, canada). birds were kept in an isolation facility at the ontario veterinary college throughout this experiment. chickens were infected with the rb b strain of very virulent marek's disease virus (passage ) (schat et al., ) which was obtained from dr. k.a. schat (cornell university, ny, usa) . twenty-four, one-day old chicks were randomly divided into two groups and were housed in the isolation facility. one group of birds (n = ) was given plaque-forming units (pfu) of the rb b strain of very virulent mdv intraperitoneally on day of age. the rest (n = ) were kept as uninfected controls. infected and uninfected control birds were kept in separate units with similar environmental conditions. on , and dpi, representing different stages of mdv pathogenesis, four chickens that were randomly selected from each group were euthanized using co inhalation. at necropsy, a portion of spleen was collected from each bird and was snap-frozen in liquid nitrogen. subsequently, frozen tissues were kept at − °c until further processing. another portion was preserved in rnalater (qiagen inc., missisauga, on, canada) . animal experiments were conducted in accordance with the guidelines provided by the canadian council on animal care. all experiments complied with institutional animal care guidelines and were approved by university of guelph animal care committee (protocol number r ). each frozen spleen tissue was briefly homogenized in a lysis buffer (ph . ) containing mm tris-cl, m thiourea, m urea and % (w/v) chaps. the volume of lysis buffer used for each tissue sample was equal to times of tissue mass. samples were further solubilized by sonication for min on ice and insoluble tissue debris was removed by centrifugation under , ×g at °c. subsequently, supernatant was collected separately from each sample and protein concentrations were determined using the bio-rad protein assay as prescribed by the manufacturer. the spleen protein sample from each chicken was analyzed separately. there were a total of chickens in two groups (infected and uninfected) i.e. protein samples for analysis by d-page. each analytical d-page gel was prepared with μg of proteins mixed with rehydration buffer ( m urea, % chaps, mm dtt, μl/ml appropriate ipg buffer, μl/ml destreak reagent (ge healthcare) and . % bromophenol blue) to a total volume of μl. the first dimension separation was performed in cm, ph - non-linear immobiline drystrips (ge healthcare) using ettan ipgphor isoelectric focusing unit (ge healthcare). after rehydration at v for h, isoelectric focusing was performed at v for h, v for h and v until a total of , volt hours was reached. each focused strip was incubated at room temperature, initially in ml of equilibration buffer ( mm tris-cl (ph . ), m urea, % (v/v) glycerol, % (w/v) sds and . % bromophenol blue) containing % (w/v) dtt for min and subsequently in a similar volume of equilibration buffer containing . % (w/v) iodoacetamide for a similar time. for the second dimension separation, each ipg strip was placed on a . % sds-polyacrylamide gel and four such gels were simultaneously run each time subjecting them to ma/gel of current at °c in a ruby apparatus until the bromophenol blue dye front reach the opposite edge of the gel. each gel was subsequently fixed for h in a solution containing % (v/v) methanol and % (v/v) acetic acid, stained with sypro ruby stain (bio-rad) overnight and destained in the fixing solution for h. gel images were digitized using typhoon variable mode imager (ge healthcare) at nm using a nm filter. preparative gels were prepared in a similar manner, using mg of protein from each sample and stained them with coomassie brilliant blue instead of sypro ruby. digitized gel images were used to estimate the expression of different proteins in each analytical gel using version of phoretix d software (nonlinear dynamics). the pixel volume of each detected spot was referred to as the spot volume and was used in the subsequent comparisons. background correction for pixel volumes of each spot was done using the mode of non-spot and normalized spot volumes were calculated as a fraction against the total volume of spots in each gel. the spots which were not present in the expected position or showed decreased intensity were considered "down-regulated", while the spots that appeared in only one group or showed enhanced intensity were considered "up-regulated". although this is an indicator of protein abundance, the possibility of post-translation modifications, hence changes in location of spots on the d-page gels, could not be ruled out in our study. there were eight d-page gels per time point: derived from infected spleens and from those of uninfected birds. only the spots that were present in all gels and those that were absent from a maximum of one analytical gel per group at a given time point were considered for the statistical comparison. statistical analysis was done using sas (version . ). normalized volumes of each corresponding spot from each group at similar time point were compared with student t-test. the resulting p values were used to calculate the fdr. spots that were having both p ≤ . and fold difference n in mean normalized volumes were considered as significantly differentially expressed. fdr for any selected spot was less than %. the spots that showed a significant difference, and those expressed only in a particular group at a given sampling point, were selected for identification by d lc esi ms/ms using a lcq deca xp plus mass spectrometer coupled with two thermo surveyor ms pump quaternary gradient pumps at the life science and biotechnology institute, mississippi state university as described below. each selected spot was excised manually from coomassie brilliant blue stained preparative gels and "in-gel" digested exactly as previously described (shevchenko et al., ) . proteins were reduced with mm dtt at °c for min and alkylated with mm iodoacetamide at °c for min. trypsin digestion was done using molecular biology grade porcine trypsin ( μg; °c; h; : ratio of protein:trypsin; promega corporation, madison, wi). liquid chromatography was done with a reverse phase (c ) lc column coupled directly in line with the mass spectrometer. peptides were loaded into a liquid chromatography gradient ion exchange system containing a thermo separations p quaternary gradient pump (thermoelectron corporation; san jose, ca) coupled with a . × mm biobasic c reverse phase liquid chromatography column of a proteome x workstation (thermoelectron). the reverse phase gradient used . % formic acid in acetonitrile and increased the acetonitrile concentration in a linear gradient from % to % in min and then % to % in min followed by % for min and % for min. the mass spectrometer was configured to optimize the duty cycle length with the quality of data acquired by alternating between a single full ms scan followed by three tandem ms scans on the three most intense precursor masses (as determined by xcalibur software in real time) from the full scan. the collision energy was normalized to %. dynamic mass exclusion windows were set at min and all of the spectra were measured with an overall mass/ charge (m/z) ratio range of - . identification of all spots was done as a single run in a randomized order. to prevent "carry-over" after the peptides generated from each spot were analyzed, and before those from the next spot were analyzed, the lc column was washed with % acn, a negative control sample was run to confirm no carryover and the lc column was washed again. resulting mass spectra were analyzed using bioworks . (thermoelectron) using a non-redundant proteome database containing protein from both chicken and mdv-rb b (build . ) downloaded from the ncbi. we used the bioworks reverse database function to create the decoy database from this proteome database. the probability of the tandem mass spectrometry match occurred by chance was calculated using; (a) the decoy database searching exactly as described by elias and gygi ( ) and (b) the orthogonal p(pep) function in bioworks . (which calculates probability based on a theoretical y and b ion spectrum calculated based on theoretical amino acid dissociation). these two probabilities were the used to calculate the fdr as described by benjamini and hochberg ( ) and only peptides with fdr b . were considered as a significant and retained for protein identification. the probability of protein identity was then calculated from the peptide probabilities exactly as described (maccoss et al., ; nesvizhskii et al., ) . to calculate the percent protein coverage by identified peptides, the protein sequence was digested in silico using "peptidecutter" (http://us. expasy.org/tools/peptidecutter/ (gasteiger et al., ) ) and the total number of amino acids in peptides between and amino acids (the size range of % of the peptides detected by the mass spectrometer) was used as the denominator; the peptides identified were used as the nominator. for spots with multiple protein identities, the protein with the highest peptide coverage and highest protein score (i.e. Σxcorr; a surrogate for the amount of precursor ion) (nanduri et al., ) was considered as the dominant protein and the most likely protein to contribute to the differential expression in the gel. subsequently, their biological relevance was discussed. spot identities were submitted to goretriever (http://www. agbase.msstate.edu/) to obtain the go annotations. if no annotation was returned, goanna was used to retrieve go annotations assigned depending on the sequence similarities. the resulting annotations were summarized based on the goa and whole proteome goslim set using goslimviewer (mccarthy et al., ) . dna extraction, conventional pcr confirmation of the presence of mdv-meq gene and subsequent determination of the absolute mdv genome copy number from infected spleen samples were essentially performed as previously described . quantitative real-time pcr reactions to determine viral genome load in samples were performed in duplicate. 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host cells infected with infectious bursal disease virus key: cord- - gahl authors: tamouza, ryad; krishnamoorthy, rajagopal; leboyer, marion title: understanding the genetic contribution of the human leukocyte antigen system to common major psychiatric disorders in a world pandemic context date: - - journal: brain behav immun doi: . /j.bbi. . . sha: doc_id: cord_uid: gahl the human leukocyte antigen (hla) is a complex genetic system that encodes proteins which predominantly regulate immune/inflammatory processes. it can be involved in a variety of immuno-inflammatory disorders ranging from infections to autoimmunity and cancers. the hla system is also suggested to be involved in neurodevelopment and neuroplasticity, especially through microglia regulation and synaptic pruning. consequently, this highly polymorphic gene region has recently emerged as a major player in the etiology of several major psychiatric disorders, such as schizophrenia, autism spectrum disorder and bipolar disorder and with less evidence for major depressive disorders and attention deficit hyperactivity disorder. we thus review here the role of hla genes in particular subgroups of psychiatric disorders and foresee their potential implication in future research. in particular, given the prominent role that the hla system plays in the regulation of viral infection, this review is particularly timely in the context of the covid- pandemic. since the first description of an association between human leukocyte antigen (hla)-b and hodgkin lymphoma (amiel, ) the highly polymorphic hla gene cluster has been linked to a wide array of immune/inflammatory disorders, including now psychiatric disorders . recently, a strong association between schizophrenia and the major histocompatibility complex (mhc), which hosts the hla gene cluster, was reported by the psychiatry genomic consortium (ripke et al, ) . this finding extends the long-standing observation of links between hla and psychiatric disorders (eberhard et al, ) . given the prominent role of hla gene cluster in the regulation of immuno-inflammatory processes, as well as in neurodevelopment and neuroplasticity, through microglia regulation and synaptic pruning (moktari and lachman, ), it was expected that hla alleles would play a major role in the pathophysiology of psychiatric disorders. convergent results now show that this is particularly relevant for autism spectrum disorders (asd), schizophrenia or bipolar disorders, all known to be associated with chronic low-grade inflammation and comorbid autoimmune diseases (pape et al, ). the hla system is expected to influence both the early development and the course of psychiatric disorders. the mhc is a four megabases region located on the short arm of the chromosome ( p . - . ) and is one of the most polymorphic and gene dense regions of the human genome (trowsdale and knight, ) ,). this region hosts the hla gene cluster, which is physically divided into three functionally distinct sub-regions: i) the hla-class i region includes the classical hla-a, hla-b, and hla-c genes as well as the non-classical hla-e, hla-f and hla-g loci. the three classical hla genes act to regulate antigen presentation to cd + t-lymphocytes, whilst the non-classical genes are mainly implicated in different immunomodulatory functions; ii) the hla class ii region encompasses the hla-dpa , hla-dpb , hla-dqa , hla-dqb , hla-dra, hla-drb , hla-drb , hla-drb and hla-drb genes which are involved in antigen presentation to and iii) the class iii region which encompasses gene loci involved in inflammatory responses, leukocyte maturation and in the complement cascade. while the encoded molecules of the hla-a, -b and-c genes play an essential role in the detection and elimination of virus-infected cells and tumoral cells through cell-mediated cytotoxic processes, their hla class ii counterparts modulate humoral immune responses (klein & sato, ) . both gene sets are highly polymorphic with more than . alleles reported to date, although each locus has only around to dominant alleles (imgt/hla database, ), the main function of which is to present self or foreign antigens to t cell receptors (tcr) on effector cells. the hla molecules have long been appreciated to be involved in fine tuning of inflammatory processes as well as in the development of immuno-mediated pathophysiological processes including autoimmunity, a frequent comorbidity of psychiatric disorders (khandaker, dantzer, jones et al, ) . recent data also show that hla molecules modulate the development and function of the central nervous system (cns), including core functions such as neuronal/synaptic plasticity, learning, memory and behavior (boulanger, ) , as well as more direct modulation of neuron-neuron interactions and neuro-signaling (sterner, weckle, and chugani, ) . the highest hla expression levels are detected in post-synaptic hippocampal neurons (goddard, butts and shatz , ) . moreover, the hla molecules are pivotal for the anatomical integrity of the cns, as exemplified by the enlarged ventricles observed in hlaclass i deficient murine models (huh et al, ) . despite evidence of prominent immune implication in a significant subset of major psychiatric disorder patients such as schizophrenia, bipolar disorder or depression (khandaker, dantzer, jones, ) or autism spectrum disorder (meltzer & van de water, ) , deciphering the mechanistic link between the hla system and these disorders was difficult, primarily because of the complex genetic architecture of the hla system. recent technological advances now allow a more precise characterization of the hla gene cluster and the identification of its etiological or protective role in psychiatric disorders. it thus timely to review the role of hla genetics in major psychiatric disorders and to describe future research directions including the regulation of the impact of viral infections on aetiology and course of psychiatric conditions in the context of a world pandemic. since the discovery of the hla system by jean dausset (dausset and brecy, ) , the analysis of hla polymorphisms has undergone successive technological improvements over years, leading to the gradual incorporation of dna-based molecular approaches, including the high throughput and cost-effective next generation sequencing (ngs) which provides reliable and extensive hla genotyping. however, fine-tuned analysis and understanding of the hla diversity in genetic studies still requires specialized training either in terms of histocompatibility testing or imputation analysis. despite such advances and expertise, hla investigation in disease association studies still faces a number of challenges, including: (i) the extreme rate of polymorphism; (ii) the ethnogeographical-dependent distribution of hla alleles; (iii) the disease-dependent variable pertinence of a given hla allele e.g. the hla-b association is prominently evident for ankylosing spondylitis while for other multi-genic and multifactorial disorders, evidence of disease association may not be that apparent as distinct alleles may have shared functions; and (iv) the variability of allele expression (ramsuran et al, ; d'antonio et al, ; aguiar et al, ) . it thus should be stressed that while the candidate gene approach, which may ignore the degree of contribution of other interacting loci to the phenotype, is not a satisfactory way to fully underscore the hla genetic diversity, genome-wide association studies (gwas) followed by hla imputation-based methods, at least in caucasian and asians, may provide clues towards understanding the hla genetic contribution to psychiatric disorders. unfortunately, at the present time, only few post-gwas hla imputation-based studies have been undertaken. an approach to overcome these difficulties is to understand the evolution-based shaping of present-day hla diversity as stemming from a limited, but manageable, number of ancestral haplotypes (ah). various genetic events, viz crossing-overs, recombination and point mutations, have participated in that evolution (price et al, ; dawkins et al, ) . these ahs were selected under diverse geographic-specific environmental pressures, then conserved and fully or partially transmitted to successive generations (dawkins et al, ) . consequently, study of ah distribution in disease association studies have clarified why apparently distinct hla alleles exhibit association with a given disorder. indeed, these alleles, linked to ahs, allow to stratify the patients into an immuno-inflammatory subset for ah associated with steady state inflammation even among healthy subjects. for example, hla- . ah is the most associated ah with immune-related disorders, including infections and autoimmunity, whilst also being characterized by a steady state pro-inflammatory background in healthy individuals (price et al, ; giambino et al, ) . such properties may explain why the . ah can be protective against pathogens and therefore positively selected, while also having a negative health impact due to the association of chronic pro-inflammatory status with high risk for autoimmunity (crespy and go, ) . hla functional diversity can also be investigated via allele-dependent expression status which reflects the influence of hla alleles per se. for example, single nucleotide polymorphisms (snp) categorize the hla-dpb and hla-c alleles into highly and lowly expressed variants (petersdorf et al, ; thomas et al, ; apps et al, ) . hla-peptide combination is mediated by the polymorphic tcrs on cd + t cells, which bind class i molecules, and on cd +t cells which bind class ii molecules. the specificity of hla-peptide-tcr tripartite interactions is fundamental in enabling the adaptive immune system to mount an efficient and appropriate response against infection, whilst simultaneously preventing auto-immune processes (creusot, mitchison, terazzini, ; woodsworth, castellarin and holt, ; morris and allen, ). different mechanisms may underpin hla associated diseases, including: ) atypical hla-peptide-tcr binding orientation; ) low affinity peptide binding that facilitates thymic escape; ) tcr-mediated stabilization of weak-peptide-hla-interaction; and ) presentation of peptides in a different binding register. other mechanisms that may generate autoreactive t cells are driven by epitope variation, including molecular mimicry (yin, li and mariuzza, ) . thus, while gwas identified the mhc/hla genetic cluster as being a pivotal region, specific hla-dedicated expertise started to uncover meaningful functional haplotypes associated with specific psychiatric entities. hla analysis in psychiatric disorders will help not only to identify homogeneous subgroups, hla based, but also to decipher their underlying mechanisms. recent gwas and haplotype-based studies have revived and strengthened interest in the roles of the immune system in psychiatric disorders. this section reviews investigations of hla gene candidate association in schizophrenia, bipolar disorders and autism spectrum disorders, focusing on the risk/protection that hla alleles/haplotypes may confer on specific sub-groups. accumulating evidence from epidemiological, immunological, genetic, and imaging studies strongly indicate a role for mhc in schizophrenia risk , dating from the 's (eberhard, franzén and löw, ; cazzullo and smeraldi, ) . previous work has shown a number of associations across different ethnic populations, including the hla-a , hla-a , hla-drb , and hla-dqb alleles . in , three gwas and a meta-analysis of these gwas were published in nature, revealing a strong association between the mhc region and schizophrenia, although without any precision as to the specific location (purcell et al, , shi et al, stefansson et al, ). subsequent studies using gwas subjected to hla imputation of classical hla alleles, revealed a marked dominant protective effect conferred by hla-a* , b* and drb * (donnelly et al, ) . these alleles are all derived from the so-called . "autoimmune" ancestral haplotype ( . ah) (a* ~b* ~cw* ~drb * ~dqb * ). the . ah is the most associated hla haplotype with inflammatory processes and autoimmune diseases, including type diabetes, celiac disease, grave's disease, and myasthenia gravis (price et al, ) , a situation that may appear at first sight contra-intuitive according to the protecting effect conferred against schizophrenia risk. a deeper exploration of the hla region further revealed a major contribution to schizophrenia risk mediated by increased complement c a gene copy number with consequent c -dependant neuro-synaptic pruning (sekar et al, ). the complement system is not only in first-line defense against pathogens but also a major contributor to synaptic pruning during neurodevelopment (druart and le magueresse, ) . however, it is important to signal that the sekar's study not only suffer from absence of replication especially in ethically-distant population groups such as asians (lam et al, ) but also from the complexity of long-range imputation statistics and absence of direct inference of c haplotypes along their respective expression status. it is evident that the full sequencing of the c cluster may help genetic dissection of this complex region. besides these considerations, recent investigations have established a link between the c locus and classical hla haplotype diversity in the modulation of schizophrenia risk. indeed, the protective status conferred by the . ah haplotype plausibly arises from this haplotype naturally lacks the c locus. we recently showed that a . ah-derived hla haplotype was significantly less frequent in schizophrenia patients with early onset, whilst gradually increasing in frequency with the age of schizophrenia onset . this would suggest that . ah, via decreased c expression, leads to less synaptic pruning and cortical thinning, thereby delaying the age of schizophrenia onset, whilst concurrently potentially favoring heightened autoimmune processes due to its proinflammatory properties. in contrast, we hypothesized that schizophrenia patients not bearing . ah-derived hla haplotypes have an active c complement and may suffer from a more severe form of the disorder, characterized by early age at onset, increased synaptic pruning and cortical thinning, whilst being less prone to develop autoimmune disorders. this is in line with previous observations that carriers of mhc-linked risk variants (rs ) have larger ventricles (agartz et al, ) . even if it is assumed that the above-mentioned mechanisms, if proven, would be not the unique disease process, overall hla data may highlight the possibility that hla genetics will help to identify homogeneous sub-groups of schizophrenia patients. although gwas clearly indicate that the mhc region confer an increased autism spectrum overall, these data give some indication of the role that hla haplotypes may play in the abnormal neurodevelopment as well as in the systemic and gastro-intestinal inflammatory processes at work in autism. we recently reported an association between an . ah derived hla haplotype and bipolar disorder, specifically in patients with severe forms of the disorder defined by rapid cycling and/or personal history of suicidal behaviors . this association might also underline the well-known high frequency of auto-immune disorders in bipolar patients, including elevated levels of anti-thyroid or other organ-specific autoantibodies (padmos et al, ; jeppesen et al, ) . we also found that two haplotypes namely hla . ah and . ah were specifically associated with bipolar disorder having first episodes defined by hypomanic episode or psychotic symptoms. this is of importance as these two haplotypes are also linked to common inflammatory conditions, and might thus contribute to the proinflammatory processes observed in bipolar disorder. although these data, observed in phenotypically well-defined subgroups of patients, may constitute interesting tags of the mhc implication in bd, here again, a recent gwas did not allowed to uncover any mhc-derived signal (stahl et al, ) . the contrasting effect of . ah, being protective in schizophrenia and autism but conferring severity in bipolar disorder, is interesting. andreasen et al. ( ) reported that the same hla allele was shared between schizophrenia and multiple sclerosis (ms), but not between ms and bd. despite the large overlaps of genetic as well as clinical features in bipolar disorder and schizophrenia, these data are suggestive of temporal differences in hla-dependent immunogenetic influences on neurodevelopmental processes (andreassen et al, ; bergen et al, ) . these two psychiatric disorders may be differentiated by opposite effects mediated by the . ah possibly through specific complement c -mediated synaptic pruning processes. the role of hla-driven changes to the temporal neurodevelopmental specificities of bipolar disorder and schizophrenia will be important to clarify in future research. beside schizophrenia, autism spectrum disorders and bipolar disorders, major depressive disorders and attention deficit hyperactivity disorders are also major, common and frequent psychiatric conditions but suffer from scarcity of published/robust data implicating hla genetics. concerning major depressive disorders results from number of small studies, using broad serological typing (now considered as obsolete), generated inconsistent data. besides, a very recent gwas failed to identify any hla-related signals in mdd (glanville et al, ) . this is also true for adhd where few previous studies indicated potential association with the mhc non-classical complement c b, but again, a recent large gwas did not allow to detect any association between adhd and hla (nudel et al, ) . nevertheless, the potential genetic association between hla polymorphism and anti-nmda-r encephalitis awaits confirmation. more consistently, other autoantibodies for brain receptor targets have recently been described. in particular, a german study described a strong association between anti-leucine-rich glioma-inactivated (lgi ) encephalitis and the hla-drb * : ~dqa * : haplotype (mueller et al, ) . in addition, another study, besides replicating the above-mentioned hla association with lgi -mediated encephalitis, described an additional association between the hla-drb * : ~dqa * : ~dqb * : haplotype and anti-contactin-associated protein- (caspr ) encephalitis (binks et al, ) . hla genetic diversity is also implicated in the modulation of treatment responses in psychiatric conditions, including in the regulation of adverse drug reaction (adrs) and treatment efficacy. both candidate gene studies and gwas have shown that clozapine-induced agranulocytosis is partly mediated by class i and ii hla alleles (numataa et al, ) . in a study of treatment response to antipsychotic, we showed that a double amino-acid change in the hla-a peptidebinding groove was associated with a better response to treatment with risperidone in patients with schizophrenia (leclerc et al, ) . a recent large survey showed that treatment response to lithium in bd is strongly influenced by both schizophrenia-linked polygenic score and the mhc/hla genetic diversity (amare et al, ) . in the latter context the authors identified signals related to antigen presentation pathway (hla-dm region), the main function of hla molecules. such observation could be in line with the notion that differences in the heritability between schizophrenia and bd may lie in the mhc cluster (andreassen et al, ) . the where two types of herv family, namely herv-w and herv-k, are respectively implicated (greenig, ) . given the role of gene and environment interactions in herv reactivation, and its capacity to induce pro-inflammatory and neurotoxic proteins, herv has been the focus of studies in psychiatric disorders. we and others have found associations between the herv-w type and both schizophrenia and bd at protein and/or at dna/rna levels, further influenced by copy number variations (perron et al, ; leboyer et al, ) . recent data show herv-k to be a potential risk component for schizophrenia. it is worth mentioning that sekar et al demonstrated that complement c long allele, harboring insertion of herv-k, expressed higher levels of c a molecules, thereby increasing the risk of exaggerated synaptic pruning, cortical thinning and early onset (sekar et al, ) . this is an interesting area of investigation as clearly different herv family members can be associated with the same disease, although with different disease pathways. all living organisms have to constantly learnt to deal with environmental insults in order to survive, including various types of pathogens. given the extreme diversity of these environmental insults, evolutionary forces have gradually shaped powerful biological systems characterized by a large number of genes with high allelic diversity adapted to handle these challenges, viz the different wings of the immune system. upon interaction with a given trigger, the immune system first mounts non-specific pro-inflammatory processes and then, if necessary, more adaptive cellular processes directed against the triggering event. in parallel with the shaping of the immune response, counteracting immune-modulatory genetic strategies, limiting uncontrolled inflammation, have emerged and have been positively selected by evolutionary constraints. the hla-class i classical and non-classical molecules represent one of the best examples of such janus-faced system. indeed, within the same hla-class i region lie (i) the classical hlaclass i -a, -b and -c loci, characterized by an extreme polymorphism essential for their antigenpresentation functions, and maintained by balancing selection (heterozygote advantage) to cope up with a large variety of environmental pathogens and (ii) the non-classical hla-e, g and f genes, remarkable due to a very low rate of diversity that reflect broader properties, such as immunomodulation. among the latter, the non-classical hla-g encode cell surface molecules exerting powerful immunomodulatory functions, demonstrated to be essential for the establishment and tolerance between the maternal immune system and the semi-allogeneic fetus at the fetal-placental interface (ferreira et al, ) . genetically determined low expression of the tolerogenic hla-g molecules at the fetal-mother interface, possibly led to prenatal immune activation, is associated with asd risk (guerini et al, (guerini et al, , a (guerini et al, , b (guerini et al, , . schizophrenia is also widely believed to be a neurodevelopmental psychiatric disorder, although the role of hla-g polymorphism and expression has been less investigated. however, available data does indicate that low levels of hla-g, either circulating or genetically determined, may influence disease onset and phenotype (rajasekaran et al, , rajasekaran et al, rajasekaran et al, ; shivakumar et al, ) . in two studies performed on bipolar disorder patient populations of distant ethnicity, namely french and south indian tamils, data shows that in contrast to autism and schizophrenia, genetically determined hla-g low expression confers protection against bipolar disorder, suggesting the role of distinct hla-g effects in the aetiology of these disorders (debnath et al, ; sundaresh et al, ) . it is hence possible that in bipolar disorder, the protection conferred by low grade immunomodulation may favor a more efficient and intense pro-inflammatory, anti-infectious response, but outside the neurodevelopmental window. at the time of writing this review, the covid- pandemic was devastating the health and economies of countries worldwide, highlighting the powerful influence that viruses have had and have on animal and plant life over the course of evolution. there is an increasing appreciation of the role of viruses in wide array of diverse medical conditions, including cancers and neurodegenerative conditions, but also in psychiatric conditions (avramopoulos et al, ) . direct impact of viruses on the central nervous system was illustrated in contemporary history by clinical situations in neuro-psychiatric settings after pandemics such as the spanish flu or the more recent h n . while an increased frequency of psychosis (yudofsky sc, ) and encephalitis lethargica/parkinsonism (lymphaibool et al, ; hoffman & vilensky, ) were observed following the spanish flu pandemic, a raised rate of narcolepsy was described after h n pandemic likely triggered by the pandemrix® vaccination (sarkanen et al, ) . more recently, comforting early reported deleterious effects of viral infections on cns, large nationwide studies demonstrated association between maternal viral infectious events during the first trimester and subsequent psychiatric diseases in the offspring (brown & derkits, ) . it is worth reminding here again, as for the vast majority of viral infections, that the hla system is pivotal in the anti-infectious immune response processes, very likely in the present pandemics too. in the current context, it is important to note that variations in hla/mhc act to regulate viral infections, including influenza infection in humans (dutta et al, ) . influenza and other infections prenatally can also increase risk of schizophrenia and autism spectrum disorders in the offspring, suggesting that hla/mhc genetic variations may interact with prenatal infection in the etiology of major psychiatric disorders, although complicated by the immune-suppression that occurs in pregnancy (shah et al, ) . the devastating influence of the severe acute respiratory syndrome coronavirus (sars-cov)- virus that has led to the covid- pandemic raises the question as to the role of hla/mhc in the regulation of the inflammatory processes which is an integral aspect of sars-cov- infection, usually referred to as the 'cytokine storm'. this will also be important to investigate in the distinct sub-groups of major psychiatric disorder patients carrying particular variations of their hla alleles. although the literature concerning the immunogenetic aspects of previous pandemic waves of sars or mers-cov (middle east respiratory syndrome) is relatively low, data do show that hla plays a major role in viral infection regulation, and this is also relevant to analyze risk/protection of major psychiatric disorders in the pandemic context. the available data on hla diversity show associations with hla-class ii alleles, notably the hla-drb * variant in two studies from taiwan (wang et al, ) , and hong kong (ng et al, (nguyen et al, ) . as acknowledged by the authors, among many other limitations, peptide-mhc binding affinity cannot be alone a predictor of subsequent t-cell responses. the psychiatric inpatient population-group is at high risk for any epidemic threat. increased vulnerability not only arises due their psychiatric condition, associated stressors, and confinement with other patients but also from their comorbid somatic disorders including, cardiovascular disorders, metabolic syndrome, diabetes, autoimmune disorders and respiratory tract dysfunctions. all of these common comorbidities are risk factors for severe infection and almost all are linked to hla genetic diversity (trowsdale and knight, ) . as with influenza viruses, the most deleterious event in covid- does not seem to be the infectious agent per se, but the overwhelmed reactive inflammation arising from the 'cytokine storm'. future research will have to determine whether variations in hla genetic diversity modulate the susceptibility to severe infection and fatality in major psychiatric disorders subjected to the sars-cov- pandemic. the data reviewed above and the long evolutionary history of hla-equivalent loci, including further investigation is required in people from africa, given that the rate of the genetic diversity is the highest in africans amongst all human ethnic groups. in this context, a recent study of a sample of sz patients from south africa, namely the xhosa population-group, revealed not only that the observed overall genetic diversity was more important than that of non-african populations, but importantly uncover mutational events relevant to the origins of schizophrenia (gulsuner et al, ) . future studies of long-established populations from where the human genome was shaped by various environmental pressures over time, including a variety of social and microbial pressures, will be pivotal for the understanding of psychiatric disorders. as highlighted in this review hla diversity is integral to variations in the immune responses that form biological underpinnings, among others and likely in subsets of patients, of a wide array of psychiatric presentations as well as to how we, and other animals, have interacted with viruses over the course of evolution. relevant papers were identified through pubmed searches of articles published in english from jan , , up to april , , using the following search terms (alone or in combination): "hla, mhc, psychiatry, polymorphism, immunogenetics, immuno-psychiatry, covid". additional studies were identified from our own files. the final reference list was generated on the basis of their relevance to the topics covered in this review. the authors declare that they have no competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. this review was written under the framework of agence nationale de la recherche (i-give anr- -sama- - ), inserm (institut national de la santé et de la recherche médicale) and fondation fondamental however without any role of the above-mentioned institutions in study design; in the collection, analysis, and interpretation of data; in the writing of the report; and in the decision to submit the review for publication. rt: designed the search strategy and performed it, wrote the manuscript and take the primary responsibility to submit the review. rk and ml: participated to the search strategy, critically reviewed and edited the manuscript. all authors approved the final version.  major psychiatric disorders are associated with genetically-determined immune dysfunctions  human leukocyte antigen (hla) molecules are prominent players of immune and neurodevelopmental processes.  hla molecules are encoded by polymorphic genes located in the major histocompatibility complex (mhc).  recent methodological approaches allowed understanding of the hla genetic complexity and reconcile findings from gwas in psychiatry.  hla haplotypes studies uncover protective and at risk markers for disease development or proxies of severity in schizophrenia, autism and bipolar disorders.  beyond disease risk, the hla genetic diversity is associated with autoimmune encephalitis, treatment response, retrovirology and neurodevelopmental immunomodulation.  hla is also involved in anti-infections processes including against the coronavirus family common sequence variants in the major histocompatibility complex region associate with cerebral ventricular size in schizophrenia expression estimation and eqtl mapping for hla genes with a personalized pipeline association of polygenic score for schizophrenia and hla antigen and inflammation genes with response to lithium in bipolar affective disorder: a genome-wide association study study of the leukocyte phenotypes in hodgkin's disease genetic pleiotropy between multiple sclerosis and schizophrenia but not bipolar disorder: differential involvement of immune-related gene loci influence of hla-c expression level on hiv control infection and inflammation in schizophrenia and bipolar disorder: a genome wide study for interactions with genetic variation hla-class ii haplotypes and genome-wide association study in a swedish population yields support for greater cnv and mhc involvement in schizophrenia compared with bipolar disorder distinct hla associations of lgi and caspr -antibody diseases combining clinical and molecular heterogeneity within caspr -antibody mediated diseases: towards the underlying disease biology immune proteins in brain development and synaptic plasticity thinning faster? 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at the interface of maternal-fetal tolerance autoimmune diseases and . ancestral haplotype: an update. hla classical human leukocyte antigen alleles and c haplotypes are not significantly associated with depression regulation of cns synapses by neuronal mhc class i identification of common genetic risk variants for autism spectrum disorder hla-g( * ) bp insertion/deletion polymorphism associates with the development of autistic spectrum disorders hla-g* coding region polymorphism is skewed in autistic spectrum disorders hla-g * bp insertion and the kir ds -hlac complex impact on behavioral impairment in children with hla-g allelic distribution in sardinian children with autism spectrum disorders: a replication study genetics of schizophrenia in the south african xhosa hervs, immunity, and autoimmunity: understanding the connection genetic and environmental influences on structural brain measures in twins with autism spectrum disorder encephalitis lethargica: years after the epidemic 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implications the role of the immune system in autism spectrum disorder the major histocompatibility complex (mhc) in schizophrenia: a review how the tcr balances sensitivity and specificity for the recognition of self and pathogens genetic predisposition in anti-lgi and anti-nmda receptor encephalitis anti-caspr clinical phenotypes correlate with hla and immunological features inflammation and its discontents: the role of cytokines in the pathophysiology of major depression involvement of innate and adaptive immune systems alterations in the pathophysiology and treatment of depression association of human-leukocyte-antigen class i (b* ) and class ii (drb * ) genotypes with susceptibility and resistance to the development of severe acute respiratory syndrome human leukocyte antigen susceptibility map for sars-cov- immunity and mental illness: findings from a danish population-based immunogenetic study of seven psychiatric and neurodevelopmental disorders clozapine pharmacogenetic studies in schizophrenia: efficacy and agranulocytosis a high prevalence of organ-specific autoimmunity in patients with bipolar disorder immunoneuropsychiatry -novel perspectives on brain disorders differences in cortical structure and functional mri connectivity in high functioning autism molecular characteristics of human endogenous retrovirus type-w in schizophrenia and bipolar disorder high hla-dp expression and graft-versus-host disease autoimmune psychosis: an international consensus on an approach to the diagnosis and management of psychosis of suspected autoimmune origin the genetic basis for the association of the . ancestral haplotype (a , b , dr ) with multiple immunopathological diseases international schizophrenia consortium, common polygenic variation contributes to risk of schizophrenia and bipolar disorder soluble human leukocyte antigen (shla)-g levels may predict early onset of schizophrenia in male patients elevated hla-a expression impairs hiv control through inhibition of nkg a-expressing cells schizophrenia working group of the psychiatric genomics consortium incidence of narcolepsy after h n influenza and vaccinations: systematic review and meta-analysis schizophrenia risk from complex variation of complement component pregnancy-related immune suppression leads to altered influenza vaccine recall responses influence of correlation between hla-g polymorphism and interleukin- (il ) gene expression on the risk of schizophrenia common variants on chromosome p . are associated with schizophrenia hla class ii allele drb * : is associated with anti-nmdar encephalitis genome-wide association study identifies loci associated with bipolar disorder common variants conferring risk of schizophrenia dynamic gene expression in the human cerebral cortex distinguishes children from adults the hla-g genetic contribution to bipolar disorder: a trans-ethnic replication hla polymorphism in regressive and non-regressive autism: a preliminary study the hla . ancestral haplotype in schizophrenia: dual implication in neuro-synaptic pruning and autoimmunity? hla genetics in bipolar disorder hla-c cell surface expression and control of hiv/aids correlate with a variant upstream of hla-c major histocompatibility complex genomics and human disease anti-nmda-receptor encephalitis in a year old patient with chromosome p . microdeletion including the hla cluster emerging roles for the gut microbiome in new insights into the genetic mechanism of iq in autism spectrum disorders human-leukocyte antigen class i cw and class ii dr genotypes are associated with resistance to severe acute respiratory syndrome (sars) infection common hla-b -dr haplotype in northern india is different from that found in europe sequence analysis of t-cell repertoires in health and disease structural basis for self-recognition by autoimmune t-cell receptors contracting schizophrenia: lessons from the influenza epidemic of - key: cord- - unu x authors: levine, beth; deretic, vojo title: unveiling the roles of autophagy in innate and adaptive immunity date: journal: nat rev immunol doi: . /nri sha: doc_id: cord_uid: unu x cells digest portions of their interiors in a process known as autophagy to recycle nutrients, remodel and dispose of unwanted cytoplasmic constituents. this ancient pathway, conserved from yeast to humans, is now emerging as a central player in the immunological control of bacterial, parasitic and viral infections. the process of autophagy may degrade intracellular pathogens, deliver endogenous antigens to mhc-class-ii-loading compartments, direct viral nucleic acids to toll-like receptors and regulate t-cell homeostasis. this review describes the mechanisms of autophagy and highlights recent advances relevant to the role of autophagy in innate and adaptive immunity. our armamentarium for fighting intracellular pathogens includes multiple facets of the innate and adaptive immune response. in the past few decades, we have witnessed an explosion in our understanding of the mechanisms underlying antigen presentation, immune recognition of infected cells, cellular sensing of pathogens, and signalling pathways that induce antimicrobial states in infected cells. surprisingly, however, microbiologists and immunologists have long overlooked one of the greatest challenges that the immune system faces in dealing with intracellular pathogens -that is, the problem of how to dispose of a microorganism without disposing of the entire infected cell. a seemingly simple strategy to tackle this challenge is now beginning to unveil itself. mammalian cells use an evolutionarily conserved lysosomal degradation pathway known as autophagy to selectively dispose of intracellular pathogens. autophagy is a fundamental cellular homeostatic process that enables cells to clean up, in a regulated manner, portions of their own cytoplasm and degrade their constituents . this primordial function is preserved in all eukaryotic organisms, from yeast to humans. during autophagy, an isolation membrane wraps around portions of the cytoplasm to form a double-membrane organelle known as the autophagosome. the engulfed cytoplasmic material in an autophagosome is degraded after fusion of the autophagosome with late endosomes or lysosomes (fig. ) . the autophagy pathway has many physiological roles, and is often used to remove damaged or surplus organelles. it is also used by cells to turn over long-lived proteins and other macromolecules, either to supply nutrients for essential anabolic needs under conditions of nutrient deprivation or growth factor withdrawal, or to rid cells of potentially toxic aggregate-prone proteins . the broad spectrum of autophagy functions is intricately linked to a wide range of health and disease states , . for example, autophagy is involved in the control of development, tissue homeostasis and the lifespan of an organism; the suppression of tumour development; and the prevention of neurodegeneration. aberrant regulation of autophagy has been mechanistically linked to cancer, huntington's disease, parkinson's disease, myodegeneration and cardiomyopathy (box ) . autophagy also functions in diverse aspects of immunity [ ] [ ] [ ] . although the term autophagy means 'to digest oneself ' , it is now clear that the autophagy pathway also eliminates intracellular pathogens, including viruses, parasites and bacteria (a process sometimes referred to as xenophagy) [ ] [ ] [ ] [ ] . the autophagic sequestration of viral components can also fuel mhc class ii presentation of endogenous antigens and the production of type i interferons (ifns) in response to toll-like receptor (tlr ) signalling . furthermore, autophagy may directly affect t-cell homeostasis , . more speculatively, autophagy may have a role in the prevention of autoimmunity and inflammatory disorders [ ] [ ] [ ] [ ] [ ] [ ] . here, we provide an introduction to the molecular mechanisms of autophagy, discuss the major recent advances in autophagy and immunity, and highlight important unanswered questions in the field. autophagy: a lysosomal degradation pathway there are several morphologically and functionally distinct forms of autophagy, including macroautophagy (herein referred to as autophagy), microautophagy, chaperone-mediated autophagy and others that involve the selective degradation of specific organelles (such as peroxisomes, mitochondria and the endoplasmic reticulum). during the initiation of autophagy, a damaged organelle or a portion of cytosol is sequestered in a structure known as the isolation membrane or phagophore (fig. a) . the phagophore then becomes enlarged during the elongation stages by the addition of new membrane -the origin of which is still unclear. the phagophore seals to form an autophagosome, an organelle that is distinguished from the conventional phagosome by the presence of a double delimiting membrane (two lipid bilayers) and intra-lumenal cytoplasmic content. during maturation, autophagosomes fuse with lysosomes to form autolysosomes in which the captured material is degraded. the capture of intracellular pathogens is thought to follow a similar path (fig. b) , although the sequestration of microorganisms during autophagy has not been studied as extensively as that of cellular contents. most cells undergo some level of autophagy while adjusting their biomass, removing protein aggregates or eliminating damaged organelles (such as mitochondria). the classical signalling pathways that regulate autophagy have been reviewed extensively elsewhere , , whereas signals of particular relevance to immunity are discussed later. key players in autophagy regulation are the serine/threonine kinase mammalian target of rapamycin (mtor; also known as frap ) and the class i and class iii phosphoinositide -kinases (pi ks). two well-characterized stimuli that induce autophagy are amino-acid starvation and growth-factor withdrawal . in response to growth-factor stimulation, class i pi ks generate phosphatidylinositol- , , -trisphosphate (ptdins( , , )p ) on the plasma membrane by phosphorylating phosphatidylinositol- , -bisphosphate (ptdins( , )p ) and in turn ptdins( , , )p activates mtor, thereby repressing autophagy. the class iii pi k vps (also known as pik c ) generates phosphatidylinositol- -phosphate (ptdins p) by phosphorylating the cellular events during digestion of self constituents or intracellular pathogens follow three distinct stages: initiation (formation of the phagophore), elongation (growth and closure) and maturation of a double membrane autophagosome into an autolysosome. a | autophagy sequesters and removes cellular constituents from the cytosol, including surplus or damaged organelles from the cytosol. b | autophagy can eliminate bacteria (free in the cytosol or inside a phagosome), viruses and protozoan parasites in a manner similar to the elimination of self constituents. the autophagy pathway has numerous adaptive functions in eukaryotic organisms. in cellular starvation settings, autophagy functions to preserve cellular bioenergetics by providing metabolic substrates (obtained through bulk cytoplasmic degradation), which maintains macromolecular synthesis and atp production. another important function of autophagy that probably underlies its protective role against diverse pathologies is its ability to perform 'routine housecleaning' and also to clean-up toxic or damaged cytoplasmic constituents, a process that may have more selectivity than autophagy induced by cell starvation. this function of autophagy contributes to its protection against neurodegenerative disease; it has a basal role in preventing the abnormal accumulation of ubiquitylated protein aggregates and it specifically degrades toxic aggregate-prone mutant polyglutamine expansion proteins. the degradation of damaged mitochondria and other organelles also may underlie the antiageing effects and the tumour suppressor effects of autophagy, by helping to reduce genotoxic stress and to prevent dna damage and genomic instability. in parallel to cleaning-up endogenous cellular constituents, autophagy cleans up intracellular microorganisms, thereby protecting against disease caused by intracellular pathogens. autophagy also can selectively deliver microbial genetic material and antigens to the innate and adaptive immune systems. some of these effects on immune regulation and bacterial clearance may explain the recently uncovered genetic linkage between autophagy genes and susceptibility to crohn's disease. given the diverse functions of autophagy in health and disease, there is now considerable interest in targeting the autophagy pathway in the treatment of different diseases, including cancer, neurodegenerative diseases, heart diseases, ageing, infectious diseases and crohn's disease. however, for some of these diseases, such as cancer and heart disease, there is intense debate as to whether autophagy should be turned on or turned off, with no clear-cut data to resolve the debate. for other conditions, such as ageing and neurodegenerative diseases, presently available data suggest that autophagy augmentation is likely to be beneficial. in the case of infectious diseases, it is also likely that, at least in most cases, autophagy induction will foster increased innate and adaptive immunity. nonetheless, the possibility that certain microorganisms may fare better in the setting of increased autophagy remains. macroautophagy (also known as autophagy). the largely non-specific autophagic sequestration of cytoplasm into a doubleor multiple-membranedelimited compartment (an autophagosome) of nonlysosomal origin. note that certain proteins, organelles and pathogens may be selectively degraded via macroautophagy. the uptake and degradation of cytoplasm by invagination of the lysosomal membrane. the import and degradation of soluble cytosolic proteins by chaperone-dependent, direct translocation across the lysosomal membrane. (sirna). synthetic rna molecules of - nucleotides that are used to 'knockdown' (that is, silence the expression of) a specific gene. this is known as rna interference (rnai) and is mediated by the sequencespecific degradation of mrna. ptdins on endomembranes and acts at several steps along the signalling pathway associated with autophagy (fig. ) . when amino acids are plentiful, vps contributes to mtor activation, thereby repressing autophagy , . by contrast, the initiation stages of autophagy depend on vps in a complex with the autophagy-associated protein beclin (also known as atg ) . the beclin- -vps autophagy complex can be activated by the beclin- -interacting partners, uvrag (uv radiation resistance associated gene) and ambra (activating molecule in beclin- -regulated autophagy) , and inhibited by another beclin- -interacting partner, bcl- (b-cell lymphoma ) . pharmacological induction of autophagy can be achieved using rapamycin, a drug that inhibits mtor activity , whereas pharmacological inhibition of autophagy can be achieved using -methyladenine, a drug that inhibits class iii pi k activity . it is noteworthy, however, that as mtor and class iii pi ks have pleiotropic cellular functions, both of these drugs are potent, but not specific, regulators of autophagy. knockdown of expression of autophagy-related genes by small interfering rna (sirna) is therefore an indispensable tool for selectively probing the role of autophagy in defined biological processes. the execution of autophagy is mediated by evolutionarily conserved proteins known as the autophagy-related (atg) proteins . the molecular mechanisms by which this group of proteins mediates autophagy has been the subject of recent reviews , - and is depicted schematically in fig. . autophagosomal membrane formation and expansion is facilitated by two specialized protein conjugation systems (fig. ) , the atg (known as lc in mammals) system and the atg system. this results in the carboxy-terminal conjugation of lc to the lipid phosphatidylethanolamine (pe) and the localization of lipidated lc (lc -ii; also known as atg -pe) to autophagic membranes. membrane-associated lc -ii has become the most universally used marker for the detection of membranes that are undergoing autophagy. interestingly, lc interacts with p (sequestosome- ; also known as sqstm ), a protein that recognizes polyubiquitylated protein aggregates (that are too big autophagy is regulated by a set of autophagy-related proteins (atg proteins). in the absence of amino acids or in response to other stimuli, atg and a complex of the class iii pi k (phosphoinositide -kinase) vps and beclin lead to the activation of downstream atg factors that are involved in the initiation (a), elongation (b) and maturation (c) of autophagy. a | in amino-acid-rich conditions, vps contributes to mtor (mammalian target of rapamycin) activation and inhibition of atg and autophagy. the sources of membrane for autophagosome initiation and elongation may include those containing the only known membrane integral atg protein atg , redistributing between a resting location to autophagosomes in an atg -and pi k-dependent manner. atg redistribution may depend on atg , which binds phosphatidylinositol- -phosphate (ptdins p). b | the elongation and shape of the autophagosome are controlled by two protein (and lipid) conjugation systems, similar to the ubiquitylation systems: the atg and lc (also known as atg )-phosphatidylethanolamine (pe) conjugation pathways, which include e -activating and e -conjugating enzymes. atg is initially conjugated to atg (an e -activating enzyme) and then is transferred to the e -like conjugating enzyme atg . this intermediate presents atg for conjugation to an atg lysine residue. the atg -atg conjugate, stabilized non-covalently by atg , triggers oligomerization on the outside membrane of the growing autophagosome, and enhances lc carboxy-terminal lipidation through the lc conjugation system. upon autophagosome closure, atg -atg -atg and lc (delipidated by atg ) are recycled. c | lc associated with the lumenal membrane remains trapped in the autophagosome and is degraded during maturation into the autolysosome, which involves fusion of autophagosomes with late endosomes, including endosomal multivesicular bodies and lysosomal organelles, and dissolution of the internal membrane. vps has a role in the formation of late endosomal multivesicular bodies and lysosomal organelles contributing to the maturation stages of autophagy. to be disposed of by the proteasome) and may deliver them to autophagosomes for degradation . although only studied so far in the context of targeting polyubiquitylated protein aggregates, an interesting question is whether lc may interact with p or other proteins to target microorganisms for degradation or antigens for presentation through the mhc class ii pathway. the first indication that intracellular bacteria may be degraded by an autophagy-like pathway emerged more than two decades ago in morphological studies of polymorphonuclear cells infected with rickettsia conorii . however, before the discovery of the components of the autophagic machinery, this was difficult to prove. there was a lack of markers to unequivocally identify autophagosomes; it was difficult to follow the dynamic fate of intracellular bacteria; and it was difficult to determine the significance of bacterial association with autophagosomal membranes in host defence. indeed, several studies proposed that autophagy was a 'microorganism-friendly process' that supported the intracellular survival of certain pathogens . however, with new tools available to label autophagosomes and to inactivate the autophagy pathway in infected cells, we now know that autophagy is an important host mechanism for the removal of intracellular bacteria and protozoans, in keeping with its primary function as a cytoplasmic clean-up process (fig. ) . in parallel, many pathogens have evolved strategies to protect themselves against autophagy or to harness components of the autophagy pathway for their own benefit, although, in general, the molecular details of such strategies are not well defined . one of the initial indications that autophagy may have a role in immunity against intracellular bacteria was provided by observations regarding the role of ptdins p in innate immunity. it was known that ptdins p participated in phagolysosomal biogenesis and microbial clearance upon macrophage phagocytosis of microorganisms , . this link prompted investigations into a potential role of increased ptdins p production and, by extension, autophagy -as ptdins p is involved in the initiation of autophagy downstream of mtor -in eliminating certain intracellular pathogens, such as mycobacterium tuberculosis, that block normal phagolysosome biogenesis. gutierrez et al. showed that the mycobacterial-imposed block in phagolysosomal maturation can be overcome by activating cellular autophagy, either through starvation or inhibition of mtor . nearly simultaneously, other studies determined that autophagy can capture intracellular bacteria that lyse the phagosome and escape into the cytosol (such as shigella spp.) or extracellular bacteria that manage to invade the host cytoplasm (such as group a streptococcus) . other studies have confirmed these initial findings and extended the list of intracellular bacteria and parasites targeted by autophagy to include listeria monocytogenes, salmonella enterica, francisella tularensis and toxoplasma gondii , , [ ] [ ] [ ] [ ] [ ] . there are some aspects of autophagy that may be particular to the control of intracellular bacteria. first, the size of autophagosomes that engulf intracellular bacteria tends to be considerably larger than 'typical' autophagosomes (that is, those clearing up cytoplasmic constituents) , raising the possibility that these autophagosomes may have a different biogenesis than typical autophagosomes. nevertheless, there are similarities in size between lc -positive structures that clear large protein aggregates and the lc -positive bacteria-containing autophagosomes . this suggests that the formation of these large autophagosomes, if distinct from classical autophagy, is not reserved exclusively for microorganisms. another area of debate is whether autophagosomes can only target intracellular bacteria that reside in the cytosol or whether they can sequester pathogens that reside in membranous or intravacuolar compartments , , , . as autophagy is designed to engulf membranous organelles , , the autophagic enclosure of a pathogen within a membranous vacuole, such as a phagosome, should not represent an obstacle to autophagic elimination. this question has been answered experimentally using the parasite t. gondii. in t. gondiiinfected cells, two different but equally potent pathways of autophagic elimination of the pathogen operate: one that disrupts the parasitophorous vacuole that harbours the protozoan and the other that does not require disruption of the parasitophorous vacuole . thus, encasement of the pathogen in a vacuole does not seem to represent a physical barrier to autophagic capture and elimination. it has been proposed that damage to the vacuolar membrane containing the organism may precede its autophagic sequestration , but it is not yet known whether such damage or a molecular modification of the target membrane leads to autophagic uptake. another pertinent question is how pathogens that are free in the cytosol are recognized by the autophagic machinery. one possibility is that microbial proteins may be marked for autophagy by modifications, such as ubiquitylation, that are already known to modify bacterial products , or by other yet to be identified molecular tags. alternatively (or in addition), patternrecognition receptors, such as tlrs, nod-like receptors and rig-i (retinoic-acid-inducible gene i)-like helicases, may recognize pathogen-associated molecular patterns (pamps) to stimulate autophagy. a related possibility is that exposure of certain epitopes at the surface of a microorganism may be involved in microbial targeting to autophagosomes. for example, exposure of a shigella epitope that is normally unexposed (due to masking by another shigella-encoded protein) leads to autophagic bacterial capture in cells infected with mutants lacking the epitope-masking protein . another possible signal that links microbial presence and autophagy could be the generation of reactive oxygen intermediates, as these are often associated with pathogen recognition by host cells and are also known to induce autophagy , . future research into the mechanisms underlying the induction of autophagy by microorganisms and their targeting to autophagosomes is likely not only to uncover the specifics of how the autophagic machinery recognizes foreign material in the cytoplasm but also may shed light on how the autophagic machinery detects the cell's own damaged organelles, aggregated proteins and other cytoplasmic targets for lysosomal degradation. the sequestration of intracellular pathogens during autophagy is not limited to bacteria and parasites. autophagy can also capture virions that are newly assembled inside their host cells. in neurons and fibroblasts infected with herpes simplex virus -a dna virus that replicates in the nucleus -viral nucleocapsids are engulfed by autophagosomes as they egress out of the nucleus into the cytoplasm (fig. ) . it is not yet known whether autophagy also targets viruses during cell entry, but this seems probable based on extrapolations from findings in bacterial systems. if true, this could explain why the ratio of virus particles per cell often needs to be very high for cells to become productively infected by viruses. the possibilities for how viruses are targeted to autophagosomes are conceptually similar to those discussed previously for intracellular pathogens in general. however, the specific molecules involved are likely to differ given the unique pathogen-specific receptors and pamps that recognize viral versus bacterial components. similar to studies in bacteria, several investigators have also proposed that autophagy may be a 'virus-friendly' pathway, as it can provide viruses with a source of intracellular membrane that serves as a scaffold for viral rna replication complexes, an event that is necessary for efficient cytoplasmic replication of certain viruses . so, similar to bacteria, viruses may have also found ways to harness the autophagic machinery for their own replicative benefit. the best-studied examples are the mammalian picornavirus poliovirus and the murine coronavirus mouse hepatitis virus , . in infections with either of these viruses, knockdown of expression of autophagyrelated genes reduces viral yields. interestingly, however, a drosophila melanogaster picornavirus also replicates in association with double-membrane vacuoles (which are morphologically similar to the 'autophagy-like' double-membrane vacuoles associated with mammalian picornavirus replication) but autophagy genes are not required for its normal replication . this suggests that the requirement for the autophagic machinery in the generation of double-membrane vacuoles that support picornavirus replication may be cell-type specific or restricted to certain phylogenetic hosts. furthermore, the replication of vaccinia virus, a dna virus that replicates in the cytoplasm in association with double-membrane vacuoles, also does not require the autophagic machinery . so, it is unclear to what extent viruses exploit the autophagic machinery to obtain membrane anchors for their cytoplasmic replication. even in circumstances in which components of the autophagic machinery are necessary for viral replication, it is unknown whether the viruses are truly subverting the host autophagic pathway or merely using a partially overlapping pathway that is involved in membrane trafficking and rearrangement. although there is perhaps less direct in vitro evidence for viruses, compared with bacteria, that autophagy functions in pathogen elimination, studies with viruses have provided the first in vivo evidence for a role of autophagy in immunity. in two different phylogenetic kingdoms, genetic manipulation of autophagy-related genes has been shown to have striking effects on viral diseases. in plants, rnai-mediated silencing of several different autophagy-related genes increases local replication of tobacco mosaic virus and results in the uncontrolled spread of programmed cell death beyond infected plant cells . in mice, enforced neuronal expression of the autophagy-associated protein beclin reduces alphavirus replication and alphavirus-induced neuronal apoptosis and protects mice from lethal virus-induced encephalitis . together, these studies suggest that autophagy functions in antiviral immunity in vivo not only by restricting viral replication but also by restricting pathogen-induced cell death. the mechanisms underlying these protective functions of autophagy are not yet defined. in principle, autophagy may function in the direct elimination of viruses (as shown in vitro), in the breakdown of host factors required for viral replication or the inhibition of innate immune signalling, and in the promotion of cell survival either by maintaining bioenergetics in virally infected cells or by removing toxic self or viral components. as discussed in more detail later, autophagy may also promote adaptive immunity by the endogenous presentation of certain viral antigens through the mhc class ii pathway. moreover, the role of autophagy in peptidome the repertoire of peptides that is presented by antigenpresenting molecules. the process in the thymus that selects thymocytes expressing t-cell receptors that have the ability to interact with self mhc molecules. the process in the thymus that eliminates t cells that express t-cell receptors with high affinity for self antigens. innate antiviral immunity may not be confined to direct pathogen elimination. lee et al. recently found that the autophagic machinery can deliver viral nucleic acids to endosomal tlrs in plasmacytoid dendritic cells in vitro, resulting in type i ifn production . perhaps autophagy has a similar role in type i ifn production during viral infections in vivo and in other cell types infected with viruses. another major line of evidence that autophagy is important in antiviral immunity in vivo is the recent discovery that, to be pathogenic, viruses may need to successfully counter autophagy. an essential herpes simplex virus neurovirulence protein, icp . , confers pathogenicity by binding to beclin and by antagonizing the host autophagy response . although this is the first example of a viral virulence factor directly targeting the autophagic machinery to elicit disease, it seems probable that viral evasion of autophagy will prove to be a more general strategy that viruses use to evade host antiviral defence. other viruses encode proteins that inhibit the autophagy function of beclin , including bcl- -like proteins encoded by the oncogenic gammaherpesviruses . in addition, numerous viruses inhibit the pkr (ifn-inducible doublestranded-rna-dependent protein kinase) antiviral signalling pathway that is required for the induction of autophagy in virally infected cells or activate the autophagy-inhibitory class i pi k-akt-mtor signalling pathway . the multiplicity of mechanisms that diverse viruses have to turn off autophagy highlights a probable fundamental role for autophagy in antiviral immunity. recent evidence indicates that autophagy functions in delivering viral nucleic acids to the innate immune system . a subset of receptors of the tlr family sense viral nucleic acids in the lumen of endosomes . however, viral nucleic acids are most often released directly into the cytoplasm after fusion of a viral envelope with the endosomal membrane or penetration of cellular membranes by a viral capsid. this poses a topological challenge for the efficient detection of viral nucleic acids by endosomal tlrs. this challenge may be met, at least in part, by using the autophagic pathway for the delivery of cytoplasmic viral nucleic acids to endosomal tlrs, which would then lead to the induction of type-i-ifn-dependent innate immune responses (fig. ) so, similar to the use of autophagy for endogenous antigen presentation in adaptive immunity (described later), autophagy may be used for the delivery of endogenous viral nucleic acids to their cognate innate immunity detectors. the precise details of how the autophagy machinery senses viral rna and targets it to endosomal tlr remain to be elucidated. moreover, it is still unclear how the nucleic acids of dna viruses, and even other rna viruses, are targeted to endosomal tlrs in plasmacytoid dendritic cells, as viral replication is not required for type i ifn production in plasmacytoid dendritic cells infected with herpesviruses (dna viruses) or influenza virus (an rna virus) , . another unexplored question is whether there is any molecular interplay between the autophagy pathway and type i ifn signalling mediated by cytoplasmic rna helicases that function as receptors for cytoplasmic double-stranded rna produced during viral infection. the role of autophagy in immunity is not limited to the direct elimination of intracellular pathogens or stimulation of type i ifn production. at least in certain contexts, autophagy promotes mhc class ii presentation of cytosolic antigens [ ] [ ] [ ] [ ] [ ] . together, these initial studies led to the generally accepted idea that the autophagy pathway allows the transfer of cytosolic antigens to late endosomal or lysosomal compartments (fig. ) , in contrast to the processing of exogenous antigens captured through endocytosis or phagocytosis in antigen-presenting cells. the autophagic delivery of cytosolic antigens to endosomes and/or lysosomes represents an attractive model for explaining why the mhc class ii peptidome contains many peptides of cytosolic or nuclear origin that cannot be delivered to mhc class ii compartments by the classical exogenous route. however, it is not yet clear how universal a role autophagy has in the delivery of self and foreign cytosolic and nuclear antigens. interestingly, high levels of autophagy activity are observed in the thymic epithelial cells of newborn mice that transgenically express a fluorescently tagged autophagy marker (green fluorescent protein (gfp)-lc ) , suggesting that autophagy may indeed enable thymic epithelial cells to present self antigens to lymphocytes during positive and negative selection. yet, the role of autophagy in mhc class ii presentation of self antigens has not yet been directly examined. moreover, whereas ebna is processed by autophagy, two other epstein-barr-virusencoded nuclear antigens, ebna and ebna c, are preferentially processed by intracellular transfer of antigenic moieties and endocytic uptake from the culture media , indicating that autophagy may only be used for mhc class ii presentation of certain endogenously produced viral antigens. a fascinating question is why, even in the same cell, some microbial antigens, but not others, gain access to the mhc class ii antigen-presentation pathway by autophagy. as some of the earlier studies that showed a role for autophagy in mhc class ii presentation of endogenous antigens were performed in conditions in which autophagy was upregulated by cell starvation, another important question has been whether cytosolic antigens are delivered to mhc class ii molecules by autophagy under normal conditions. recently, figure | functions of autophagy in innate and adaptive immunity during infection with intracellular pathogens. a | intracellular pathogens (bacteria, parasites and viruses) that are either free inside the cytosol, inside phagosomes or inside pathogen-containing vacuoles are surrounded by isolation membranes, engulfed into autophagosomes, which fuse with lysosomes, and then degraded inside autolysosomes. b | viral nucleic acids are transferred by autophagy from the cytoplasm to intracellular compartments containing toll-like receptor (tlr ), which signals the induction of type i interferon (ifn) production. c | viral antigens (and potentially other endogenously synthesized microbial antigens and self antigens) are engulfed into autophagosomes that fuse with mhc-class-ii-containing late endosomes (miics), and then loaded onto mhc class ii molecules for presentation to cd + t cells. cytosolic antigens that contain a kferq recognition motif may also be directly imported into miics by chaperone-mediated autophagy. clip, class ii-associated invariant chain peptide. (t h cell). the term used for a cd + t cell that has differentiated into a cell that produces the cytokines interferon-γ, lymphotoxin-α and tumour-necrosis factor, and supports cell-mediated immunity. (t h cell). the term used for a cd + t cell that has differentiated into a cell that produces interleukin- (il- ), il- and il- , supports humoral immunity and downregulates t h -cell responses. p gtpase family a group of - -kda proteins that are produced in response to interferons (ifns) and that are involved in resistance to intracellular protozoa, bacteria and viruses. members of this family include ifnγ-induced gtpase (igtp), immunity-related gtpase family, m (irgm; also known as lrg ) and t-cell-specific gtpase (tgtp). . in these cells, at least one half of all autophagosomes intersect or fuse with mhc-class-ii-loading compartments. this trafficking pathway may be highly relevant for antigen presentation, as the targeting of an influenza virus matrix protein (mp) to autophagosomes by fusion with the autophagosomal protein lc led to a -fold enhancement of mhc class ii presentation to mp-specific cd + t-cell clones. this has exciting implications for vaccine development; targeting proteins for autophagic delivery to mhc-class-ii-loading compartments may be an effective means to improve t helper (t h )-cell responses. however, the contribution of autophagic delivery of viral antigens to adaptive immunity during natural infections has not yet been explored. the 'individualized' form of autophagy termed chaperone-mediated autophagy also has a role in endogenous mhc class ii presentation . chaperonemediated autophagy imports individual cytosolic proteins containing specific pentapeptide recognition motifs into the lysosome via a particular isoform of lysosome-associated membrane protein (lamp a) and an accessory chaperone, the heat-shock protein hsc . importantly, targeting to the chaperonemediated autophagy pathway is intrinsic to a large fraction of self proteins, as the targeting signal (kferq) is present in roughly % of all cytosolic proteins . although the relative contribution of autophagy and chaperone-mediated autophagy in endogenous mhc class ii antigen presentation is not yet known, zhou et al. have shown that chaperone-mediated autophagy may regulate mhc class ii presentation of several cytoplasmic antigens . overexpression of lamp a or hsc increases cytoplasmic self antigen presentation, and diminished hsc expression reduces mhc-class-ii-restricted t-cell responses to these antigens. now that it is known that autophagic pathways may have a role in mhc class ii presentation of endogenous antigen, many important new questions arise. how important are these pathways for adaptive immunity to intracellular pathogens? what is the relationship between autophagic elimination of intracellular pathogens and mhc class ii presentation of microbial antigens? does autophagic degradation of pathogens provide a source of antigens for loading into mhc class ii compartments and/or does the autophagic machinery independently capture newly synthesized microbial peptides? how are microbial (and self) antigens targeted for autophagic delivery to mhc-class-ii-loading compartments? beyond immunity against infection, what is the broader significance of autophagic antigen processing and presentation in an mhc-class-ii-dependent manner? it will be interesting to unravel the role of this pathway not only in immunity against infection, but also in cancer immunology, central and peripheral tolerance, autoimmunity and transplant rejection. autophagy regulation by immune signals the relationship between autophagy and immunity is bidirectional. not only does autophagy, at least in certain contexts, enhance innate and adaptive immune responses, but in parallel, cytokines, receptors and ligands involved in innate and adaptive immunity enhance autophagy. immune signalling molecules that have been shown to positively regulate autophagy in some contexts include pkr , ifnγ (and its downstream effector immunity-related gtpases) , , , , , tumour-necrosis factor (tnf) , , , and the cd -cd l (cd ligand) interaction . by contrast, autophagy is negatively regulated by the t h -type cytokines, interleukin- (il- ) and il- , although this has been shown so far only in a non-immune cell line - . in general, there is a correlation between activation of autophagy by immune mediators and the control of infection with intracellular pathogens. the pkr signalling pathway is an important arm of the innate defence pathway against viruses and is required for virusinduced autophagy , . cell-mediated immunity can induce autophagy through cd -cd l stimulation and protect target cells against the vacuolar parasite t. gondii . ifnγ and tnf are crucial for protection against infection by mycobacteria and other pathogens that replicate in macrophages, and are potent inducers of autophagy in both macrophages and other cell types , , , , [ ] [ ] [ ] [ ] . it is interesting to note the contrasting roles that the t h -type cytokines ifnγ and tnf, and the t h -type cytokines il- and il- (ref. ) may have on the regulation of autophagy. perhaps, t h -cell responses activate autophagy, thereby affording protection against intracellular microorganisms, whereas t h -cell responses dampen the autophagic response, thus, potentially explaining the negative role that the t h -cell response has in the control of intracellular pathogens . p gtpase-mediated regulation of autophagy. recent advances have been made in identifying the molecular mechanisms that underlie the antimicrobial action of ifnγ-induced autophagy. the mouse genome contains different immunity-related gtpases that are responsive to ifnγ and that have been long known to have a role in defence against a wide range of intracellular pathogens . however, until recently, the mechanisms by which these immunity-related gtpases control intracellular pathogens have remained unclear, as has the question of whether human immunity-related gtpases have a similar role in defence against intracellular pathogens. this has been partially resolved by the recent discoveries that both the mouse and human p gtpase family member immunity-related gtpase family, m (irgm; also known as lrg ) are required for ifnγ-induced autophagy and antimycobacterial activity in macrophages , , and that mouse irgm is also required for antiparasitic activity associated with macrophage autophagy . although the expression of human irgm is not regulated by ifnγ, cells must be stimulated with this cytokine, or other physiological or pharmacological inducers of autophagy, for irgm to exert its action. the exact mechanisms by which irgm promotes autophagy are not known, but they may depend on direct or indirect interactions with organelles, regulators or effectors of the autophagic pathway. alternatively, in view of the putative function of irgm as a dynamin-like membrane remodelling protein, autophagy induction may be indirectly promoted by irgm-induced changes to the parasitic vacuole membrane. autophagy and t-cell homeostasis autophagy has a central role in life and death decisions of numerous cell types across diverse phyla, functioning both as a pro-survival mechanism during nutrient deprivation and other forms of cell stress and as a cell-death mechanism in other contexts, such as in cells defective in apoptosis and in cells with very high levels of autophagy , . recent studies indicate that this homeostatic role of autophagy extends to t cells. pools of mature t cells in the periphery are subject to tight regulation that must balance naive t-cell flux following thymic selection with effector t-cell proliferation, cell death and differentiation. a role for autophagy in t-cell survival and proliferation has been shown in vivo using lethally irradiated mice repopulated with haematopoietic cells from fetal livers of atg -/mice . cd + and cd + t cells from atg -/mice fail to undergo efficient proliferation after t-cell receptor stimulation. moreover, atg -/-t cells develop normally in the recipient thymus, but fail to repopulate the periphery due to overwhelming cell death. one interpretation of this finding is that t cells, on exit from the thymus, become exposed to nutritional stress owing to limitations in trophic factor support (such as il- ) and require autophagy to sustain them during this period. at present, it is not known how autophagy affects immunological memory, but based on its role in the maintenance of other long-lived cells, such as neurons, the prediction is that autophagy may also have a role in maintaining memory t cells. in contrast to its function as a t-cell survival process, excessive autophagy has been linked to the cell death of effector t cells under conditions that model normal homeostasis. li et al. found that t h cells become more resistant to cell death induced by growth-factor withdrawal when autophagy is blocked using pharmacological or genetic methods . this cell death process may be exploited by viruses such as hiv, as the hiv envelope glycoprotein has been shown to induce autophagic cell death by binding to cxc-chemokine receptor (cxcr ) in uninfected bystander cd + t cells . however, in cd + t cells, the natural ligand of cxcr , cxc-chemokine ligand (cxcl ; also known as sdf α), induces lymphocyte activation and homing rather than cell death, indicating divergent outcomes in t-cell physiology in response to engagement of the same cellular chemokine receptor by a viral glycoprotein versus its endogenous ligand. future studies are needed to determine the role of this phenomenon in cd + t-cell depletion in patients with aids and to better define the factors that regulate whether autophagy has a pro-survival or pro-death role in lymphocytes. given the extensive molecular interplay between autophagy and apoptosis , it is not surprising that autophagy might have a dual role in t-cell homeostasis, executing both life and death decisions. furthermore, there is no reason to think that the homeostatic role of autophagy will be confined to t cells; as research in the field progresses, we are likely to witness the unfolding of crucial roles for autophagy in maintaining not only homeostasis but also proper differentiation and function of other populations of immune cells. an unexpected link between autophagy and the removal of apoptotic cell corpses has recently been reported , which raises some intriguing possibilities about a role for autophagy in the prevention of inflammation and autoimmunity. qu et al. found that autophagy provides apoptotic cells with signals to ensure their clearance during programmed cell death . it is well established that the rapid removal of apoptotic cell corpses is crucial for the prevention of tissue inflammation , and indeed, autophagy-deficient atg -/embryos have increased inflammation in tissues that have impaired clearance of apoptotic cells . moreover, it is now believed that defective clearance of apoptotic cells overcomes tolerance to self antigens and leads to autoimmune diseases such as systemic lupus erythematosus (sle) , . so, it is also possible that defective autophagy may contribute to the pathogenesis of sle or other autoimmune diseases. of great interest, a strong genetic link has recently been uncovered between autophagy and crohn's disease, a chronic inflammatory disease of the intestine. several recent genome-wide scans have identified a strong association between a non-synonymous single-nucleotide polymorphism (snp) in the autophagy gene atg l (t a variant) and susceptibility to crohn's disease [ ] [ ] [ ] [ ] . in addition, the gene encoding the autophagy-stimulatory gtpase irgm has been identified as a susceptibility gene for crohn's disease . together, these studies are suggestive of a role for autophagy dysregulation in the pathogenesis of crohn's disease. this hypothesis will be strengthened if the atg l variant (t a) associated with crohn's disease is found to lead to defective autophagy function. it is not yet known how autophagy might be mechanistically linked to susceptibility to crohn's disease. the pathogenic mechanisms of crohn's disease are poorly understood but are speculated to involve a dysregulated immune response to commensal gut bacteria and possibly defects in mucosal barrier function or bacterial clearance , . it is therefore possible that defects in autophagy lead to altered clearance of and/or altered immune responses to commensal gut bacteria. given the possible role of autophagy in peripheral tolerance, another speculation is that, in the setting of decreased autophagy, tolerance induction might fail and produce gut-reactive immune responses. as the intestine is a site of constant epithelial-cell shedding owing to apoptosis and regeneration, defective autophagy might also contribute to the pathogenesis of this inflammatory disorder by interfering with apoptotic cell clearance. studies in targeted mutant mice with conditional deletions or mutations of autophagy genes should help to elucidate the pathogenetic mechanisms of crohn's disease. autophagy probably originated to degrade cellular constituents, recycle nutrients and maintain cellular survival during starvation. yet perhaps, with confrontations between primitive eukaryotes such as amoebae and bacteria, this ancient lysosomal degradation pathway evolved and became exquisitely adapted to orchestrate a multipronged defence against intracellular pathogens. the autophagy pathway degrades intracellular pathogens, and delivers microbial genetic material and antigens to the necessary cellular compartments for activation of innate and adaptive immunity. in addition to its role in defence against pathogens, it also is involved in immune-cell homeostasis and potentially, in preventing inflammation and autoimmunity. the journey forward -in deciphering how autophagy executes these and, similarly, other not yet identified immune functions -will be an exciting challenge for immunologists. note added in proof while this manuscript was in press a new link was reported between pattern-recognition receptors of innate immunity and stimulation of autophagy. xu et al. found that the gram-negative bacterial lipopolysaccharide induces autophagy in macrophages through a tlr signalling pathway . autophagy as a regulated pathway of cellular degradation autophagy in metazoans: cell survival in the land of plenty development by selfdigestion: molecular mechanisms and biological functions of autophagy references - provide excellent general reviews on autophagy and its molecular mechanisms, physiological functions and roles in disease autophagy in innate and adaptive immunity autophagy in innate and adaptive immunity against intracellular pathogens autophagy as an immune defense mechanism cd induces macrophage anti-toxoplasma gondii activity by triggering autophagy-dependent fusion of pathogencontaining vacuoles and lysosomes vacuolar and plasma membrane stripping and autophagic elimination of toxoplasma gondii in primed effector macrophages autophagy in innate immunity against intracellular bacteria autophagy in mhc class ii presentation: sampling from within autophagy-dependent viral recognition by plasmacytoid dendritic cell this is a landmark paper describing a new function of autophagy in innate immunity; this paper provides the first evidence that the autophagic machinery delivers viral genetic material to endosomal tlrs a critical role for the autophagy gene atg in t cell survival and proliferation autophagy is induced in cd + t cells and important for the growth factor-withdrawal cell death this paper describes a cell-autonomous essential role for autophagy in generating signals for apoptotic cell corpse removal, raising the possibility that autophagy may help to prevent inflammation and autoimmunity a genome-wide association scan of nonsynonymous snps identifies a susceptibility variant for crohn disease in atg l genome-wide association study identifies new susceptibility loci for crohn disease and implicates autophagy in disease pathogenesis a nonsynonymous snp in atg l predisposes to ileal crohn's disease and is independent of card and ibd the wellcome trust case control consortium genome-wide association study of , cases of seven common diseases and , shared controls references - provide the first genetic evidence that human autophagy genes are linked to susceptibility to inflammatory disease regulation and role of autophagy in mammalian cells references - provide excellent, comprehensive reviews of the signalling pathways that regulate autophagy hvps is a nutrient-regulated lipid kinase required for activation of p s kinase amino acids mediate mtor/raptor signaling through activation of class phosphatidylinositol oh-kinase beclin-phosphatidylinositol -kinase complex functions at the trans-golgi network autophagic and tumour suppressor activity of a novel beclin -binding protein uvrag ambra regulates autophagy and development of the nervous system bcl- antiapoptotic proteins inhibit beclin -dependent autophagy tor signaling in growth and metabolism -methyladenine: specific inhibitor of autophagic/lysosomal protein degradation in isolated rat hepatocytes a unified nomenclature for yeast autophagy-related genes molecular dissection of autophagy: two ubiquitin-like systems autophagy: molecular machinery for self-eating molecular machinery of autophagosome formation in yeast, saccharomyces cerevisiae p /sqstm forms protein aggregates degraded by autophagy and has a protective effect on huntingtin-induced cell death glycogen autophagosomes in polymorphonuclear leukocytes induced by rickettsiae this is an excellent review of the complex relationships between autophagy and microorganisms eating oneself and uninvited guests; autophagy-related pathways in cellular defense role of phosphatidylinositol -kinase and rab effectors in phagosomal biogenesis and mycobacterial phagosome maturation arrest mycobacterium tuberculosis phagosome maturation arrest: selective targeting of pi p-dependent membrane trafficking mycobacterium tuberculosis phagosome maturation arrest: mycobacterial phosphatidylinositol analog phosphatidylinositol mannoside stimulates early endosomal fusion autophagy is a defense mechanism inhibiting bcg and mycobacterium tuberculosis survival in infected macrophages escape of intracellular shigella from autophagy references - are landmark papers that formed the foundation for the principle that autophagy is important in defence against intracellular bacterial pathogens (such as m. tuberculosis and shigella spp.) and extracellular bacterial pathogens that invade the cytosol this paper identified an important signalling mechanism (involving irgm) that activates autophagy-dependent control of mycobacteria cytoplasmic bacteria can be targets for autophagy autophagy limits listeria monocytogenes intracellular growth in the early phase of primary infection autophagy controls salmonella infection in response to damage to the salmonella-containing vacuole autophagy-mediated reentry of francisella tularensis into the endocytic compartment after cytoplasmic replication autophagy-mediated clearance of huntingtin aggregates triggered by the insulin-signaling pathway intracellular survival of shigella the ubiquitin ligase cbl-b limits pseudomonas aeruginosa exotoxin t-mediated virulence nf-κb activation represses tumor necrosis factor-α-induced autophagy reactive oxygen species are essential for autophagy and specifically regulate the activity of atg pkr-dependent autophagic degradation of herpes simplex virus type subversion of cellular autophagosomal machinery by rna viruses references - indicate a role for components of the autophagic machinery in the establishment of viral replication complexes copi activity coupled with fatty acid biosynthesis is required for viral replication cellular autophagy machinery is not required for vaccinia virus replication and maturation this paper provided the first demonstration that loss-of-function mutations of autophagy genes increases susceptibility to microbial infection in vivo protection against fatal sindbis virus encephalitis by beclin, a novel bcl- -interacting protein this paper provided the first evidence suggesting that autophagy inhibition is a mechanism by which viruses evade innate immunity and cause disease autophagy in immunity and infection: a novel immune effector innate immune recognition of viral infection toll-like receptor -mediated recognition of herpes simplex virus- by plasmacytoid dendritic cells excessive degradation of intracellular protein in macrophages prevents presentation in the context of major histocompatibility complex class ii molecules major histocompatibility complex class ii-restricted presentation of a cytosolic antigen by autophagy processing and presentation of hla class i and ii epitopes by dendritic cells after transfection with in vitro-transcribed muc rna autophagy promotes mhc class ii presentation of peptides from intracellular source proteins this paper provided the first genetic evidence that autophagy components can be required for efficient mhc class ii presentation of an endogenous antigen this paper describes the characterization of transgenic autophagy reporter mice (a tool that has greatly facilitated the study of autophagy in vivo) and the presence of high levels of autophagy a role for intercellular antigen transfer in the recognition of ebv-transformed b cell lines by ebv nuclear antigen-specific cd + t cells this paper provided the first evidence that autophagy occurs constitutively in antigenpresenting cells and also demonstrated that presentation of an influenza virus antigen can be enhanced by specifically targeting it to autophagosomes. the latter finding has important implications for vaccine design chaperone-mediated autophagy in aging and disease autophagy: many paths to the same end lamp- a facilitates mhc class ii presentation of cytoplasmic antigens this paper demonstrated that the ifn-inducible, antiviral signalling molecule pkr is required for virus dap kinase and drp- mediate membrane blebbing and the formation of autophagic vesicles during programmed cell death essential roles of atg and fadd in autophagic cell death: dissection of autophagic cell death into vacuole formation and cell death insulin-like growth factor- and tnf-α regulate autophagy through c-jun n-terminal kinase and akt pathways in human atherosclerotic vascular smooth cells cd -traf and autophagy-dependent antimicrobial activity in macrophages activation of phosphatidylinositol -kinase by interleukin- . an inhibitory signal for inducible nitric-oxide synthase expression in epithelial cell line ht- the tumor suppressor pten positively regulates macroautophagy by inhibiting the phosphatidylinositol -kinase/protein kinase b pathway distinct classes of phosphatidylinositol ′-kinases are involved in signaling pathways that control macroautophagy in ht- cells molecular mechanisms of interferon resistance mediated by viral-directed inhibition of pkr, the interferon-induced protein kinase autophagy is an effector of th -th polarization: th cytokines inhibit autophagic control of intracellular mycobacterium tuberculosis p gtpases: regulators of immunity to intracellular pathogens autophagy in cell death: an innocent convict? autophagy: paying charon's toll autophagy is involved in t cell death after binding of hiv- envelope proteins to cxcr phagocytosis of apoptotic cells and the resolution of inflammation removal of dying cells and systemic lupus erythematosus inefficient clearance of dying cells and autoreactivity mechanisms of disease pathogenesis: pathogenesis of crohn's disease and ulcerative colitis inflammatory bowel disease: cause and immunobiology toll-like receptor is a sensor for autophagy associated with innate immunity the original work from the authors' laboratories was supported by the national institutes of health, usa (v.d. and b.l.) and the ellison medical foundation (b.l.). the authors declare no competing financial interests. key: cord- -j t rjkc authors: odales, josué; guzman valle, jesus; martínez-cortés, fernando; manoutcharian, karen title: immunogenic properties of immunoglobulin superfamily members within complex biological networks date: - - journal: cell immunol doi: . /j.cellimm. . sha: doc_id: cord_uid: j t rjkc antibodies, t cell receptors and major histocompatibility complex molecules are members of the immunoglobulin superfamily and have pivotal roles in the immune system. the fine interrelation between them regulates several immune functions. here, we describe lesser-known functions ascribed to these molecules in generating and maintaining immune response. particularly, we outline the contribution of antibody- and t cell receptor-derived complementarity-determining region neoantigens, antigenized antibodies, as well as major histocompatibility complex class i molecules-derived epitopes to the induction of protective/therapeutic immune responses against pathogens and cancer. we discuss findings of our own and other studies describing protective mechanisms, based on immunogenic properties of immunoglobulin superfamily members, and evaluate the perspectives of application of this class of immunogens in molecular vaccines design. idiotypic antibody the immunoglobulin (ig) superfamily is a large functionally diverse group of proteins which bear a common ig domain, consisting of two anti-parallel β-sheets stabilized with a disulfide bond [ ] . antibodies (abs), t cell receptors (tcrs) and major histocompatibility complex (mhc) class i or class ii molecules are members of ig superfamily that play critical roles in immune response network [ , ] . since the discovery of the therapeutic potential of serum from animals exposed to attenuated pathogens, more than a century ago, huge progress in the development of ab-based therapeutics has been made. many abs are currently being used to treat several major pathological conditions: cancer, autoimmune, cardiovascular, infectious and neurodegenerative diseases, and the mechanisms of action of ab treatment has been reviewed elsewhere [ ] [ ] [ ] [ ] [ ] [ ] . tcr and ab molecules contain three complementarity-determining regions (cdrs), per variable domain, which are responsible for ag recognition. cdr is the most diverse region, in terms of sequence and length, and is considered the most important in determining the specificity of a given ab or tcr [ ] . b lymphocytes often undergo affinity maturation after initial encounter with ag, subsequently they produce new, slightly modified abs with increased affinity. this implies that the ag-ab interaction is almost perfect, i.e., the six cdrs of a given ab would be the "specular image" of the interaction established with a specific ag. for decades, there has been compelling evidence of cdr-specific t and b lymphocyte activation. this concept is partially based on the proposed ab network, pioneered by jerne in ( fig. a) , which suggests that one antibody (ab ) will induce an effective immune response that generates a second ab (ab : ab α targeting the region close to ag-recognizing site of ab proposed to describe the optimal affinity during pmhc-tcr interaction: kineticproofreading and serial triggering models; along with the prediction that there is an upper and lower limit to the half-life during pmhc-tcr complex binding, which narrows the range of optimal affinities leading to t cell activation [ ] . during organ transplantation, determining mhc compatibility between the donorderived organ and recipient is a crucial step to reduce rejection mediated by graftversus-host interaction. today, the presence of donor-derived hla-specific ab and t cell responses are suggested as part of the leading causes of organ rejection [ ] . preexisting organ-specific ab responses lead to acute ab-mediated rejection, even though ab responses could be elicited any time after organ transplantation; furthermore, direct or indirect t cell-mediated organ rejection provides other mechanisms that act in conjunction with ab responses leading to the damage of the transplanted organ or finally, to the patient death [ , ] , (fig. b) . also, autoimmunity, mediated by non-hla ab responses following solid organ transplantation, contributes to transplant rejection [ ] . cancer cells generally present modified versions, or abnormal expression of mhc molecules, as seen through loss of heterozygosity or loss of expression, which are associated with immune editing of tumors, and may contribute to cancer evolution and immune escape [ , ] . in line with the abovementioned immune mechanisms of organ rejection, and with the potential immunogenicity of mhc molecules ( fig. b) , we were the first, to our knowledge, to generate cancer vaccine immunogens, based on peptides derived from mhc qa- and h -k molecules, and to show their protective effects targeting the tumor as a transplanted organ in a mouse model of breast cancer [ ] . we have demonstrated significant inhibition of the tumor growth and the reduction of metastatic lesions in the lungs of immunized animals [ ] . therefore, harnessing the immunogenic properties of mhc molecules might provide an entirely new direction to treat cancer. furthermore, newly synthetized mhc class i α chains contain signal peptides that do not form part of the mature protein. these signal peptides remain in the endoplasmic reticulum after cleavage from the α chain, but afterwards are processed by proteolytic cleavage via signal peptide peptidase, and their amino-terminal portion is released into the cytosol. the mhc class i-derived signal peptide reenters the normal mhc class i antigen processing and presentation pathway and is finally loaded, specifically, on the non-classical hla-e molecule whose function is immunosurveillance [ ] . this mechanism indirectly evaluates mhc class i protein level translation. the nk and cd + t cell cd /nkg- a inhibitory and cd /nkg- c activating receptors oversee the production of peptide-hla-e complexes [ , ] . in addition, inhibitory kir family receptors on cytotoxic cells directly recognize mhc class i molecules and function by protecting normal non-stressed cells from cytotoxic lysis [ ] . thus, mhc molecules have several immunological functions beyond the immunological synapse with t lymphocytes. muromonab-cd (okt- ) was the first anti-human cd monoclonal antibody approved for human use by the food and drug administration (fda) in [ ] . it was used to prevent organ rejection after transplantation by primarily reducing t cell functions. in % of patients treated with the murine ab, human anti-drug antibodies (adas) were induced, a phenomenon known as human anti-mouse antibody (hama) response [ ] . the ada response decreases the efficacy of the treatment and induces adverse effects such as hypersensitivity-type reactions [ ] . these findings encourage efforts to reduce ab-related drug immunogenicity in order to develop better and safer drugs. this led to ab chimerization which is a process where xenogeneic ig constant regions are replaced by human ig constant sequences, in this manner ab-associated immunogenicity is reduced. rituximab (rituxan) was the first fda-approved chimeric ab in and is a cd -specific ab used to treat several b lymphocyte-related conditions [ ] . unfortunately, an ada response was also generated against [ , ] . importantly, sera obtained from mice immunized with vels were able to neutralize half of a tier- hiv- reference panel [ ] . we also confirmed the anti-id nature of these abs by isolation of an ab-binding peptide motif that resembles the original hiv- epitope after screening of immune sera against phage display random peptide libraries [ ] . in cancer-related research, we generated for over years, ab-based therapy has been approved by medical regulatory agencies worldwide and has been used in various clinical settings. ab-based therapy is widely considered a safe and effective medical treatment; however, adverse effects in patients have been reported [ , ] . to our knowledge, long-term studies on the undesirable effects of ab treatment have not been conducted, thus we are most likely ignorant to the consequences of manipulating the immune system. although several mechanisms of action have been described for this treatment modality, in most cases, these can be reduced to (i) an interaction-blocking agent and/or (ii) a targeted drug with fc-related immune effector functions. however, other unorthodox therapeutic approaches have also been explored such as anti-idiotype vaccines and intravenous immunoglobulin (ivig) treatment. the anti-idiotype vaccine concept is based on the idiotype/anti-idiotype cascade proposed by jerne ( fig. a) showed longer median survival time [ ] . these results led vaxira to be the first approved anti-idiotype vaccine, with permission granted in cuba and argentina. a phase iii clinical trial using vaxira is currently undergoing (www.clinicaltrials.com). ivig is a blood product consisting primarily of a mixture of iggs from thousands of healthy donors. the main indication for ivig is in replacement therapy, where low doses ( mg/kg, every weeks) are administered to patients with immunodeficiencyrelated conditions, with the purpose of providing passive immunity against pathogens [ ] . furthermore, ivig has immunomodulatory effects at high concentrations ( g/kg/month), where it has been used for the treatment of autoimmune or inflammatory disorders [ , ] . interestingly, positive preliminary data on ivig therapy in pediatric covid- patients have been reported recently, and its use in other groups of patients is under consideration [ ] . the mechanism of action for the immunomodulatory effects are not well established but many fab-and fc-dependent mechanisms are presumed to be involved, e.g., ab neutralization, cytokines, complement molecules, blockade of neonatal fc receptor and fc activating receptors [ , ] . also, the presence of t cell epitopes for natural regulatory t cells (ntreg) in the primary igg sequence is presumed to be involved in increasing the ntreg population, concomitant to reduction of the proliferative t cell response [ ] . the induction of these ntregs that recognize highly promiscuous mhc class ii t-cell epitopes or "tregitopes" in the fc fragment of igg, as a possible mechanism for the immunosuppressive activity of igg, may have clinical implications; for example, for hemophilia treatment, to avoid immunogenicity and induce immune tolerance, the recombinant factor viii (rfviii) and rfix, fused to the fc domain of igg, have been developed as therapeutic agents with longer-lasting circulating half-life [ , ] . regarding tcrs, their αβ protein chains are limited to expression as a membrane anchored complex, not in a soluble form, and are functionally restricted to mhc molecules. for these reasons, the tcr has not been exploited in the biotechnology field as much as abs. however, based on the similarity in immune functions and structure with the b cell receptor (bcr)/ab, we could expect similar immunogenic properties to be mirrored by the tcr. indeed, it has been proven that a morbillivirus nucleocapsid protein-specific cd + t cells, stimulated in vitro, process and present its own tcr-derived peptides, to both anti-idiotypic cd + and cd + t cells, confirming that ig-derived cdr epitopes are immunogenic [ , ] . interaction amongst these three members of the ig superfamily goes beyond their roles in lymphocyte activation or aiding the adaptive arm of the immune system (fig. ) . one possible implication is contraction of the immune response, the final phase after efforts of the immune system to eliminate a pathogen and achieve homeostasis. after initial encounter with an immunogen, t cells and b cells undergo clonal proliferation, this implies a simultaneous expansion in cdr neoantigens that may elicit an effective antiidiotypic response. this natural anti-idiotype cellular and humoral immune response might function as a contraction element of the immune system, eliminating effector lymphocyte clones. the specific elimination hypothesis for contraction of the immune response could explain experimental evidence for epitope-specific ctl elimination, after prolonged exposure to a lymphocytic choriomeningitis virus (lcmv)-derived np ctl epitope but not to exposure to other lcmv ctl epitopes [ ] . if elimination occurs in differentiated effector or memory cells, it would be subject to further research. other t cell dysfunctional processes may be involved such as exhaustion of epitope specific ctls, which has been also demonstrated in the same model [ ] . either clonal deletion, exhaustion or other t cell dysfunction processes may be occurring in cancer, which may reduce the repertoire of effector lymphocytes. experimental evidence came from studies in where tumor-infiltrating lymphocytes were only able to recognize of tumor-derived neoantigens from a stage iv melanoma patient [ ] . the authors demonstrated that an outsourced naïve pool of t cells from healthy donors, indeed, has idiotypes that respond to those neoantigens, in contrast to the patient-derived own t cells [ ] . other experimental clues concerning the idiotype-anti-idiotype network came from two separate experiments, which demonstrated specific idiotypic response inhibition after anti-idiotype intervention against two hapten groups, azophenylarsonates and phosphorylcholine, respectively [ , ] . the proposed mechanism involved a haptenspecific idiotype bcr-ab blocking interaction for hapten-specific inhibition of b lymphocyte responses [ , ] . a similar theoretical mechanism might occur in the event of ab recognition and blockade of either the tcr or the peptide-mhc complex; this could explain the reduction of the functional t cell repertoire, described above. furthermore, experiments in which t cells interact with naïve or resting anti-idiotypic t cells demonstrated the induction of anergy or apoptosis in the idiotypic t cell [ ] . analogous with such immune inhibition by anti-idiotypic responses, it has been shown in autoimmune diseases that it is not the presence of the autoantibody against selfproteins, but the lack of ab which is the underlying characteristic amongst patients [ , ] . hypotheses concerning the maintenance of immune memory without the presence of ags have been proposed. uytdehaag and colleagues first established that cd + b lymphocytes could be activated by increases in cdr epitopes, derived from the expansion of ag-specific b memory cells [ ] . theses cd + b lymphocytes may undergo affinity maturation to increase affinity to the ag-specific b cell idiotype, then, after ag clearance the v region of the anti-idiotype cd + b lymphocyte may serve as an ag mimic to maintain memory cells [ ] . another hypothesis suggests that memory responses could be maintained, not only in the absence of persisting ag, but also without long living memory cells. the interaction of idiotypic and anti-idiotypic responses could be an indefinite interaction, with no need for long-living memory b cells [ ] . the same group found that peptidomimetics of the antigen in v regions of ab are recognized by antigen-specific t cells and that maintenance of memory by the idiotype-anti-idiotype network could be extended to t cells as well [ ] . the previous hypotheses agree with the need to generate t cell responses to help to achieve and regulate memory responses; furthermore, they propose mechanisms for affinity maturation based on idiotype-anti-idiotype interactions. experimental designs and more importantly, results to confirm these hypotheses will be difficult to obtain but we cannot discard this phenomenon as a possible mechanism for immune memory maintenance [ ] . we believe that along with the already well-known functions of abs, tcrs and mhc molecules there are still several not fully appreciated nor completely understood but related immunological phenomena. interactions established by these molecules could expand our understanding of the immune system by adding non-canonical immune functions to already well-known molecules. of interest, is the possibility to explore the immunogenicity of ig superfamily members, as a promising way to find novel molecular vaccine candidates. furthermore, we believe that the "internal image" of an originally encountered ag, represented by a collection of polyclonal ab molecules, may potentially bear resemblance to an even larger pool of epitopes/mimotopes, present within pathogens or cancer cells; these may possibly reflect their entire antigenic landscape. if this is true, then it could aid in the development of much needed vaccines against antigenically variable pathogens and cancer. the authors have no potential conflict of interest to declare. idiotype-anti-idiotype network alters immune system memory compartment antigenized antibodies are efficient vaccine immunogen immunoglobulin superfamily, in: els the goldilocks model for tcr-too much attraction might not be best for vaccine design therapeutic antibodies: successes, limitations and hopes for the future the therapeutic monoclonal antibody market monoclonal antibodies: the new magic bullets for allergy: iuphar review recombinant antibody fragments for neurodegenerative diseases passive immunotherapy of viral infections: "superantibodies" enter the fray therapeutic antibodies: their mechanisms of action and the pathological findings they induce in toxicity studies an analysis of the sequences of the variable regions of bence jones proteins and myeloma light chains and their implications for the drivers of mhc restriction of t cell receptors outstanding questions in transplantation: b cells, alloantibodies, and humoral rejection antibody-mediated rejection of solid-organ allografts response to treatment and long-term outcomes in kidney transplant recipients with acute t cell-mediated rejection humoral autoimmunity after solid organ transplantation: germinal ideas may not be natural mhc-i genotype restricts the oncogenic mutational landscape allele-specific hla loss and immune escape in lung cancer evolution generation of cancer vaccine immunogens derived from major histocompatibility complex (mhc) class i molecules using variable epitope libraries intramembrane proteolysis of signal peptides: an essential step in the generation of hla-e epitopes the inhibitory nk cell receptor cd /nkg a and the activating receptor cd /nkg c bind the top of hla-e through mostly shared but partly distinct sets of hla-e residues the inhibitory receptor cd /nkg a on cd + tumor-infiltrating lymphocytes in colorectal cancer: a promising new druggable immune checkpoint in the context of hlae/β m overexpression human nk cells: surface receptors, inhibitory checkpoints, and translational applications okt monoclonal antibody plasma levels during therapy and the subsequent development of host antibodies to okt side-effects of a monoclonal antibody, muromonab cd /orthoclone okt : bibliographic review rituximab in b-cell hematologic malignancies: a review of years of clinical experience cells expressing an h chain ig gene carrying a viral t cell epitope are lysed by specific cytolytic t cells major histocompatibility complex class irestricted presentation of influenza virus nucleoprotein peptide by b lymphoma cells harboring an antibody gene antigenized with the virus peptide targeting gp and trp- with a dna vaccine: incorporating t cell epitopes with a human igg antibody induces potent t cell responses that are associated with favourable clinical outcome in a phase i/ii trial phage-displayed t-cell epitope grafted into immunoglobulin heavy-chain complementarity-determining regions: an effective vaccine design tested in murine cysticercosis variable epitope library-based vaccines: shooting moving targets variable epitope libraries: new vaccine immunogens capable of inducing broad human immunodeficiency virus type -neutralizing antibody response variable epitope library carrying heavily mutated survivin-derived ctl epitope variants as a new class of efficient vaccine immunogen tested in a mouse model of breast cancer adverse effects of immune checkpoint inhibitors (programmed death- inhibitors and cytotoxic t-lymphocyte-associated protein- inhibitors): results of a retrospective study immune-related adverse events of checkpoint inhibitors murine dendritic cells pulsed with an anti-idiotype antibody induce antigenspecific protective antitumor immunity dendritic cells pulsed with an anti-idiotype antibody mimicking carcinoembryonic antigen (cea) can reverse immunological tolerance to cea and induce antitumor immunity in cea transgenic mice a randomized, multicenter, placebo-controlled clinical trial of racotumomab-alum vaccine as switch maintenance therapy in advanced non-small cell lung cancer patients use of intravenous immunoglobulins for prophylaxis or treatment of infectious diseases a controlled trial of high-dose intravenous immune globulin infusions as treatment for dermatomyositis intravenous immunoglobulin for guillain-barré syndrome an outbreak of severe kawasaki-like disease at the italian epicentre of the sars-cov- epidemic: an observational cohort study clinical uses of intravenous immunoglobulin mechanisms of action of ig preparations: immunomodulatory and anti-inflammatory effects activation of natural regulatory t cells by igg fc-derived peptide "tregitopes igg fc immune tolerance group, tolerogenic properties of the fc portion of igg and its relevance to the treatment and management of hemophilia idiotypic t cells specific for morbillivirus nucleocapsid protein process and present their tcr to cognate anti-idiotypic cd + t cells activated mouse t cells downregulate, process and present their surface tcr to cognate anti-idiotypic cd + t cells viral immune evasion due to persistence of activated t cells without effector function induction and exhaustion of lymphocytic choriomeningitis virus-specific cytotoxic t lymphocytes visualized using soluble tetrameric major histocompatibility complex class i-peptide complexes targeting of cancer neoantigens with donor-derived t cell receptor repertoires suppression of idiotypic specificities in adult mice by administration of antiidiotypic antibody specific inhibition of plaque formation to phosphorylcholine by antibody against antibody the lack of anti-idiotypic antibodies, not the presence of the corresponding autoantibodies to glutamate decarboxylase, defines type diabetes anti-idiotypic antibodies prevent the serologic detection of antiribosomal p autoantibodies in healthy adults maintenance of immunological memory: a role for cd + b cells? perpetuation of immunological memory: a relay hypothesis a cd + t cell clone specific for antigen also recognizes peptidomimics present in anti-idiotypic antibody: implications for t cell memory maintenance of antigen-specific immunological memory through variable regions of heavy and light chains of anti-idiotypic antibody key: cord- -fcno z authors: nan title: molecular aspects of viral immunity date: - - journal: j cell biochem doi: . /jcb. sha: doc_id: cord_uid: fcno z nan mechanisms of t-cell mediated clearance of viruses from the central nervous system are poorly understood, but likely to differ from those employed in the periphery because the cns lacks lymphatic drainage and constitutive expression of mhc class i antigen, and the unique structure of the cns vasculature imposes constraints on access by leukocytes and soluble immune mediators. to study the mechanism by which viruses are cleared from neurons in the central nervous system, we have developed a mouse model involving infection with a neurotropic variant of mouse hepatitis virus (oblv ). grew preferentially in the olfactory bulbs of balbk mice. using in situ hybridization, we found viral rna localized primarily in the outer layers of the olfactory bulb, including neurons of the mitral cell layer. virus was cleared rapidly from the olfactory bulb between and days. athymic nude mice failed to eliminate the virus demonstrating a requirement for t lymphocytes. immunosuppression of normal mice with cyclophosphamide also prevented clearance. both cd + and cd + t-cell subsets were important as depletion of either of these subsets delayed viral clearance. gliosis and infiltrates of cd + and cd + cells were detected by immunohistochemistry at days. the role of cytokines in clearance was investigated using an rnase protection assay for il-la, il-lp, il- , il- , il- , il- , il- , tnfa, tnfp and ifny. in immunocompetent mice there was upregulation of rna for il-la, il-lp, il- , tnfa and ifny at the time of clearance. nude mice had comparable increases in these cytokine messages with the exception of ifny. induction of mhc-i molecules on cells in infected brains was demonstrated by immunohistochemistry in normal and nude mice, suggesting that ifny may not be necessary for induction of mhc-i on neural cells in vivo. luca g. guidotti, kazuki ando, tetsuya ishikawa, lisa tsui and francis v. chisari. the scripps research institute, la jolla, ca although cytotoxic t lymphocytes (ctl) are known to clear viral infections by killing infected cells, recent studies suggest that they can also suppress the replication of certain viruses by noncytolytic mechanisms. we have examined this area by monitoring the immunopathological and antiviral consequences of antigen recognition by hepatitis b virus (hbv) specific ctl in hbv transgenic mice that express the viral gene products in their hepatocytes. we have shown that intravenously injected ctl rapidly trigger their target hepatocytes to undergo apoptosis, but that the direct cytopathic effect of the ctl is minimal in comparison with the cytopathic effects of the antigen-nonspecific intrahepatic inflammatoly response that they activate. in addition to killing the hepatocyte, the same ctl also downregulate hbv gene expression and completely abolish hbv replication in the hepatocytes that they don't destroy. this noncytolytic antiviral ctl effect is mediated by at least two distinct processes in these animals. first, the ctl cause a quantitative reduction in the steady state content of all hbv mrna species in the hepatocyte, and this is followed by disappearance of all of the corresponding viral proteins in the liver and serum. the ctl initiate this process by secreting ifny and tnfa when they are activated by antigen recognition. since the regulatory effect of the ctl can he prevented completely by prior administration of the corresponding antibodies. nuclear run-on experiments reveal that viral mrna transcription is unaffected despite the profound reduction in hbv mrna content in the liver, suggesting that the ctl-derived cytokines accelerate viral mrna degradation in the hepatocyte. a second noncytolytic antiviral pathway is also activated by the ctl. we have recently shown that hbv nucleocapsid particles, and the replicative hbv dna intermediates that they contain, disappear from the transgenic mouse liver following either ctl administration or partial hepatectomy. the latter of which triggers hepatocellular regeneration without any change in hepatocellular hbv mrna content. these results suggest that preformed hbv nucleocapsid particles may be actively degraded during hepatocyte turnover, and they raise the possibility that similar events might also occur in nondividing hepatocytes that are activated by noncytolytic signals delivered by the ctl. we propose that, in addition to their pathogenetic effect, the comhined effects of the ctl response at die hbv mrna. nucleocapsid and rcplicative dna levels may represent a curative antiviral stimulus during hbv infection. since the virus must contain molecular elements that iespond to these ctl-induced antiviral signals. inactivating mutations at these loci could be very efficiently selected by immune pressure, because a single mutation could abrogate the antiviral effect of a wide spectrum of t cell responses, irrespectrve of epitope specificity. identification of these viral response elements and the intracellular pathways that interact with them may lead to the development of new strategies for antiviral drug design. human fibroblasts infected with hsv are resistant to lysis by cd + cytotoxic t lymphocytes (ctl), yet human b cell lines can be efficiently lysed by these ctl. the effect on human fibroblasts is rapid (within hr of infection of cells), occurring before synthesis of mhc class i is altered by virus infection. a recombinant hsv, f-usbmhc, which expresses mouse mhc class i proteins does not render human fibroblasts sensitive to lysis by mouse ctl. mhc class i molecules are retained in the er of hsv-infected fibroblasts i n a misfolded, unstable form and stability of the mhc complex can be restored by addition of exogenous peptides. using a panel of hsv mutants and ad expression vectors we demonstrated that the hsv ie protein icp was both necessary and s f i c i e n t to cause retention of class i and icp expression in fibroblasts caused the cells to resist lysis by cd + t lymphocytes. icp is a soluble, cytosolic protein and we have found no evidence of membrane association. therefore, it appears that icp inhibits cytosolic stages of the antigen presentation pathway so that antigenic peptides do not reach the er. to date, polyclonal and monoclonal antibodies directed to icp have not specifically precipitated any of the previously described components of the antigen presentation pathway and we have not found icp associated with tap transporter proteins or proteosomes i n these experiments. the effects of icp are being assessed in proteosome and tap transporter assays. gst-icp fusion proteins tightly bind a . kda cytosolic cellular protein which is found in a number of adherent human cell lines but not lymphocytes. the protein has been purified and sequencing is in progress. in addition, radiolabelled icp binds to a single cellular protein of = kda on ligand blots. these proteins are good candidates as cellular targets of icp and as novel components of the antigen presentation pathway. preliminary experiments support the hypothesis that icp is very effective i n blocking cd + t lymphocyte responses in vivo, perhaps explaining the predominance of cd + vs. cd + anti-hsv ctl i n vivo. we expect that icp may be very useful, not only to elucidate antigen presentation pathways, but also to prevent immune recognition of gene transfer vectors and a s a immunosuppressive agent. susceftibility to polyoma virus-induced tumors is conferred by an endogenous mmtv superantigen. aron e. lukacherl, yupo ma , john p. carroll , sara r. abromson-leeman , joseph c. laning , martin e. dorf , and thomas l. benjamin . idepartment of pathology, emory university school of medicine, atlanta, ga , and department of pathology, harvard medical school, boston, ma . susceptibility to tumors induced by mouse polyoma virus varies among inbred mouse strains. we have previously shown that polyoma tumor susceptibility is controlled by products of mhc as well as non-mhc genes. in crosses between mhc-nonidentical strains differing in tumor susceptibility, resistance correlates with dominantkodominant inheritance of the resistant h- haplotype. we have observed the opposite pattern of inheritance of susceptibility in crosses between mhc-identical strains. in crosses between the highly susceptible c wbida mouse and the highly resistant but mhc-identical (h- k) c bwcd.i mouse, polyoma tumor susceptibility is conferred by a single autosomal dominant gene, which we have designated pyvs. pyj does not encode cell receptors for the virus, affect viral dissemination or anti-viral antibody responses, or affect intracellular events essential for productive infection or cell transformation by the virus. whole-body irradiation renders cs bwcd.i mice fully susceptible to polyoma-induced tumors, indicating an immunological basis for this strain's resistance. we hypothesized that p y j encodes an mtv superantigen (sag) that confers susceptibility to c wbida mice by deleting precursors of polyoma-specific t cells. we found that tumor susceptibility in (c wbida x c bwcd.i) x c bwcdj backcross mice cosegregated with mtv- . inheritance of mtv- showed perfect concordance with absence of peripheral vp + t cells. genotyping of backcrossmice using markers of simple sequence repeat polymorphisms flanking mtv- showed no evidence of recombination between pyvs and mtv- . strongly biased usage of vp by (a) polyoma-specific cd + ctl from virus-infected c bwcdj mice and by @) cd + t cells infiltrating a polyoma tumor in a virus-immune c bwcd.i host provide further evidence that t cells bearing this mtv- sag-reactive vp domain are critical anti-polyoma tumor effector cells. these results indicate identity between p y j and mtv- sag, and demonstrate a novel mechanism of inherited susceptibility to virus-induced tumors based on effects of an endogenous superantigen on the host's t cell repertoire. infection of mice with lymphocytic choriomeningitis virus (lcmv) causes a transient to longlasting immunosuppression dependent upon virus-isolate dose of virus and age, h- , non h- , level of cd + t cells, of cd + t cells and kinetics of neutralizing antibodies of the host. the immunohistological analysis suggests that cd + t cell dependent disappearence of marginal zone macrophages of follicular dendritic cells and of virus infected cells in general correlates with immunosuppression. the details of mechanisms responsible for these findings are now being analysed. a role of this cd + t cell dependent immunosuppression in the establishment of a lcmv carrier state in immunocompetent mice is suggested by the following experiments: the otherwise slow and low neutralizing antibody response agamst lcmv is accelerated and enhanced by cd + t cell depletion at the time of infection, suggesting virus-specific immunopathology being responsible at least partially. the elisa antibody response is not significantly altered under the same conditions but is abrogated if lcmv-specific t cell receptor transgenic mice are infected with high doses of lcmv, indicating, that suppression of the specific antibody response depends upon the relative kinetics of ctl versus antibody responses. whether exhaustion of specific ctl responses is enhanced by similar mechanisms remains to be tested. the role of interleukins of the relative distribution of virus in the mouse and in the various aspects of immunosuppression are now being studied. immunosuppression, caused by cd + t cell-dependent immunopathology, may also be operational in hiv infection in humans. such a pathogenesis of hiv-triggered aids could explain several aspects of the disease process not readily fitting the (unproven) conventional idea that hiv is causing immunodeficiency via direct viral pathogenicity. the cellular immunity against two dna tumor viruses (i.e. human adenovirus type (ad ) and human papillomavirus type (hpv )) was studied with respect to possible immune escape mechanisms and to the development of ctl epitope based peptide vaccines. after identifying an immunorelevant ctl epitope in the ad e a protein to which ctl clones were directed that could eradicate ad e induced tumors in nude mice, an amino acid replacement study of this epitope revealed a point mutation that totally eliminated the possibility to recognize the mutant peptide by the ctl clones directed against the wild-type peptide sequence. new viral constructs were made that contained this point mutation and used to transform mouse embryo cells. however, these mutant tumor cells were still immunogenic and ctl clones specific for these mutant tumor cells were shown to react with a peptide derived from the ad e b protein. these ad eib specific ctl clones, however, were as effective as the ad e a specific ctl clones in the eradication of ad e induced tumors in nude animals, indicating that a choice can be made of immunorelevant epitopes to which an immunization strategy could be developed. in addition, we discovered that by supertransfection of ad e induced tumor cells with the activated ras oncogen the possibility of ad e b specific ctl to recognize the ad e induced tumors was eliminated whereas the ad e a specific ctl could still kill these tumor cells. this might indicate a new mechanism of tumors to escape ctl. in an hpv induced mouse tumor model an immunosubdominant ctl epitope was identified in the e protein that could, upon immunization with that peptide,protect mice against a subsequent challenge with hpv induced tumor cells. by changing the anchor residues in that peptide an even more immunoprotective peptide could be generated. combined, these data indicated a successful use of a ctl epitope based peptide vaccine in the prevention of hpv induced tumors in mice. subsequently this led us to identify relevant ctl epitopes of hpv , that is highly associated with cervical carcinoma in humans, for the major hla-a alleles (i.e. hla-a * , a * , a" , a* and a * ). together these alleles cover a majority of all humans. ctl epitopes were identified through peptide-mhc binding assays followed by in v i m peptide immunizations with high affinity binding peptides to induce primary ctl responses and immunogenicity studies in hla-a transgenic mice. thereafter, memory ctl responses were measured in cervical cancer patients against selected peptides. combined, these data led us to develop a ctl epitope based peptide vaccine that could be of use in hpv induced cervical cancer patients. a clinical trial for this disease is scheduled to start in the fall of . class ii presentation of an endogenously synthesized glycoprotein. carol s. re is^'.'.^, shirley m. bartido', miriam stein', and stephanie diment . , biology department', and center in contrast to class i presentation which is well characterized to use peptide fragements of proteins sythesized in the cytoplasm, exogenously administered experimental antigens enter the class ii mhc pathway through endocytosis. we have been studying the recognition of the glycoprotein of vesicular stomatitis virus (vsv) which can enter either the exogenous or endogenous pathways for presentation to cd + t cells. investigations of the intracellular sites involved, the proteolytic processes involved in the epitope generation, will be discussed. the glycoprotein studied in detail is a truncated form of the wt type glycoprotein, termed poison tail (gpt) . expressed with a vaccinia virus vector, the gpt remains endo h sensitive and never becomes endo d sensitive, indicating that it is restricted to the endoplasmic reticulum. gpt is degraded in the er, and w e believe the degradation products include the immunogenic epitopes recognized by a panel of lad and i-ed t cell clones and hybridomas. lmmunofluorescence studies have confirmed the er localization. flow cytometric evaluations s h o w that the gpt never appears on the cell surface, in contrast to the wt g. the peptides generated are not secreted; using an innocent bystander assay, gpt-infected cells are incapable of sensitizing 'cr-labeled uninfected apc. this contrasts with the rapid ability of supernatants from wt g-vaccinia virus-infected cells to sensitize apc for t cell recognition. investigations of the characteristics of the enzymes contributing to the degradation of the gpt have shown that a reducing environment is essential, as diamide treatment of cells prevents degradation. lysosomotropic drugs (eg. nh,ci and leupeptin) d o not alter the half-life of the protein, but do prevent presentation of the peptides; this is inconsistent with an autophagic component to the proteolysis. ph optima are physiological, as ph environment inhibits the enzyme activity. inhibitors of enzyme classes are consistent with a trypsin-like, and not cystein-, cathepsin b-, or chymotrypsin-like class. supported by nih grant al to csr. (emcv) and mengovirus are related members of the cardiovirus genus of picomaviruses. their rna genomes encode a large polyprotein which is cleaved proteolytically in co-and post-translational reactions to yield all mature viral proteins necessary to establish an infection. although originally thought to be exclusively murine in host range, both viruses actually infect a wide range of mammals. emcv has caused devastating epizootics in captive primates (eg: macaques, chimps and baboons), domestic pigs and exotic zoo collections (elephants, lions and tigers). death, following ingestion of virus-contaminated material, is rapid, and caused by extensive meningoencephalitis and virus-induced damage to the cns. myocarditic lesions are common in older animals. when administered intracerebrally, the ld,, for emcv strain r is about pfu. we are studying the pathogenesis of emcv and mengo with engineered cdna plasmids containing infectious viral sequences. many plasmids contain h'uncated versions of the unusual ' noncoding homopolymeric poly(c) tract that is a hallmark of these cardioviruses. short poly(c) mengoviruses grow very well in tissue culture but are - 'z fold less pathogenic to mice than the wild-type strains. animals receiving sublethal doses of short-tract mengo strains develop high titers of neutralizing antibodies, exhibit potent ctl responses and acquire lifelong protective immunity against challenge with wild-type virus. the genetic stability of the short-tract strains, even upon serial brain passage, mark them as safe, efficacious live vaccines. currently, we believe the poly(c) phenomenon is due to interference by the wild-type virus sequences (long poly(c) tract) with normal cellular cytokine induction mechanisms (ie: ifa and ifp) during the initial stages of animal infection. the targeted cells are probably macrophages, and their singular ability to correctly respond or not respond to poly(c) tract length during the first few hours of infection determines whether an inoculated animal will live (protectively vaccinated) or die. the short-tract viruses probably induce if in the macrophages, and are consequently killed then rapidly cleared from the host in related experiments we've found that attenuated mengo strains can easily carry large heterologous insertions within their genomes, and express these sequences into protein them during replication in animals. the resulting immune response (b cell and ctl) to the chimeras is directed towards the foreign sequences (epitopes) as well as towards the mengo proteins. a chimeric hiv vaccine, a rabies vaccine and an lcmv vaccine have been developed and tested. the lcmv chimera seems especially effective, as a single pfu of this engineered mengo strain, administered orally to a mouse, is sufficient for complete immunogenic protection against intracerebral challenge with wild-type lcmv virus. rsv is the most common cause of serious viral lower respiratory tract disease in infants and children. we have recently renewed our efforts to generate a safe and effective live attenuated rsv vaccine for topical administration that will overcome the deficiencies of previously studied live and non-living rsv vaccines. this vaccine will be a bivalent vaccine consisting of subgroup a and b live attenuated virus components. since the peak incidence of severe disease caused by rsv is in the -month old infant, an rsv vaccine will need to be effective when given to -month old infants. based on the success of live poliovirus vaccines given early in infancy, it is anticipated that the intranasally administered live virus vaccine will infect and induce a protective local and systemic immune response even in infants with passively acquired maternal antibodies. the main approach that we have taken in this effort to develop the live rsv vaccine is to introduce one or more ts mutations by chemical mutagenesis into a cold-passaged virus (cprsv) that had been partially attenuated by the acquisition of host-range mutations selected by passage in cells of a heterologous host species. we have developed a large set of cprsv subgroup a rs mutants (termed cprs mutants) that contain the host-range mutations selected during cold passage and two or more ts mutations introduced by chemical mutagenesis. these mutants have been evaluated in virro for their level of temperature sensitivity and in vivo in rodents, chimpanzees, and humans. a large set of rsv subgroup b cpts mutants has been similarly produced and evaluated. the immunogenicity and protective efficacy of three candidate live attenuated rsv vaccine strains that represent a specaum of attenuation were evaluated for protective efficacy in chimpanzees. prior to infection some of these animals were given rsv immune globulin by the iv route to simulate the condition of the very young infant who possesses passively-acquired maternal rsv antibodies. the three candidate vaccine strains were immunogenic and induced significant resistance to rsv challenge in both groups of chimpanzees. interestingly, the chimpanzees infused with rsv antibodies prior to immunization were primed more effectively for an unusually high serum neutralizing antibody response to infection with challenge virus than chimpanzees which did not receive such antibodies. this high booster response occurred despite marked reshiction of replication of the challenge virus. the evaluation of two candidate vaccines in seronegative human infants will also be described. rs virus is immunologically interesting for at least t w o reasons: ) upper respiratory reinfection occurs despite previous exposure and demonstrable immunological memory: ) humans or rodents previously immunised against virus infection can show enhanced disease during reinfection. others have shown that passive transfer of antiviral antibody either protects against virus infection or has no effect, and there is no evidence of antibody enhancement of disease in vivo. by contrast, t cell immunity appears closely associated with disease augmentation. we have focused on examining the immunological mechanisms of disease enhancement in mice. initial studies showed that transfer of cd + cytotoxic t lymphocytes (ctl) causes rapid virus clearance from the lungs of rs virusinfected mice, but also increased disease severity with alveolar haemorrhage and polymorphonuclear (pmn) cell recruitment to the lung. this disease (reminiscent of shock lung) could sometimes be fatal, whereas normal mice recover well from similar doses of rs virus. next, we compared the effects of cd ' and cd + t cells, using polyclonal t cells separated immunomagnetically from mixed lines grown in vitro with viral antigen. cd ' t cells were more pathogenic than cd + t cells in a dose-for-dose comparison, but that the type of pathology varied depending on the type of cell injected. while testing recombinant vaccinia viruses expressing single rs viral proteins for their ability to protect mice against infection, we observed that animals sensitised t o the major surface glycoprotein g (attachment protein) developed lung eosinophilia after challenge with rs virus intranasally. t cell lines from the spleens of mice sensitised with various recombinant vaccinia viruses were established. those form mice primed with the m ( k) protein were predominantly cd ' ctl, and that produced few cytokines. those from mice primed with fusion protein (f) generated mixed t cell lines with both t h l cd + t cells, and ctl. mice primed to g protein gave rise to predominantly cd ' t cells producing th cytokines. ln vivo transfer of these cell lines into na'ive rsv infected mice reproduces the patterns of disease seen in mice sensitised in vivo with the respective antigens. the mouse model of rs virus disease therefore has excellent potential for illustrating mechanisms of lung immunopathology. the eye is a complex organ whose function is to transmit light images through different cell and tissue layers and liquid media to a neurosensory retina. elements as could occur when invading pathogens arrive and an inflammatory response with its swelling, plasma protein extravasation, leukocyte infiltration and tissue damage results. inflammatory responses when possible and rely on immune defenses which do not involve tissue distortion and damage. restricting tissue damaging responses is not always effective and the process is best developed in response to agents delivered to locations such as the anterior chamber. where an inflammatory response is initiated which may result in ocular impairment. such herpetic stromal keratitis (hsk) is a common cause of blindness in man. animal model studies indicate that hsk is a multi-step process initiated by virus in an avascular structure. hsk fails to occur in the absence of t cells or replicating virus. disappears several days before a visible inflammatory response becomes evident. evidence will be presented that the secondary agonists which drive the inflammatory response may not be viral antigen(s) per s e . multiple cell types are involved in hsk, with the respective role of functional sets of lymphocytes changing according to the clinical phase of the disease. in addition, nonspecific inflammatory cells such as neutrophils and nk cells also influence the severity of lesions. basically the reaction begins with t cells that produce type one cytokines, particularly ifn-y, dominating the scene, but during remission type cytokines, notably il- , appear as mechanistically involved. from the use of knockout mice for various immunological parameters, evidence will be presented that numerous mechanisms of pathogenesis may be at play during hsk. damage to corneal tissues in all systems appear to involve tnfo. a second ocular damaging event in which immunopathology is at least partially involved is herpetic retinal necrosis. evidence that this disease may involve the immunopathological role of cd t cells and protective effects by cd ' t cells will be presented, as will be suggestions by which the pathological events are mediated at the molecular level. thus it is in the eye's functional interest to limit acute viral infections and live vaccines often confer long-term immunity the nature of t and b cell memory is different. b cell memory is manifested not only by the presence of memory b cells but also by continuous antibody production in contrast, the effector phase of the t cell response i s shortlived and long-term t cell memory is due to the presence of 'quiescent' antigen specific memory t cells that are present at higher frequencies and are able to respond faster upon re-exposure to virus due to increased levels of adhesion molecules in this talk i w i l l present our results on. (i) the bone marrow as a major site of long-term antibody production after acute viral infection, (ii) the role of c d ' t cells and b cells (immune complexes) in maintaining cd + t cell memory, (iii) the role offas antigen in regulating t cell responses, and (iv) the efficacy ofvarious antigen delivery systems in inducing long-term t cell memory sendai virus is a natural respiratory viral pathogen of mice. intranasal infection of mice with the virus provokes a virus specific antibody-forming-cell reaction that exhibits a distinct kinetic pattern in the lymph nodes that drain the respiratory tract, in the spleen, and in the bone marrow. the bone marrow afc population is extremely long-sustained, and supports an active humoral response that essentially persists for the lifetime of the infected animal. thus the conventional categories of "primary" and "secondary" response may not apply to the humoral response of mice naturally exposed to respiratory viruses. paradoxically, the population of b cells that reacts most rapidly to sendai virus infection does not itself secrete antibody, but can he demonstrated by the recovery of hyhridomas that secrete "polyspecific" antibodies. the activation of this polyspecific b cell population is, like the humoral response, extremely persistant. viral infection thus sets in train multiple b cell "memory" processes. variation in the rules of development and turnover of different b cell populations constrains the mechanisms that may operate to generate these different forms of memory. establishment and maintenance of t cell memory to respiratory viruses, peter c. doherty, sam hou, christine ewing, david topham, anthony mcmickle, james houston, and ralph tripp, department of immunology, st. jude children's research hospital, memphis, tn . the analysis of the development and memory phases of the cd + "helper" n h ) and cd + cytotoxic t lymphocyte (ctl) responses to the respiratory pathogens, influenza virus and sendai virus (parainfluenza type ) have been characterized by a combination of limiting dilution analysis (lda) for determining th and ctl precursor @) frequency and facs separation of lymphocytes with different activation phenotypes. the interpretation at this stage, largely based on the analysis of the ctl response, is that the development phase of t cell memory and the primary response are synonymous. virus-specific ctlp are produced in considerable excess of the numbers required to provide the effector ctl that terminate the primary infection, with only a fairly small proportion localizing to the target organ (the lung) that supports virus growth. even when many of the proliferating ctlp are killed by administration of a small dose ( mgkg) of the dna-targeted drug cyclophosphamide (cy), there is no indication of immune exhaustion. the cd + th response has, at this stage, not been analyzed through the course of the primary infection. use of the lda approach to determine thp frequencies is inherently more difficult, as the "read-out'' is lymphokine production and there is considerable "bystander" activation in these primary responses to respiratory viruses. memory thp and ctlp are characterized initially by the expression of an "activated" phenotype: cd -high, l-selectin-low, cd d (vla- ) high. after some months, an increasing proportion of the memory t cells revert to the l-selectin-high cd d-low form typical of naive ctlp. the change, which is never absolute, seems to occur first with cd d and the rate varies for different viruses. current experiments are addressing the possibility that intercurrent infection, particularly with the mouse y-herpesvirus which causes persistent infection of lymphoid tissue, may be inducing a switch back to the activated pattern, as a consequence of "bystander" effects, or "low affmity" stimulation via the clonotypic tcr in responding lymphoid tissue. the question of such cross-reactivity and/or exposure to "high lymphokine" environments for the long-term maintenance of memory is also being addressed. to study the factors which regulate the generation and persistence of specific t cell memory we have used model systems utilizing t cell receptor transgenic mice as a source of enriched naive cells which can be either cultured in vitro to generate effector populations or restimulated in adoptive hosts. in either case one can visualize the development of an expanded effector population. we have documented that the proliferation and il- production of the naive t cells depends on their activation by apc expressing high levels of co-stimulatory molecules. we find that b . and icam-i as costimulators strongly synergize and that increased t cell receptor triggering can both increase the magnitude of the response and decrease its dependence on costimulation. when cytokines il- vs il-i /ifny are present at the initiation of the response of either cd or cd cells they dictate that the effectors generated will be polarized either towards il- and il- secretion or il- and ifny secretion, respectively. the fate of the effector population generated and followed in vitro, also is tightly regulated by ag, cytokines and probably by costimulation. cd effector cells not re-exposed to ag, produce no cytokines and they die within - days. effectors restimulated with ag make massive amounts of cytokines, regardless of the presence of cytokines, at low densities of ag and with little dependence on costimulation. when there is little il- produced and no cytokines added, effectors die rapidly by apoptosis. however the combination of il- and tgfp block apoptosis and support expansion of the effector population which is greatly enhanced by periodic ag stimulation. some conditions favor the reversion of effector-like cells to a more resting memory phenotype and these are being further explored. we have also examined the development and maintenance of memory after transfer of effector cells to adoptive hosts. long-lived polarized memory populations are generated from the polarized effectors and these persist for prolonged period in the absence of apparent ag stimulation. this supports the idea that factors other than antigenic stimulation, present in situ can support the expansion and maintenance of memory cells. the rabies glycoprotein (g) is the only external protein of the virion and is therefore responsible for any interaction that rabies makes with the host cell during the first steps of the virus cycle. the g protein is also the target of neutralizing antibodies. there are around trimers of g at the virion surface which constitute the spikes visible by electron microscopy. upon exposure to slightly acidic ph, the glycoprotein undergoes a conformational change which results in ion er and less regular spikes. strikingly and quite differently from influenza hemagglutinin, this conformational change is reversible: if the p d is risen back to . , the s ikes re ain their neutral configuration ( ). probably as a consequence, the viral infectivity is totally preserved after an exposure of hours at p . an cf t, which induces the conformational change, followed by an incubation at neutral ph. since the conformational change is reversible, there is a ph-dependant equilibrium between the native and the low-ph conformation: the higher the ph, the more spikes are in their native configuration. two main antigenic sites and several minor sites have been identified on the native rabies glycoprotein ( ). specific amino acids belonging to each of the two major antigenic sites are important or essential for viral virulence. for instance a lysine in position , which is part of antigenic site , is important, although not essential, for the viral virulence. similarly, the arginine , which belongs to antigenic site , is essential for pathogenicity while dispensable for multiplication in cell culture (reviewed in ). viral strains mutated at arginine have lost the capability to penetrate certain categories of neurons, suggesting that this mutation affected the recognition of specific receptors or subsequent interactions necessaly for the penetration of the virus at nerve terminals. therefore the two main antigenic sites are regions of the glycoprotein which also interact specifically with neurons in the animals. we have found that neutralization requires the fixation of at least one or two igg for every three spikes, irrelevant of the anti enic site recognized by the antibody ( ). most neutralizing antibodies recognize conformational epitopes which are accessible on the native configuration of the protein. some epitopes remain accessible also on the acidic configuration while others are not. in addition, a minority of antibodies recognize epitopes which are only accessible on the acidic conformation. this is not unlikely in view that each spike has a certain probability to undergo a conformational change, even at neutral ph. in consequence the surface of the virus probably fluctuates and g epitopes which are not accessible on the native glycoprotein could be transiently exposed. conformational flexibility at neutral ph and physiological temperatures has also been observed for poliovirus ( ). structural flexibility of external proteins could have important implications in virus-host interactions. katpus, norlhwestern university medical school, chicago, il theiler's murine encephalomyelitis viruses (tmev) are endemic enteric pathogens of wild and colony-reared mice. lntracerebral inoculation of susceptible mouse strains leads to a chronic, progressive inflammatory demyelinating disease of the central nervous system (cns) characterized clinically by an abnormal gait, progressive spastic hind limb paralysis and urinary incontinence, and histologically by parenchymal and perivascular mononuclear cell infiltration and demyelination of cns white matter tracts. demyelination is related to persistent cns viral infection. due to the similarity in clinical and histological presentation, tmev-induced demyelination is considered to be a highly relevant model of multiple sclerosis (ms). our current interests are in determining the phenotype, fine specificity, lymphokine profile and tcr usage of cns-infiltrating cells involved in the effector stages of tmev-induced demyelination. based on a variety of experimental evidence, it is clear that demyelination induced in sjuj mice by infection with the bean strain of tmev is a thl-mediated event: (a) disease induction is suppressed in t cell-deprived mice and by in vivo treatment with anti-i-a and anti-cd antibodies; (b) disease susceptibility correlates temporally with the development of tmev-specific, mhc-class il-restricted dth responses and with a predominance of anti-viral lgg a antibody; (c) activated (le., ll- rc) t cells infiltrating the cns are exclusively of the cd + phenotype, and (d) proinflammatory cytokines (ifnq and tnf-p) are predominantly produced in the cns. we have mapped the predominant thl epitope on the virion to amino acids - of the vp capsid protein. a thl line specific for vp - exacerbates the onset of demyelination in recipient mice infected with a suboptimal dose of tmev. tmev-infected sjuj mice fail to exhibit peripheral dth and t cell proliferative responses to the major myelin proteins, mbp and plp, and pre-tolerization with neuroantigens has no affect on the incidence or severity of tmev-induced demyelinating disease, whereas neuroantigen-specific tolerance prevents the induction of relapsing experimental autoimmune encephalomyelitis (eae). in contrast, tolerance induced with intact tmev virions specifically anergizes virus-specific thl responses and results in a dramatic reduction of the incidence and severity of clinical disease and cns dernyelination in sjuj mice subsequently infected with tmev. these results have important implications for a possible viral trigger in ms as they indicate that chronic demyelination in tmev-infected mice is initiated in the absence of demonstrable neuroantigen-specific autoimmune responses and are consistent with a model wherein early myelin damage is mediated via primarily by mononuclear phagocytes recruited to the cns and activated by pro-inflammatory cytokines produced by tmev-specific thl cells. the concept that prions m novel pathogens which are different fium both viroids and viruses has received increasing support from many avenues of investigation over the past decade. enriching fractions from syrian hamster (sha) brain for scrap= prion infectivity led to the discovery of the prion protein . prion diseases of animals include scrapie and mad cow disease; those of humans present as inherited, sporadic and w o r n neurodegenemive disorders. the inhecited human pion diseases m genetically linked to mutatim in the prp gene that result in non-conswative amino acid substitutions. transgenic v g ) mice expressing both sha and m o w @lo) prp genes were used to demonstrate that the "specie bank?' for -pie prions resides in the primary structure of pip. this concept was strengthened by the results of studies with mice expressing chimeric mdsha transgenes &om which "artificial" prions have been synthesized. similar chimeric mdhuman (hu) rp transgenes were constructed which differ from m o w by amino acids between residues and . au of the tg(mhu m) mice developed neurologic drsease - days after inmulation with brain homogenates from three patients who died of creutzfeldt-jakob disease (cjd). inoculation of tg(mhu m) mice with cjd prims produced mhu mprpsc, inoculation with mo prions produced moprw. ihe patterns of meluzmprpc and mom% accumulation in the brains of tg(mhu m) mice wen differenl about % of tg(huprp) mice expressing huf" and non-tg mice developed neurologic diseane > days after inoculation with cn, prions. the different susce@uies of tg(hw) and tg(mhu m) mice to human prions indiate that additional species specific factors such as chaperone proteins are involved in prion replicaton. diagnosis, prevention and treament of human @on diseases should be faciliated by tg(mhu m) mice. in other sindies, tg mice were compared expressing wt and mutant moprp. overexpression of the wtmoprp-a aansgene - -fold was not deleterious to themiw but it did shorten scrapie incubation times from - d to - d after inoculation with murine m p i e pnons. in contrast, overexpression at the same level of l morp-a transgene mutated at codon (corresponding to codon in hurp) pmdnced spontaneous, fatal neurcdegeneration between and d of age in two lines of tg(mohp-pio l) mice designated and . genetic crosses of tg(moprp-p l) mice with gene targeted mice lacking both rp alleles ( p m -p ) produced anhats with a highly synchronous onset of illness between and days of age. the t g~o p r p -p l o l l ) ~~ mice had numerous prp plaques and widespread spongiform degeneration in contrast the tg and mice that exhibited spongifonn degeneration but only a few prp amyloid plaques. another line of mice designated tg overexpress the mutant transgene - -fold and develop fatal neurodegeneration behveen and d of age. tg mice exhibited the most severe spongiform degeneration and had numerous, large pip amyloid plaques. while mutant moprpccploll) clearly produces neurodegeneration, wtmoprpc profoundly modifies both the age of onset of illness md the mumpathology for a given level of transgene expression. our tidigs and those from other smdies suggest that mutant and wtprp interact, phaps through achaperone-like protein as noted above in sndies of tg(mhu m) mice, to modify the pathogenesis of the dominantly inhe&ed prion diseases. anton, heidi t. link, and jonathan w. yewdell, laboratory of viral diseases, niaid, bethesda, md - . cd ' lymphocytes (tcd +) play an important role in host immunity to viruses and other intracellular parasites. virus-specific tcdi+ recognize mhc class i molecules in association with peptides of to residues derived from viral proteins. this presentation will focus on how and where antigenic peptides are generated by cells. to begin to characterize the nature of proteases involved in the generation of antigenic peptides from cytosolic proteins, we used a panel of recombinant vaccinia viruses expressing different forms of influenza virus nucleoprotein (np). we found that the efficiency of generation of two np peptides is related to the metabolic stability of the source gene product. there has been considerable speculation that such short lived proteins are degraded by proteasomes in a ubiquitin-targeted process. our observations, however, call into question the importance of ubiquitin targeted-proteolysis in generating antigenic peptides from exogenously provided or endogenously synthesized viral proteins. we also examined the extent to which antigenic peptides can be generated in the endoplasmic reticulum (er). we found that antigenic peptides could be produced from short precursors ( residues) hut not from a number of full length proteins (influenza virus hemagglutinin, np, ovalbumin) that are targeted to the er by a nh -terminal signal sequence. peptides were generated much more efficiently from the cooh-terminus of the residue precursor than from the nh -terminus. these findings indicate that the er has a much more limited capacity than the cytosol to generate antigenic peptides, but that er proteases (particularly aminopeptidases) could perform the final proteolytic steps in the generation of class i binding peptides from precursors imported from the cytosol by tap, the mhc encoded peptide transporter. potential advantages of synthetic peptide or engineered recombinant vaccines are that they can be limited to contain only the specific antigenic determinants for desired responses without other determinants that elicit unwanted responses, and that the sequences of the determinants themselves can be modified to enhance potency or breadth of crossreactivity. however, they can have the disadvantage that any single determinant may be presented by only a limited selection of major histocompatibility complex (mhc) molecules of the species. to overcome the problem of mhc polymorphism, we have identified determinants presented by multiple mhc molecules, and have also located multideterminant regions of the hiv- envelope protein that contain overlapping determinants each presented by different class i mhc molecules, so that the whole multideterminant region is presented by multiple mhc molecules of both mouse and human. we have made use of "cluster peptides" spanning these multideterminant regions of the hiv- envelope to provide help for neutralizing antibody (ab) and cd + cytotoxic t lymphocyte (ctl) responses to peptides attached to these helper regions. these synthetic peptide vaccine constructs containing the p peptide from the v loop of hiv- iiib or mn, elicited both neutralizing ab and ctl in multiple strains of mice. the cluster peptides inducing helper t cells were essential for elicitation of ab and ctl to the p segment of both iiib and mn strains of hiv- in mice of several mhc haplotypes. several adjuvants were compared for their ability to elicit both ctl and ab simultaneously, without one response inhibiting the other. a single formulation in incomplete freund's adjuvant (ifa) could elicit all responses, neutralizing ab, ctl, and th helper cells. the ctl specific for the mn strain p peptide crossreacted with strains sc, sf , , and cdc . the peptides in f a also elicit high titers of antibodies in rabbits. boosting was found to enhance ctl responses as well as ab responses. these constructs are being prepared for a human immunotherapy trial. these vaccine constructs are potent and also avoid sites on gp that are known to elicit enhancing antibodies or autoimmune responses that might conmbute to disease pathogenesis. however, we can potentially improve on these by tinkering with the internal structure of the individual epitopes. we have found that replacing a negatively charged glutamic acid residue with an uncharged amino acid in one of the helper determinants makes it to -fold more potent in binding to the class i mhc molecule and in eliciting murine helper t cells that still recognize the natural hiv- sequence. thus, such a modified peptide should be more potent as a vaccine, while retaining the ability to elicit t cells that will respond to hiv proteins that of course do not have the altered sequence. we are currently mapping the critical residues for presentation of one of these peptides by human hla-a , with the intent of developing modified peptides that will be more potent as components of a human vaccine. thus, by leaming how these peptides bind to mhc molecules and tcell receptors, we can design internally modified determinants to construct more potent or more crossreactive second generation vaccines. we are testing these vaccine approaches in a mouse model in which mice can be protected against tumor cells expressing hiv proteins as would an hivinfected cell. dna vaccines, comprised of non-replicating plasmids encoding viral proteins, are capable of generating protective immunity in animal models of several viral diseases. in preclinical models of influenza infection, reduced viral shedding was observed in dna-vaccinated ferrets after challenge with the human clinical virus strain, a/georgia/ . cross-strain protection was conferred by dna encoding the major internal proteins (nucleoprotein, np, and matrix, m ) and the surface protein haemagglutinin (ha) from the antigenically-distinct previous virus strains, a/beijing/ and a/hawaii/ . this protective efficacy was greater than that seen by immunization with the widely-used clinical vaccine composed of killed a/beijing/ virus. thus, compared to a killed virus vaccine, protection seen with the dna vacane against a drifted virus strain was greater. we previously demonstrated that immunization of mice with np dna generated mhc class i-restricted cytotoxic t lymphocytes. mice likewise were protected from death and morbidity following cross-strain challenge'. ha dna vaccines generated neutralizing antibodies in mice, ferrets and primates, and provided protection in m i d and ferret models of influenza. in animal models of other viral diseases, immune responses and protection against viral challenge have been seen after immunization with dna encoding viral proteins. dna encoding hiv gp generated ctl and neutralizing antibodies in monkeys. antigen-specific proliferative responses and, in mice, secretion of high levels of yifn relative to levels of il- , months after immunization were also observed . immunization of rabbits with dna encoding l , the major viral capsid protein of cotton tail rabbit papilloma virus (crpv), resulted in neutralizing antibodies and protected against the development of warts after inoculation with crpv. mice immunized with dna encoding the glycoprotein gd from herpes simplex virus type (hsv- ), developed neutralizing antibodies and were protected from death when subsequently challenged with hsv- . dna vaccines were protective in animal models of various viral diseases. neutralizing antibodies, helper t cells (thl) and cytotoxic t cells were generated. cross-strain protection due to cellular immunity was demonstrated. ' science, - , dna cell biol, the profile of a neurovirulent virus is determined by its mechanism of entry into the cns (neuroinvasion), the type of cns cell in which it replicates (neurotropism) and its ability to cause pathologic effects in the brain (neurovimlence). whereas neuroectodermal cells, especially neurons, are the target cells of most neurovirulent viruses, the main target cell in the brain for siv and other lentiviruses is the macrophage. infection in, expression of viral antigens by and products of siv replication exported from these cells result in inflammation and degenerative changes in the brain and concomitant loss of neurons. siv strains that are mainly t-cell tropic cause transient activation of t-cells and during this period, infected t-cells cross the blood brain barrier and localize in the brain causing persistent hut minimally productive infection and minimal neuro pathologic effects. viral proteins but not virions are produced continuously. by virtue of the tropism of the virus for cd t cells, many infected animals eventually become immunosuppressed and develop aids, but not classical ueurological disease. viruses which are macrophage tropic invade the brain presumably also in t lymphocytes and the viruses infect macrophages in the brain. however, productive virus replication is minimized by antiviral cd t cells which suppress (kill?) all virus producing cells throughout the body, including the cns. productive virus replication in brain macrophages and accompanying inflammatory changes develop only when cd cells fail i.e. after profound immunosuppression sets in. the neurological disease that results from productive virus replication in macrophages in the brain therefore depends on presence of an appropriate macrophage-tropic viral phenotype invading the neuropil and development of immunosuppression in the host. the neurological disease could therefore be defined as one of the aids syndromes. the adenovirus (ad) early transcription region (e ) codes for more than polypeptides, four of which have already been shown to alter the immune response to ad infection. the amount of the class i major histocompatibility complex (mhc) on the plasma membrane can be reduced by the binding of the ad e gpl k protein to the mhc heavy chain, which prevents transport of the complex out of the endoplasmic reticulum. this process interferes with presentation of viral peptides to cytotoxic t lymphocytes. cytolysis by tumor necrosis factor-o (tnf) is inhibited by distinct viral polypeptides, of which (the ad e . k or the complex of the . k and . k proteins) are coded in the e region. the e polypeptides are translated from a family of viral mrnas, that are synthesized from a single viral promoter and processed by alternative splicing. we have studied the functions of the e polypeptides in several murine models. the goals of these experiments were to determine the effects of the ad e polypeptides in acute and persistent viral infections as well as in a transplantation model designed to measure whether these viral immunoregulatory proteins would abrogate allogeneic graft rejection. in a vaccinia virus (v.v.) pneumonia model, in which the isolated ad e . k or ad e gpl k genes were inserted into the v.v. pathogen, the ad anti tnf polypeptide increased viral virulence but the ad anti mhc had no effects. in addition to manipulating the ad e genes in viral constructs, several transgenic mouse lines containing the ad e genes have been constructed for these experiments. the e genomic dna behind the rat insulin promoter (rip) has been used to generate transgenic animals. islets from rip-e transgenic animals (h- b'd) have been transplanted allogeneically to h- d recipients and remained viable, secreting insulin until the end of the experiment at days; in contrast, control nontransgenic islets of the same genotype were rejected by - days. the e genes behind the native e promoter have been inserted into mouse embryos to generate transgenic animals, and the expression of the transgene monitored in multiple organs. the e promoter of the transgene is responsive to stimulation by the ad e a following infection with an e minus ad and can also be upregulated by administration of bacterial lipopolysaccharide. the effects of this transgene on ad pathogenesis are currently being studied. thus, these viral immunoregulatory genes have been shown to alter viral pathogenicity during acute infection and to downregulate the host immune response sufficiently to permit islet cell transplantation. these results on manipulating the ad e genes for the control of the host immune response also have implications for designing adenovirus vectors for gene therapy. "emerging" infections can be defined as infectious diseases that either have newly appeared in the population, or that are rapidly increasing their incidence or expanding their geographic range. recent viral examples include aids, ebola, and hantavirus pulmonary syndrome (fnst identified in a outbreak in the southwestern u.s.). emerging viral infections show a number of common features. most "new" viruses derive from existing viruses that move into new areas or acquire new hosts ("viral traffic"). many are zoonotic (originating from animal sources) (even pandemic influenza appears usually to be a reassortant originating in wildfowl). ecological or environmental changes (either natural, or, often, man-made) may precipitate emergence of new diseases by placing people in contact with a previously unfamiliar zoonotic reservoir or by increasing the density of a mtud host or vector of a pathogen, increasing the chances of human exposure. upon introduction into a human population from a zoonotic reservoir, the newly introduced virus may cause localized outbreaks of disease. some may show rapid variation and evolution upon introduction, and some evidence suggests a role for immune selection in this process. a few viruses (such as hiv) may succeed in establishing themselves and disseminating in the human population, becoming truly "human" infections. human activities can also play an important role in establishment and dissemination. migrations from rural areas to cities, now an accelerating worldwide phenomenon, or other displacements, can introduce remote viruses to a larger population; the virus may then spread along highways and (globally) by air travel. the development of an effective system of surveillance and rapid response is essential, but resources for this are presently inadequate. vaccine development, production, and deployment problems also need to be addressed. immunopathology may be a key feature of many of these infections, a number of which manifest as hemorrhagic fevers. many of the life threatening complications are due to increased vascular permeability. the resemblances to septic shock suggest that cytokines (such as tnf) are likely to be important in the pathogenesis of these infections. the response of cells, such as the macrophage, that induce or synthesize key cytokines, may be an important element, and the ability to infect these cells may be one common denominator. why some viruses elicit this response, while other closely related viruses do not, cannot yet be predicted from molecular data. better understanding of these aspects of the immune response should lead to additional therapeutic strategies. (supported by nih grant roi rr .) genetic approaches have been used to detect and characterize numerous previously unidentified hantaviruses. puumalarrospect hilvsin nombre-like viruses or virus variants are present throughout north and south america, europe and russia. several of the american viruses identified are associated with the newly recognized hantavirus pulmonary syndrome (hps), a severe respiratory illness with high mortality. the genetic relationships of these and previously characterized hantaviruses have been studied by phylogenetic analysis of the nucleotide sequence differences located in pcr bgments amplified from the g encoding region of the virus m segments. the relationships observed are consistent with a long-term association of viruses with their primary rodent reservoirs and suggestive of coevolution of host and virus. a sin nombre virus isolate is now available and its genetic characterization has been completed. various virus antigens have been expressed and are being used to probe the interaction of the virus with the host immune system. hantaviruses cause significant morbidity and mortality throughout the world. more than , cases of hemorrhagic fever with renal syndrome (hfrs) are reported annually in asia, europe and scandinavia. the etiologic agents of hfrs are hantaan, seoul and puumala viruses, with hantaan virus causing the most severe form of the disease. in , a new hantavirus was discovered in the united states (initially termed four comers virus), and was identified as the etiologic agent of hantavirus pulmonary syndrome (hi's). vaccines for hantaviruses are not readily available, although a number of inactivated viral preparations have been made and tested in asia. recurrent problems with inactivated hantaviral vaccines have been lot to lot variability, the need for repeated immunizations, and their inability to elicit long-lasting neutralizing antibody responses in immunized volunteers. because of such limitations on traditional vaccine development for these viruses, as well as the viruses' hazardous nature and slow, low-titer replication in cell culture, we used a recombinant dna approach to develop a vaccine for i-ifrs. our vaccine is a recombinant vaccinia virus expressing the m segment of hantaan virus under control of the vaccinia virus . k promoter and the s segment under control of the k promoter. the m segment, which encodes the g and g envelope proteins, was included because of our findings that: ( ) immunization with vaccinia or baculovirus-expressed g and g induced a neutralizing and protective immune response in hamsters; and, ( ) neutralizing antibodies to g or g could passively protect hamsters from challenge with virulent virus. the s segment, which encodes the nucleocapsid protein (n), was included because of our finding that hamsters immunized with baculovirus-expressed n also were protected from subsequent infection. although the protective immune response to n is probably cell-mediated, the importance of such a response is presently not well defined. assessment of our vaccine in preclinical studies, indicated that immunized hamsters developed neutralizing antibodies and were protected from displaying viral antigen in their lungs after challenge. in a phase i, dose escalation, clinical study, the vaccine induced neutralizing antibodies in individuals immunized subcutaneously with approximately lo pfu of the recombinant virus. in addition to humoral responses, immunized volunteers developed a cell-mediated immune response as indicated in lymphocyte proliferation assays. larger clinical studies, including alternate routes or booster immunizations, are planned. based on these studies, we anticipate that the vaccine will be efficacious for preventing hfrs caused by hantaan and the antigenically closely related seoul virus. we are studying the cross-protective properties of this vaccine with more distantly related hantaviruses such as puumala virus. although we expect this vaccine to be safe as well as effective, we also are investigating the use of more attenuated pox-viruses as vaccine vectors. infection of mice with lymphocytic choriomenigitis virus (lcmv) results in a profound expansion in the number of spleen cd t cells and in the induction of virus-specific ctl activity. thereafter, the cd t cell number declines, and the ctl activity diminishes, though the frequency of lcmvspecific precursor ctl per cd cell, as assessed by limiting dilution assays (lda), is remarkably stable throughout long-term immunity. the decline in t cell and total spleen leukocyte number at the late stages of acute infection is associated with high levels of apoptosis, as detected by the in situ nucleotidyl transferase assay. apoptosis occurred in both the t cell and b cell populations, with the b cells dying in clusters. this apoptosis was also seen in tfansgenic mice ectopically expressing bcl- in the t and b cells and in c bl/ ipr/@r mice, which have a mutation in the fas gene. t cells from the infected animal underwent apoptosis in vitro when stimulated through the tcr with anti-cd , thereby explaining some of the immunosuppression seen during acute viral infections. memory cells persisted for over a year and could be found in blast-size cell populations. challenge of lcmv-immune mice with either pichinde virus, vaccinia virus, or murine cytomegalovirus led to the reactivation of the lcmv-specific ctl response. lda analyses showed unexpectedly that these heterologous viruses crossreacted with subpopulations of lcmv-specific memory t cells. this memory t cell response to virus from an earlier infection was associated with enhanced immunopathology and enhanced clearance of virus during a heterologous virus challenge. over the course of the acute infection, ctl specific for the second virus were preferentially expanded over the crossreactive ctl, and after the acute infection, when the t cell response had subsided, ctl memory to the first infection had decreased. there is therefore a network of memory t cells which contribute to and are modulated by infections with putatively unrelated viruses, and apoptosis plays a homeostatic role in the course of these t cell responses. immune responses to live attenuated retroviral vaccines, r. paul johnson*?, cara wilsont, kelledy mansons, michael wyands, bruce walker?, ronald c. desrosiers* *new england regional primate research center, southborough, ma thfectious disease unit, massachusetts general hospital, boston, ma §tsi/mason, worcester, ma immunization of rhesus macaques with live attenuated retroviruses deleted in nef can induce protective immunity against challenge with pathogenic siv. development of protective immunity in these vaccinated animals occurs only after several months of infection, with maximal protection observed after one year. the specific immune responses responsible for mediating protection have not been defined, and little is known about the cellular immune responses in animals vaccinated with these live attenuated retroviruses. we have analyzed cellular and humoral immune responses in rhesus macaques and chimpanzees infected with live attenuated retroviruses. siv-specific neutralizing antibodies were present in vaccinated animals, but did not clearly correlate with protection against challenge. ctl specific for envelope and gag were identified in vaccinated macaques studied or more months after vaccination. quantitation of siv-specific ctl activity in one of these animals using limiting dilution analysis revealed a relatively high precursor frequency of cytotoxic t lymphocytes, up to moo for gag and / for envelope. cd + lymphocytes obtained from vaccinated macaques were also able to suppress siv replication in autologous cd + cells. suppression mediated by unstimulated cd autologous cells was maximal when cells were in direct contact with siv-infected lymphocytes, but cd + cells activated by an anti-cd -specific monoclonal antibody were able to release. a potent soluble inhibitor of siv replication. in contrast to the relatively vigorous ctl response present in vaccinated macaques, we were not able to detect consistent ctl activity in chimpanzees infected with a hiv- molecular clone (nl ) or attenuated viruses at periods up to one year after infection, despite the use of a variety of stimulation techniques. proliferative responses to hiv p and gp were observed in chimpanzees infected with n u and attenuated variants. although the relative contribution of these immune responses to protective immunity is not known, the relative vigor of the cellular immune responses observed in vaccinated macaques suggest they may play a role in mediating resistance to challenge. obiectives: to analyze the magnitude and specificity of the ctl response to hiv- , and to determine the tcr usage by clonal ctl responses in infected persons, including persons with documented infection of up to years with cd cells > /mm . methods: hiv-l-specific ctl activity was evaluated in pbmc as well as in pbmc stimulated in vitro with hn- infected autologous cd cells, using target cells infected with recombinant vaccinia viruses expressing hn- proteins. ctl epitopes recognized by these individuals were determined using cloned effector cells. quantitative cultures were performed by endpoint dilution, and viral quantitation was determined by qc-pcr tcr analysis was performed by pcr, using both family-specific primers and anchored pcr, followed by sequencing. sequence analysis of ctl epitopes in autologous viruses was determined by pcr amplification and sequencing. clonal frequency was analyzed in pbmc by oligonucleotide probe to the cdr region of the tcr. studies performed in long-term non-progressing persons indicate the presence of a vigorous and broadly directed ctl response. detailed epitope mapping in a person infected for years, who by qc-pcr had <i viral rna molecules per ml and who tested negative by bdna assay, revealed ctl clones directed at six different epitopes, with detectable recognition of three of these epitopes using freshly isolated pbmc as effector cells. the ctl clones were broadly reactive against many hown hiv- variants, and both the p and p epitopes were cross reactive with siv. sequence analysis of autologous virus within the three dominant ctl epitopes revealed the lack of immune escape variants in pbmc, plasma, and virus obtained from coculhue. supernatant. analysis of tcr usage by clones to defined epitopes in persons at different disease stages revealed heterogeneity in tcr usage and heterogeneity in recognition of virus variants withii these epitopes. analysis of clonal frequency using an oligonucleotide probe to the cdr region of the tcr in an individual with a vigorous response to gp suggested that approximately % of circulating t cells were a single hiv-l-specific clone. conclusions: our results indicate that cytotoxic t lymphocytes are part of the immune response in persistent non-progressive hiv- infection, and that prolonged infection can occur without the development of viral immune escape variation within defined ctl epitopes. in addition, vigorous circulating ctl responses can occur in the setting of an e x m e l y low viral load. heterogeneity in the ability of clones from different individuals to recognize sequence variation within ctl epitopes may be important in the pathogenesis of infection. ctl to conserved epitopes or ctl which are broadly reactive with multiple viral mains may be important in conmbuting to maintenance of the asymptomatic state. factor-i (irf- ) gene is essential for the transcriptional activation of inducible nitric oxide synthase (nos) in murine macrophages. it also plays an important role in the activation of interferon and il- and il- receptor genes. to investigate the role of irf- related systems of defense against viral myocarditis, the transgenic mice were innoculated with l o pfu of coxsackie virus (cvb ). homozygous irf- (+) and heterozygous irf- (+/-) mice were randomized into groups to determine heart and spleen viral titres, cardiac pathology and mortality rates at early and late stages of the disease. the mortality was dramatic in the irf- -imice. the hearts of the homozygous animals also had increased mononuclear cell infiltration, necrosis and viral replication. we condude that irf- regulated gene products such as inos and ifn are essential for the early defense against cvb -induced myocarditis by attenuating viral replication and inflammatory responses. the flu season is a yeariy problem, especially in the elderly population. annual vaccination lacks broad protection and antibody titers induced in the elderly are frequently lower than those acheived by younger vaccinees. using the mouse model, we are investigating the differences in immune response between old and young mice to influenza vaccine and at the same time testing the ability of a potent adjuvant to boost the response in the the elderly. our studies show defects in the immune response of the elderly. antibody titers after immunization are lower in the elderly for both seronegative and seropositive mice. cmi studies show that helper t-cell response (proliferation) to in-vitro antigen stimulation is also decreased in the elderly. facs analysis of wbcs show the elderly mice have lower leukocyte and lymphocyte populations, lower cd and b-cell populations and a higher proportion of macrophages and cd cells than young mice. it was also noted that the young mice had very consistent wbc profiles while the elderly profiles had greater variability. serum cytokine studies show lower levels of il , il and il , but higher levels of il and ifng in the elderly as compared to the young mice. the addition of mf to the immunization increased the antibody titers of both naive and seropositive young and old mice. the helper t-cell response of the young mice was unaffected, while the response of the elderly group was increased. cytokine profiles show an increase in il , il and il levels for both young and old, while il and ifng levels went up for young animals but remained constant for the elderly mice. coxsackievirus (cvb ), a member of the picornavirus family, is a common cause of acute or chronic human myocarditis. however, whether cell damage results primarily from direct virusmediated effects or from immune-mediated destruction of the heart tissue during the infection remains unclear. we used a cardiovirulent variant of cvb which is able to infect several strains of mice. the infection results in a disease which strongly implicates immunopathogenetic mechanisms in myofiber necrosis. to characterize the role of the immune system in this disease we used various transgenic knockout (ko) mice with different immune defects. cvb is able to infect normal c bl/ mice and immunocompromised (cd ko and m ko) mice and causes a lethal disease after i.p. injection in a viral dose dependent manner. we found elevated ld os in immunocompromised mice, which also die slightly later than normal mice. this virus replicates in the hearts of all mice used with maximal titers - days pi.. large pathological changes were detectable only in heart tissue of cd ko mice, and were maximal at - weeks after infection. these results suggest the possibility that cd ' t cells may limit tissue damage by an as yet undefined mechanism; the roles of cd + and cd ' t cells in this disease are under investigation. research supported by a grant of the daad, germany. mice lacking p lck are resistant to coxsackievirus b induced heart disease. tamara martino, karen aitken, joseph penninger, jan nikhilanandhan, tak mak, fayez dawood, wen-hu wen, lily wee, martin petric, and peter liu, the toronto and princess margaret hospitals, university of toronto, canada. coxsackievirus (cvb ) causes severe heart disease in adult mice, and is a useful model for the human cardiac conditions of myocarditis and dilated cardiomyopathy. in this study, we have demonstrated that transgenic mice lacking the t-cell signalling molecule p kk are resistant to cvb induced heart disease. the virus replicates in the heart, and is cleared from the myocardium at a rate comparable to "normal" littermates, but there is only minimal mortality, or damage to the cardiac tissue. however, in ick-deficient mice depleted of natural killer (nk) cells prior to viral infection, there is increased cvb replication, and some infiltration by mononuclear cells in the myocardium. to further elucidate the role of the immune system in cvb induced heart disease, cytokine expression in the hearts of cvb infected normal, ick-deficient, and nk-cell depleted mice is under investigation. we conclude that t-cell activation is critical to produce substantive myofiber necrosis after cvb infection, that nk-cells play an fundamental role in protecting the mice from severe virus infection, and that ick transgenic mice serve as an important model for understandina the dathogenic mechanisms involved. we now show that cd + t cells from m-/-mice exhibit antigen specific release of serine esterase and interferon-gamma. further, as quantitated by flow cytometry, the early decrease in cd + t cells seen in normal mice days after lcmv infection, previously presumed to be due to the activity of cd + ctl, still occurs in m-/-mice. however, mice show an earlier rise in percentage and absolute numbers of cd + t cells by days after infection. cd + t cells from am-/mice rise to above baseline levels with kinetics paralleling the early rise in cd + t cells in normal mice after lcmv infection. cd + ciz activity is lower in m-/mice and have a later peak than cd + ctl from normal mice. these differences may explain the lower mortality and longer time course of immunopathologic meningitis in -mice. mice can assume some of the cytotoxic functions of the cd + cell population from normal mice, including immune-mediated pathology. further, cd + t cells from m-/-mice have the capacity to release serine esterase, and show kinetics similar to cd + t cells from normal mice. mice genetically engineered to be deficient in -microglobulin in conclusion, these data suggest that cd + t cells from am-/- australia. molecule is an essential component of the t cell help required for an antibody response. the interaction between cd and its ligand, in the presence of b cell acting cytokines, such as interleukin (il- ), is sufficient to trigger b cells to proliferate and differentiate and to induce immunoglobulin class switching. a recombinant vaccinia virus encoding both factors, cd l and il- , did not, however, stimulate an enhanced antibody response in mice infected with the construct. instead, the antibody levels were lower than those of mice infected with a control virus. the reduced antibody response reflected the diminished growth of cd ol-expressing viruses, to the extent that immunocompromised mice were able to resolve these infections. the kinetics of virus clearance indicate that the anti-viral mechanism of cwol is rapidly activated and has generalized tissue distribution. the cwolmediated clearance of virus is independent of the anti-viral cytokines, tnf and ifn-y. the anti-viral activity of c w l may represent a surprising and potent effector mechanism of t cells activated during a virus infection. mary's medical school, norfolk place, london w lpg, u. k. and *nationallnstirute formedica research, london, nw aa. respiratory syncytial (rs) virus is the most important respiratory pathogen of infants, causing the majority of cases of bronchiolitis, it can be life threatening in very young infants and those with underlying cardiopulmonary disease or immunodeficiencies. t cell immunodeficiency can lead to prolonged infection in children and t cell transfers clear virus in the murine disease model. mice can be readily infected with rs virus and have proved a valuable model of its pulmonary pathology and the t cell response to it. il- has a broad spectrum of activities on proliferation and terminal differentiation of early blast cells and committed progenitors of many lineages. less is known about the effects of il- on t cell development and proliferation and its effect on the host response to infection by common pathogens. the il- gene was disrupted in /sv embryonic stem (es) cells by homologous recombination after transfection with omega-type constructs flanked with a herpes simplex thymidine kinase gene. these cells were used to produce a mouse strain deficient of il- . mice were infected intranasally with a strain rs virus; pathological changes in the lung were monitored by cytology of bronchial lavage, and virus persistence by nested rt-pcr of lavage fluid and lung homogenate. no differences were found in pathology or virus persistence between knockouts and normal mice. il- deficiency is therefore compatible with apparently normal responses to viral infection. recombinant adenoviruses are an atuactive vehicle for gene therapy to lung in the treatment of cystic fibrosis (cf). first generation viruses deleted of ela and elb transduce genes into airway epithelial cells in vivo, however, expression of the transgene is transient and associated with substantial inflammatory responses, and gene transfer is significantly reduced following a second administration of the virus. in this study, we have used mice deficient in immunological effector functions in combination with adoptive and passive nansfer techniques to define antigen-specific cellular and humoral immune responses that underlie these important limitations. our studies indicate that mhc class i resmcted cd + cytotoxic t lymphocytes (ctls) are activated in response to viral antigens leading to destruction of virus infected cells and loss of transgene expression. mhc class ii associated presentation of viral antigens activates cd +t helper (th) cells of the - subset and, to a lesser extent, of the tm subset. ldv establishes a life long viremic infection in mice which is not controlled by anti-ldv immune responses. anti-ldv antibodies are rapidly generated but they neutralize ldv infectivity only poorly. here we show that ldv-specific cytotoxic t cells (ctls)are generated within days pi. c t l s were detected by h- specific lysis of ldv-specific target cells. these target cells were generated by transfection of t -nm cells with a retrovirus vector carrying the gene (ow ) for the ldv nucleocapsid (n) protein. spleen cells from ldv-infected mice were incubated in vitro with ldv-infected macrophages or transfected t -nih cells and cultured for days in the presence of il- . the ctls had largely disappeared in mice by days pi. the following experiments were conducted to further explore the mechanism of immune escape. in situ hybridization was used to localize ldvinfected cells in tissues of infected mice. these were found in persistently infected mice only in the liver. testis, and lymphoidal tissues. in addition large amounts of virion or virion debris accumulated in the germinal centers of the spleen and lymph nodes beginning about days p.i. the accumulation of large amounts of ldv antigen in the germinal centers may be causally related to the suppression of the ctl response as well as to the polyclonal activation of b cells associated with ldv infection. to determine whether immune selection plays a role in ldv immune escape, ldv was reisolated from nude and immunocompetent balb/c mice at various times pi. the ows for the nand the two envelope glycoproteins were r t k r amplified and sequenced. the role of cytokines in initiating the immune response to venezuelan equine encephalitis (vee) was investigated. mice were infected in the left rear foot pad with either cloned virulent vee (v ) or an isogenic single-site attenuated vee mutant (v ) (single amino acid change at e glycoprotein position from glu-lys) and at , , , , , and hours post-infection, the popliteal lymph nodes and spleen were harvested and examined for tnf-a, il- , ifn-y, il- , il- , il- , and il- gene expression using a quantitative rt-pcr. in both strains elevations of tnf-a, il- , ifn-y, il- , and il- were detected in the lymph node, with no changes in either il- or il- ; however, whereas cytokine elevations were detected by hours after injection of v , elevations were delayed until hours following infection with v . cytokine mrna elevations in the spleen were less substantial, but elevated levels of ifn-y, il- , and il- were also observed. these findings suggest that vee infection induces an early highly specific cytokine pattern associated with simultaneous elevations in both ifn-y and il- and that a single amino acid mutation can induce temporal changes in this pattern without alterring either the actual cytokines induced or the degree of their elevation. we are currently performing intervention experiments to investigate the effect of intravenous anti-il- andor anti-ifn-alp antibody administration on this early in vivo cytokine response to examine in particular whether these cytokines may be required for the observed elevations in either ifn-y or il- . to confirm the importance of the immune system in clearing rotavirus infection we infected rag knockout mice with murine rotaviruses. both knockout p u p s and adults developed chronic infections. to determine if a t cell independent antibody could be mediating the clearance of rotavirus infection in nude mice we measured igg, igm and iga in faeces and serum of rotavirus infected mice. a low and transient igm response was found in serum but not faeces of infected nude mice but not in age matched naive nude mice. the protein specificity of these antibodies is being determined. to determine if a primary infection in n u d e mice would induce protection (memory) against a secondary infection, we challenged six week old mice that had been infected as pups with the murine ew strain of rotavirus with the murine cambridge strain of rotavirus. the previously infected mice, as well as naive nude mice, both shed very low but equal levels of rotavirus in the stools. interestingly adult naive heterozygous littermates shed more virus than the nudes. the factors responsible for this relative resistance of adult nude mice to rotavirus infection as well as the mechanism for viral clearence in nude mice will be discussed. symp. , : - ) . the great majority of the poxvirus encoded proteins actively mediating the evasion of host defense are secretory proteins termed "virokines". viruses produce proteins which can either compete with or antagonizz molecules critically involved in generating immune responses. the vaccinia virus complement control protein (vcp) was the first virokine to be reported that has structural similarity to humadmouse complement control proteins, with the greatest similarity to the human complement b binding protein (bc -bp) and the first protein to have a postulated role in the viral evasion of host defense (kotwal and moss, nature , : - ) . bioactive vcp, purified from serumfree medium has been shown to be more potent than the hc bbp ( kotwal, am. biotech. lab., ) and has also been shown to bind to two important complement components, c and c , and block the complement cascade at multiple sites in vitro (kotwal et al., science , - ; mckenzie, kotwal et al., j. inf. dis. , - . the importance of this protein was demonstrated in vivo using recombinant virus lacking an intact dna coding for vcp (isaacs, kotwal and moss, pnas , : - ) and further underscored by the smallpox virus genomic sequence analysis, indicating the presence of a highly conserved homolog of vcp ( massung et al., nature , : - ) . in order to determine the precise effects of vcp at the site of infection, a mouse model has been developed. measurement of the specific swelling response and microscopic examination of the histological changes in balb/c and dbn mice has revealed that vcp is capable of regulating inflammatory response in vivo (jayaraman and kotwal, mol. immunol. : ) . in order to ensure that the possiblity of the unrelated mouse strains sharing identical h- haplotypes but differing at numerous other loci was not overlooked, we performed similar experiments in congenic strains. well characterized c deficient mice injected with poxviruses in comparison to normal mice, produce a significant inflammatory response, accompanied with severe ulceration that persists for several days. the data suggests that the host complement response is a critical factor in egulating poxvirus pathogenesis. previous studies have suggested that immune response was one of the major factors that contributed to the host resistantlsusceptible mechanism to sendai virus infection (brownstein, ) . various immune regulatory molecules were investigated in inbred susceptible /j ( ) mice and resistant c bl/ j (b ) mice which share the same mhc haplotype. when they were given eid, of sendai virus intranasally, mice showed histologically diffuse interstitial pneumonia compared to the local moderate inflammation in mice. virus was eliminated from the infected lung days earlier in b mice than in mice. the cytokine levels and cytokine-producing cells were investigated in the acute pneumonic lung. also the recall cytokine production in the draining mediastinal lymph node (mln) were studied. the maximal numbers of il- , ifn-y, il- , and il- producing cells were comparable for each strains of mice, while more il- producing cells were present in the lung of mice. however, the numbers of these cytokine producing cells peaked in mice days later than in b mice. analysis of soluble cytokines in bronchoalveolar lavage fluid showed that at least two-fold higher levels of ifn-y, il- , and il- were present in mice than in mice. recall cytokine production in the draining mln showed only the presence of il- , ifn-y, il- , and il- , while no il- was detected. generally, higher levels of these cytokines were detected throughout the whole infection process in mice. therefore, the susceptibility of mice to sendai virus is not due to insufficient cytokine production in these two anatomical sites, yet heavier and prolonged virus burden in susceptible mice may drive the more rigorous and protracted induction of immune regulatory molecules. cytokines play a decisive role in determining the type of immune response elicited by an antigen, both in driving th cell differentiation along thl or th pathway and, ultimately, the effector and regulatory activity of these subsets. we have studied the role of cytokines in virus infections by constructing recombinant virus vectors encoding cytokine genes. during replication in mice, these vectors produce the encoded factor which is secreted from the infected cells with profound immunological consequences. cytokines studied todate fall into two categories; either altering viral pathogenesis or selectively stimulating specific immune responses. the expression of il- , ifn-y, tnf-a or il- in recombinant vaccinia virus (rvv) dramatically alters pathogenicity, such that immunodeficient mice resolve the normally lethal infection. on the other hand, expression of il- by rvv suppresses the induction of cytotoxic t cells (ctl) inhibiting the clearance of virus which leads to an increase in viral pathogenicity. the expression of il- by rvv was shown to inhibit the host il- response required for the development of ctl's. no production was also significantly suppressed by il- . rvvs expressing il- or il- , although not affecting pathogenicity, preferentially stimulates iga reactivity to coexpressed antigens. encoding cytokines in recombinant viruses has clearly demonstrated the important role of cytokines as anti-viral effector molecules and in regulating appropriate immune responses. in mice infected with lymphocytic choriomeningitis virus (lcmv), interleukin-i (il- ) doses of >i ngld induce profound immunotoxicities characterized as almost complete inhibition of virus-induced cd + t cell expansion and ctl activation, and up to log increases in viral replication [orange, wolf, and biron, j. immunol. : , . serum tumor necrosis factor (tnf) is also observed under these conditions. the studies reported here further characterize the expression and function of tnf in this context. northern blot and in sifu hybridization analyses demonstrated that il- induced tnf-cx expression and that lcmv infection synergized with il- for this induction. administration of antibodies neutralizing tnf reversed the il- -induced immunotoxicities in lcmv-infected mice and restored anti-viral defenses. the tnf-mediated immunotoxicities appeared to result from an induced cellular sensitivity to the factor, as splenic leukocytes and cd + t cells isolated from lcmv-infected mice were more sensitive to tnfmediated cytotoxicity in culture than were equivalent populations prepared from uninfected mice. additional physiological changes were observed in il- -treated uninfected mice and were dramatically elevated in il- -treated virus-infected mice, including: ) decreases in body weights; ) elevation of circulating glucocorticoid levels: and ) decreases in thymic mass. these changes were also reversed by anti-tnf. the results delineate a unique tnf-mediated immunotoxicity and have significant implications concerning detrimental consequences of in vivo tnf andlor il- for protective anti-viral responses. lactate dehydrogenase-elevating virus (ldv), a naturally occurring virus, causes a persistent infection in mice and presents an ideal model for the study of immune modulation during acute and persistent virus infections. within a few days following infection with ldv there is a pronounced polyclonal activation of b cells followed by the suppression of primary b cell responses to t-dependent ag. we investigated the effect of acute and persistent ldv infection on the development of a memory b cell response to the model protein antigen, horse cytochrome c (cyt), by employing a modification of the splenic fragment assay. about a % decrease in the frequency of responding agspecific memory b cells was observed in balb/c mice infected with ldv, whether the mice were immunized with cyt at the time of ldv infection or three weeks later. this may be due in part to a defect in t cell help, since in cultures of normal memory b cells and t cells derived from ldv acutelyinfected mice the frequency of responding b cells was also decreased two-fold. in situ hybridization using a cdna probe specific for ldv revealed two patterns of ldv rna within the spleen. twenty-four hr p.i. ldv rna was located within the marginal zone, surrounding each follicle. this pattern is consistent with permissive macrophages. during persistence viral rna could no longer be detected in the marginal zone, but was located within the follicles. the absence of ldv-permissive cells within the follicular region suggests that the source of ldv rna is not due to ongoing viral replication. one possibility is that circulating virus is trapped by a specific cell population within the follicle. the effect of virus trapping within the spleen provides a mechanism by which ldv and other viruses can modulate immune cell function during persistent infections. ifn-y can be produced by activated nk cells. this cytokine enhances immune responses by augmenting macrophage antigen presentation. viral infection induces ifn-dp and nk cell activation. changes in splenic architecture, cell trafficking, and cytokine expression were examined during viral infections of c bu mice. at times coinciding with ifn-dp production and nk cell activation, there was a redistribution of nucleated cells from red pulp to white pulp regions in spleens isolated from mice infected with either lymphocytic choriomeningitis virus (lcmv) or murine cytomegalovirus (mcmv). cell transfer experiments with dioctadecyl- , , ', '-tetramethyl indocarbocyanine perchlorate-or pkh -gl-labeled bone marrow cells isolated from normal mice demonstrated an infection-induced accumulation of non-t/non-b cell populations along recipient splenic marginal zones. flow cytometric analyses demonstrated that approximately % of the transferred bone marrow cells accumulated in spleens after hrs and % of these expressed the nk cell marker, nkl.l+. in vivo antibody treatment procedures, to eliminate cell subsets in donor mice, demonstrated that the cells localizing at the marginal zone were derived from agmi+ and n k l . l + populations. a small subpopulation of marginal zone cells in infected mice were shown to be expressing high levels of ifn- mrna by in sifu hybridization. treatment with anti-agmi or anti-nk .i antibodies eliminated both endogenous nk cells and the ifn-y mrna positive cells. these data demonstrate that newly derived nk cells accumulate along marginal zones. the results also suggest that this trafficking pattern may act to enhance immune responses by facilitating delivery of cytokines to specialized antigen presenting cells. david segal, janet ruby, alistair ramsay and ian ramshaw. depamnent of cell biology, john curtin school of medical research, po box , canberra, act, australia cytokine expression has been shown to correlate with protective or ineffective immune responses in a number of disease models. recently there has been the suggestion that immunity to some retroviruses is associated with the production of ceaain patterns of cytokines. to explore this further we have have used rauscher murine leukemia virus (r-mulv) infection of c bu (resistant) and balb/c (susceptible) mice to elucidate the role of cytokines immunity to retroviruses. initially the in viho proliferation of spleen and lymph node cells from infected mice was examined. in response to stimulation with immohilised anti-cd antibodies the proliferation of spleen hut not lymph node cells from infected mice. was found to he rapidly suppressed. suceptible balb/c mice exhibited a much greater suppression than resistant c bu mice. the cause of this suppression is under investigation however, the immunosuppressive molecules nitric oxide and prostaglandins are not involved. in vitro cytokine production by spleen and lymph node cells from r-mulv infected mice was determined. in response to stimulation with immobilised anti-cd antibodies, spleen cells from infected balb/c mice produced diminishing amounts of ifn- and il- . in contrast spleen cells from infected c bu mice produced ifn-y and l- to levels that were only slightly less than uninfected controls. a- production by spleen cells from infected mice of both strains was at levels higher uninfected controls. anti-cd stimulated lymph node cells from infected mice produced elevated ifn- suggesting that suppressed cytokine production is spleen specific. expression of cytokine genes in vivo is currently being investigated using rt-pcr to detect cytokine mrna in the spleens of infected mice. we have previously shown that primary resting murine b lymphocytes are non-permissive for vesicular stomatitis virus (vsv), however, a productive infection can be induced when infected b cells are activated with anti-immunoglobulin (a-lg) plus il- or lipopolysaccharide (lps). we posit vsv in unactivated primary b cells provides a paradigm of persistently infected lymphocytes and activation dependent recall of an active infection. analysis of the behavior of virus in unstimulated b cells during long term culture and the requirements for subsequent induction of productive infection has been limited by the poor survival of primary cells in culture. we circumvented this limitation by using highly purified small b cells from mice transgenic for the bcl- proto-oncogene, expression markedly extends in vitro survival of unstimulated primary b cells. overexpression of bcl- does not alter b cell infection or induction of a productive infection by activators during acute infection. infection does not effect b cell survival in culture. unstimulated virus infected b cells produce primary viral mrnas but not viral proteins or infectious particles (pfu) during culture. persistently infected b cells stimulated with a-lg plus il- produced a fully productive vsv infection at all times analyzed, up to weeks post infection. in contrast, vsv production in persistently infected b cells activated with lps markedly declined relative to acutely infected activated cells ( - fold by week and , fold by week ). cells were not completely refractory to lps activation as vsv protein was produced. the selective lps deficiency is unique to persistently infected cells as uninfected cultured b cells proliferate and differentiate to produce antibody upon lps activation. these data show that a persistent infection may selectively alter the host cell response to previously productive activators which may as a consequence interfere with immune regulation. rsv-g glycoprotein specific t cells preferentially secrete il- and predispose to pulmonary eosinophillia., anon srikiatkhachorn. and thomas j. braciale, the beirne b. carter center for immunology research and the departments of microbiology, pathology, and pediatrics, university of virginia health sciences center, charlottesville, va we studied the immune responses to two different glycoproteins of respiratory syncytial virus (rsv) in a murine model. balb/c mice were immunized with recombinant vaccinia virus expressing either rsv-fusion glycoprotein ( vac-f), attachment glycoprotein (vac-g) or -galactosidase (as a control). these mice were given rsv intranasally three weeks after priming and then sacrificed or days later. spleens and bronchial lymph nodes were harvested for in vitro culture and lungs were harvested for histologic studies . we found that bulk cultures obtained from both vac-f and vac-g immunized animals secreted both thl and th type cytokines when stimulated with rsv infected spleen cells . however, the levels of - and fn-y were higher in bulk cultures derived from vac-g primed animals while the levels of il- were higher in the bulk culture from vac-f primed animals. the il- and il- production was relatively short lived since spleen cells and bronchial lymph node cells obtaind form mice sacrificed days after intranasal inoculation produced much lower levels of il- and - while the levels of il- and ifn-y production were comparable to bulk cultures obtained from mice at the peak of infection. there was little inflammatory response in the lungs obtained from mice immunized with the control vaccinia. in contrast , lungs from mice immunized with vac-f or vac-g showed significant infiltration of inflammatory cells. there was a striking infiltration of eosinophils in the lungs from mice primed with vac-g. these eosinophils could be detected aroud major bronchi and blood vessels, as well as, in some cases, in lung parenchyma. this study suggests that the immune responses to different viral glycoproteins may be distinct and may play important roles in viral pathogenesis. during infection of normal mice with lymphocytic choriomeningitis virus (lcmv), nk cell responses peak on day and subside as cd + t cell responses are activated at day post-infection. in contrast, m-/-mice, lacking cd + t cells, have dramatically elevated nk cell responses on day postinfection. the m-/-response is evidenced by increased nk cell activity, as well as up to -fold increases in blast and total nki.i+cd -cell numbers. nk cell responses in normal mice are cyclosporin a (csa)-resistant and interleukin (il)- independent, whereas day nk cell responses in m-/-mice are csa-sensitive and il- -dependent. to investigate the role of additional cytokines in regulating cellular responses during acute viral infections, production and function of il- and transforming growth factor (tgf- ) were examined. induction of il- mrna, at late times post-infection of normal mice, was shown by in situ hybridization of t cell-enriched splenic leukocytes and polymerase chain reaction (pcr) amplification of cdna from rna. ellsas of media cor.aitioned with cells isolated on days , , , , , and post-infection demonstrated delayed induction of il- protein as compared to ctl activation. tgf- , evaluated in biological and elisa assays, was induced maximally at days to post-infection. the kinetics of tgf- production by cells from infected m-/mice was similar to that of normal mice. however, cells from m-/-mice produced il- at early but not at late times postinfection. together, these results suggest that either il- is a critical cytokine for shutting off nk cells during normal responses to viral infection, or that the m-l context modulates responsiveness of nk cell subsets to other late cytokines. studies are in progress to distinguish between these two possible mechanisms. the induction of fever in response to infection is an important host defense mechanism that enhances aspects of the immune response and restricts the replication of some microorganism. vaccinia virus, a member of the poxvirus family, is a complex cytoplasmic dna virus that encodes a variety of proteins that interfere with host immune functions, such as complement regulatory factors and soluble receptors for il-lp, tnf and ifny. here we show that expression of the vaccinia virus il- p receptor (vil-lpr) in the w r strain prevents the febrile response and reduces the severity of infection in intranasally inoculated mice. fever was recorded on days - after infection of mice with a vil-lpr deletion mutant, but not in animals infected with wild type wr or a virus revertant. these studies were extended to other virus strains that were used as smallpox vaccines, and expression of the vil-lpr was consistently found to prevent the onset of fever. vaccinia virus induced a severe hypothermia after days in infected mice that was independent on vil-lpr expression and correlated with virus replication in the brain, the organ that controls body temperature. these results represent the first example of a virus mechanism to inhibit the host febrile response and suggest a central role for soluble il-lp in the induction of fever in poxvirus infections. measles virus (mv) infection can depress cell-mediated immune responses for months following clinical disease. mv is known to infect the thymus during human illness and this may contribute to immune suppression. we have used the scid-hu m o w with co-implants of human fetal thymus and liver to determine the effect of virulent and avirulent strains of mv on the thymus. scid-hu mice were. infected by direct inoculation of the graft with pfu of either a wild type strain of mv(chicago- ,chi- ) or an attenu-ated strain (moraten, mor) and sacrificed at intervals over days. peak viral titers, as judged by plaque assay on vero cells, were reached by chi- on d ( . pfu/ third of implant), and moron d ( . pfu/ third of implant). hematoxylideosin stained sections of chi- -infected thymuses showed marked distortion of the cortex and medulla by d with thymocyte poilolosis and decreased cellularity. by d , these. implants were mostly devoid of normal thymocytes. mor-infected thymuses showed relatively preserved architecture and cellularity. suspensions of the cells from implants stained with mabs to cd ,cd and cd were analyzed by flow cytomehy. there were significant decreases in the cd +cd + cell pop-ulation by d with complete loss of all such cells by d with chi- , and only modest reductions with mor. immune fluorescence staining of sections with a mv mab to hemagluttinin(ha) and abs for either human cytokera-tins(ael/ae ) or cd co-localized mv predominantly to epithelial and monocytic cells. additionally, mv antigen was present diffusely by d in both cortex and medulla in chi-i infection whereas mor-infected implants had only patchy distribution by d . only rare cells stained both with mv ha and cd or cd . mv ha was not expressed over background on any cd + cells judged by facs. we conclude that mv replicates in the scid-hu thymic implant primarily in epithelial and monocytic cells, and that the attenuated virus reproduces more slowly and with less cellular disruption. little mv ha could be demonstrated in thymocytes, therefore the data suggest that significant infection of the thymic epithelial stroma disrupts the thymic microenvironment which normally supports and aids in selection of immature t cells. part of the long-term immune suppression seen in mv infection may be due to infection of the thymic epithelial stroma with subsequent loss of thymocytes. it is becoming increasingly evident that many poxviruses contain genes that enable the virus to evade the host's immune system. myxoma virus is a leporipoxvirus and is the causative agent of myxomatosis, a rapidly lethal disease in the european rabbit (oryctolagus cuniculus). one possible mechanism of immune evasion is virus-induced downregulation of cell-surface receptors important for an immune response. cell-surface levels of several receptors on a rabbit t cell lymphoma cell line (rl- ) were monitored by flow cytometry. following infection with myxoma virus, cellsurface levels of cd were found to drop dramatically. other cell surface antigens such as cd , cd , and cd were unaffected during infection with myxoma virus. further more, the downregulation of cd by myxoma virus could be inhibited by treating cells for an extended period of time with pma, suggesting that the downregulation was not simply a masking of the epitope via viral antigens. analysis of cd levels in the presence of cytosine arabinoside indicates that late gene expression is not necessary for the modulation. since the tyrosine specific protein kinase p lck associates with the cytoplasmic domain of cd we have also examined the association of p lck with cd as well as steady state levels of p lck during viral infection. the modulation of surface cd has also been described in hiv infected t cells suggesting that the loss of cell-surface cd may be a common viral immune evasion tactic by lymphotrophic viruses. i n addition, stably-transfected cell l i n e s expressing e i t h e r u s o r us - gene products s i g n i f i c a n t l y reduced l e v e l s of mhc class i heavy chain. studies are i n progress t o f u r t h e r d e f i n e t h e mechanism by which t h e s e v i r a l gene products a l t e r immune recognition. cytotoxic t lymphocytes (ctl) may play a significant role in containing the spread of hiv in infected individuals. although hiv-infection is associated with immune suppression, a vigorous ctl response has been detected in infected adults. hiv can be transmitted from mother to child. one third of vertically infected children has a rapid evolution toward disease, with onset of aids before months. the other two thirds remain asymptomatic for years. the bimodal course of disease evolution in hiv-infected children could be related to differences in the host immune control of viral replication. hiv-specific ctl response from fresh and in vitro activated pbmc of hiv-infected children was measured. the vast majority of infected chidren had detectable hiv-specific ctl, which where cds+cd +. we previously showed that among children with a slow disease progression, fresh ctl were more frequent in the p a(paucisymptomatic) group than in the pl(asymptomatic) and the p b-f groups (symptomatic group). the cohort of children has now been followed during years, and children have been tested at least once. we found that ctl responses were less frequent in the children with a rapid disease progression than in the children with a slow disease progression at the same age. our data suggest that ctl response is an important factor in delaying disease evolution. we, as well as others. have proposed that sag function is critical to the ability of milk-borne m m n to infect mice. to determine whether this is the case, we created transgenic mice (hyb pro/cla) with a frameshift mutation int the sag gene. young hyb pro/cla mice (c weeks of age) showed no deletion of their cognate vp * t cells, unlike transgenic mice carrying a functional sag gene however, a slow, progressive loss was seen in the hyb prolcla mice as they aged, indicating that it was due to expression of wild type sag protein. thus, as the hyb pro/cla mice aged, there was production of virus that appeared to lose the cla mutation. the hyb pro/cla mice produced transgene rna in their lactating mammary gland and shed virus in their milk. their nontransgenic offspring of showed infection with transgene-encoded mmtv because they had the typical slow deletion of vp + t cells characteristic of c h mmtv infection and because we detected transgene-derived m m n rna in their mammary glands. cloning and sequencing of the viral rna produced by the nontransgenic offspring of the hyb pro/cla mice showed that recombination between the mtv- endogenous viral rna and the transgene-encoded rna occurred, such that the frameshift introduced by the cia mutation was repaired. these results show that there is selection of infectious virus that contains a functional sag gene. thus, it appears that the only virus that is capable of being transmitted by the milk borne infection pathway is that which encodes a functional sag protein. hepatitis b virus (hbv) causes acute and chronic liver diseases and is closely associated with hepatocellular carcinoma. in order to understand the cellular immune response against hbv in chronic hbv infection, t cell proliferation, cytotoxicity and cytokine production were studied. we found that although the majority of asymptomatic hbsag carriers and patients of chronic hepatitis b (chb) had no proliferative response to hbsag, some individuals in both groups showed significant t cell proliferation against hbsag. in contrast, the proliferative t cell response to hbcag in asyrnpatomatic hbsag carriers was significantly stronger than that in patients of chb with acute exacerbation. in addition, the frequency of hbcag-reactive t cell precursors measured by limiting dilution assay was much higher in asymptomatic hbsag carriers than in patients of chb. therefore, t cell responses against hbsag and hbcag are regulated differently in chronic hbv infection. furthermore, we demonstrated hbsag-and hbcag-specific cytotoxic t lymphocyte (ctl) activity in asymptomatic hbsag carriers, using autologous hbsag-and hbcag-expressing lymphoblastoid cell lines (lcl) as target cells, respectively. the cloned ctl were able to produce ifn-y, tnf-a or gm-csf after stimulation. these findings demonstrate that t cell response to hbv is not completely suppressed in asymptomatic hbsag carriers. most of them have strong hbcag-specific response and some of them have hbsag-specific response. transcription and tax the human t-lymphotropic virus type i (htlv-i) promoter contains the structural features of a typical rna polymerase i (pol ) template. the promoter contains a tata box bp upstream of the transcription initiation site, binding sites for several pol i transcription factors, and long poly a+ rna is synthesized from the integrated htlv-i proviral dna in vivo. consistent with these characteristics, htlv-i transcription activity was reconstituted in v i m using tbp, tfiia, rtfiib, rtfiie, rtfiif, tfiih and pol . in hela whole cell extracts, however, the htlv-i ltr also contains an overlapping transcription unit (otu). htlv-i otu transcription is initiated at the same nucleotide site as the rna isolated from the htlv-i-infected cell line, mt- , but was not inhibited by the presence of a-amanitin at concentrations which inhibited the adenovirus major late pol i promoter ( pglml). htlv-i transcription was inhibited when higher concentrations of a-amanitin were used ( pglml), in the range of a typical polymerase in (pol ) promoter (va-i). purified tax, transactivates this promoter -to -fold in v i m . interestingly, basal and tax,-transactivated transcriptional activity of the htlv-i ltr could be reconstituted with the . m phosphocellulose fraction. these observations suggest that the htlv-i ltr contains overlapping tax,responsive promoters, a typical pol i promoter and a unique pol i promoter which requires a distinct set of transcription factors. tax, further in vifro transactivates a polymerase i template containing the base pair repeats cloned upstream of the ovalbumin promoter and g-free cassette. tax,-transactivated transcription was concentration dependent and inhibited by low concentrations of a-amanitin. flaviviruses are arthropod-borne viruses whose route of infection is via the skin. they are mostly neurotropic and responsible for significant human morbidity and mortality. the classic cell-mediated immune response to a viral infection may be influenced by the ability of these viruses to modify expression of cell-surface molecules involved in the presentation of antigen to, and activation of, t cells. the skin langerhans cell is the prototypic nonlymphoid dendritic cell and as such is uniquely placed to participate in a response against epidermally-acquired viral infections. the migratory properties of these cells contribute to their role as initiators of t cell-mediated immune responses within the draining lymph node. we have previously shown infection of epidermal cells in vifro by the flavivirus west nile (wnv) results in an increase in mhc class i and i expression on the majority of epidermal cells and langerhans cells respectively. in this study a technique for infecting the epidermis with wnv in vivo was developed. tme-dependent increases in the surface expression of a number of antigens which are involved either directly or in a co-stimulatory capacity in initiating a cell-mediated immune response, were detected on both the majority of epidermal cells and the langerhans cell population using flow cytometry. these increases were detectable as early as hours after infection. a significant decrease in the percentage of langerhans cells remaining in the epidermis was observed within hours of infection. the phenotypic changes observed in vivo are analogous to those described following in vifro culture of langerhans cells. these results, together with the reduction in langerhans cell numbers, may represent the in situ maturation and concomitant migration of these. cells as a consequence of virus-induced cytokines within the skin microenvironment. which cause a wide variety of illnesses with high morbidity and mortality in humans throughout the world. their high genomic stability argues for a survival strategy related more to interaction with the vertebrate host immune response, than a dependence on viral genetic mutation. our previous work has shown that west nile virus (wnv) infection of many cell types directly induces functional increases in class i and mhc expression. we report here that wnv infection of human embryonic fibroblasts (hef) results in the increased expression of cd by two distinct mechanisms. an early, direct cytokine-independent mechanism operates within h of virus infection, while an indirect mechanism, regulated by type interferon (ifn), operates within h of virus infection. cd expression increased by - fold within h of wnv infection on hef, and by - -fold within h. wnv-inactivated, conditioned supematants removed from infected hef cultures after h incubation did not alter cd expression on unqimulated hef. whereas conditioned supernatants from h-infected cultures increased cd expression by about . - -fold after incubation for h, but not after h, similar to cd induction by ulml of ifn-p. increased cd expression on hef by wnv was also cell-cycle dependent. cd increased only in quiescent, contact-inhibited infected hef in go phase. in contrast, induction of cd by types and ifn was not cell-cycle dependent. other viruses, including double-stranded dna viruses, vaccinia, and adenovirus and , and the single, positive-stranded rna alphavirus, semiliki forest virus, did not induce cd expression on hef after h. another alphavirus, ross river, was able to induce cd but only by the indirect mechanism of type ifn-dependent release. poly i.c, also, increased cd expression to the same extent as ifn-p after h, making it unlikely that the early increase was due to a nonspecific viral effect. the closely related flavivirus, kunjin, induced increased cd expression in a manner similar to wnv. the ability of flavivhses to induce increased cd expression directly within a few hours of infection may be an important virus-host survival strategy promoting cell-cell adhesion and hence possible further viral infectiodreplication. recognition of viral peptides presented on the cell surface in association with class i mhc molecules leads to lysis by cytotoxic t cells (ctl) and forms an important part of the immune response to hiv infection. hiv virus has a high mutation rate and variation in the region of the viral epitope may allow evasion of this immune response. variation could theoretically affect processing of the antigen, binding of the epitope to the hla molecule or recognition of the presented epitope on the cell surface. we have studied proviral sequence variation in gag and ctl responses in a number of hla b patients infected with hiv. amino acid substitutions, such as a lysine to arginine change at position of the pi gag nonamer cckkkyklk, lead to loss of recognition of the peptide by ctl from the patient whose provirus contained this sequence. these variant peptides bind to hla with comparable affinity to the index peptide suggesting that this loss of recognition is likely to be caused by changes in the interaction between the hla-peptide complex and the t cell receptor. other changes, such as lysine to arginine or glutamine at position , not only cause loss of recognition, but also lead to inhibition of lysis of targets bearing the index peptide. thus it appears that in addition to loss of recognition by cytotoxic t cells, naturally occurring epitope variants may act as "antagonists", as has been demonstrated in mhc class ii systems. antagonism may be an important mechanism allowing immune escape by the hiv virus. genes. subsequent complex formation between peptide, class i and p microglobulin in the er results in stable cell surface expression of the trimeric mhc- molecule. in previous studies we showed that in hpv- positive cervical carcinomas there was a loss of mhc- protein expression, which correlated at the single cell level with loss of tap protein. in this study we investigated whether loss of tap and mhc- is mediated by an hpv- encoded protein. human keratinocytes were transfected withvarious hpv- constructs including pat , the full length genome, pat esx the full length genome with a premature stop codon in e , puc.et , the e and e oncogenes only, and pkve , expressing e from mouse moloney ltr the different constructs were transfected into primary keratinocytes, cloned cells grown in medium supplemented with and without y-interferon ( y -a r ) for hours. cells were harvested and total rna and protein harvested for northern and western blots respectively. western blots showed very low steady state levels of tap- and mhc- heavy chains in the cells with pat as well as those containing es alone, which was marginally increased by y-lfn. in contrast, primary keratinocytes, pat esx and puc.et lines showed comparable tap- and mhc- protein levels, which increased a & y-ifn treatment. northem blots showed no differences in the amounts of tap- and mhc- mrna between the different cell lines. the data indicate that expression ofhf'v- e leads to post-transcriptional loss of mhc- , presumably by interfering with tap. to map and characterize functional differences between e a of ad and adl , we previously constructed a series of hybrid ad / e a genes and used them with ad e b to transform primary hooded lister rat kidney cells. at least two regions within the first exon of ad e a were identified which influenced tumorigenicity. this study further examines the role of these regions in tumorigenicity by analyzing their affect on cell surface mhc class i expression and sensitivity to class i-restricted cd + as well as to non-class irestricted nks. the bcrfl open reading frame of epstein-barr virus exhibits remarkable sequence homology with the coding sequences of interleukin- from a variety of organisms. many of the numerous immunological properties ascribed to interleukin- are shared by the product of bcrfl and this has led to it being termed viral interleukin- . in order to investigate the activity of viral interleukin-i (vil- ) and its interactions with the human interleukin- receptor we have expressed the protein in a bacterial and the eukaryotic cos- expression systems. the bacterially expressed vil-i was partially purified and used to set up two assays to measure i l l o activity: i)the increase in igm secretion from an ebv transformed b cell line -mt .l and ii)the downregulation of class ii hla expression on the human monocytic cell line thp- . a series of deletion mutants (both n-and c-terminal as well as an internal deletion to remove a putative heparin binding domain) were constructed to identify possible domains within the vil- protein that interact with the hil- receptor and confer its biological activity. a number of these mutants have been expressed in the cos- expression system and their structure and biological activity are currently being assessed. the identification of the domains within vil- that interact with the receptor or accessory proteins may aid in the understanding of the possible role of vil-i within the ebv life cycle and in the pathogenesis of the numerous diseases associated with the virus. generation. to further test the role of ctl in ad pathogenesis, viruses lacking the cll epitopes were tested when mutants that lack the immunodominate ctl epitope in eia where used, a second immun-ssive epitope in elb becorns the predominate target of clu. these findings arc important since human ad is currently being tested as a vector for gene therapy of cystic fibrosis. our data suggest that when consuucting ad vectors to be. used for gene therapy, one must retain either the . k or . k genes to decrease pathology and that meting the genes that encode the antigens that a n recognized by clu does not prevent the generation of ad specific clu. the interferons (ifns) a n ? a family of cytokines whose functions include the protection of cells against viral infection. type i ifns include the ifna subtypes and ifnp that compete for binding to the same cell surface receptor, while type ii ifn (ifny) binds to a different receptor. the orthopoxviruses, of which vaccinia virus (vv) is the prototypic member, have developed a number of anti-ifn strategies. the vv e l protein competitively binds dsrna and prevents the activation of ifninduced and dsrna-activated protein kinase (pkr), while the vv k l protein shows sequence similarity to the eukaryotic initiation factor a (eif a) that is phosphorylated and inactivated by pkr. the k l protein competitively binds the kinase and blocks host eif a phosphorylation and hence ifn-induced inhibition of host protein synthesis. onhopoxviruses also suppress cytokine action by expressing soluble cytokine receptors that bind and sequester the ligand; to date soluble receptors for interleukin- , tumour necrosis factor and ifny have been described. supernatants from vv-infected cells were found to contain a soluble inhibitor of type i ifn that was conserved in most of the orthopoxviruses tested. the inhibitor was produced early in infection and did not inhibit ifny. the ifna/p inhibitor was mapped and the gene expressed from recombinant baculovirus. the inhibitor blocked the binding of i-ifna to u cells and binding of i-ifna to supernatants from baculovirus and vv-infected cells demonstrated that the inhibitor functioned as a soluble receptor for fnc fp. direct binding of -ifna to vv wr supernatants revealed that the soluble ifna/p receptor had a high affinity for type i ifn. deletion of the gene from the vv genome and ligand blotting of the soluble receptor demonstrated that ifn binding was encoded by a single protein. competitive binding curves using ifna from other species revealed that the poxvirus soluble ifndp receptor bound human and bovine ifn with high affinity but murine ifn with relatively low affmity. interestingly, the soluble ifncrip receptor is highly conserved in variola virus. given the importance of ifn in antiviral defense it is likely that the soluble ifndp receptor plays an important role in the virulence of the orthopoxviruses. endogenous processing of a viral glycoprotein for presentation t o cd + t cells has defined a previously under-investigated pathway in antigen processing and presentation. it may be important not only for pathogens, but also for self-proteins, and thus may be involved in self-tolerance. we have been characterizing the processing o f the er-restricted gpt glycoprotein of vesicular stomatitis virus (vsv) biochemically and enzymatically, by cellular localization using confocal immunofluorescence, cellular fractionation, and by t cell recognition assays. by flow cytometry, gpt is undetected on the plasma membrane; in contrast, the wild type protein (g) is readily found following infection of a cells with a vaccinia virus vector, leading t o endogenous synthesis. the gpt can be found exclusively in the er compartment using co-localization with markers for er (signal peptide binding protein, calnexin), and not in the golgi compartment (a-mannosidase , wheat germ agglutinin), endosome, lysosome, or surface plasma membrane. this is consistent with the characteristics o f the localization of the proteases which appear to be responsible for its degradation. work is in progress to localize the site of peptide binding to mhc heterodimers. supported by nih grant a t o csr. presentation of an out-of-frame class i restricted epitope. t.n.j.bullock and l.c.eisenlohr, department of immunology, thomas jefferson university, philadelphia, pa . antigen presentation by class i mhc molecules is thought to require the degradation of fully formed proteins in the cytosol. this degradative process supplies oligopeptide epitopes for transport into the endoplasmic reticulum (er) where they can interact with and stabilize class i molecules. stable class i molecules, associated with p -microglobulin, can then proceed to the cell surface where they present the epitopes to t cell receptors. the generally accepted model for protein translation, the scanning hypothesis proposed by ko&, is thought to describe the traditional method of translation for the majority of proteins. we wished to test the hypothesis that any internal methionine that is in good translation initiation context can be a source of short peptides, which may then be processed into class i epitopes. nucleoprotein gene (np), the target of the ctl response of several inbred mouse strains. np contains three class i restricted epitopes at amino acids - (h -kk), - (h -kd) and - (h -db). the frameshift was introduced amino acids upstream of the h -kd epitope. the mutated genes were then recombined with vaccinia virus and tested for presentation using ctl restricted to each of the epito s described above. we found that, whilst presentation of the h -i@ epitope was unaffected by the frame shift, the epitope proximal to the frameshift (h -kd) was no longer presented to appro riately restricted ctl. however, presentation of the distal h -dg epitope was retained. therefore we have shown, using a viral protein and a viral expression system, that out-of-frame epitopes can be processed and presented to ctl. work is ongoing to c o n f m that internal methionines are capable of providing a platform for the initiation of translation for in-frame and out-of-frame epitopes. we have created a frameshift mutation in the influenza pr the fine specificity of t cell recognition of peptide analogues of the influenza nucleoprotein epitope np - srywairtr was studied using hla b -restricted influenza-specific cytotoxic t cell (ctl) clones, of defined t cell receptor (tcr) usage, derived from unrelated individuals following natural infection. synthetic analogue peptides were synthesized containing single amino acid substitutions, and tested both for binding to hla b' in vitro, and for presentation to ctl clones by hla positive targets. even conservative amino acid substitutions of the peptide residues p , , and profoundly influenced ctl recognition, without affecting binding to hla ' . these amino acid side chains are thus probably directly contacted by the tcr. ctl clones which used the tcr v a l gene segment (but not those using tcr va ) were also sensitive to p substitutions, suggesting that the tcr alpha chain of these clones lies over the n terminus of bound peptide, and that the "footprint" of certain tcrs can span all exposed residues of a peptide bound to mhc class . these results, taken together with previous structural and functional data, suggest that, for nonarner peptides bound to hla , p i , p and p are "flag" residues with tcr accessible side chains. the e / k protein of human adenovirus type (ad ) is a resident transmembrane glycoprotein of the endoplasmic reticulum. its capacity to associate with class i histocompatibility (mhc) antigens abrogates cell surface expression and the antigen presentation function of mhc antigens. at present, it is unclear exactly which structure of the e / k protein mediates binding to mhc molecules. apart from a stretch of approximately conserved amino acids in front of the transmembrane segment, e / k molecules from different adenovirus subgroups (b and c) share little homology. remarkably, the majority of cysteines is conserved. in this report, we examined the importance of cysteine residues (cys) for structure and function of the ad e / k protein. we show that e / k contains intramolecular disulfide bonds. by using sitedirected rnutagenesis, individual cysteines were substituted by serines and alanines, and mutant proteins were stably expressed in cells. based on the differential binding of monoclonal antibody tw . and cyanogen bromide cleavage experiments, a structural model of e / k is proposed, in which cys and cys as well as cys and cys are linked by disulfide bonds. both disulfide bonds (all four cysteines) are absolutely critical for the interaction with human mhc antigens. this was demonstrated by three criteria: loss of e / k coprecipitation, lack of transport inhibition and normal cell surface expression of mhc molecules in cells expressing mutant e / k molecules. mutation of the three other cysteines at position , and had no effect. this indicates that a conformational determinant based on two disulfide bonds is crucial for the function of the e / k molecule, namely, to bind and to inhibit transport of mhc antigens. previous studies have suggested that several abundant cmv proteins are major immunogenic targets in seropositive adults. we are interested in defining the major viral protein targets of a cd ' ctl response, in order to derive a vaccine strategy for individuals who are unable to mount immune responses which are lymphokinedependent because of immunosuppression. hla-typed and cmv-pgsitive normal volunteers who have hla-a alleles that represent - % of the u.s. population are being tested to determine which of abundant cmv proteins they recognize by a cd ' ctl response: p , p , p , ie, and gb. t cell lines will be derived in order to unambiguously determine the hla restriction of the cd ' ctl response to each of these proteins. proteins which are recognized by the most hla diverse population will be further characterized in terms of mapping of class epitopes through the use of t cell clones derived from the polyclonal cell lines by limiting dilution. the defined epitopes will form the basis of a vaccine strategy to augment the memory responses of seropositive volunteers against cmv. these epitopes will be used to boost the ctl precursor frequency of bone marrow transplant donors as a means to transfer cellular immunity to immunosuppressed hematologic transplant recipients. an alternative strategy is to immunize seropositive individuals with recombinant viral proteins as a means to boost immunologic memory. we are pursuing that strategy in a transgenic murine model of hla-a . developed by dr. l. sherman (scripps institute, la jolla). we are vaccinating the transgenic mice with two well defined cmv proteins, p and gb together with either of two lipid-based adjuvants, commercially available d tapm (bcehringer-mannheim) or mf gth (chiron, emeryville, ca). our preliminary studies with hsv- gb demonstrate that both adjuvants are effective at eliciting murine class i restricted responses against the protein. current studies are evaluating the recognition properties of the adjuvant-cmv protein complexes by hwa as a restriction element in the transgenic model. the ctl response to sendai virus in c by mice is directed almost exclusively to a single h- kb-restricted epitope derived from the virus nucleoprotein, npj - (sev- ). analysis of independent t cell hybridomas generated from c by mice following primary sendai virus infection has shown that a very diverse repertoire of tcr is selected in response to this epitope. crystallographic analysis of sev- bound to kb has shown that the side chaiis of peptide residues phpi, gl , a d s , and alaps protrude towards the solvent and are potentially available for recognition by the tcr notably, residues gi and a d protrude prominently from the peptide binding site due to their l o c a l i o n on a bulge in the center of sev- . to determine the importance of each of these residues for t cell recognition, we analyzed hybridoma responses to sev- analogs substituted at each of these four positions. preliminary data showed there generally appeared to be dominant recognition of glyp and asnm. however, individual hybridomas exhibited distinct patterns of fine specificity for residues phep and alaps. thus, individual hybridomas were dependent on one, both, or neither of these residues for recognition of sev- . these data are consistent with a critical role for the gi and a d in governing tcr-sev- eb recognition and suggest a structural basis for the diversity of the tcr repertoire selected by this @tope. previous results from this laboratoty demonstrated that the dominant influenza a epitope recognized by hla . restricted ctl from hla-a . uansgenic mice was the m peptide epitope that is immunodominant in human ctl responses. however, analysis of a large number of ctl lines revealed a subset of influenza a/pr/ / -specific murine ctl that recognized an hla-a . restricted epitope distinct from m . using recombinant vaccinia viruses encoding werent influenza gene segments, the epitope recognized by these ctl was shown to be derived from the a/pr/ nsl protein. because these ctl did not recognize targets infected with the a/alaska/ / saain of influenza, candidate peptide epitopes were synthesized based on sequences that included an hla-a . specific binding motif and that differed between a/pw and nalaska all of these ctl recognized a nonamer and a decamer peptide which contained a common amino acid sequence and two distinct sets of bmding mtif residues. however, the n name.r peptide was able to sensitize ctl for half maximal lysis at - fold lower doses than either the octamer or decamer. the homologous peptide derived from nalaska nsl contained conservative amino acid changes at positions and and was not recognized at any tested concentration, although it bound with higher &ity to hla-a . than the peptide from a/pw . the a/pr/ nsl nonamer epitope was also recognized by human influenza a specific ctl derived from two individuals. these results substantiate the general utility of hla class i aansgenic mice for the identification of human cn epitopes for other pathogens. furthemore, the recombinant dhfr was functional in the induction of gb epitope-specific ctl response upon immunization of c bv mice. these results indicate that an viral epitope expressed in a cellular protein can be. efficiently processed, presented and recognized by epitope-specific ctl, and suggest that the cellular proteins can be used to express ctl epitopes for induction of cd + immune responses. virus-specific cytotoxic t lymphocytes (ctl.) were generated a day later at this site. to determine which apc was capable of stimulating virusspecific ctl precursors in the mln, b, t and dendritic cells from the mln of influenza-infkcted mice were separated and examined for the presence of virus. the predominant cell type which contained infectious virus was the dendritic cell. b and t cells from the mln contained little, ifany, virus. the apc capacity ofthese populations was tested by their ability to stimulate vir~~-~pecific t cell hybridomas. only dendritic cells from the mln of influenza-infected mice were able to stimulate virusspecific t cell hybridomas, althwgh all apc populations from both naive and influenza-infected mice were effective stimulators after in y h pulsing with the appropriate intluenza peptide. potential apc populations were also separated from the lung. v i s was detected in bronchioalveolar macrophages and dendritic cells but not b or t cells. both macrophages and dendritic cells isolated from intlum-infected lungs could stimulate virus-specific t cell hybridomas. the ability of the mln and lung apc populations to stimulate naive cd ' t cells and generate virus-specific ctl is currently being examined. virus infected cells present only a very limited number of peptides intracellularly processed from a viral protein to ctl even when many peptides hearing the mhc class i-restricted binding motif are present in the protein. infection of h- b mice w i t h lymphqtic choriomeningitis virus (lcmv) induces a cd + ctl response directed against three wellcharacterized epitopes presented by h- db molecules: " - (fqpq-ngqfi), gp - (kavynfatcgi) and gp - (sgven-pggycl). the h- db motif is characterized by a sequence of to a.a. with two anchor residues: asn at position and hydrophobic (met, ile, leu) at the c-terminus. the lcmv np and gp proteins contain thirly-one other peptides exhibiting the db motif. however, no ctl response against one (or more) of these peptides has been characterized. peptide binding to mhc is a critical step in antigen presentation. the aim of this study was therefore to analyze the binding properties of the potential db lcmv peptides. the lcmv peptides and known db-selective peptides were synthesized and their mhc binding affinities measured in two db-specific binding assays. most of the lcmv peptides ( / ) did not bind to db. the other (including the epitopes) and all the known db peptides showed good affinity. comparison of the sequences (good vs. non binders) allowed the identification of auxilliary anchors required for high binding affinity or of negative elements hampering mhc binding. in addition to the main anchors, the positive and negative factors at secondary residues play a crucial role in governing peptidemhc interactions. knowledge of such factors might he of importance for the prediction of mhcrestricted ctl epitopes. etienne joly, andrea gonzalez, carol clarkson, jonathan c. howard and geoffrey w. butcher. laboratory of immunogenetics, department of immunology, the babraham institute, cambs cb at, uk. tap transporters from rats can be divided into two allelic groups, depending on their capacity to provide the rt .aa molecule with an appropriate level of suitable peptidesl. recent results suggest that this might correlate with the rt .aa molecule requiring arginine-ended peptides (powis et al., manuscript submitted), which the tapb allele of the transporter is unable to translocate across the er membrane efficiently ~ . rt .a alleles are naturally linked with the tapa or the tapb allelic group . we have set out to characterise various alleles for the rt .a molecule, and find that, for the majority of tapaassociated rt .a molecules, acidic residues line the c/e pocket, dictating arginine as c-terminal anchor residue for the bound peptides. on the other hand, in tapb-associated rt .a molecules, one acidic residue at the most is found in the c/e pocket, which certainly results in a different anchor residue for the bound peptides. the selective pressure of viral infections must have driven this coevolution which affects dramatically the array of peptides presented to cytotoxic t lymphocytes. cytotoxic t lymphocyte responses in hiv infection can be impaired due to variation in the epitope regions of viral proteins such as gag. we show here an analysis of variant epitope peptides in three gag epitopes presented by hla b . seventeen variant peptides were examined for their binding to hla b ; all but one bind at concentrations comparable to known epitopes. all except two could be seen by ctl clones grown from hla b positive hiv- infected patients and were therefore immunogenic. however, in one haemophiliac patient studied in detail, there was a failure to respond to some of the peptides that represented virus present as provirus in his peripheral blood. in one case his ctl had previously responded to the peptide. thus there was a selective failure of the ctlresponse to variant epitopes. this impaired reaction to new variants and failure to maintain responses to some epitopes late in hiv infection could contribute to the loss of immune control of the infection. pira, anna ferraris, daniele saverino, peifang sun and annalisa kunkl; dept. immunology, san martino hosp. univ. of genoa, genoa, italy. th epitopes present on viral proteins can be recognized by specific th cells if appropriately expressed by antigen presenting cells (apc) as a result of uptake and processing. since viral epitopes are not simply present in the context of viral proteins, but also in the context of whole viral particles, it is important to determine the role of the molecular and/or structural context on antigen uptake-processing-presentation. therefore we have generated panels of cd + human t cell lines and clones specific for different hiv antigens (gp , p , p ), in order to test their ability to respond to the same epitopes present within synthetic peptides, recombinant proteins or inactivated virions (provided by g. lewis, dept. microbiology, univ. maryland, baltimore). we could identify t cell lines and clones that were able to discriminate the molecular and structural context of the epitops. certain t cells, in fact, responded to peptides and proteins, but not to viral particles, whereas other t cells were also able to proliferate when challanged in vitro with autologous apc and viral particles. the data suggest that in the human th cell repertoire specific for viral antigens t cells exist that can discriminate the molecularstructural context of th epitopes. it will be interesting to ascertain whether t cells specific for epitopes that can only be recognized when provided in the context of a soluble molecule, but not of a viral particle, have any relevance in viva protection, or are a simple by-product of the cellular immune response. eric g. pamer, merceditas s. villanueva, section of infectious diseases, yale university school of medicine, new haven, ct listeria monocytogenes is a gram positive bacterium that infects macrophages and secretes proteins into host cell cytosol. the murein hydrolase p is secreted by l. monocytogenes and is required for complete bacterial septation. in the infected macrophage secreted p is processed by the host cell into the nonamer peptide p - and is presented to cytotoxic t lymphocytes by the h- kd mhc class i molecule. we have used strains of l. monocytogenes that secrete different amounts of p to show that the rate of p - production is proportional to the amount of antigen secreted into the host cell cytosol. p is degraded in the host cell cytosol with a half life of minutes. the appearance of p - is coupled to the degradation of newly synthesized p . we have determined the rate of intracellular p secretion and by accounting for the rate of p degradation we estimate that approximately p molecules are degraded to produce one p - epitope. this ratio is maintained over a range of intracellular antigen concentrations. our findings provide an estimate of the efficiency of antigen processing and demonstrate the remarkable capacity of the mhc class i antigen processing pathway to accommodate new epitopes. we have isolated and characterized three cytotoxic t lymphocyte (ctl) clones from the peripheral blood of two acute seroconversion patients and one patient in the first trimester of pregnancy. these clones were cd + and class i hlarestricted by the b molecule. all three clones recognized lllb and rf but not mn strains of hiv- . using vaccinia vectors expressing truncated versions of the hiv- envelope, the clones were found to recognize an epitope within amino acids - , but not including - of gp . further mapping of the epitope with synthetic -mer peptides overlapping by , or -mers overlapping by , was unsuccessful. the sequence of the region of gp recognized by these clones was compared to the predicted hla- peptide binding motif and a possible matching region was found. using shorter peptides corresponding to this potential epitope recognition site, the minimum epitope recognized by the clones was determined to be the aa sequence rpnnntrksi spanning amino acids we have further pursued a strategy to define a minimal cytotoxic epitope for a vaccine against cmv infection using t cell clones derived from individuals who have the mhc gene (kind gifts of drs. riddell and greenberg, fred hutchinson cancer research center and dr. robert siliciano, johns hopkins university medical center). we tested by chromium release assay (cra) the recognition of a series of allelic variants of ebv-lcl. by restricted and cmv or hiv-specific t cell clones. several conclusions quickly became apparent. the previously described ' peptide epitope from pp was not able to prime the autologous ebv-lcl for killing by the pp -specific ctl, whereas a recombinant vaccinia virus expressing whole pp could cause the same cell line to be recognized and killed in the same experiment. in addition, an hiv gp -specific cd ' ctl which has a defined minimal cytotoxic epitope will only recognize and kill a subset of ebv-lcl. the two t cell clones will not recognize each other's autologous ebv-lcl. the resolution of this interesting phenomena comes from sequence analysis of the hla class i b genes from both ebv-lcl. ebv-lcl which contain the b' allele are recognized and killed by the pp -specific t cell clone, and cell lines carrying ' alleles are recognized by the hiv gp -t cell clone. we conclude that the reported cmv pp b" restricted epitope is not correct, since the ctl in question will only recognize ' alleles in combination with the correct pp epitope. fragments with or without a signal sequence sensitize rma-s/kd to a similar limited extent. this data i s consistent with an inefficient movement of peptides from the cytoplasm into the er by a tap independent mechanism and does not reveal a processing competent compartment within the secretory pathway. peptide transport by the transporter associated with antigen processing (tap) was studied using a microsome system as previously reported by heemels et. al.. in this system, a radiolabeled synthetic peptide which can be n-link glycosylated is used as the indicator peptide for the transport studies. the transport efficiency of synthetic peptides corresponding to antigenic peptides restricted to the murine kd molecule was measured by inhibition of labeled peptide transported into the microsomes. the transport efficiency of three kd epitopes in the type a influenza virus " - , ha - and ha - was found to be similar. an amino acid peptide corresponding to ha - which contains the - epitope was transported at a similar efficiency as the amino acid minimum epitope. however, when the peptide sequence is further extended by one amino acid to residue , this peptide is poorly transported. these results suggest that the flanking region of an epitope can dramatically influence the transport of the epitope. when the transport kinetics of tap was studied using the microsome system, the vmax for transporting the indicator peptide (a variant of np epitope that has the sequence tynrtrali) was found at . fmolelminute (+/- . ). the km for this peptide was found to be . nm(+/- . ). bypassing a block in antigen processing for class i-restricted cytotoxic t cell recognition. amy j. yellen-shaw and laurence c. eisenlohr. thomas jeferson universitv. hiladelphia, pa., . previous work from our laboratory showed that processing of an influenza nucleoprotein (np) epitope (amino acids - ) expressed endogenously from a recombinant vaccinia virus "minigene" is severely impaired when a flanking sequence (the dipeptide threonine-glycine) is appended to the cterminus of the construct ( - /r-). the inhibition of processing is overcome by placing the unprocessed peptide in the context of the fulllength np molecule, demonstrating that regions of a protein outside the epitope itself critically affect the ability of the proteolytic machinery to fragment the protein appropriately. to determine the requirements for bypassing the block in antigen processing, we have constructed an array of "minigene"-expressing vaccinia recombinants in which the unprocessed epitope is extended by varying lengths toward either the c-terminus or the n-terminus of the np molecule. our results show that while an extension of the c-terminus by only one amino acid restores processability, a much longer extension of the n-terminus ( < n < amino acids) will also allow the substrate to be processed. it is therefore clear that a full-length, properly folded molecule is not required for liberation of the blocked epitope, and that probably more than one mechanism can contribute to enhancement of substrate proteolysis. we hypothesize that the c-terminal extension allows recruitment of an endopeptidase versus exopeptidase ("trimming") activity which is capable of cleaving the difficult bond. we considered the possibility that the n-terminal extension rescues processing by recruitment of the ubiquitin-dependent degradation system. to address this possibility we replaced all available ubiquitination sites (lysine residues) in one of the rescued constructs ( - /r-) to see if the construct would still be processed and presented. the six available lysine residues were changed to arginine using pcr-based mutagenesis. the resulting construct (termed r) was recombined into vaccinia virus and tested for presentation to np-specific ctl. the r construct was presented at a level equivalent to that seen with the wild-type - /rconstruct. this result provides clear evidence that entry into the ubiquitindependent degradation pathway is not responsible for rescue of presentation in this system and more importantly, that ubiquitination is not required for processing of all large substrates. chia-chi ku, li-jung chien ,and chwan-chuen king, institute of epidemiology, national taiwan university, taipei, taiwan, r.o.c. dengue virus (den) can cause dengue fever (df) and dengue hemorrhagic fever (dhf) i dengue shock syndrome (dss) and den- was the most common serotype found in dhf outbreaks globally. current hypotheses suggested that dhf may be associated either with antibodydependent enhancement (ade) or with viral virulence. den can replicate predominantly in monocytedmacrophages (mim), but whether peripheral blood lymhocytes (pbls) are the target cells of den still remain controversial. in order to compare whether various clinically derived den- will interact with mim and lymphocytes in different manners, we used two isolates --plo strain (obtained from a df patient during taiwan outbreaks) and strain (isolated from a dhf patient in thailand by cdc, usa) to infect primary mim and lymphocytes as well as several types of cell lines. primary lymphocyte culture was nonadherent cells obtained after hr adherence of pbmcs, whereas the primary mim culture was collected by depletion of lymphocytes using anti-cd icdi mab and complement prior to adherence procedure and the purity of mim culture was checked by cd surface marker staining. supernatants (sn) of virus were harvested at various time points post infection after with several or without treatments. our prelimanary data showed that dhf-associated den- strain had higher viral yield in certain age of mim and a promonocytic cell line (hl-cz) than taiwan df-associated den strain. in addition, this dhf-den strain was more likely to infect the promonocytic (hl-cz) than well differentiated monocytic (ctv- ) and lymphocytic (h ) cell lines and also had higher peak yields than den-i virus in hl-cz cells. interestingly, dhf-den strain replicated much more efficiently in primary lymphocytes no matter these cells were activated with pha or not, whereas taiwan df-den strain virus was hardly detectable in sn of both activated and non-activated lymphocyte cultures. therefore we conclude that ( ) different strains of dengue virus could orchestrate quite differently with immune cells, ( ) different stage of mim differentiation might be an important permissive determinants for dengue virus infection and replication, and ( ) den virus strain virulence -a more important factor than lymphocyte activation status -seemed to determine whether this strain would infect human pbls. further studies should be focused on searching for detaied mechanisms of virus and immune cell interactions. ( ) when viral yields were enhanced early than day post infection, it provided tremendous opportunity to attack the immune system and finally may lead to severe disease. hiv- using recombinant immunoglobulin molecules, marie-claire gauduin, graham p. allaway, paul j. maddon, carlos f. barbas, dennis r. burton, and richard a. university school of medicine, new york. ny . primary isolates of hiv- have been shown to be less sensitive to neutralization by immune sera, monoclonal antibodies and cd -based molecules than t cell line-adapted strains of hiv-i. we studied two immunoglobulin molecules for ability to neutralize primary isolates of hiv-i. lgg is an immunoglobulin molecule created from a combinatorial phage expression library and reacts with the cd binding site (cd -bs) on gp . cd -lgg is a recombinant molecule in which the variable domains of both heavy and light chains of lgg were replaced with the first and second immunoglobulin-like domains of human cd . both molecules have been previously shown to effectively neutralize hiv-i in vitro. ex vivo neutralizations were performed as follows: lgg and cd -lgg were added at pg/ml to wells containing serial dilutions of plasma from hiv-i-infected patients and phastimulated peripheral blood mononuclear cells from seronegative donors. p production was measured over days of culture and an end-point titer of hiv- in the presence and absence of added antibody was determined. both igg and cd -lgg were found to reduce the original hiv titer from seven plasma samples with high virus titer (> tcid /ml) by up to -fold. this is in comparison to soluble cd which only reduced viral infectivity by -fold at the same concentration. in vitro binding and neutralization assays on isolates recovered from plasma confirm the potency and breadth of neutralization by these two molecules. these studies suggest that recombinant antibodies directed at the cd -bs of hiv- gp are able to effectively neutralize primary isolates of hiv- and may be useful in dissecting the mechanisms of resistance to neutralization by other antibodies. dillner and p. heino, microbiology & tumor biology center, karolinska institute, stockholm, sweden hpv the major cause of anogenital precancers in man. the search for neutralizing epitopes that could form the basis for a preventive vaccine has shown that the surface-exposed imunodominant epitopes of the capsid are strongly conformationdependent, which has precluded detailed epitope analysis. similarly, immunization with whole, denatured capsid proteins has only identified linear immunodominant epitopes positioned on the inside of the capsid. reasoning that linear surface-exposed epitopes should exist, but might be cryptic, a set of overlapping synthetic peptides corresponding to the entire hpv capsid proteins was used to generate hyperimmune sera. several antisera against different peptides were reactive with intact hpv capsids at titers up to : . . hiv- serum antibodies and mucosal iga. basil golding, john inman, paul beining, jody manischewitz, robert blackburn and hana golding. div. of hematology and viral products, cber, fda, and lab. of immunology, niaid, bethesda md . previously, we showed that hiv- proteins conjugated to . abortus (ba) could generate anti-hiv- neutralizing antibodies in mice even after depletion of cd * t cells. in this study a -mer peptide from the v loop of hiv- (mn) was synthesized ) and coupled to ba and klh. balb/c mice were immunized twice i.p. with these conjugates at two week intervals. v -klh induced mainly igg , whereas v -ba induced all igg isotypes but lgg a predominated. fecal extracts from mice immunized with v -ba were shown by elsa to contain iga antibodies. sera from these mice bound gp , expressed on the surface of infected cells. sera from mice immunized with v -ba inhibited syncytia formed between cd ' t cells and chronically infected [hiv-i (mn)] h cells. inhibition of syncytia, formed by other hiv- lab. strains correlated with the degree of their homology with the v region of hiv-i (mn). to mimic the efffect of hiv- , mice were depleted of cd ' cells using anti-l t at the time of primary or secondary immunization. following primary immunization, cd + t cell depletion abrogated v -klh antibody responses, whereas responses to v -ba were retained and sera from these mice were able to inhibit gp- mediated syncytia. in secondary responses, cd ' t cell-depletion prevented boosting to v -klh, but v -ba increased anti and syncytia-inhibiting antibodies. these results suggest that: . . abortus, can provide carrier function for a peptide and induce both serum and mucosal antibody responses, and . that infection with hiv- with subsequent impairment of cd ' t cell function would not abrogate anti-hiv- antibody responses if . abortus is used as a carrier to stimulate memory responses. nucleotide sequence analysis of the vh genes revealed the usage of one particular vh germline element (vh - p) in all clones. this finding allowed the determination of somatically mutated positions in the vh regions. two vsv-ind neutralizing antibodies expressed vh and vl genes in complete germline configuration whereas the rest of the clones showed somatic mutations which obviously were antigen dependently selected for. however, binding affinities of mutated and unmutated antibodies were comparably high. in order to determine the influence of somatic point mutations on one single antibody we generated a monovalent single chain antibody (fv-ck) of a mutated clone and reversed it stepwise to germline configuration by means of site directed mutagenesis. surprisingly, already the germline configuration of fv-ck could neutralize vsv-ind, even though the binding affinty was lower than that of the mutated fv-ck. every single somatic point mutation tested improved the binding avidity although some mutations reduced affinity. thus, during the course of vsv-ind infection some antibodies are subjected to avidity maturation although this is not required for the generation of high affme, efficiently virus neutralizing antibodies, lisa hyland'", sam hou'.~, and peter c. doherty'. 'department of immunology, st. jude children's research hospital, memphis, tn i, departments of immunology and microbiology, and 'pathology, university of otago,dunedin, new zealand. the b and t cell responses in c bl/ j(b ) mice treated with the mab mel- to l-selectin have been analysed following i.n. infection with sendai virus. mel- treatment caused a - % decrease in the lymphocyte recruitment to the mediastinal (h ln) and cervical (cln) lymph nodes following infection with sendai virus. the cellularity of the spleen was unchanged. the clonal expansion of cd + ctl precursors in the mln was slightly delayed, but potent ctl effectors were present in the virusinfected lung by day after infection and the overall magnitude of the response was not compromised. the prevalence of iga antibody forming cells (afcs) was greatly increased in both the mln and the cln of the mice given the mel- antibody. the igm response was prolonged and the igg response, particularly iggl, was delayed compared to controls. the altered pattern of the antibody response may reflect the limited availability in mel- -treated mice of th cells secreting lymphokines which are involved in ig class switching, by blocking the entry of cd + th precursor cells into lymph nodes. facs sorting for l-selectin+, +, and l-selectin-, b + cell populations from the mln and the cln of normal b mice days post sendai virus infection, showed that the afcs were from the l-selectin-, b + cell population, a population which comprised - % ofthe total cell population. we have distinguished targets of broadly neutralizing antibodies present in hiv- infected individuals by imunoselection in vitro and by the use of chimeric virus. one target of neutralizing antibodies, defined by an escape mutant with an ala to thr substitution at position in gp , is resistant to human monoclonal antibodies that map to a site closely congruent with that for cd binding. substitution of gly, ser, and val fail to confer resistance. a second, defined by an ala to val substitution at position , upstream from the v loop, does not involve the same site and does not involve v . substitution of thr or ile also confers resistance. replacement of the v loop of hiv-l(mn) into a clone of hiv-l(iiib) allows the detection of two other broadly neutralizing targets. one recognizes the v peptide of mn but is affected by regions outside v . the other appears to be conformational and outside v , but its functional recognition is influenced by the v loop. all of these sites seem to depend on the overall conformation of the envelope protein rather than a single discrete linear epitope. antibodies against amino acids - of the hiv transmembrane (tm) glycoprotein have been shown to enhance hiv infection in vitro in the presence of complement. there has been no study demonstrating that enhancing antibodies to this region of hiv, despite increasing levels of infectious virus to fold in vitro, adversely affect disease pathogenesis. in two separate studies reported herein, it is shown that animals which have high levels of antibody against this region of siv, amino acids - of the envelope, fair poorly compared to animals with lower antibody levels against this region when subsequently challenged with siv. when actively immunized with a synthetic peptide from this region of siv, animals died earlier and failed to clear antigen at two weeks after infection compared to animals that received a control peptide (p< . ). when animals were passively immunized with antibodies from a longterm survivor of siv infection, those animals that received higher levels of antibody against the tm peptide died within six months compared to longer intervals for those animals that had lower levels of antibody to this region. when taken together, these data suggest that antibody to the tm region of siv and hiv in general, and to this highly conserved peptide in particular, are detrimental to the host. therefore, immunization strategies that minimize the immune response against tm or treatment protocols that decrease antibody levels against tm may lead to prolonged survival following exposure to lentiviruses. we have developed a mouse model to examine the immune response to hpv proteins when these proteins are presented to the immune system via the epithelial route. in this model animals are grafted with keratinocytes expressing hpv e a n d e genes using a transplantation procedure which permits epithelial reformation. animals so grafted when challenged intradermally with e either as protein or via a recombinant vaccinia virus exhibit a delayed type hypersensitivity response which is e -specific and cd + t cell mediated. animals grafted with a sub optimal priming inoculum of cells develop immune non-responsiveness and have an abrogated dth response when challenged subsequently with a priming cell graft. in the present study w e have examined the antibody status in these animals. the e protein of hpv was expressed in e. coli as a maltose binding fusion protein using the plasmid vector pmalc. after cleavage and affinity purification this protein was used in a n elisa assay to measure antibody levels in groups of mice ( ) those not challenged with e ( ) mice not grafted but challenged with e protein in the ear ( ) mice primed by grafting with hpv e expressing cells and challenged with e protein ( ) mice primed by grafting with x hpv e cells on day , grafted again with lo hpv e cells on day and challenged with e protein in the ear. mice optimally grafted and challenged (group ) exhibited high titres of igg antibodies, particularly elevated levels of iggza. mice sub-optimally grafted (group ) exhibited igg antibody levels comparable to the control group ( ). the possible mechanisms of this immune attenuation are discussed. the hepatitis c virus is a frequent cause of chronic liver disease. a proposed mechanism responsible for virus persistence is evasion of the host immune response through a high mutation rate of crucial regions of the viral genome. the portion of hcv genome coding for the amino-terminal part of the putative envelope protein (gp ) undergoes frequent mutation during the course of infection. we have cloned and sequenced the hypervariable region (hvri) of the virus isolated from an hcv asymptomatic patient at three time points during months follow up. sequence analysis has allowed the identification of variants of this region and multiple antigenic peptides (map), corresponding to three hvrl variants, sequentially foundin the blood stream of the patient, have been synthesized. maps have been used as antigens for detection of specific antibodies in elisa. our results show that anti-hvri antibodies and their cognate viral sequence coexist in the blood stream but a viral sequence becomes undetectable when the specific antibodies reach maximum levels of reactivity. thus humoral immunity against the hvrl may play a role for virus clearence. the presence of anti-hvr antibodies was also investigated in hepatitis c viremic individuals and non-viremic patients. a high frequency of positive reaction ( %) against at least one of the three hvrl variants analysed in this study was detected in the viremic patients. finally, competition experiments show that antibodies crossreacting with more than one hvrl variant are produced by hcv infected individuals. this results suggest that complex cross-reactivity exist between hcv isolates for antibodies against the hvrl region as described for antibodies against the gp v loop of hiv. we propose as mechanism for viral escape in hcv chronic infections the one described as the "original antigenic sin", observed firstly in influenza, in togavirus, paramixovirus, enterovirus, and recently in hiv infection. using an adult mouse model to study active immunity against rotavirus infection, it was previously shown that oral immunization with some, but not all, animal rotavirus strains induced protection against subsequent infection following oral challenge witb the murine rotavirus strain edim (ward et al., ) . to determine i f a specific rotavirus protein could be associated with protection in this model, mice were immunized with a series of reassortants between the fully protective edim strain and a partially protective heterologous rotavirus strain (rrv-g). reassortants that contained genes for edim proteins responsible for protection were anticipated to provide complete protection; however, no edim proteins were found to be both necessary and sd cient for full protection. instead, protection was found to be highly correlated with viral shedding (p = , ) and with serum rotavirus iga titers stimulated by the different reassortants (p < , ). this indicated that protection was related to the intestinal replication properties of the different reassortants rather than to specific immunogenic properties of edim proteins. this conclusion was supported by the finding that the titers of serum rotavirus i& but not igg, stimulated in mice following oral immunization with a series of animal rotaviruses was directly related to protection against edim. if these findings can be extended to humans, they suggest that the efficiency of intestinal replication following oral inoculation with a live rotavirus vaccine candidate may be the primary determinant of successful immunization. h a l l medical center, individuals with adequate serum samples were identified as either rapidly progressing (rp) or slowly progressing (sp) by clinical and surrogate marker criteria. anti-v profiles were determined using synthetic proteins derived from the amino acid sequences of the v region of laboratory strains of hiv- in standard capture elisa format. serum obtained from each patient at multiple different time points was screened against these peptides. the majority of individuals in both groups demonstrated broad recognition, with reactivity to peptides corresponding to the v regions of mn, sf , ny and han/sc. less than % of individual in each group recognized the v peptide derived from iiib, @=ns, between groups). as the rp progressed to aids there was significant nonspecific narrowing of response, while the sp remained broadly reactivity (p< . ). in v i m neutralizing activity of the homologous laboratory isolates was determined with cytotoxicity, cytopathic effect and p ag inhibition assays. although most patient serum was capable of inhibiting p ag production in homologous lab strains while aids-free, there was no relationship with the ability to inhibit homologous virus effects on target cells and anti-v profiles. model, we show that after resolution of the acute infection, when antiviral plasma cells in the spleen decline, a population of virus-specific plasma cells appear in the bone m w and constitute the major sou~ce of longterm antibody production. following infection of adult mice, wspecific antibody secreting cells (asc) peaked in the spleen at days postinfection, but were at this time undetectable in the bone marmw. the infection was essentially cleared by days and the asc numbers in the spleen rapidly declined while an increasing population of lcmv-specific asc appeared in the bone marrow. when compared to the peak response at days post-infection, timepoints from days to more than one year later demonstrated greater than a l@fold reduction in splenic asc. in contrast, jltvlv-specific plasma cells in the bone marrow remained at high numbers and correlated with the high levels of antiviral serum antibody. the prewnce of antiviral plasma cells in the bone marrow was not due to a persistent infection at this site, since virus was cleared from both the spleen and bone marrow with similar kinetics as determined by infectivity and pcr assays. the igg subclass profile of antibody m e t i n g cells derived from bone manuw and spleen correlated with the igg subclass distribution of lcmv-specific antibody in the serum. upon rechallenge with w , the spleen exhibited a substantial increase in virus-specific plasma cell numbers during the early phase of the secondary response, followed by an equally sharp decline. bone marrow asc populations and lcmv-specific antibody levels in the serum did not change during the early phase of the reinfection but both increased about -fold by days post-challenge. after both primary and secondary viral infection, lcmv-specific plasma cells were maintained in the bone marrow showing that the bone marrow is a major site of long-term antibody production after acute viral infection. "memory" t cells, and associated with responsiveness to soluble and recall antigens. cd + lymphocytes staining bright, dim, or negative (equivalent to an isotype control) for cd were evaluated in uninfected controls (group l), hiv- positive patients with % cd + t cells (group ), and hiv-i-infected patients with ~ % cd + t cells (group ). most of these subjects also had -color staining for cd \cd ro\cd ra. the appearance of positive cd and cd ro on hivinfected and uninfected cells correlated well (r=. p<.ool). the percentage of cells staining cd +\cd +.(bright plus dim) was . ( %cl . - . ) in group , . ( . - . ) in group , and . ( . - . ) in group . the respective values for these groups that were cd +\cd gbwm was . ( . - . ), . ( . - . ), and . ( . - . ). values for cd +\cd ro+ were . ( . - . ), . ( . - . ), and . ( . - . ), respectively. in single factor discriminate function tests, the %cd +\cd + cells best predicted subject group ( % correct), proving to be a better discriminator than %cd +\cd b'h' ( % ~orrect),cd +\cd ~"" ( l%), cd +\cd ro+ ( %) and cd +\cd ro+\cd ra-( %). overall, no advantage was seen to splitting the cd +\cd + cells into bright and dim positive subsets in the subjects studied for the purpose of stratifying early vs. late hiv infection. likewise, splitting the cd +\cd ro+ compartment into cd ra+ subsets did not improve the ability to distinguish between uninfected and early or late hiv- infected patients. the relationship between the virus-specific cytotoxic response in hiv infected patients and disease progression support the concept that a vaccine candidate should also induce a virus-specific ctl activity. immunization of uninfected adult volunteers by a hiv-gpl recombinant canarypox virus was carried out in a phase i trial.two injections of a recombinant canarypox expressing the hiv-l/mn gp were performed at month and and two boosts of recombinant gpl mn/lai at month and in alum or incomplete freund adjuvant( fa). hiv-envelope specific cytotoxic activities were detected from ctl lines derived from pbmc stimulated by specific stimulation with autologous hiv infected blasts. ctl lines were obtained from out of donors : seven out of eighteen ( %) were found to present envelope specific cytotoxic activity at months , , or post immunization ; this activity was characterized as a cd +,cd +, mhc class-i restricted cytotoxic activity, and for at least two volunteers, this activity was still present two years after the first canari-pox/env injection. because avian poxviruses are incapable of complete replication and undergo abortive replication in mammalian cells , this is a n example of the persistence of long term memory cd + cytotoxic t lymphocytes in the absence of the priming antigen, indicating that t-cell memory might be independent of continued antigenic exposure. the university of alabama at birmingham, al . mhc class i restricted cd ' ctl activity plays an important role in the control of influenza virus infection as indicated in studies in mice and humans. cytokines such as il- and ifn-y regulate the generation of virus-specific ctl responses. we recently demonstrated a good correlation between the induction of influenza virus-specific ctl activity and the production of ifn-y by the cd ' t cells at the single cell level using an if?-specific elispot assay, secreted ifn-y by an elisa, and ifn-y specific mrna expression by rt-pcr. several recent studies have characterized cd + and cd ' t cells by their expression on the surface of distinct d r isoforms. cd ra is expressed on naive or virgin t cells, while cd ro is expressed on memory t cells. in the present study, pbmc of healthy young adult subjects were stimulated with influenza a virus and then enriched for cd + t cells. the cd ' cells were stained for cd ro' (pe) and cd ra' (fitc) cells and sorted. ctl activity against virus-infected autologous target cells was determined in a hour 'lcr release assay while ifn-y production and expression was assessed by elispot and quantitative rt-pcr, respectively. cds+/cd ro+ (memory) cells exhibited significant mhc class i ctl while cds+/cd ra+ cells exhibited no lytic activity. no activity was exhibited by freshly isolated or unstimulated cd +/cd ro+ t cells. similarly, cd +/cd rot t cells contained significantly higher numbers of ifn-y spot forming cells and higher quantity of ifn-y-specific mrna than cd +/cd rac cells. these data support our previous findings that ifn-y may serve as a useful surrogate marker for influenza virus-specific ctl activity in humans. in studying the kinetics of the cd + t cell response in lcmv infection we have observed a profound activation and proliferation of cd + t cells with a - fold increase in total number peaking at day - post infection. in c bw mice, most of the viral antigen is cleared by day seven, and after day the total cd + number per spleen drops about -fold. however, the relative specificity of the viral peptidespecific precursor ctl frequencies @ctwf) per cd + cell remains remarkably stable between day - of the acute infection and for many months thereafter. thus, the decline in the cd ' t cell number is not a function of the tcr specificities but is rather an across-the-board event. in contrast, we found that subsequent to the decline of the ctl response to a second heterologous virus infection such that the mouse was in a "resting, immune'' state, there often was a reduction in pctl/f to the first virus. for example, infections with w or mcmv substantially reduced the pctuf to lcmv or pv in all memory compartments, including spleen, lymph nodes, peritoneal exudate cells. reinfection with the original virus substantially elevated its pctuf and restored the pctuf that had been reduced by a heterologous viral infection. analyses of the progression of ctl responses during a heterologous virus challenge of a virus-immune mouse indicated a high frequency of crossreactive ctl appearing early during infection, but as the infection progressed there was a higher proportion of ctl specific only for the second virus. thus, we believe that when the across-the-board apoptosis of t cells occurs late in the infection, ctl specific for the first virus are diluted by those responding to the second virus. this may cause the reduction in memory to the first virus and may be one of the mechanisms contributing to the waning of secondary immune responses to certain viruses over time if there is no re-exposure to the original infectious agent. t-cells which arise after virus infection will aid our understanding of tcell memory and be useful in the design of vaccines which augment the memory response. to estimate the sendai virus specific precursor frequency in memory mice, cd + cells from c bl female mice which had been infected with sendai virus intranasally (i.n.) more than two months earlier were subjected to limiting dilution analysis. responder cell populations were enriched for cd + cells either by magnetic bead depletion of non-cd + cells, or by facs after staining with anti-cd monoclonal antibody these enriched (> % cd +) responders were cultured with sendai virus-infected, irradiated, t-cell depleted splenic antigen presenting cells (apc). supernatants from these cultures were tested for activity on the cytokine-dependent ctll cell line. duplicate cultures of responders on uninfected apc were used to set the level of rejection (mean cpm + x std. dev.). using this type of analysis we were able to demonstrate a frequency of memory thp at cd + cells, compared to a frequency greater than / ooooo in naive controls. the memory cd + cells were further characterized as cd rb-low ( / ) , cd -high ( / ), lselectin-low ( / ), and cd d-high (vla- -high) (v ). this is close agreement with other phenotyping studies on cd + memory cell specific for soluble antigens. t cells, ralph a. tripp, sam hou, anthony mcmickle, james houston and peter c. doherty, department of immunology, st. jude children's research hospital, memphis, tn . the immune response of influenza a and sendai-virusspecific, memory cd ' cytotoxic t lymphocyte precursors (ctlp) have been analyzed in c bu mice infected intranasally with unrelated or cross-reactive respiratory viruses. the numbers of influenza a-specific memory t cells increased in the regional lymph nodes (ln), spleen and bronchoalveolar lavage through the course of an irrelevant infection (influenza b). memory t cells showed evidence of enhanced steady-state activation. profiles of ctlp recruitment were analyzed in association with t cell proliferation and activation to determine whether signaling via the t cell receptor is necessary to induce "bystander" stimulation of the memory t cell pool. the extent of t cell proliferation was addressed by treating mice with low doses of cyclophosphamide (cy). "resting" sendai virus-specific memory t cells were unaffected by cy treatment, however upon challenge with influenza and treated or days later, the emergence of influenzaspecific ctlp was severely diminished. cell cycle analysis showed that cy eliminated the majority of cd ' t cells from the ln and spleen resulting in dna fragmentation of - % ofthis lymphocyte subset. a decrease (though smaller) in the numbers of sendai virus-specific ctlp indicated that some of the cycling cells killed by cy were memory t cells, presumably activated in a "bystander" manner. the decrease in ctlp numbers for both influenza and sendai virus-specific ctlp was still apparent days after cy treatment, long after the viral elimination. thus, immune responses to unrelated antigens may be a mechanism involved in maintaining the pool of memory t cells. experimentally vsv can result in an acute cns infection of mice. data from our in vitro experiments indicate that no has inhibitory effect on productive vsv infection. vsv infection at neuroblastoma nb a cells was significantly inhibited by loopm of a no donor s-nitro-n-acetylpencillamine (snap), while oopm of the control compound n-acetylpencillamine (nap) had no effect. when vsv infected nb a cells were treated with pm of a constitutive no synthase (cnos) activator n-methyl-d-aspartate (nmda), a significant inhibition of vsv production was observed. inhibition by pm of nmda was reversed by pm of nos inhibitor n-methyl-l-arginine (l-nma). work is in progress to determine the effects of inducible nos (inos) in a glioma cell line c on vsv infection. levels of no and expressions of both cnos in neurons and inos in glial cells in the cns following vsv will be further investlgated. supported by nih grant a to carol s. reiss. pediatrics, university of iowa, iowa city, ia. mouse hepatitis virus, strain jhm (mhv-jhm), is a neurotmpic coronavirus which causes acute encephalitis and acute and chronic demyelinating encephalomyelitis in susceptible rodents. . % of suckling c bu (kbdb) mice inoculated intranasally with mhv-jhm at days and nursed by dams immunized against the virus develop a chronic demyelinating encephalomyelitis characterized clinically b hindlimb paralysis, at - weeks postinoculation. the chronic demyelinating encephalomyelitis nor the clinical symptoms. recently, it was shown that lymphocytes isolated from the central nervous system (cns) of c bu mice both acutely and persistently infected with mhv-jhm display a cytotoxic t lymphocyte (ctl) response to the s protein of mhv-jhm. this response was further characterized by identifying the ctl epitopes that are recognized by a bulk population of ctls from the cns of mhv-jhm infected c bv mice. three epitopes were identified using synthetic peptides and truncated forms of the s protein in primary ciz assays. the epitopes recognized were amino acids - (cslwngphl, db), - (rcqifani, kb), and - (nfcgngnhi, db). thus, the results indicate that cytotoxic t lymphocytes responsive to the s protein of mhv-jhm in c bu mice recognize both kb and db-restricted cil epitopes. ctl lines and clones specific to these peptides and the entire s protein are being developed to test their biological significance in vivo with respect to the acute encephalitis and chronic demyelinating disease caused by mhv-jhm. a marked change in susceptibility to some neurotropic viruses during the first few postnatal weeks has long been recognised in rodents. infection of neonatal or suckling mice with the neurotropic alphavirus, semliii forest virus results in lethal encephalitis. infection of weaned animals is not lethal. earlier investigations focusing on changes in specific immunity have shown this not to be the explanation. infection of - week old mice with severe combined immunodeficiency does not result in acute rapidly fatal encephalitis. we have studied mortality, neuroanatomical distribution and spread of infection in mice of different ages and the effect of gold compounds on rendering infection of - week old mice lethal. neuroanatomical distribution of infection correlates with synaptogenesis. as this is completed in different systems within the first two weeks postnatal, systems no longer transmit virus and infection switches from disseminated to focal and restricted. complete productive replication and transmission of infection require smooth membrane synthesis which is present in neurones undergoing synaptogenesis, absent in mature neurones but inducible by administration of gold compounds. infection of neurones undergoing synaptogenesis is productive and virus is transmitted along neuralpathways, infection spreads rapidly around the brain, destroys cells and animlas die of a fulminant encephalitis. in mice infected after days of age replication in mature neurones is restricted, nonproductive, cannot be transmitted, does not spread, is non-destructive and non-lethal. as a consequence, in the absence of immune responses virus can persist in isolated cns cells for life and can even be detected by reverse transcriptase pcr in immunocompetent mice months after infection. in the presence of an immune response, cd + t-cells recognise and destroy infected glial cells leading to dem yelination. a~k e r m a n n ,~ virology swine,' virology cattle,' and avian diseases research units, national animal disease center, usoa, agricultural research service, ames, ia a recombinant pseudorabies virus (prv) (lltbap) was constructed which contains a . kb deletion spanning the standard recombination junction of the unique long and internal repeat sequences replaced by e lacz expression cassette. this deletion interrupted the large latency transcript gene (llt) and truncated one copy of the diploid immediate early iel gene. replication and viral gene expression of lltbaz in madin-darby bovine kidney cells was similar to that of the parental virus and a virus rescued for the deleted sequences (lltbres). when inoculated intranasally in -week-old or -day-old pigs, lltba replicated efficiently at the site of inoculation yet caused markedly reduced fatality when compared to the parent or lltbres viruses. in particular, the lltba -infected pigs did not exhibit neurological symptoms characteristic of prv infection. to further examine the pathogenesis of lltba , -day-old pigs were infected intranasally with lltpa or lltbres and necropsied at various times postinfection. virus isolation from the nasal turbinate, tonsils, and trigeminal ganglia was comparable between the two viruses. although both viruses spread to the brain and induced an inflammatory response in cns tissues, virus isolation from brain tissues was reduced about -fold for lltpa . abundant prv antigen was detected in the cerebrum and cerebellum of lltpresinfected pigs, but only a few antigen positive neurons were observed in the cerebrum of lltba -infected pigs. while replication of lltbres in the brain progressed until death at days post-infection, replication of lltpa in the brain ceased by days post-infection and the pigs exhibited only mild clinical signs. since lltba is capable of spread to the cns, reduced neurovirulence of lltbaz is likely the result of its decreased ability to replicate in cns tissues. the cns is a target for hiv infection, and in individuals with aids this can lead to a devastatin dementia. only certain viral variants appear capable invading the cns and infecting microglia and brain macrophages. in order to determine whether the virus entering the brain may be particularly pathogenic to the cns, we isolated microglia from the brains of siv-infected rhesus monkeys. transfer of these cells into naive animals indicated that productive siv infection could indeed be transferred. furthermore, cns infection occurred within a relatively short time span, and was associated with viral gene expression in the brain and pathology characteristic of hiv encephalitis. serial transfer of microglia into additional animals also resulted in successful transfer of infection, neuroinvasion, and neuropathology. behavioral analysis in a trained group of animals is ongoing. this result demonstrates that neuropathogenic virions partition into the cns during natural siv infection, likely driven by mutational events that occur during the course of infection. molecular characterization of the microglia-associated virus has revealed that a distinct pattern of sequence changes in the envelope gene occurs concomitantly with this in vivo selection. our approach will allow the dissection of functional neuropathogenic elements present in these viruses. in non-specific host defense mechanisms. ifn-y-induced nitric oxide (no) in murine macrophages was previously shown to inhibit the replication of poxviruses and herpes simplex virus type (hsv- ) . we now demonstrate that murine macrophages activated as a consequence of vaccinia virus (vv) infection in viva express inducible nitric oxide synthase (ios). the vvelicited macrophages were resistant to infection with w and efficiently blocked the replication of w and hsv- in infected bystander cells of epithelial and fibroblast origin. this inhibition was arginine dependent, correlated with no production in cultures and was reversible by the nos inhibitor nqjmonomethyl-l-arginine. the mechanism of no mediated inhibition of virus replication was studied by treating vv-infected cells with the noproducing compound, s-niuoso-n-afetyl-penicillamine. antibodies specific for temporally expressed viral proteins, a vv-specific dna probe and transmission electron microscopy were employed to show that no inhibited late gene protein synthesis, viral dna replication and virus particle formation, but not expression of the early proteins analyzed. further, we have also identified putative enzymatic targets of inactivation by no that results in inhibition vv replication. although antiviral ctl are important for virus elimination. they can only halt further virus spread, and cannot reduce the number of infectious particles already present. the beneficial effect of ctl-mediated lysis is apparent only if infected cells are lysed before assembly of progeny virus. if infectious virus was released from infected cells in solid tissues before the generation of neutralizing antibody or in sites where antibody did not readily penetrate, then recruitment of mononuclear phagocytes, which phagocytose and destroy infectious material and/or become non-productively infected, would definitely help control virus dissemination. in this context, inos induction in macrophages may be an important antiviral strategy. in addition, the inhibition of virus replication in infected contiguous cells by inos-expressing macrophages at infectious foci would prevent release of mature viral particles after lysis by nk cells and ctl. since viral early proteins are expressed in such infected cells, their recognition and subsequent lysis by ctls will not be hindered. cns persistence, tropism and genetic j. pedro s i s , anthony a. nash and john k. fazakerley, department of pathology, university of cambridge, cb iqp, uk theiler's murine enchephalomyelitis virus, a natural occuring enteric pathogen of mice, is a picomavirus belonging to the curdovirus genus. following intracerebral inoculation of - week old cba or balb/c mice, the bean strain causes a chronic persistent cns demyelinating infection in a proportion of the cba that survive acute infection. balb/c mice are resistant to chronic demyeliating disease. we have studied the tropism, persistence and genetic variability of bean, in cba and balb/c mice in the chronic phase of this disease. by in situ hybridisation and reverse transcription (rt) pcr and southern blot analysis, no viral rna could be detected in the cns of any balb/c mice later than day post-infection. in contrast, in a large group of cba mice studied up until days post-infwtion, viral rna could be detected by both techniques in % of mice until as late as days post-infection. by employing a combination of, in situ hybridisation for viral genome followed by immunocytochemistry for cell phenotypic markers, bean rna was observed predominantly in oligodendrocytes and occasionally in astrocytes during persistent infection, in both brain and spinal cord. in the persistently infected mice, the striking total destruction of the pyramidal layer of the hippocampus, substantia nigra and anterior thalamic nuclei indicated that these were the mice that had had greatest dissemination of virus and highest virus titers during the preceeding acute phase of infection. direct pcr t h d cycle sequencing of uncloned rt-pcr products, revealed that during persistent infection, loops i and ii of the vpi capsid protein gene did not undergo any genetic variability. furthermore, no changes were detected in this region in sequenced pcr products amplified from the cns of mice with severe combined immunodeficiency in which no selective immunological pressure would have been operative. infection, thomas e. lane, michael j. buchmeier, dorota jakubowski, debbie d. watry, and howard s. fox, department of neuropharmacology, the scripps research institute, la jolla, ca our laboratory is interested in the effects of siv infection in the central nervous system of rhesus macaques. to enrich for neuroinvasive and neurovirulent viruses, microglia were isolated from infected monkeys and used t o infect new, uninfected monkeys. such microglia-mediated infection resulted in the production of neuropathological changes, including giant cells, macrophage infiltrates and microglial nodules in recipient animals within months. microglial cells isolated from siv-infected monkeys produced virus in vitro as measured by reverse transcription (rt) and p production. treatment of microglia with recombinant human interferon alpha (rhulfn-a) resulted in a sharp decrease in viral activity (both rt and p production) suggesting that rhulfn-a is able t o modulate viral activity in infected microglia. we have analyzed slvenv sequences by pcr amplification directly from microglia dna preparations from monkeys. nucleotide sequence analysis results in an enrichment of unique sequences in the v region of the siv env gene. the majority (> %) of nucleotide changes encoded amino acid changes, indicating that these envelope sequences evolved as a result of selection. moreover, sequential passage of sivassociated microglia resulted in an increase in potential n-linked glycosylation sites within the v region of the env gene when compared with the parental virus. these data suggest that sequential passage of microgliaassociated siv may select for neuroinvasive, neurovirulent variants. the adoptive transfer of ctl specific for an ld-restricted epitope within the nucleocapsid protein of the jhmv strain of mouse hepatitis virus both protect from acute infection and reduce virus replication in the mhc class positive cells within the cns. the source of these ctl and the route of their delivery is critical in the outcome of this protection. for example, fold less spleen cells activated in vitro with the pn peptide are required for protection via the direct i.c. route than the i.v. route. in addition, ctl clones are unable to protect via the i.v. route and are very efficient via the i.c. route. these data suggested the possibility that the cd + t cells within the polyclonal activated spleen cell population derived from in vitro culture on the pn peptide were facilitating access to the cns. to examine this question, polyclonal pn-specific t cells were either depleted of cd + t cells prior to transfer to infected recipients or untreated cells were transferred to recipients depleted of cd + t cells with monoclonal antibody gk . . both of these treatments eliminated the ability of the ctl to reduce virus replication within the cns, suggesting that cd + t cells in the peripheral compartment are required for the entry of ctl into the parenchyma of the cns during acute cns encephalomyelitis. division of retrovirology, walter reed army institute of research and henry m. jackson foundation, rockville, md ; department of retrovirology, armed forces research institute of medical sciences, bangkok, thailand background the hn- epidemic in thailand is largely due to two highly divergent subtypes of virus, b and e. dual infection with distinct hn- subtypes, which has not been reported previously, would suggest that antiviral immunity evoked by one subtype can be incompletely protective against a second. merhoak: pcr typing and serologic typing were used to screen a panel of non-random convenience specimens from hiv- infected subjects in thailand. specimens that showed dual subtype reactivity in these assays were subjected to differential pmbe hybridization and nucleotide sequence analysis of multiple molecular clones to c o n f m the presence of dual infection. results. two individuals were shown to simultaneously harbor hiv- of env subtypes b and e (table) . additionally, both subtypes were identified in co-cultured pbmc from one individual. conclusions. these data provide the fmt evidence of dual hiv-i infection in humans and reinforce the need for polyvalent vaccines. infection by herpes simplex virus wsv) induces in man and in mice cytolytic t lymphocytes (ctl) which recognize the immediateearly protein icp . because of its early expression during the hsv replication cycle, lcp represents a prime target for specific t cell responses susceptible of controlling virus replication. we have expressed in e. coli a remmbinant construct coding for a fusion protein consisting of a fragment of influenza virus non-structural protein (nsi) and the lcp sequence of hsv- . the nsi-icp protein was purified by preparative eleclmplmresis and formulated in oil-in-water emulsions with monophosphoryl lipid a (mpl) and qszl adjuvants. balwc mice were immunized by two intrafootpad injections of formulations containing pg of nsi-icp . responder cells obtained from draining lymphnodes were re-stimulated in vitro with p cells lransfected with icp and then lesled for cytolytic activity on icp -p and control p . the induction of icp specific ctl by different formulations was observed and will be discussed. the induction of heterologous cytotoxic t lymphocytes (ctl) using cassettes of multiple conserved t cell epitopes derived from different proteins and/or virus strains is envisioned as a promising vaccine approach. to study the effects of antigen processing on peptide presentation from chimeric epitope precursors we are using a model system comprising two distinct viral epitopes which are immunodominant in the h- d haplotype: a dd restricted epitope from the gp protein of hiv- and an ld restricted epitope from the murine hepatitis virus nucleocapsid protein (mhv n). the influence of proximity and flanking sequences of epitopes on antigen presentation was analyzed using vaccinia virus (vv) recombinants in which the epitopes were expressed as chimeras containing the individual epitopes in reverse order or separated by different spacer residues. whereas individually expressed epitopes were efficiently recognized by protein-specific ctl, recognition of peptides derived from tandem constructs varied significantly with closer epitope proximity and sequential order. following immunization with the recombinant viruses, the chimeras were all able to induce antiviral ctl specific for the native proteins. however, cn, frequency analysis indicated that the number of responder cells to the same epitope dramatically depends on its context within the chimera and correlates with antigen recognition in vitro. the profound effect of flanking regions on ctl induction suggests that the context of an epitope will require careful evaluation in the design of recombinant multivalent minigene vaccines to induce an optimal t cell mediated immune response. and robert e. johnston', departments of 'microbiology & immunology and %iochemistxy, univ. of north carolina, chapel hill, nc a hll-length cdna clone of venezuelan equine encephalitis virus w e ) has been altered to contain two strongly attenuating mutations and a second subgenomic rna promoter immediately downstream of the structural gene region. expression ofthe influenza ha protein from this second promoter in baby hamster kidney (bhk) cells was approximately ?? of the level in influenza virus-infected cells, as measured by immunoprecipitation. fourweek-old cd- mice were inoculated subcutaneously with x ' p h of the ha vector, vector alone or diluent. expression of ha mrna was detected in the draining lymph node of ha vector-inoculated mice by in situ hybridization, consistent with the organ tropism of vee. mice were challenged three weeks after imnmization by intranasal administration of lo ed, of influenza h s . au corn mice suffered severe disease and % died. only one of ha vector-inoculated mice died, and another exhibited signs of disease for one day and recovered. the geometric mean elisa titer of anti-ha serum igg in the ha-vector inoculated mice was , while only three control mice had measurable serum reactivity, and that was at the lowest dilution tested, . in a parallel experiment, no influenza infectivity was detected in the lungs of ha vector immunized mice at days postchallenge. in contrast, / pbs-inoculated mice and / inoculated with vector alone were positive for influenza infectivity and had geometric mean titers of . and . x lo pwgm, respectively. this vector also has been used to express the h n w c a protein in a form recognized by patient sera and a specific antibody on western blots. these experiments demonstrate the feasibility of using vectors based on attenuated vee cdna clones for protective immunization against heterologous human and animal pathogens. dose/response curves have been used to compare different routes of immunization with plasmid dna encoding the h hemagglutinin glycoprotein of influenza virus. routes of inoculation included intramuscular, intradermal and gene gun delivery of dna. from to . ug of dna was inoculated by intramuscular and intradermal routes. from . ug to . ug of dna was inoculated by gene gun. each route was evaluated for single and boosted immunizations. antibody titers were followed over a week period, following which animals were evaluated for protection against a lethal challenge. each of the routes raised both antibody and protective responses. gene gun-delivery of dna required to , times less dna to raise responses than the intramuscular and intradermal inoculations. boosts did not have much of an effect on antibody titer or protection except at low dose inoculations ( ng and lower for the gene gun). for each of the routes, antibody responses showed good persistence over the weeks of the experiment. inoculation of mice with plasmid vectors carrying a microbial gene under the control of an appropriate promoter results in a full spectrum of immune responses to the vectorencoded antigen. using a murine rabies model a plasmid termed psgsrab.gp expressing the full-length rabies virus glycoprotein regulated by an sv promoter was shown to induce upon inmuscular inoculation a rabies virus specific t helper cell response. of the thl type, cytolytic t cells and virus neutralizing antibodies resulting in protection against a subsequent challenge with live rabies virus given either peripherally or directly into the cenaal nervous system. a response comparable in magnitude was also induce upon inoculation of a vector expressing a secreted form of the rabies virus glycoprotein. the immune response to the dna vaccine could be modulated by co-injection of the rabies virus glycoprotein-expressing vector with plasmids expressing mouse cytokines. inoculation of mice with the psg rab.gp vector and a vector expressing granulocyte/macrophage colony stimulating factor (gm-csf) enhanced both the t helper and the b cell response to rabies virus thus improving vaccine efficacy. co-inoculation with vectors expressing interferon-g failed to improve the response. co-inoculation of the antigen-expressing vector with a plasmid encoding mouse i l caused a reduction of both the t helper cell response and the b cell response to rabies vlns. hpvl e hpv associated cervical cancer cells express hpv e protein and antibody to hpv e can be detected in the blood of cancer patients, yet the twnours are. not rejected. a mouse transgenic for the e protein of hpv , and expressing e protein in the skin, has recently been described ( ) and these mice develop spontaneous humoral immunity to e protein similar to patients with cervical cancer( ). to determine whether immunisation could induce immunity to e sufficient to allow tumour rejection, we firstly demonstrated that immunisation of h-zb mice with hpv e protein with quil a as adjuvant could induce cytotoxic t cells able to kill hpv e expressing tumour cells in nrro. we then used similar immunisation with e iquil a to induce e specific immunity in fvb (h- ) mice. h- qskin @s expressing e were not rejected by e immunised h-zq mice, though immunisation induced antibody to e , and similar grafts were rejected, as expected, across an doantigen mismatch in h-zb mice. we conclude either that hpvl e lack a tc epitope in the context of h-zq, or that expression ofe in the skin from the e transgenic mice is insufficient for recognition by primed effector cells, and further experiments will address this distinction. cervical carcinoma is strongly associated with infection by human papillomavirus (hpv) types or , and continued expression of the e and e gene products. this provides an opportunity for an immunotherapeutic approach to the treatment of cervical carcinoma by activation of immune reponses directed against these virally encoded tumour specific antigens. we have constructed a recornbinant vaccinia virus expressing e and e from hpv and with the aim of inducing e and e specific hla class i restricted cytotoxic t lymphocytes (ctl). the sequences have been inserted into the wyeth vaccine strain of vaccinia virus at a single locus in the form of two separate fused e /e reading frames, each under the control of an early v a d n i a promoter, and each modified to inactivate the rb binding site. the virus has been characterised with respect to its ability to synthesise the expected hpv proteins, its genetic stability, and growth and virulence in a mouse model prior to use in human clinical trials. analysis of hpv € specific ctl from c bu mice immunised with this recombinant virus show the response to be equivalent to that generated by a control vaccinia recombinant expressing non-modified hpvi e alone, with similar recognition of the defined immunodominant h- db restricted epitope, e residues - . ability of mice to resist influenza challenge, arthur friedman, douglas martinez, john j. donnelly and margaret a. liu, department of virus and cell biology research, merck research laboratories, west point, pa mice infected with the laboratory strains of a/pr/ / (hln ) or the mouse adapted a/hk/ (h n ) show complete protection against challenge with a different strain of influenza a. humans, however, undergo multiple influenza infections as previous infections appear to provide weak or short-lived protection against the continual antigenic change of strains. we have previously shown that immunization of naive mice with dna encoding the conserved internal antigen nucleoprotein (np) provides protection against both h and h strains of a/influenza. although such mice became infected they were resistant to weight loss and death this differed substantially from a/pr and a/hk recovered mice which were resistant to subsequent infection. to produce a more representative model of human infection, we infected the lungs of mice with currently circulating strains of human influenza. mice that had been given lung infections with a/beijing/ were susceptible to subsequent infection with the a/hk/ strain although they were resistant to weight loss and death. other strains such as a/beijing/ or a/georgia/ provided only marginal protection against weight loss and death against a/hk challenge. mice that were immunized with np dna had greater resistance to weight loss and death after a/hk/ challenge than mice previously infected with a/bei/ and a/ga/ , and were similar to mice that had been previously infected with a/bei/ . thus, infection with different virus strains provide various levels of cross strain protection and the level of protection provided by immunization with dna can exceed that induced by live influenza infection. the development of sendai virus-specific cytotoxic t lymphocyte (ctl) effectors and precursors (p) has been compared for mice that are homozygous (-/-) for a disruption of the h- -ab class ii major histocompatibility complex (mhc) glycoprotein, and for normal (+i+) controls. the generation of cd + ctlp was not diminished in the (-/-) mice, although they failed to make virusspecific igg class antibodies. while the cellularity of the regional lymph nodes was decreased, the inflammatory process assayed by bronchoalveolar lavage (bal) of the infected lung was not modified and potent ctl effectors were present in bal populations recovered from both groups at day after infection. there was little effect on virus clearance. as found previously with cd -depleted h- b mice, the absence of a concurrent class il-mhc-restricted response does not compromise the development of sendai virus -specific cd + t cell-mediated immunity. the importance of cytoxic t lymphocytes in defense against acute and chronic viral infections is gaining increasing recognition. our approach to investigating the structure-function relationship between immunogens and their in vivo ability to elicit cytotoxic t lymphocyte responses has been to formulate simple, well-defined structures that vary in their ability to introduce associated antigens directly into the cytoplasm of antigen presenting cells. we have introduced methods for the preparation of unique, lipid-matrix based immunogens, which are highly effective in mice and monkeys for stimulating strong cd + cytotoxic t cell responses, (ctl). antigens used have been proteins or peptides derived from influenza, parainfluenza, and hiv viruses, and whole formalin-fixed siv. ctl can be induced by parenteral as well as oral administration. comparing the physical and chemical nature of our formulations with those from other laboratories which have reported the use of subunit preparations to induce cd + ctl, leads us to propose that a minimal immunogenic formulation capable of eliciting cd +, mhc class i restricted cytotoxic t lymphocytes includes: i) a peptide that represents a mhc class i epitope; ii) a component that enhances the aftinity of the immunogen for mhc class i positive antigen presenting cells ; iii) properties that can compromise the integrity of a lipid bilayer, facilitating delivery of the antigen directly into the cytoplasm for class i presentation. cd + responses to peptides, glycoproteins, and even whole fixed viruses, makes them attractive candidates for diseases where clearance of infected cells is important in protection and recovery. cani ne rabies is uncontrolled. rabies also is epizootifally active in several species in most areas of the wor!d. thm, vaccination of animals, both wild and domestic, as well as postexposure treatment of humans remains a global concern. unfortunately, in those countries in which,people most need postexposure prophylaxis, the best vaccines are expenswe and in limited supply, whereas available vaccines are of questionable immunogenic efficiency, are otten contaminated and may produce neurological complications. the goal of this study was to determine whether a rabies vaccine for global use is complete one round of replication have the potential to be used as vaccines. we have previously reported the abiliity of a ghdeleted herpes simplex virus type (hsv- ) to protect mice and guinea-pigs from subsequent challenge with wild-type hsv. this virus, which we have called disc (disabled infectious single cycle) virus, can infect normal cells but the absence of gh in the progeny virus prevents further rounds of infection. as disc hsv clearly has potential as a vaccine, it is important to determine the durability of the immune response elicited by this virus. we have investigated the ability of disc hsv- to protect mice from a wild-type virus challenge six months post vaccination using the ear model of hsv infection. two immunisations on day and day resulted in a considerable reduction in virus titres in the challenged ears, and an almost complete absence of virus in the dorsal root ganglia. hsv-specific antibody titres as determined by neutralisation and ellsa were maintained for the six months period. it was possible to demonstrate an hsvspecific cytotoxic t-cell response in the disc hsv- vaccinated mice following challenge; this ctl activity was similar to that observed in mice vaccinated with wild-type virus and challenged after the same time period. animals vaccinated with inactivated virus or control mock-vaccinated mice showed a low level of ctl activity typical of a primary ctl response following challenge. these results indicate that an effective cell-mediated and humoral anti-hsv immune response can be maintained for at least six months following vaccination with disc hsv- . viruses which lack an essential gene and thus can only the lmmunogenicity of two ctl @topes. influenza npl - and plasmodium berghei cs protein - were studled in balblc mice. paptides were formulated as a) a iipopepudepeplkle conlugated to irlpalmltoyl-sgly~~l cysteine (pam cyj) and dissolved in a % dmsolglycerol solution. b) micmparticles prepared with poly @.l ladide-coglywlide) using a solvent evaporation technique. the micropaltides were administered as a suspension in phosphate buffered saline or c) an emulsion prepared wilh egg lecithin and % soya oil in water. wpg of peptide or controls (the welght equivalent of blanks) were administered to groups of mice intra-peritoneally or subcutaneously at . and days. days following the last immunization splenocytes were cukured in vlro in the presence of appropriate pepwe or wntml m h rat con a supematant as a source of omwth fadors. ctl adivity was measured in a standard hour chromium release assay and results expressed as % specific lysis. ctl could be elicited in vivo with all three formulations. at an ewedoctarget ratio of w:l the plasmodium berghei peptide encapsulated in micmpartides gave % iysls on peptide pulsed target calls. levels of lysis were similar for the peptide in emulslons. the iipopeplide p ccs - gave a level of lysis of % at an e:t ralioof w . these results demonstrate that peplides edminldered in a variety of formulations can induce a systemic ctl response in vivo. peplide vaccines using such formulations wuld be used to stimulate ctl responses as part of a prophyladic vaccines or as immunotherapeulics. attenuated the attenuated sabin strains of poliovirus have been used for many years to elicit protective immunity to poliovirus. oral vaccination with replicating polioviruses generates both mucosal and systemic immunity. therefore, use of recombinant polioviruses expressing heterologous antigens as vaccine delivery vectors should provide a system for generating protective immunity to those antigens. cdna copies of the poliovirus genome has been used to construct vectors containing a multiple cloning site for insertion of heterologous genes. a pilot enhanced-potency inactivated poliovirus vaccine (ejpv) with assumably improved immunogenicity containing win-treated type poliovirus (shah sauketf) together with the regular type and type canponena was subjected to s*mdard safety and potency tesls in the labmatory and laken through wase i and i clinical aials. in balb/c mice, the lrypin-mcdit%d e-if' v cfryipv) was found to induce antibodies targeted ouaide the uypsin-sensitive bc-loop of capsid protein vp . as previously shown for hypsin-mated type poliovirus @vm alone. trypsin used to modify the type component at the bulk phase was removed by the vaccine manufacturer (rivm) in the regular purification process. absence of uypsin in the final product was further confumed by immunizing mice and rabbits with -fold concentrated type component of tryipv. assays for lrypin antibodies using eia and westem blot techniques were newve. in the clinical phase aial six adult volunteers with existing immunity to poliovirus were given increasing doses of tryipv. already one tenth of the regular dose induced a booster effect in neuarlizing antibodies to both intact and mypsin-treated type poliovirus. no unexpected sideeffects were recorded phase i trials comprised adult volunteers with at least years since the last dose of poliovirus vaccine and children who were due to receive the third dose of the regular immunization schedule at about years. in both groups. individuals received tryipv and were injected with the regular enhanced potency ipv (e-ipv). serum specimens drawn before injection and one month after were tested for neunalizing antibodies using standard microneuwlization assays (all mutypes) and the racina test (intact and uypsin-mated type poliovirus). in all volunteers tryipv was at least as immunogenic t w the regular e-ipv according to all assays. no statistically significant differences in side effects were reported. a murine/influenza virus model has been used to evaluate the longevity of antibody and protective responses raised by gene gun delivery of a hemagglutinin-expressing dna. mice were immunized and boosted at one month with . ug of an h expressing plasmid dna (pcmvri ). antibody responses and protection against a lethal challenge were followed over the next year. antibody responses had good longevity exhibiting comparable titers at one year post boost as at days post boost protection against the lethal challenge was complete at days, month and four months post boost, but only partial at one year. a transgenic mouse model for identifing htlv- t-cell epitopes: generation of hla-b* -restricted ctl directed against synthetic peptides and naturally processed viral antigens, christian schiinbach*, ai kariyone*, kiyoshi nokiharaa+, karl-heinz wiesmulle and masafumi takiguchil, departments of tumor biology* and immunology#, institute of medical science, university of tokyo, tokyo "tokyo university of agriculture and technology, tokyo +biotechnology instruments department, shimadzu corp., kyoto, japan $natural and medical science institute at the university of tubingen, reutlingen, germany the majority of human t-cell leukemia virus type-i (htlv-l), hla class i-resmcted t-cell epitopes have been identified by cloning htlv- patient-derived t cells. here we describe for the frst time a rapid method (reverse immunogenetics) for identifing t-cell epitopes, together with a transgenic mouse model as a guide for testing the cellular immune response to a mixture of the lipohexapeptide immunoadjuvant pamgcys-ser-(lys) and synthetic htlv- peptides which seem suitable for vaccine design. htlv- amino acid sequences were searched for eight to mer patterns carrying the anchor residues of the hla-b* peptide motif at positions two and eight to fourteen. candidate peptides were synthesized according to the matched sequence patterns. their hla-b* affinity was quantitatively analyzed in an indirect immunofluorescence peptide binding assay using rma-s-b* cells. the fourth group (controls) were inoculated with h n (in) thereby providing heterotypic ctl immunity in the context of a natural infection without the confounding effects of humoral immunity against surface antigens. all four types of inoculations have been shown to protect normal (class i expressing) mice from a lethal challenge with influenza, presumably mediated by class i restricted cytotoxic t cells. the two groups inoculated via the intranasal route may gain additional protection by activating the mucosal immune system (iga). none of these types of inoculations has been evaluated in the context of class i restricted cytotoxic t cells, the only ctls found in class i deficient mice. for all four types of inoculations, mhc class i deficient mice lost significantly more weight than the class i expressing control groups (seven mice per group) indicating the importance of class i restricted t-cells in protection. within the class i expressing groups, there was no significant difference between the four types of inoculations; within the class i deficient groups the vac-np im immunized mice lost significantly more weight than the h n group;the other two groups, vac-np in and genetically immunized groups had intermediary results. these data lend support for a protective role for mucosal immunity. results on both class i and class i ctl activity for the four types of inoculations will also be presented. we tested the pbmcs of patients participating in two vaccine therapy trials for their ability to recognize overlapping peptides of the gp la sequence. seventeen patients participating in a phase i gp protocol and patients participating in a phase i gp protocol had their pbmcs isolated by ficoll separation of heparinized venous blood. the fresh pbmcs were plated, in triplicate, into well plates containing peptides overlapping the la sequence of gp , pulsed on day with tritiated thymidine and harvested and counted on day . results: the percentage of patient's pbmcs from each trial with an lsi to each peptide are depicted below. conclusions: the pbmcs of hiv-infected volunteers who have been multiply immunized with either gp or gp proliferate to multiple peptides within the gp molecule. reactivity from the end of c through early c (lai #i - ) is particularly prominent and contains previously undescribed th epitopes (asterisks). conspicuously missing is reactivity to the v loop peptide (la # ). although the percent reactivity to the entire gp molecule is similar between the immunization groups, there is differential recognition of some of the individual peptides, particularly peptides in early c (lai # - ). the intracytoplasmic lifecycle of listeriu mmcytogenes (lm) enables it to be a convenient vaccine vehicle for the introduction of foreign proteins into the mhc class i pathway of antigen presentation. taking advantage of these properties, we have inserted the nucleoprotein (np) gene from lymphocytic choriorneningitis virus (lcmv) into the lm chromosome by site specific homologous recombination. infection of mice with recombinant lm expressing lcmv-np elicited a virus-specific ctl response. we were able to recover lcmv-np specific ctl precursers from recombinant lm vaccinated mice as shown by vigorous secondary ctl responses after in vitro stimulation. in contrast to mice immunized with wild type lm, mice vaccinated with np-recombinant lm were protected against challenge with immunosuppressive lcmv variants. protection was demonstrated by reduced viral titers or complete clearance of lcmv from serum and various organs including, spleen, liver, lung, kidney, and brain. the kinetics of the lcmv challenge indicate that mice vaccinated with recombinant lm were able to arrest viral growth early in the infection due to a strong ctl response and did not exhibit the immunopathology associated with infection of naive mice. since lm not only delivers antigens into the mhc class i pathway but also induces il- production, it has the potential to function simultaneously as a vehicle for expressing foreign antigens and as an adjuvant promoting cell mediated immunity. , latent membrane protein [lmp] and a) in chromium release assays. we were fortunate in identifying one child from whom cryopresetved pbmc samples were available before. and during ebv seroconversion. ebv-specific ctl activity was demonstrated concurrent with initial detection of virus in the peripheral blood by ebv-dna pcr. in the absence of detectable serum antibody. ctl lines from all nine children recognized one or more ebv latent gene prcduct(s). all children demonstrated ctl responses against one or more ebna proteins ( a, b, c). and ebna c was recognized most frequently. no ctl responses were detected against the ebv latent proteins ebna , , lp or lmp . the ebv-specific ctl lines expressed cd /cd and mab blocking experiments demonstrated that the majority of target cell lysis was inhibited by antibody against mhc class-i but not antibody against mhc class- . these results represent one of the first reports characterizing ebv-specific ctl responses in young children. the striking similarity between ebv-specific ctl responses described here in young children and those reported for adults suggests that the ebna family of proteins and lmp a should be considered for inclusion in candidate ebv vaccines. evaluation of cellular immune responses to adenovirus vectors in the cotton rat. soonpin yei,' gary kikuchi,' ke tang' and bruce c. trapnell.' departments of virology' and immunology, genetic therapy, inc., gaithersburg, maryland replication deficient recombinant adenovirus (av) vectors are efficient gene delivery vehicles currently being developed for a variety of in vivo gene therapy strategies such a s for the fatal pulmonary component of cystic fibrosis. the cotton rat (sigmodon hispidus) is one of the most widely accepted animal models for studying these av vectors because wild type human adenovirus replicates in cotton rats and because of the histopathologic similarities of infected respiratory epithelial tissues from humans and cotton rats. despite this, methods for studying immunologic responses in the cotton rat have not been developed. importantly, recent studies in the cotton rat (gene tber. : - ; ) in our laboratory suggest that a dose-dependent specific immune response to av vectors can limit expression of the transgene. in this context, w e have established methods to evaluate cytotoxic lymphocyte (ctl) responses to av vectors in the cotton rat. to accomplish this, a ctl target cell line was established consisting of primary cotton rat lung fibroblasts (crlf). splenocytes from cotton rats exposed previously to an av vector were harvested, cultured in virro with irradiated, addb nfected crlf. cultured (effector) splenocytes were then incubated with s'cr-labelled crlf (target) cells a t effectoctarget (e:t) ratios of , and . in parallel, splenocytes from naive cotton rats served as negative controls. results demonstrated vector-specific ctl lysis of target cells significantly greater than controls: . f . % vs . * . %. . * . % v s . * . %. and . * . % vs . * . % (meanrts.e.m., n= ; p<o.ol, all comparisons) a t e:t ratios of , and , respectively. thus, a specific ctl response does develop in cotton rats following in vivo av vector administration. this observation will be useful in guiding vector modifications in the rational development of improved vectors for clinical use. the identification of ctl epitopes is essential for subunit vaccine design against aids. a major portion of the anti-viral ctl responses after infection with htv in humans or siv in rhesus macaques is made up by anti-gag specificities. here, effector cell populations were established from hiv seropositive individuals by specific stimulation of pbl with pfa fixed autologous b-lcl infected with tw gag. expanded cultures were then tested for ctl activity against autologous b-lcl pulsed with overlapping -mer peptides derived from p . two individuals ( and ) had ctl against p . (a(mino) a(cids) - , krwiilglnknrmysftsi), two others ( and ) recognized p . (aa - , frdwdrfyktlraeqasqd). similarly we found two siv infected rhesus macaques reacting against the analogous aa sequences of siv; it. p . ( ) and p . (ivy). this led us to further fine map the human and rhesus ctl reactivities using sets of aa overlapping -mers and to test for hiv- isiv crossreactivity. both two p . epitopes (possibly restricted by hla-a ) and the p . epitope were non-overlapping, and all three epitopes were non-crossreactive. finemapping of the p . epitopes is in progress. the sn p . epitope was also non-crossreactive, despite only one aa difference (position , s+t) between the siv and hiv sequence. others have also mapped cl'l epitopes to the same regions of p in humans (johnson ji , , , buseyne jv , , and to p . in cynomolgus macaques (gotch jmprim . , ) . in conclusion, multiple non-cross reactive ctl epitopes are clustered within corresponding regions of hiv and siv gag. ctl hotspots may be important for inclusion into vaccines. forming virus) with cd- and cd- -cells, b-cells and adherent cells, while an average of % of virus was associated with lps-stimulated -cells and adherent cells. in the second experiment the combined function of antigen presenting cells, -helper cells and -cells (humoral immune response) to a third party antigen (srbc) during the acute phase of enterovirus induced disease was increased at day , and decreased at days , and post-cvb , infection relative to unlnlected animals. conclusions: these data indicate that cvb , associates with and replicates in rnurine splenic immune cells, and that infection of susceptible mice with cvb , modifies the immune response to a third party antigen during the acute stages of disease. an understanding of such cvb ,-i mmune cell interactions is criitical for gaining insight into the pathogenesis and persistence of enteroviral infection both within and outside of the immune system. understanding the pathogenic mechanisms of virus infections depends upon identification of viral gene products involved in host-virus interactions in vivo. many viral gene products which affect pathogenesis in vivo are nonessential for virus replication in vitro. therefore, we constructed insertion or deletion mutants in the hind i j and i regions of murine cytomegalovirus (mcmv) to assess the importance of these genes in in vivo replication of virus as well as interaction with immune regulatory and effector cells. three mutants replicated in nih t fibroblasts as efficiently as wild-type virus, thus confirming the nonessential nature of the genes. deletion of . kb in hind i j and of . kb in hind i j and i resulted in mutants with reduced replication in target organs in vivo. an insertion in hind i j had no significant effect on replication in mouse salivary gland, but curtailed growth of the virus in footpad fibroblasts and subsequent priming of mcmvspecific cpl precursors in the draining lymph node. in addition, the . kb deletion mutant was unique in its failure to replicate in a permissive, differentiated macrophage cell line. current studies aim to further define the role of this gene region in hcmv pathogenesis. that in vv - progression o f v v life cycle is blocked at the the uncoating ii or replication stage. furthermore, host protein synthesis in v v - cells is not shut-off after v v infection suggesting uncoatingll/replication is important for determining cell fate. since this mutant clone vv - was isolated b y retroviral insertional mutagenesis. a cellular gene that was integrated by single retrovirus was isolate and codes for a kb transcript in northern blot analysis. a kb cdna was isolated and codes f o r a novel polypeptide o f amino acids that show no homology with known proteins. experiments are in progress to understand t h e role of this cellular gene in vv infection. in addition, a two-hybrid system will be employed to identify a n y viral gene product interacting with this cellular target u s i n g o u r laboratory's s t a n d a r d s t r a i n o f a/japan/ / in in uitro assays of viral infectivity of the mouse b-lymphoma a - . . we have found that although infection sensitizes the a cells for recognition by both class i and class i restricted ctl.'s and leads t o high surface expression levels o f hemaglutinin (ha), the infected a cells grow through t h e infection, ha expression declines, and few of the infected cells die. in contrast, using a second s t r a b of a/japan/ / from a different passage history, we find that n o t only are the infected a cells sensitized for lysis by both class i and class ii restricted ctl's, exhibiting even higher levels o f ha surface expression, but in addition, most cells in the infected population die with characteristics of apoptosis. the two virus strabs appear to be highly related because in various assays we have found t h e m antigenically indistinguishable in both their t-and b-cell epitopes. using these two virus strains in a n in uiuo dose response assay, we have found that o u r laboratory's standard strain will kill all mice infected with a - the therapeutic and prophylactic antiviral efficacy of interleukin- (il- ) was studied using murine models of herpes simplex virus (hsv) and murine cytomegalovirus (mcmv) infection. therapeutic intraperitoneal administration of il- commenced hours after mice were infected w i t h hsv, and was continued daily for a total of days. il- therapy improved the survival rates of mice w i t h systemic hsv infection, compared to that of placebo treated infected mice. since neutrophilia and thrombocytopenia were common in dengue patients,we are investigating whether bm may be accompanied with abnormal hematopoiesis. using ficoll-hypaque separation, mononuclear cells prepared from the total bm cells. . x we~ells/well of both adherent and nonadherent cell types were infected with two different den- virus strains : ( ) thiland strain isolated from the dengue hemorrhagic fever(dhf patient and ( ) taiwan strain isolated from the dengue fever(df) patient at moi=l and collected the suprnatants at various time points for assay. the preliminary data showed that : (a) adherent cells were infected with den and dhf virus strain replicated much more efficiently ( . x pfu/ml on day p.i. than df strain; (b) nonadherent cells were times less efficient to produce viral yields than adherent cells (dhf and df strains peaked at . ~ ' pfu/ml and . ~ pfu/ml on day p.i., respectively); (c) addition of gm-csf to nonadherent cells increased virus infectivity and productivity. in conclusion, den viruses were able to infect bm cells and the more virulent virus strain isolated from dhf patients had better replication capability in human bone marrow cells. future studies on cytokines and immunologic evaluation are in progress. number of gene products whose function is to modify host responses. recent evidence suggests that a subclass of poxvirus-encoded host response modifiers interferes with host defense responses, and thus can be termed immune or inflammatory response modifiers (irms). several poxvirusencoded irms possess homology to mammalian proteins which are associated with host defenses, while other poxvirus irms have little sequence homology to immune-effector molecules or their receptors. the ultrecht strain of rabbitpox (rpv) virus induces a large reddish pock on the chorioallantoic membrane (cam) of the chicken embryo. rpv mutants lacking the spi- /crmn k gene trigger an inflammatory response by - hr after inoculation, similarly to that found for cowpox virus (brighton red strain) mutants. rpv mutant viruses with a deletion of the ps/hr gene, a gene which has recently been associated with virus host range and virion maturation, elicited an inflammatory response on the chicken embryo cam at - hr after virus inoculation. thus, another poxvirus-encoded gene product has been identified that has irm functional activity which would not have been predicted by sequence analyses. poxviruses have recently been found to possess a herbert w. virgin iv, center for immunology and division of infectious diseases, washington university school of medicine, st. louis, mo murine cytomegalovirus (mcmv) follows three stages of infection in the immunowmpetent host. following the initial acute infection, a persistent infection remains in which lytic virus resides in the acinar cells of the salivary glands. finally a third stage of infection exists which is characterized by a lack of detectable infectious virus. it is not known whether mcmv remains persistent at some low level during this thiid stage or establishes molecular latency. to test this, spleens, kidneys and salivary glands from mice - months post infection were evaluated by two sensitive assays. sensitivity was tested using virus co-cultured on murine embryo fibroblasts (mefs) for days. by this method, pfu mcmv was reproducibly detected in the presence of sonicated spleen, kidney and salivary glands diluted :lo. scid mice were used as a second detection assay for mcmv. using scid mice and different doses of virus, the lds, of mcmv in scid mice was - pfu. mcmv infections were detected when - pfu were added to sonicated spleens, kidneys, and salivary glands prior to injection into scid mice. using both co-culture with mefs and injection of tissue into scid mice, we have tested a total of spleens, kidneys, and salivary glands and have detected no persistent virus. while no preformed virus existed in these tissues, reactivation from explanted spleen, kidney and peritoneal exudate cells occurs in culture after - days. in addition, lytic mcmv was detected in scid mice days after injection with ( )' kidney cells from latently infected mice. following identical transfers of latent spleen cells, mcmv was not detected in scid mice although explants from the same organs reactivated in vitro. using facs sorting, panning, magnetic bead separation, and reactivation assays, individual cell types have been analyzed for the presence of mcmv genome and the ability to reactivate virus. heat shock proteins (hsp) are expressed in all organisms in response to a variety of stresses, including virus infection. however, since viruses are not known to encode hsp genes, this represents expression of cellular hsps. we have found a dramatic induction of the kda hsp in the ovaries of w-infected mice, and using primary ovarian fibroblasts, demonstrated hsp mrna within virus-infected cells. the physiological role of hsps expressed during virus infection is unknown, although hsps act as molecular chaperones to facilitate protein folding and assembly in unstressed cells, and a number of bacterial hsps are essential for bacteriophage replication and morphogenesis. in order to determine whether hsp expression was beneficial to vv replication, we constructed a recombinant vv which encoded murine hsp , but found that this virus grew to similar titres as control viruses, both in vitro and in vivo. in particular, kinetic studies of virus growth in an hsp -negative cell line showed no difference from control viruses, and the presence of hsp did not influence the virulence of infection in immunocompromised nude mice. these results are interesting given that hsp proteins act as molecular chaperones and that hsp can be found in association with vv proteins. we suggest that this association may simply reflect the inherent affinity of hsp for non-native proteins, and the close physiological proximity of hsp and nascent viral proteins within the virus infected cell. our results indicate that the functional significance of these interactions are minimal with respect to the outcome of virus infection. furthermore, although the expression of hsw in vv-infected mice coincides with the peak anti-viral cellular immune response, hsp does not appear to be immunologically significant, as we cannot demonstrate any immunological responses to hsp during the course of vv infection. the effect of in vivo neutralisation of ifn-y on cytokine production during influenza infection was compared in cs /bl and balb/c mice. similar cytokine profiles were obtained on in v i m restimulation of mln or spleen cells from virus-infected mice of each strain. at days and postinfection, mln or spleen cells from both strains of mice produced il- , il- , il- and ifn-y, but little or no il- or il- . however, following in vivo treatment with anti-ifn-y antibody, production of l- and il-s was induced in mln and spleen cells of balb/c mice whereas those from cs /bl mice showed little change in cytokine profile. an increase in il- and l- production was also observed on in virro restimulation of splenocytes from balb/c mice. however, significant amounts of l- and ifn-y were still secreted in these cultures. in v i m neutralisation of ifn-y in the restimulated cultures had little effect on the production of other cytokines, regardless of whether this cytokine had also been neutralised in vivo .despite the change in cytokine profile, anti-ifn-y treatment had no effect on the kinetics of viral clearance in balb/c mice. a similar situation was seen for c /bl mice. congenic strains of mice are currently being used to determine whether the effect on cytokine profiles is associated with the mhc haplotype. wunner, and steven l. spitalnik, department of pathology and laboratory medicine, university of pennsylvania and the wistar institute, philadelphia, pa rabies virus glycoprotein (rgp) is the only glycoprotein on the viral surface, is the target of viral neutralizing antibodies, and is the viral protein that interacts with the host cell receptor. rgp of the era strain is a amino acid type i membrane glycoprotein with potential n-glycosylation sites at asn , asn , and asn . due to rgps central role in the immunology and biology of rabies, it is important to determine its three-dimensional structure. towards this end we produced a recombinant form of rgp that may be more amenable to analysis by x-ray crystallography. to avoid the use of detergents, we constructed a soluble amino acid form of rgp lacking a transmembrane domain (rgpt ). rgpt was secreted by transfected cells and was appropriately glycosylated, assembled, antigenic, and immunogenic. since n-glycosylation was required for rgpt secretion, we minimized the number of n-glycans by site-directed mutagenesis. interestingly, rgpt was secreted only when asn l was n-glycosylated. in contrast, full-length rgp was expressed at the cell surface a any of the three potential sites was n-glycosylated. soluble inhibitors of n-glycosylation were used to simplify the structures of the n-glycans on rgpt . although inhibiting alpha-glucosidases with castanospermine blocked secretion, inhibiting any further processing with deoxymannojirimycin had no effect on secretion. finally, to simplify the purification process, a tail of his residues was added to the cooh-terminus of rgpt . this modification did not affect secretion, antigenicity, of assembly of the recombinant glycoprotein, but did allow purification using ni+ -agarose chromatography. in summary, we constructed a soluble form of rgp with a minimal number of homogeneous n-glycans and an "epitope" tag. this form of rgp is structurally similar to the extracellular domain of the full-length protein and may be amenable to analysis by x-ray crystallography. endogenous retroviral genes (ev) are well characterized in chickens. our laboratory has established chicken lines with single and multiple ev loci and, compared to ev negative birds, these lines exhibit a delayed and suppressed titer of neutralizing antibody in responses to avian leukosis virus (alv) challenge. to address the effects of ev genes on the cell mediated immune response we have developed an in-vitro assay to monitor the in-vivo induction of mhc restricted cytotoxic t cells (ctl) induced by alv. using this assay, we find the ctl response in birds expressing the ev- locus is reduced to % compared to ev negative birds having nearly identical (eighth backcross) genetic backgrounds. other ev loci ( , , and ) also reduce the ctl response to alv in mhc matched chickens with different genetic backgrounds. ' national public health institute. helsinki. finland universities of tampere, qulu and 'helsinki, finland coxsackie b virus infections have been associated with clinical iddm in seveml previous studies but their initiating role in the slowly progressing beta-cell damage was for the fmt time directly implied in our recent prospective. nationwide dime study. siblings of the index iddm cases who progressed to clinical iddm experienced enterovim infections more frequently than nonprogressing siblings during prospective follow-up period. enterovim infections were initially demonstrated by detecting significant increases in serum class-specific cross-reactive enteroviral antibodies by using radioimmunoassay (ria) with the heavy-chain capture principle. causal association benveen these infections and the pathogenesis of iddm was suggested by temporal association of sercconvenions of viral antibodies with increases in islet cell antibody levels which are considered to be markers ok ficell damage. the possibility of the existmce of specific diabetogenic s & s of enternviruses was then considered. to identify the serotypzs responsible for the infections, successive sera from subjects exhibiting seroconvenion of autoantibodies coinciding with an increase in enteroviral antibodies were anlyzed by plaque n e u n a t i o n assay using a set of different enterovhes. the results indicate that several enternvirus serotypes may be associated with the progressive prediabetic p~ocess. not only different coxsackie b but also coxsackievirus a -infections were found to coincide with seroconvenions of islet-cell and insulin auwantibodies. it is known that an immunogenic region of gad . one of the major isletcell antigens, shows a high degree of homology with the c protein of cbv- . studies on potential immunological cross-reactions between enteroviruses and two significant autoantigens, hsp and gad , are in progress. this is carried out by using immunohistochemistry in infected cell lines and in frown sections of human pancreas with antibodies induced by synthetic peptides and natural and expressed viral proteins, the role of p alterations in human malignant pleural mesotheliomas (mpm) is unclear. immunostaining (ipox) of snap frozen mpm specimens revealed p expression in of ( %). p detection by ipox is usually associated with mutated p . sscp for exons - revealed p alterations in of specimens, and loh at exon was seen in only of evaluable specimens. thus, most of these specimens appeared to contain wild-type (wt) p . we recently reported that sv induces mesotheliomas in rodents, and that sv -like sequences are present and expressed in mpm. of note, sv large t antigen (tag) binds and inactivates wt p i n ! , abolishing its tumor suppressor function. we theorized that the immunohistochemical persistence of p may result from its physicdl binding with tag. tag was detected by ipox in ( %) of these mpm specimens. expression of tag was significantly associated with coexpression of wt p (p = . , fisher's exact text), and preliminary experiments suggest co-precipitation of p and tag. finally, we demonstrate that waf- is not detectable in mpm expressing wt p suggesting that p is inactive. we speculate that tag expression in mpm binds to and inactivates p contributing to the malignant phenotype. human papilloma virus (hpv) infection is involved in % of cervical carcinomas and possibly % of cervical intraepithelial neoplasia (cin). at present it is unclear whether patients infected with the transforming hpv types can mount efficient t cell responses. to address this issue we examined the ctl response of peripheral blood lymphocytes from patients attending a cervical neoplasia outpatient clinic. all were diagnosed hpv positive with various stages of c m and nine patients were selected for hla-a expression by facs analysis. pbmc were stimulated with caski, a cervical carcinoma cell line demonstrated to be hf' v type ' and hla-a '. culture media consisted of rpmi- % human serum supplemented in three cases with u/ml il- , in another three cases with u/ml il- and u/ml il- , and in the final three cases with u/ml il- and u/ml il- . the ctl were screened for reactivity to caski and in addition the cervical carcinoma cell line c a (hpv, hla-a ') transfected with either the hpv type e or e genes. one patient's cells maintained with uiml il- and ulml il- showed hpv e protein specific cytotoxicity in a hla-a restricted fashion. these ctl lysed the caski stimulator cell line but not the nk sensitive target k- . the ctl efficiently lysed a c a-e clone but in contrast a vector only transfectant was not lysed. the lysis of c a-e was blocked with a-cd and a-cd antibodies at % and % respectively. facs analysis determined that this ctl population was . % cd 'cds' and . % cd 'cd '. limited e recognition was also observed but has not been hlly characterized to our knowledge this report represents the first demonstration of hpv e responsive ctl isolated from the peripheral blood of hf' v infected patients. we have previously shown that proviral variants found in the peripheral blood mononuclear cells (pbmc) of hiv- infected individuals can interfere with recognition by autologous cytotoxic t-lymphocytes (ctl). abolition of recognition could be caused by interference with t h e processing of viral peptides, by a failure of the variant peptides to bind to mhc class-i molecules or by a failure of the mhc class-vpeptide complex to efficiently activate the tcr (t-cell receptor). these mechanisms could allow the virus to escape the immune surveillance by ctl. most studies so far have addressed the question of viral variation in t-cell epitopes by analysing proviral dna, extracted from the pbmc of hiv- infected individuals. however, the analysis of virion-associated rna offers several advantages. sequences found in viral rna are likely to be functional, a t least u p to the point of packaging into virus particles. furthermore, rna sequences represent virus which is currently actively produced. here, we present the sequence variation i n two hla-b restricted epitopes: p - gag and amino acids - of the pol gene. both proviral dna from pbmc and viral rna sequences from plasma are discussed. the ability of viral variants to hind to mhc class-i molecules was assessed and the recognition of variant peptides by ctl was tested. variants were also tested for their ability to antagonize recognition of the index sequences. we show that viral variants with the ability to interfere with recognition by ctl are not only found in proviral dna but also in virion associated rna. objective: clinical course of hiv- infection may be influenced by an effective host immune response against hiv- and/or by viral properties. to investigate the cellular immune response to hn- we analyzed ctl activity against different hiv- proteins in long-term asymptomatic hiv- infection as well as during rapid progression to aids. methods: longterm asymptomatic individuals with cd counts > celllpl after more than years of infection were selected from the amsterdam cohort study on aids versus subjects who progressed to aids < years. ctl activity was measured on "cr labelled hla matched or autologous b-lcl, infected with rvv expressing hiv- ag. both bulk and limiting dilution ctl assays were performed longitudinally with pbmc after ag-specific stimulation. sequences of ctl epitopes were determined in homologous virus isolates resulrs: different kinetics of anti-gag ctl responses were observed in rapid progressors. in any case ctl responses disappeared during progression to aids. in long-term asymptomatic subjects persistent ctl responses were observed together with low viral load. conclusions: sustained, broad anti-hiv cellular immunity may correlate with maintenance of the asymptomatic state in long-term survival by controlling viral replication. enteroviruses are a large group of positive stranded rna viruses known to be responsible for a number of distinct disease entities. recombination is thought to be capable of generating new enterovirus strains that cause significant morbidity. for example, enterovirus which was responsible for a pandemic of haemorrhagic conjunctivitis and poliomyelitis is thought to have originated by recombination between a coxsackie b like virus and another unidentified enterovirus. we are studying a group of echovirus isolates from an outbreak of disease in southem india. sequence analysis within the ' untranslated region reveals that these isolates fall into two groups that differ by - % (equivalent diversity to that seen between between published sequences of poliovirus and coxsackie a virus). these two groups of viruses also differ in their cell tropism. isolates defined as group by their 'utr sequence grow equally well on ht cells (a human colon carcinoma cell line) and vero cells. isolates of group , with one exception, grow only on ht cells. analysis of the structural proteins of these isolates revealed differences in migration that correlated with their cellular tropism. thus, significant genotypic and biological diversity exists amongst these virus isolates. one virus isolate had the ' untranslated region sequence of a group virus but the protein profile and cellular tropism of a group virus. the best explanation of these findings is that this anornolous isolate is a natural recombinant between the parented strains. both the ease with which viable recombinants are generated and the diversity present within this one enterovirus serotype increase the potential for the production of novel pathogenic enterovirus strains. dominant susceptibility to polyoma tumors in inbred wild mice, sharon r. nahill, yupo ma, john carroll and thomas l. benjamin, department of pathology, harvard medical school, boston, ma polyoma virus (f' y) is a mouse dna tumor virus which, under appropriate conditions, causes tumors in a wide variety of cell types. generation of tumors is a function of both the viral and host genomes. lukacher et al. have recently described a dominant gene, pyv', carried by the c -i mouse strain, which confers susceptibility to py-induced tumors mapping and immunological analyses indicate that py is the mouse mammary tumor virus superantigen (mtv sad gene, which deletes t cells required for py tumor immunosurveillance in h- ' mice. to determine the generality of endogenous superantigens as determinants of susceptibility and to reveal potentially novel mechanisms of susceptibility, we have looked for dominant susceptibility @ s ) gene(s) id newly established and genetically diverse inbred wild mouse strains, czech i and pedatteck (peru). both strains are susceptible to py as % of infected animals develop a full profile of tumors. crosses between cs br, whose resistance is contributed by the major histocompatibility (mhc) locus, and susceptible peru or czech , yield f progeny which are fully susceptible, indicating a dominant inheritance pattern of susceptibility the incidence of tumor-bearing backcross animals [((peru x cs br) x c br) and ((czech i x cs br) x c br)i suggests that ds is due to at least one, but not more than two genes. amplification of genomic dna from the czech i and peru mice by pcr using primers specific for mtv sag indicates that both strains are negative for proviral mtv sag. furthermore, the mechanism ofds in these mice may be independent of all mtv sag as pcr using primers specific for the highly conserved region of mtv sag is unable to amplify mtv dna from peru or czech i genomic dna. these results indicate that, like the c hibi, the pedatteck and czech i contain gene(s) which overide the resistance to py-induced tumors contributed by the mhc of the c br parent and which may cause tumors via a novel, mtv sag-independent mechanism. we have initiated efforts to map the ds in peru and czech i mice using pcr and primer pairs flanking simple sequence length polymorphisms. fis- is a low leukemogenic, but relatively strong immunosuppressive variant of friend murine leukemia virus (f-mulv). this variant was originally isolated from t-helper cells of flc-infected adult nmrl mice. compared to f-mulv, fis- suppresses primary antibody response more efficiently in infected mice. some of the fts- infected adult nmri mice developed a disease resembling the acquired immunodeficiency syndrome induced by hiv. restriction mapping and nucleotide sequence analysis of fis- show a high degee of homology between this variant and the prototype f-mulv clone . in this study we have attempted to localize the genomic determinant of fis- which is responsible for induction of a strong suppression of primary antibody response. six chimeric viruses of fis- and f-mulv were constructed. the primary antibody response of the mice infected with these chimeric viruses were investigated. the results of these experiments will be presented. anti-fmdv antibodies, as measured in an elisa capture assay, were cross reactive. b) cellular: proliferative (cd ) t cell responses of peripheral blood mononuclear cells (pbmc) were low or undetectable during primary responses to vaccine or virus, and frequently low during secondary responses. for good t cell proliferation in vitro, multiple immunisation is required. this may reflect preferential stimulation of the th cd t cell subset. interestingly, when cd responses were observed, cd tcell responses were also detectable. . recognition of individual viral proteins a) expression cloning: structural and non-structural protein pseudogenes were cloned from cdna by pcr. expressed in pgex- xuc. and purified by sds-page. b) humoral: structural and non-structural proteins were recognised by infected animals. a good anamneetic antinon-structural response was only observed when the boosting serotype differed from the serotype stimulating the primary response. c) cellular: both structural and non-structural proteins were recognised and some were cross reactive. interestingly, vp was strain specific, and the polymerase ( d) was the most immunogenic and cross reactive. d) a construct comprising d and the immunodominant vp epitopes was prepared and tested. in common with other herpesviruses, the envelope glycoproteins of equine herpesvirus (ehv- ; equine abortion virus) are major determinants of the infectious process and pathogenicity, and are inducers of humoral and cell-mediated immune responses. as such, they are candidates for components of subunit vaccines against ehv- . to generate useful amounts of individual ehv- glycoproteins, we have constructed recombinant badoviruses capable of expressing glycoproteins c, d, h (gc, gd, gh ) in insect cells, and have evaluated the recombinant products as innnunogens in a murine model of ehv- infection. au three glycoproteins induced serum (elisa) antihodies to ehv- , and ehv- gc and gd also induced neutralizing antibody responses. following intranad challenge with infectious ehv- , protective immunity, as demonstrated by acelerated clearance of virus fiom respiratory tissues to below detectable levels, was evident in mice immunized with either recombinant gc or gd. in contrast, gh-immmkd mice did not develop detectable neutralizing antibody, and did not clear challenge virus more rapidly than controls. delayed type hypersensitivity and lymphoproliferation responses to ehv- antigen were observed for each of the ehv- glycoproteins, and in experiments with gdimmunized mice, a role for cell mediated immunity in protection was confirmed by adoptive transfer and t-cell depletion experiments. the data provide support for the potential of glycoproteins c and d as a subunit vaccine against ehv- . molecular pathogenesis of ural infeetiom - enterovirus-immune cell interactions: implications in enterovirus-induced diseases we have also evaluated the effect of virus infection on the humoral immune response to cvb , infection in adolescent c h/hesnj mice. antigen presenting cell, -helper cell and -cell function were evaluated utllizlng a sheep red blood cell (srbc) plaque assay. mice were injected intrapentoneally (ip) with lo plaque forming units of cvb , at day and with lo srbc's at days , , and post-cvbb, infection. splenocytes were harvested days post-srbc injection, mixed with target srbc's and guinea pig complement and incubated. plaques were then quantitated. results: cvb , was associated with . % to . % of cd- positive t-cells and w a % to % of adherent splenocytes. after mitogen (lps and con a) stimulation, b-cells and adherent cells were demonstrated to be permissive for viral replication. a % and % under non-stimulated conditions. an average of % of virus is cell-associated (plaque north america. bruce anderson, teny yates, norah torrez-martinez, wanmin song, brian hjelle. university of new mexico, albuquerque, n.m.we recently identified a new species of hantavirus (hmv) associated with the harvest mouse reithrodontomys megalotis (hjelle b et al, j. viroj. , in press ). an arizona woodrat (neotoma mexicana) was found to he infected with hmv, presumably through "spillover". hmv is most closely related to the four comers hantavirus (fcv) of deer mice (genus peromyscus). the nucleocapsid gene and protein of hmv differ from those of fcv by % and % of residues, and the nt s genome is shorter by nt. we surveyed reithrodontomys animals captured in the u.s. and mexico for hantavirus antibodies; ( . %) were positive. s segment cdnas were amplified and sequenced from seropositive animals captured in california ( ), arizona ( ), new mexico (l), and mexico ( ). a monophyletic clade of hmv-like agents was identified at all sites, although an r. megalotis infected with an fcv-like virus was also identified in the state of zacatecas, mexico. nucleotide sequence distances among members of the hmv clade were up to . %. but amino acid distances were less than %. hmv is enzootic in harvest mice throughout much of north america, and can also infect wood rats. htlv i-associated myelopathy/tropical spastic paraparesis (hamnsp) is a slowly pro ressive neurological disease characterized by perivascuaar mononuclear infiltrates in the cns. htlv i-specific cd + ctl are found in pbl and csf of infected patients with htlv i-associated neurological disease but not in htlv i seropositive individuals without neurological involvement. previous studies have shown that in hla-a + patients, htlv i-specific cd + ctl restricted by hla-a recognize a peptide derived from the htlv i tax protein (tax - llfgypvw). in the present study, we have analyzed the potential of these tax-specific ctl to recognize addtional peptides. our results demonstrate that a subpopulation of high affinity cd ' tax - specific ctl clones cross-react on a self peptide derived from the se uence of myelin-associated glycoprotein (mag - vl&sdfri) presented by hla-a . these obsenatlons suggest that the demyelination process in hamltsp may be,due, in part, to virus-specific ctl recognition of a self myelin component that is independent of htlv i infection. development of pathology varies widely between different strains of mice after intracerebral inoculation with the so-called 'docile' isolate of lymphocytic choriomeningitis (lcm) virus. the c hebfej and blo.br/sgsnj mouse strains have been of special interest because they display autoimmune hemolytic anaemia with varying degrees of apparent immunological involvement. in this study, we examined the role of cd + t helper cells in this autoimmune response by treating mice with the cw-specific gk . monoclonal antibody. we also determined if polyclonal activation of b lymphocytes, induced either by lcm virus or by lactate dehydrogenase-elevating virus, another well known b cell activator, correlated with the development of anaemia in these mice. our results strengthened the central role of the immune system in the anaemia in c h mice by showing that depletion of cd + cells largely, if not completely, abrogated this anti-erythrocyte autoimmune reaction. as reported by others, we found that the anaemia was more mild in b o.br mice than in c h mice. however, we could not confirm the difference in the degree of b lymphocyte polyclonal activation between these mice. furthermore, lactate dehydrogenase-elevating virus had no apparent effect on erythrocytes, even though this virus also induced a sharp increase in plasma igg levels. one of the two class i mhc (h-pkd)-restricted immunogenic sites identified on the influenza strain aijapanl (h n ) hemagglutinin (ha) encompasses two distinct partially overlapping epitopes, mapping to residues - and . . when we investigated the magnitude of the ctl responses of balwc mice to the two overlapping epitopes, we found that while the nhrterminal nonamer epitope is immunodominant, eliciting vigorous ctl responses in njapanl -immunized balb/c mice, the ctl responses to the cooh-terminal decamer epitope are weak and variable. the c-terminal epitope subdominance seems to be due to factors other than inefficient processing of the epitope in vivo because ctls generated by priming mice with recombinant sindbis viruses expressing only one of the ha - subsites displayed patterns of responsiveness similar to that of influenza virus primed ctls. limiting dilution ctl assays showed that the ctl precursor frequency (pctl) of the nterminal epitope is at least ten fold higher than the pctl of the cterminal epitope, implying that the low and variable pattern of cterminal specific responsiveness was due to the limited t cell precursors in the c-terminal specific ctl repertoire. this was further confirmed by the limited heterogeneity in the cross reactivity patterns displayed by the c-terminal specific ctl for an ig vh fragment and the ha - epitope of influenza strain a i m in short term bulk cultures, and the facs analysis of tcr vg chain usage. taking these together with our previous observation that some jha - specific ctls can also crossrecognize an ig vh fragment. these studies had provided a strong evidence that ig gene products may influence t lymphocyte function and repertoire development. we have previously described the identification of homologous regions in the c-terminus of hiv- gp and in the n-terminus of hla class i beta chains. forty percent of patients infected with hiv-i virus were shown to have antibodies which bind to the homologous sequences, as well as to native hla class i molecules. affinity purified crossreactive antibodies (crab) were shown to have direct blocking effects on normal t cell responses to recall antigens, and could mediate adcc of hla class ii+ cell lines.in order to determine the contribution of such antibodies to disease progression, we obtained longitudinal plasma samples from patients in the macs study. in a first study, it was found that the presence of high titers crabs correlated with a more rapid disease progression (p = . by fisher two tail analysis)in a second, year-longitudinal study of progre.ssors and stable patients we found: ( ) the production of crab was seen in - % of rapid progresson, while the true stables produce only infrequent low-titers crab. ( ) in rapid pmgressors, production of crab preceded by - years the marked drop in cd counts. ( ) crab production did not correlate with the degree of hyperglobulinemia in these patients. ( ) the presence of crab during the asymptomatic stage correlated with early loss of t-helper responses to recall antigens.we are currently establishing whether periodic measurements of crab in patients sera could be valuable in predicting a drop in cd counts and disease progression. the lymphokine ifn-y is i pleiotropic insnunomodulator and possesses intrinsic antiviral activity. we studied its significance in the development of antiviral immune responses using ifn- receptor deficient (ifn-yr-'.) mice. after inoculation with live attenuated pseudorabies virus (prv) the mutant mice showed no infectivity titers in various tissues and transient viral ag expression only in the spleen similar as in wild-type mice. however, the absence of the ifn-yr resulted in increased proliferative splenocyte responses. the prv-immune animals showed a normal ifn- and - production, without detectable - , and with decreased - secretion in response to viral ag or con a. immunohistochemically, an increased ratio of ifny/i - producing spleen cells was found. after immunization with either live attenuated or inactivated prv, ifn-yr"' mice produced significantly less antiviral antibody (ab), and more succumbed to challenge infection than the intact control animals. the reduction in ah titers in the mutant mice correlated with lower protection by their sera in transfer experiments. thes? findings are in line with the strong enhancing effect of exogenous ifn-y on rabies virusand prv-specific igg responses. our data demonstrate that a physiological ifn-y system is surprisingly not critical for the generation of antiviral th-i-type and the suppression of th- -type cytokine responses. the lymphokine, however, is an important mediator in the generation of protective antiviral ab. key: cord- -zjq cscm authors: de moura, ronald rodrigues; agrelli, almerinda; santos-silva, carlos andré; silva, natália; assunção, bruno rodrigo; brandão, lucas; benko-iseppon, ana maria; crovella, sergio title: immunoinformatic approach to assess sars-cov- protein s epitopes recognised by the most frequent mhc-i alleles in the brazilian population date: - - journal: j clin pathol doi: . /jclinpath- - sha: doc_id: cord_uid: zjq cscm aims: brazil is nowadays one of the epicentres of the severe acute respiratory syndrome coronavirus (sars-cov- ) pandemic and new therapies are needed to face it. in the context of specific immune response against the virus, a correlation between major histocompatibility complex class i (mhc-i) and the severity of the disease in patients with covid- has been suggested. aiming at better understanding the biology of the infection and the immune response against the virus in the brazilian population, we analysed sars-cov- protein s peptides in order to identify epitopes able to elicit an immune response mediated by the most frequent mhc-i alleles using in silico methods. methods: our analyses consisted in searching for the most frequent human leukocyte antigen (hla)-a, hla-b and hla-c alleles in the brazilian population, excluding the genetic isolates; then, we performed: molecular modelling for unsolved structures, mhc-i binding affinity and antigenicity prediction, peptide docking and molecular dynamics of the best fitted mhc-i/protein s complexes. results: we identified immunogenic epitopes in the sars-cov- protein s that could interact with different mhc-i alleles (namely, hla-a* : ; hla-a* : ; hla-a* : ; hla-a* : ; hla-a* : ; hla-a* : ; hla-a* : ; hla-a* : ; hla-a* : ; hla-b* : ; hla-b* : ; hla-b* : ; hla-b* : ; hla-b* : ; hla-c* : ; hla-c* : and hla-c* : ) in the brazilian population. conclusions: being aware of the intrinsic limitations of in silico analysis (mainly the differences between the real and the protein data bank (pdb) structure; and accuracy of the methods for simulate proteasome cleavage), we identified epitopes able to interact with mhc-i more frequent alleles in the brazilian population that could be useful for the development of strategic methods for vaccines against sars-cov- . immunoinformatic approach to assess sars-cov- protein s epitopes recognised by the most frequent mhc-i alleles in the brazilian population in december , a novel coronavirus strain was detected and isolated in the city of wuhan, hubei province, china. this emerging viral infection was associated with severe human respiratory disease with a fatality rate of ~ %- %. despite the strict containment measures, severe acute respiratory syndrome coronavirus (sars-cov- ) has spread rapidly worldwide, with cases now confirmed in multiple countries and propagation still ongoing. on january , the who declared covid- a public health emergency of international concern. brazil is actually one of the epicentres of the pandemic. day by day, the epidemic advances with a high daily rate of cases per million people. nonetheless, these data may be underestimated because of the reduced number of sars-cov- molecular detection tests being made. the sars-cov- is an enveloped positivestrand rna virus belonging to the betacoronavirus genus of the coronaviridae family of the nidovirales order. sars-cov- genome has ~ kilobases that encode structural and nonstructural proteins. the ′ encodes two large genes (orf a and orf b), which are translated into two polyproteins (pp a and pp ab), which are cleaved in a set of non-structural proteins essential for virus replication. the ′-terminal region of the genome encodes four structural proteins, namely spike (s), nucleocapsid (n) envelope (e) and membrane (m). the protein s on the virion surface mediates receptor binding and membrane fusion. for sars-cov- to infect a host cell, it is mandatory that protein s is cleaved (s /s cleavage) by host cell proteases into two units: the n-terminal ectodomain (s ) subunit and the c-terminal membrane-anchored (s ) subunit. during the receptor binding process, s binds via its receptor binding domain to the extracellular peptidase domain of the host receptor ace- , which is mainly expressed in the lung, gastrointestinal tract, kidney and heart tissues, although it is also present in other tissues, dictating viral tropism. after that, s subunit mediates membrane fusion. although cell entry is not yet fully understood, it is likely that a second proteolytic site (s ′) at subunit s is required for viral entry. viral entry triggers the host's immune system and initiates an inflammatory cascade that starts with the mechanism of antigen presentation. during the process of presentation of antigenic peptides to cd + (cytotoxic) t cells by class i major histocompatibility complex (mhc-i) molecules, peptides are generated by proteasomal cleavage in the cytosol and transported to the lumen of the endoplasmic reticulum by original research a transporter associated with antigen processing protein before they can bind mhc groove and trigger an immune response. the correlation between mhc-i and the severity of the disease in patients with covid- has been previously hypothesised. the understanding of which sars-cov- epitopes are immunogenic may help advances in the development of diagnostic kits and prophylactic vaccines. an immunoinformatic approach is suitable for an initial screening, since it may predict algorithms and test for the immunogenicity of a vast quantity of peptides and alleles in a cost-effective manner, reducing the costs and time on workbench. here, we analysed sars-cov- protein s peptides aiming to prospect epitopes and their ability to elicit an immune response mediated by the most frequent mhc-i alleles in the brazilian population. we searched for the most frequent mhc-i alleles in the brazilian population through the human leukocyte antigen (hla) allele frequency database. we selected allele frequency data from overall brazilian populations, excluding genetic isolates, such as indigenous tribes or quilombos. after that, we calculated the average allelic frequency for each hla-a, hla-b and hla-c allele with data from more than one population data. finally, we chose the most frequent class i alleles. the protein data bank (pdb) structure of some alleles was not available (table ) . therefore, we used swiss-model to perform modelling by homology. the best model was chosen using the default parameters. model refinement was made using drefine software. the quality of the refinement was evaluated by the molprobity score. for model validation, errat and rampage softwares were used with their default parameters. for immunogenicity inference, we used the netmhcpan v. . server, by importing the fasta sequence of the sars-cov- spike protein retrieved from pdb (pdb id: vsb) that will be subsequently cleaved in peptides with a length of -mer. the interrogation of each -mer peptide was made querying the selected alleles ranked in the previous section. we accepted as possible candidates, those allele:peptide complexes with strong binding affinity, that is, those with %rank score < . . after that, we tested the strong binding peptides for probable antigenicity using vaxijen v. . , using a threshold of score < . to filter out possible non-antigenic peptides. peptide docking was carried out using the cabs-dock server. we submitted to a docking simulation, those peptides that showed strong binding affinity and probable antigenicity to their specific mhc-i allele using the default parameters. after the docking, we excluded the complexes that showed root mean squared deviation (rmsd) values above Å to follow the molecular dynamics (md). the complexes from the docking simulations (rmsd values below Å) were further investigated by md simulations using gromacs . . the complex was inserted in a . nm cubic box, filling the voided space with spc-e water molecules. calcium and sodium ions were added for reaching the system electronic equilibrium. energy minimisation was carried out using default parameters. the equilibrium phase was made during ns (for temperature and pressure), while the production phase lasted ns. these steps were also performed using default parameters. in order to investigate the stability of the complexes, we calculated rmsd, root mean squared fluctuation, radius of gyration (rg), solvent accessible surface area and the determination of hydrogen bonds. table shows the most frequent hla-a, hla-b and hla-c alleles, according to the hla allele frequency database. the top hla-a alleles correspond to . % of the a* alleles, whereas the top hla-b alleles represent % and the hla-c alleles . %. these values may not be sufficient to depict the mch-i allelic variation, but it can give an estimation of the major diversity of the class i genes. we modelled the mhc-i alleles, whose pdb structures were not available. the templates used for model prediction, refinement and validations are reported in online supplementary table . overall, the models were considered suitable for docking analysis since they presented significant amino acid sequence similarity to the templates and were above rampage and errat thresholds ( % and %, respectively). two hundred twenty-eight peptides were considered with strong binding affinity, according to netmhcpan v. . results (online supplementary table ); of these, were also considered probably antigenic based on the vaxijen v. . results. we carried out peptide docking simulations for all of them except for two (pep and pep ), which could not be docked by cabs-dock server, possibly due to some issues with the mhc-i structure. as result, peptides had good docking predictions, as they showed average rmsd < Å (online supplementary table analysing the trajectories for hla-c, it was possible to observe that the most stable complexes were hla-c* : , with pep (online supplementary figure ) ; hla-c* : , with pep (online supplementary figure ) and hla-c* : , with pep (online supplementary figure ). the latter complex, though, appeared to be stable only by the end of the simulation. there are a few works in the literature using a similar approach to evaluate possible immunogenic peptides in the sars-cov- protein s. one study identified five cd + t cells epitopes (ylqprtfll, gvyfastek, epvlkgvkl, vvnqnaqal and wtagaaayy) and eight b cell epitopes that bind the mhc class i and ii alleles more frequent in china. a second study found mhc-i (sqcvnlttr, gvyyhknnk, gkqgnfknl, giyqtsnfr, vsptklndl, kiadynykl, kvggnynyl, egfncyfpl, gpkkstnlv, sprrarsva, lgaensvay, fknhtspdv and deddsepvl) and three mhc-ii possible protein s antigenic peptides. joshi et al reported the mhc-i itlcftlkr as a possible candidate for vaccine development. comparing these previous results with our simulations, only gwtagaaayy complexed with hla-a* : and hla-a* : presented good netmhc-pan and vaxijen scores, as well as good docking stability and rmsd during the md. in conclusion, being aware of the intrinsic limitations of in silico analysis (differences between the real and the pdb structure, accuracy of the methods for simulate proteasome cleavage, as well as molecular modelling, docking and dynamics' shortcomings), we described epitopes present in the sars-cov- protein s that could interact with different mhc-i alleles in the brazilian population. these epitopes can elicit an effective cd + t cells immune response and could be useful to develop strategic methods for vaccines against covid- . finally, our immunoinformatic approach could be a useful tool to determine a guided starting point to design and develop epitope-based vaccines. take home messages ► the understanding of which severe acute respiratory syndrome coronavirus (sars-cov- ) epitopes are immunogenic may help in the development of diagnostic kits and prophylactic vaccines. ► an immunoinformatic approach is suitable for major histocompatibility complex class i (mhc-i) screening, to predict the immunogenicity of a vast quantity of peptides and alleles in a cost-effective manner. ► sars-cov- protein s peptides have been analysed in silico aiming to prospect epitopes and their ability to elicit an immune response mediated by the most frequent mhc-i alleles in the brazilian population. ► twenty-four epitopes present in the sars-cov- protein s have been observed, which could interact with different mhc-i alleles identified in the brazilian population. handling editor runjan chetty. early transmission dynamics in wuhan, china, of novel coronavirus-infected pneumonia real estimates of mortality following covid- infection covid public health emergency of international concern (pheic) who. coronavirus disease (covid- ) situation reports coronavirus pandemic (covid- ) -the data sars-cov- and coronavirus disease : what we know so far covid- , sars and mers: are they closely related? emerging novel coronavirus ( -ncov)-current scenario, evolutionary perspective based on genome analysis and recent developments preliminary identification of potential vaccine targets for the covid- coronavirus (sars-cov- ) based on sars-cov immunological studies structural basis for the recognition of sars-cov- by fulllength human ace a novel coronavirus associated with severe acute respiratory syndrome the spike glycoprotein of the new coronavirus -ncov contains a furin-like cleavage site absent in cov of the same clade characterization of the binding profile of peptide to transporter associated with antigen processing (tap) using gaussian process regression total predicted mhc-i epitope load is inversely associated with mortality from sars-cov- allele frequency net database (afnd) update: gold-standard data classification, open access genotype data and new query tools swiss-model: homology modelling of protein structures and complexes drefine: an interactive web server for efficient protein structure refinement verification of protein structures: patterns of nonbonded atomic interactions structure validation by cα geometry: ϕ,ψ and cβ deviation netmhcpan- . : improved peptide-mhc class i interaction predictions integrating eluted ligand and peptide binding affinity data vaxijen: a server for prediction of protective antigens, tumour antigens and subunit vaccines cabs-dock web server for the flexible docking of peptides to proteins without prior knowledge of the binding site heterologous expression of plasmodium vivax apical membrane antigen (pvama ) for binding peptide selection gromacs: high performance molecular simulations through multi-level parallelism from laptops to supercomputers immunoinformatics-aided identification of t cell and b cell epitopes in the surface glycoprotein of -ncov development of epitope-based peptide vaccine against novel coronavirus (sars-cov- ): immunoinformatics approach epitope based vaccine prediction for sars-cov- by deploying immuno-informatics approach acknowledgements cas-s and amb-i thank capes (coordination for the improvement of higher education personnel, brazil) and cnpq (brazilian national council for scientific and technological development) for financial support and scholarships. patient consent for publication not required.provenance and peer review not commissioned; internally peer reviewed.data availability statement data are available upon reasonable request. all data relevant to the study are included in the article or uploaded as supplementary information. raw data generated in this study may be requested by sending an email to lpm. ccm@ ufpe. br.this article is made freely available for use in accordance with bmj's website terms and conditions for the duration of the covid- pandemic or until otherwise determined by bmj. you may use, download and print the article for any lawful, non-commercial purpose (including text and data mining) provided that all copyright notices and trade marks are retained. ronald rodrigues de moura http:// orcid. org/ - - - key: cord- -ulu y authors: lee, su hae; jee, seung wan; hwang, dae youn; kang, jong koo title: characterization of changes in global gene expression in the hearts and kidneys of transgenic mice overexpressing human angiotensin-converting enzyme date: - - journal: lab anim res doi: . /s - - -y sha: doc_id: cord_uid: ulu y human angiotensin-converting enzyme (hace ) has recently received a great attention due to it play a critical role as sars-cov receptor in the infection of human body. however, no further analysis for gene regulation has been performed in target tissues of model mice during hace overproduction. to characterize changes in global gene expression in the hearts and kidneys of rtta/hace double transgenic (dtg) mice in response to hace overexpression, total rna extracted from these tissues from dtg mice after doxycycline (dox) treatment was hybridized to oligonucleotide microarrays. briefly, dtg mice were generated by cross-mating pα-mhc/rtta tg mice with ptre/hace tg mice. the expression level of hace protein was determined to be high in hearts, kidneys, and brains of dtg mice, whereas lung, liver, and testis tissues expressed low levels. the level of hace was significantly enhanced in hearts and kidneys of the dox+dtg group compared to that in vehicle+dtg mice although consistent levels of mouse ace (mace ) remained in the same tissues. based on the microarray analysis of heart tissue, genes were differentially expressed, including upregulated and downregulated, when comparing non-tg and vehicle+dtg mice, whereas genes were differentially expressed, including upregulated and downregulated, between vehicle+dtg and dox+dtg mice. in the kidneys, genes were differentially expressed, including upregulated and downregulated, between non-tg and vehicle+dtg mice. dox-treated dtg mice exhibited the differential expression of genes including upregulated and downregulated. taken together, these findings suggested that several functional groups and individual genes can be considered biomarkers that respond to hace overexpression in dtg mice. moreover, our results provided a lot of useful information to predict physiological responses when these dtg mice are applied as a susceptible model for novel coronavirus (sars-cov, covid- ) in both vaccine and drug development. angiotensin-converting enzyme (ace ) is the first known human homolog of ace and was cloned from a human heart failure and human lymphoma cdna library [ , ] . ace might play a pivotal role as a new element of the renin angiotensin system (ras) by reducing ang ii and increasing levels of ang - [ ] [ ] [ ] [ ] and is distributed in a wide variety of tissues, including the brain, lung, heart, liver, kidney, and testis, as well as most cardiovascular-relevant tissues [ ] [ ] [ ] [ ] [ ] . in the heart, myocardial infarction increases ace expression, which is localized to the vascular endothelium, smooth muscle, and cardiomyocytes of both rats and humans [ ] . ace participates in the regulation of blood pressure and cardiac and renal functions and is associated with major cardiac and renal pathophysiological processes. especially, it has been reported that ace gene expression is upregulated in humans with heart failure [ ] . it is also predominantly expressed in the proximal tubular brush border, distal tubules, and glomerular epithelial cells in the kidneys of humans, rats, and mice [ ] [ ] [ ] [ ] . the colocalization of ace with ang - in renal tubules reveals the functional ability of ang - to counteract the action of ang ii, and these vasodilator peptides might be a critical link, mediating regulatory feedback between ace and ace [ ] [ ] [ ] . although the function of ace in the brain is poorly understood, there is considerable evidence of a role for ang - . previous studies have shown that ang - is an important neuromodulator of cardiac baroreflex mechanisms [ ] . the ace / and neutral endopeptidase or neprilysin (nep) are zinc metallopeptidases [ ] . however, more recently, ace has aroused considerable attention as a receptor for the coronavirus that causes severe acute respiratory syndrome and a protector against severe lung failure [ , ] . in the past decade, functional studies on hace have continued using transgenic animals to clarify the mechanism related to heart and renal failures, as well as other pathophysiological conditions. first, in knockout mice, the genetic disruption of ace leads to severe cardiac contractile dysfunction, increases in ang ii levels, the upregulation of hypoxia-induced genes, and decreases in ace transcript and protein levels in the heart [ ] . however, it is necessary to additionally study the effects related to hace expression levels to clarify whether increased levels can have a beneficial effect on cardiac and renal functions. myosin heavy chain (mhc), which is found in the contractile apparatus and is a major protein, is composed of two heavy chains and four light chains. in the cardiac muscle, two distinct mhc genes, which encode αand β-mhc isoforms, have been identified [ ] . previously, the α-mhc gene promoter was used to direct tissue-and developmental-specific expression of the transgene in transgenic (tg) animals [ ] . further, there are many transcriptional regulation systems, and tetracycline regulatory systems have been widely used for conditional gene expression. the tetracycline-controlled transactivator (tta) is generated by fusing the dnabinding domain of the tetracycline-resistance operon (tetr) encoded by tn of e. coli with the transcriptional activation domain of vp of herpes simplex virus [ ] . there are two basic variants of this, tta and reverse tta (rtta) systems. to compensate for the tta system, which requires long-term administration of doxycycline (dox) for induction of the transgene, the other inducible system, namely rtta (tet-on system), has been developed. this system requires two dna constructs, a transcriptional regulatory unit and the responsive element teto sequences linked to a p cmv -derived target gene. in the presence of dox, rtta binds teto sequences and p cmv , which activates the target gene. in contrast, rtta does not bind teto and the target gene is not transcribed in the absence of dox [ , ] . the doxinducible gene regulatory system allows for the tight and adjustable control of a transgene of interest to study organ development and disease pathogenesis. previously, tg rats expressing human ace under the control of the rat cardiac myosin light chain (mlc ) promoter and tg mice expressing human ace under the control of the mouse cardiac α-mhc promoter have been generated [ , ] . these tg mice had a high incidence of sudden death and rhythmic disturbances with sustained ventricular tachycardia including terminal ventricular fibrillation and heart block. however, in surviving older mice, spontaneous downregulation of the ace transgene was observed, which was associated with the restoration of nearly normal conduction, rhythm, and connexin expression. because sudden death occurs earlier in higher-expressing transgenic lines, a promoter that can be regulated, such as the tet-on or off system, might be useful to examine cardiac and renal pathophysiology at the basal level. the aim of this study was to characterize changes in global gene expression in the hearts and kidneys of dtg mice, created with pα-mhc/ rtta and ptre/hace vectors, in response to the overproduction of hace protein. first, pα-mhc/rtta was constructed by fusing the rtta-m gene with the α-mhc promoter (gene bank accession no. u ). the puhrt - was a gift from dr. wolfgang hillen at the university of heidelberg, germany [ ] . this plasmid contains a mutagenized rtta-m fragment harboring the s g, e g, and a p mutations. the α-mhc sequence was amplified by pcr, with the genomic dna as a template, which was isolated from the tail of bdf mice. the following primers were used for the amplification: the sense primer, ′-ctcct tcctt gttgc atctt cc- ′ (corresponding to nucleotides , - , of α-mhc), and the antisense primer, ′-cagga ggaag atgga gaaga cag- ′ (corresponding to nucleotides , - , of α-mhc). the amplified α-mhc product was cloned into pgem-t (pα-mhc-t). the α-mhc fragment obtained by the digestion of pα-mhc-t with saci and sacii was cloned into puhdrtta s-m -splice, in which the human cmv promoter has been eliminated via digestion with xhoi and sacii (pa-mhc/rtta). second, ptre/hace was constructed by inserting the hace gene into the noti site within the multiple cloning site of ptre hyp (clontech laboratories inc., mountain view, ca, usa). the ptre hyp gene contains a tet response element (tre), which consists of seven copies of the -bp tetracycline operator sequence (teto) and hygromycin resistance genes. the hace cdna was amplified by pcr, using a sense primer ( ′-gacga tgtca agctc ttcct g- ′) with nucleotides - and an antisense primer ( ′-gccta cagat cttct tcaga aataa gtttt tgttc aaagg tctga acatc atcag tg- ′; lowercase letter, c-myc tag) with nucleotides , - , based on hace (gene bank accession no. nm_ ). full-length rna was used as the template, which was isolated from hek- cells (atcc crl- ). the amplified hace product was inserted into pgem-t (phace -t; fig. a and b). the experimental protocol for dtg mice was carefully reviewed based on ethical and scientific care guidelines and approved by the national institute of food and drug safety evaluation-institutional animal care and use committee (nifds-iacuc; approval no. nitr ). all c bl/ nkorl and dba/ korl mice at weeks of age were provided by the department of laboratory animals resources in nifds (cheongju, korea). all mice used in this study were provided with ad libitum access to water and an irradiated standard chow diet (purina mills inc., pyeongtaek, korea). mice were housed in cages under specified pathogen free (spf) condition under a strict light cycle (light on at : h and off at : h). all mice were handled in an accredited nifds animal facility in accordance with the aaalac international animal care policies (accredited unit-the ministry of food and drug safety: unit number- ). for the first lineage of tg mice, a linear . -kb α-mhc/ rtta fragment was microinjected into the pronucleus of fertilized eggs of bdf mice, which had been obtained by mating c bl/ nkorl (males) and dba korl (females) mice. for the second lineage of tg mice, the linear . -kb fragment of tre/hace was microinjected into the pronucleus of a fertilized embryo after dilution to a concentration of ng/μl. microinjection was performed using microscopy (nikon, tokyo, japan) and a micromanipulator (narishge, tokyo, japan). each tg line was established by back crossing the founder mice with a parental strain of c bl/ nkorl mice. the dtg mice were obtained by crossing the first lineage of α-mhc/rtta tg mice with the second lineage of tre/ hace tg mice. the single and double tg founder mice were back crossed onto the parental strain of the c bl/ nkorl background to establish homogenous tg lines. the transgene was identified by dna-pcr analysis of the genomic dna isolated from the tail of -week-old founder mice. the precipitated dna was separated by centrifugation at , rpm for min. after washing in μl % et-oh, dna was dried for min and dissolved in μl of distilled water. the α-mhc/rtta gene was amplified as a template using the sense primer ( ′-ctgtc ttctc catct tcctc ctg- ′) with a complementary α-mhc promoter ranging from to nucleotides and an antisense primer ( ′-caggg taggc tgctc aactc- ′) with a complementary rtta gene ranging from to nucleotides. the tre/hace gene was also synthesized as a template using a sense primer ( ′-gacga tgtca agctc ttcct g- ′) and antisense primer ( ′-catat aatgg cctca gctgc- ′) with a complementary hace gene ranging from to and from to nucleotides, respectively. pcr amplification was carried out in a thermal cycler (perkin elmer, norwalk, ct, usa) using the following cycling conditions: one cycle: °c, min; cycles: s at °c, min at °c, and s at °c; one elongation step of min at °c. the dtg mice were distributed into two groups ( - mice per group), namely vehicle and dox treatment. drinking water containing mg/ml dox (sigma-aldrich co., st. louis, mo, usa) was administered to mice of the dox-treated group for weeks, whereas tap water was administrated to mice of the vehicle group for the same period. the brain, heart, lung, liver, kidney, and testis tissues were collected from mice of subset groups and homogenized with % nnonidet p- in mm nacl, mm tris hcl, and mm edta supplemented with a protein inhibitor mixture (roche, basel, switzerland), which was followed by centrifugation at °c for min. for western blot analysis, the proteins were separated by electrophoresis using a - % gradient sds-polyacrylamide gel for h and then transferred to a nitrocellulose membrane with transfer buffer containing mm tris-base, mm glycine, and % methanol at v for h. membranes were blocked with % (w/v) non-fat dried milk in pbs ( mm nacl, . mm kcl, mm na hpo and mm kh po ) solution containing . % tween- (sigma-aldrich co.). the membrane was washed with pbs and incubated overnight °c with primary antibodies as follows: anti-human ace (santa cruz fig. reverse tet promoter-controlled transactivator (rtta) and hace vectors and identification of α-mhc/rtta and tre/hace transgenes. a construction of the pα-mhc/rtta and ptre/hace expression vectors. rtta and hace (human ace ) were placed under the control of the α-mhc and tre promoter, respectively. two independent lineages of transgenic (tg) mice were produced and mated together to obtain the double tg (dtg) mice. in the dtg mice, the expression of the hace is induced by rtta in the presence of doxycycline (dox). the arrow (--->) indicates transcription. b features of the tet response element (tre) promoter sequence. the tre promoter is contained with seven copies of the teto sequence, a tata box, and an hcmv promoter. c the genomic dna was isolated from the tail of the founder mouse, and the -bp and bp products were shown in the dual transgenic mice carrying the α-mhc/rtta and tre/hace transgenes, respectively biotechnology inc., santa cruz, ca, usa), anti-human ace (santa cruz biotechnology inc.), and anti-α-tubulin (sigma-aldrich co.). after washing, each antigen-antibody complex was visualized with biotinylated secondary antibodies as follows: goat anti-mouse igg (h+l) hrpconjugated antibody, rabbit anti-goat igg (h+l) hrpconjugated antibody, and goat anti-rabbit igg (h+l) hrp-conjugated antibody (zymed laboratories inc., san francisco, ca, usa) at a : dilution in pbs buffer containing % non-fat dried milk/pbs buffer at room temperature for h. immunoreactive proteins were detected by an enhanced chemiluminescent substrate (ecl, amersham pharmacia biotech, inc., amersham, england) reaction, which was followed by exposing the membranes to hyperfilm ecl. the hearts and kidneys from non-tg, vehicle+dtg, and dox+dtg mice were used for the isolation of total rna using rnazol (tel-test inc., friendswood, texas, usa) according to the manufacturer's instructions. the frozen tissues were minced with scissors and homogenized in rnazol b solution using a teflon glass homogenizer. the rna pellet was suspended in depc-treated dh o and purified using a qiaquick purification kit (qiagen inc., chatsworth, ca, usa). the integrity of the s/ s rrna was analyzed using a bioanalyzer (quality agilent technology inc., santa clara, ca, usa), with the rna quality checked based on the ratio of absorbance at nm to that at nm using a biophotometer (hamburg-eppendorf, hamburg, germany). the integrity of each rna sample was confirmed by % agarose gel electrophoresis, which showed the presence of intact s and s ribosomal bands. crna synthesis and labeling were performed using a chemiluminescent rt-ivt labeling kit (applied biosystems, foster city, ca, usa) according to the manufacturer's instruction. individual samples were submitted in randomly assigned pairs representing tissues from non-tg and tg mice. three micrograms of total rna was used to synthesize cdna. double stranded cdna was synthesized for h at °c in a reaction mixture containing nuclease free water, × nd strand buffer, and nd strand enzyme and then purified using a purification column. finally, the biotinylated crna was generated from the cdna using a bioarray crna high yield rna transcript kit containing purified double stranded cdna, × ivt buffer mix, dig-utp and ivt enzyme mix. changes in global gene expression in the hearts and kidneys of mice overexpressing human ace were analyzed using the mouse genome survey microarray (applied biosystems) containing oligonucleotide probes for , genes. the crna was fragmented in fragmentation buffer for min at °c prior to chip hybridization. fifteen micrograms of fragmented crna was then added to the hybridization cocktail. each sample was hybridized to a separate oligonucleotide array for h at °c in the genechip hybridization oven . after hybridization, the solution was removed and the slides were washed twice with × ssc containing . % sds for min at °c. the slides were incubated for min with anti-dig-ap in cl blocking buffer and then developed using the chemiluminescence substrate. thereafter, the hybridized arrays were scanned using an abi analyzer. scanning and basic analyses were performed using geneplex software release . (istech inc., seoul, korea). logged gene expression ratios from the fluorescent intensity of each spot were normalized based on regression. tests for significance were performed using a one-way anova test of variance (spss for windows, release . , standard version, and chicago, il, usa). all values are reported as the mean ± standard deviation (sd). microarray data and the statistical significance of differential expression were assessed by hypergeometric distribution analysis. p values less than . were considered significant. first, we produced one male founder mouse carrying the pα-mhc/rtta construct and one female founder mouse carrying the tre/hace construct using microinjection techniques. these showed different rates of transgene insertion into the chromosome at and %, respectively. moreover, α-mhc/rtta and tre/hace single tg mice with a bdf background were backcrossed with c bl/ nkorl mice to change their background. during this process, α-mhc/rtta and tre/hace constructs were transmitted into the genomes of their offspring of both sexes at a rate of approximately % hemizygous animals based on mendelian inheritance. furthermore, both construct genes were transmitted into the genomes of dtg mice at a . % efficiency. to test whether the hace transgene was expressed under the control of rtta in a tissue-specific manner, its expression level was detected in various tissues including the brain, heart, lung, liver, kidney, and testis of dtg mice treated with mg/ml dox for weeks. the expression level of hace was higher in the heart, kidney, and brain than in other organs, although the highest level was detected in the heart. a low level of hace protein expression was observed in the lung, liver, and testis (fig. ) . however, any significant expression of hace protein was not detected in heart and kidney of non-tg and c bl/ mice (data not shown). therefore, these results indicate that the hace protein might be successfully expressed in various tissues of dtg mice through regulation of the dox-controlled rtta system. to compare the expression level of mace and hace proteins in the heart and kidney with or without dox treatment, both proteins were detected with specific antibodies in these tissues of non-tg, ve-hicle+dtg, and dox+dtg mice at the age of weeks. a similar expression pattern for both proteins was observed in the heart and kidney. the hace gene was highly expressed in only dox+dtg mice, whereas in non-tg and vehicle+dtg groups, expression was maintained at low level. however, the expression level of mace protein remained constant regardless of hace expression and dox treatment ( fig. a and b) . thus, these results suggest that hace protein can be successfully overexpressed in the hearts and kidneys of dtg mice after dox treatment. further, it was suggested that these organs would have great potential for microarray analysis to characterize the changes in global genes during overexpression of the hace protein. to analyze the differential expression of global genes during hace overproduction in the heart and kidney, microarray data were normalized based on the lowest values obtained from the experiments repeated in triplicate. the normalized log ratios of various genes in each experiment were used to compare relative expression between two independent experiments. based on these arbitrary differences, a substantial number of genes were expressed at elevated or reduced levels in the tg group before and after dox treatment. in the heart, transcripts were selected as differentially expressed genes between non-tg and vehicle+dtg groups based on a -fold change in expression, whereas differential transcripts were identified between vehicle+dtg and dox+dtg groups. among the former genes, were upregulated and were downregulated in the heart tissue of the dtg group as compared to levels in the non-tg group before dox treatment. moreover, genes, including upregulated and downregulated, were differentially expressed in the dtg group after dox treatment (table ). in the kidney, a total of genes, including upregulated and downregulated genes, were changed in the vehicle+dtg mice compared to levels in non-tg mice. furthermore, after dox treatment for weeks, genes, comprising upregulated and downregulated, were altered in the dtg group based on a -fold change in (table ) . overall, these results suggest that hace overproduction is more closely related to differentially expressed genes in the kidney than in the heart. to analyze the kegg pathways associated with hace regulated gene expression in the heart and kidney of non-tg and dtg mice based on dox treatment, genplex software release . was used. the results are presented in tables and . genplex identified pathways that were associated with significant changes in the heart ( table ). as presented in table , the largest numbers of genes in the heart were mainly associated with cytokine-cytokine receptor interaction, the calcium signaling pathway, apoptosis, and ecm-receptor interaction. in the kidney, pathways were identified (table ) . especially, genes involved in wnt signaling were highly changed, followed by those related to oxidative phosphorylation, prostate and colorectal cancer, the tgf-beta signaling pathway, and apoptosis. furthermore, we characterized the genes that were downregulated and upregulated by hace overproduction in the hearts and kidneys of dox+dtg mice. of the upregulated genes in the heart, the highest difference was detected for cap , followed by pik c g, syt , hecw , and tmod , whereas downregulated genes included calgranulin a, calgranulin b, paip , ptx , and hspa a. in the kidney, gabrg was associated with the highest increase, followed by wdr , ppp r c, bin , and mef c among upregulated genes, but slc a , fgfr , slc a , stam , and mrps were significantly decreased after hace overproduction. taken together, this showed that hace overexpression is mainly associated with cellular pathways related to cytokine-cytokine receptor interactions, the calcium signaling pathway, apoptosis, ecm-receptor interactions, wnt and tgfbeta signaling, cancer, and oxidative phosphorylation in the hearts and kidneys of dtg mice. recently, advances in molecular biology have enabled functional analyses of interesting genes by using transgenic animals and stable cell lines with constant gene expression. however, if the transgene is related to the embryogenesis, the animal might be genetically predisposed to tolerate the effects of the transgene products [ ] . to overcome this problem, it is necessary to develop transgenic animals in which transgene expression can be induced at selected time points but kept silent for an extended period. conditional gene expression has been achieved using a variety of model systems [ , ] . non-regulatable promoters in tg animals cannot direct the genetic switches to upregulate or downregulate expression from the promoter-linked target gene, and therefore, it is impossible to know how the target gene interacts and regulates the pathophysiological processes at both the basal and inducible level. the tet-on and tet-off expression systems are the most widely used inducible regulatory systems. in the tet-on system, the reverse tetracycline-controlled transactivator (rtta) acts as an activator of gene transcription [ , ] . dox binds the dox-binding site of the rtta protein, which represents random mutagenesis of the tta fusion protein between the tet repressor dna-binding domain ( amino acids) and the vp- transcriptional activation domain ( amino acids) of the herpes simplex virus. the dox-rtta complex then binds the tet sequence, which brings the vp- activation domain in close proximity to the minimal human cmv promoter, thereby activating the target gene in the presence of dox. indeed, rtta under the control of the cmv promoter was previously shown to activate the expression of a target gene in various organs [ , ] . in this study, the inducible tg mice expressing α-mhc-controlled, rtta-regulated hace were generated to address the hypothesis that unregulated expression of the hace transgene leads to the generation of defects including cardiac contractile dysfunction, rhythmic disorders, and sudden death. first, single tg mice expressing α-mhc-controlled rtta and hace were successfully developed by directly introducing each gene into fertilized eggs. tg mice were mated to induce rtta-regulated hace expression, which generated founder mice in which hace expression could be increased by dox. in the established tg line, neither the location of the transgenes in the genome nor the locus was impacted by gestation or neonatal imprinting. because of this, all offspring in this experiment exhibited the transmission of α-mhc/rtta and tre/hace genes into their genomes in approximately % of hemizygotes. ace is considered the central enzyme in the ras, converting ang i to ang ii. however, the identification of novel ras components such as ace and collectrin, a homologue of ace, capable of degrading ang ii and forming ang - , has emphasized the increasing complexity and multiplicity of biochemical pathways forming the ras. in a previous study, non-regulatable promoters have been used to create transgenic rats or mice expressing ace or ace genes under control of the rat cardiac mlc or mouse cardiac α-mhc promoter [ , ] . unexpectedly, the loss of ace in mice results in profound contractile dysfunction. however, the complete rescue of the heart phenotype in ace/ace double mutant mice indicates that ace expression has a causative role in the onset of heart dysfunction [ ] . because ace is expressed in the vascular endothelium and not in cardiac myocytes, local increases in ang ii might lead to vasoconstriction, resulting in hyperperfusion and hypoxia in the myocardium. it has been established that ang ii can induce oxidative stress in endothelial cells, and thus, its increase could result in dysfunction of the vascular endothelium via the induction of oxidative stress in the heart [ , , ] . in this study, a heart-specific promoter system was employed to induce rtta-regulated hace expression at a physiologically relevant site. hace protein was abundantly expressed in heart and kidney tissue of dox+dtg mice, although this protein was also detected in several other tissues. these results suggest that the binding of dox to the rtta protein might successfully induce hace expression in the heart, kidney, and other organs of dtg mice. however, any significant histopathological changes was not observed in heart and kidney of dox+dtg mice after induction of hace expression for only weeks (supplement fig. ) . interestingly, the ras can be seen as a dual function system in which vasoconstrictor or vasodilator actions are primarily driven by the ace/ace balance [ ] [ ] [ ] . elevated ace activity concomitant with reduced ace activity leads to a decrease in ang ii levels by converting it into ang - , which in turn promotes vasodilatation [ ] . according to this concept, dox-driven hace expression resulted in a decrease in ace levels in both hearts and kidneys of dox-inducible tg mice compared to that in non-tg mice. these results imply that the effects of hace expression, via the formation of ang - , have a counter regulatory role in ace activity in heart and kidney functions. the present study also investigated gene profiles to offer critical insight into complexity of the ras, as well as heart and renal failure related to hace overexpression. inducible tg mice overexpressing hace and non-tg mice were used to address the hypothesis that genes many genes involved in cardiovascular and renal disorders are modulated as compared to levels in dox+dtg and non-tg mice. the results identified genes from the hearts of dox+dtg mice that were significantly upregulated and genes that were downregulated compared to levels in the hearts of vehicle+dtg mice. a total of genes associated with cellular pathways comprising categories mainly related to ecmreceptor interaction, cytokine-cytokine receptor interaction, apoptosis, the calcium signaling pathway, and the tgf-beta signaling pathway were identified. of these genes, s a and s a , encoding s calciumbinding proteins that bind several types of proinflammatory cytokines, such as tnf-alpha, il- , and il beta, to form the proinflammatory cytokine complex in acute inflammation, play a role in calcium-mediated signaling [ , ] . ptx , a pentraxin-related gene, is induced in vascular smooth muscle cells via atherogenic modified low density [ ] and acts as a nonredundant regulator of tissue damage in acute myocardial ischemia and reperfusion [ ] . thus, these results suggest that the overexpression of hace might be involved in acute myocardial infarction and ischemia in the hearts of inducible rtta-regulated tg mice. in the kidney, genes in dox-treated tg mice were upregulated and genes were downregulated. these genes were also associated with cellular pathways comprising categories related to apoptosis, the calcium and wnt signaling pathway, oxidative phosphorylation, cancer, and the tgf-beta signaling pathway. especially, hace expression was mainly associated with the cellular pathway related to cytokine-cytokine receptor interactions, the calcium signaling pathway, apoptosis, ecm-receptor interactions, wnt and tgf-beta signaling, cancer, and oxidative phosphorylation, and rtta-controlled hace expression regulates the expression of genes related to binding, receptor, transferase, oxidoreductase, and hydrolase activities in the hearts and kidneys of dtg mice. however, our study provides limited information since only mrna level were analyzed to characterize global gene expression in response to hace overexpression. furthermore, protein expression analyses and molecular mechanism studies are necessary to conform the effects of identified genes in future study. taken together, we produced rtta/hace dtg mice that overexpress the hace gene based on a doxinducible system and analyzed the changes in global gene expression in heart and kidney tissue using a microarray. the results of the present study suggest that hace protein was successfully expressed in the heart, kidney, and brain of dtg mice after dox treatment. moreover, our microarray analysis was able to identify several functional groups of genes and individual genes that respond to hace overexpression in the hearts and kidneys of tg mice. however, additional work should address the extent to which these changes are correlated with hace expression levels to determine whether beneficial effects can be obtained by reducing expression levels or if the increased expression of ace at any level is deleterious with respect to cardiac and renal disease. furthermore, it will be necessary to study the functions of differentially expressed genes, which could represent targets for the development of novel drugs, using pharmacoproteomics. supplementary information accompanies this paper at https://doi.org/ . /s - - -y. additional file . additional file . abbreviations ace : angiotensin-converting enzyme ; mhc: myosin heavy chain; rtta: reverse tetracycline-controlled transactivator; tetr: tetracyclineresistance operon; tre: tet response element a novel angiotensin-converting enzyme-related carboxypeptidase (ace ) converts angiotensin i to angiotensin - a human homolog of angiotensin-converting enzyme. cloning and functional expression as a captopril-insensitive carboxypeptidase counter regulatory actions of angiotensin-( - ) angiotension-( - ) and antihypertensive mechanisms the renin-angiotensin system and diabetes: an update. vasc health risk manag prevention of angiotensin ii-induced cardiac remodeling by angiotensin differential expression of neuronal ace in transgenic mice with overexpression of the brain renin-angiotensin system angiotensin ii at receptors regulate ace and angiotensin-( - ) expression in the aorta of spontaneously hypertensive rats tissue distribution of ace protein, the functional receptor for sars coronavirus. a first step in understanding sars pathogenesis distribution of angiotensin-( - ) and ace in human placentas of normal and pathological pregnancies age-and gender-related difference of ace expression in rat lung myocardial infarction increases ace expression in rat and humans ace gene expression is upregulated in the human failing heart organ-specific distribution of ace mrna and correlating peptidase activity in rodents characterization of renal angiotensin-converting enzyme in diabetic nephropathy glomerular localization and expression of angiotensin-converting enzyme and angiotensinconverting enzyme: implications for albuminuria in diabetes angiotensin metabolism in renal proximal tubules, urine, and serum of sheep: evidence for ace -dependent processing of angiotensin ii ace and ace : their role to balance the expression of angiotensin ii and angiotensin temporal-spatial expression of ang-( - ) and angiotensin-converting enzyme in the kidney of normal and hypertensive pregnant rats the role of ace in pulmonary diseases--relevance for the nephrologist pressor and reflex sensitivity is altered in spontaneously hypertensive rats treated with angiotensin exploring the structure and function of zinc metallopeptidases: old enzymes and new discoveries a crucial role of angiotensin converting enzyme (ace ) in sars coronavirus-induced lung injury angiotensinconverting enzyme is a functional receptor for the sars coronavirus angiotensin-converting enzyme is an essential regulator of heart function molecular characterization of two myosin heavy chain genes expressed in the adult heart tissuespecific regulation of the alpha-myosin heavy chain gene promoter in transgenic mice tight control of gene expression in mammalian cells by tetracycline-responsive promoters transcriptional activation by tetracyclines in mammalian cells tetracycline-controlled transcriptional regulation systems: advances and application in transgenic animal modeling over-expression of angiotensin converting enzyme- augments cardiac hypertrophy in transgenic rats heart block, ventricular tachycardia, and sudden death in ace transgenic mice with downregulated connexins exploring the sequence space for tetracycline-dependent transcriptional activators: novel mutations yield expanded range and sensitivity conditional control of gene expression in the mouse tools for targeted manipulation of the mouse genome tetracycline-inducible expression systems: new strategies and practices in the transgenic mouse modeling generation of cells expressing improved doxycycline-regulated reverse transcriptional transactivator rtta s-m doxycycline-mediated quantitative and tissue-specific control of gene expression in transgenic mice generation of the regulatory protein rtta transgenic mice vascular protective effects of angiotensin converting enzyme inhibitors and their relation to clinical events endothelial dysfunction in cardiovascular diseases: the role of oxidant stress liver fibrosis: a balance of aces? emerging evidence for a functional angiotensin-converting enzyme -angiotensin-( - )-mas receptor axis: more than regulation of blood pressure? angiotensin-( - ) and the renin-angiotensin system the therapeutic potential of angiotensin-( - ) as a novel renin-angiotensin system mediator possibility of formation of the s a /a -proinflammatory cytokine complexes in vivo in acute inflammation and their functional roles molecular basis of the complex formation between the two calcium-binding proteins s a (mrp ) and s a (mrp ) modified atherogenic lipoproteins induce expression of pentraxin- by human vascular smooth muscle cells cardioprotective function of the long pentraxin ptx in acute myocardial infarction publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations we thank the animal technician, seon m. choi, and mee k. jang for directing the animal care and use at the division of laboratory animal resources, njfds at korea. availability of data and materials available. the authors declare that there are no financial conflicts of interest with respect to the publication of these results. key: cord- - yl bdq authors: oldstone, m. b. a. title: viruses and autoimmune diseases date: - - journal: scand j immunol doi: . /j. - . .d - .x sha: doc_id: cord_uid: yl bdq nan the autoimmune response is often an immune response that attacks one's own tissues and causes disease. most autoimmune diseases are organ-(tissue-) specific, and they develop when lymphocytes or their products (cytokines, antibodies, perforin, etc.) react with a limited number of antigens in that tissue. the molecular mechanisms leading to autoreactive immune responses resemble those generated against foreign antigens such as bacteria, parasites or viruses. however, in autoimmune disease either incomplete clonal deletion or formation of clonal anergy of t cells establishes a population of cells that is potentially intolerant but under special circumstances able to react with the host's antigens. autoimmune disorders are, then, characterized by the breaking of immunological tolerance or unresponsiveness to self antigens. this review focuses on evidence suggesting that some infectious agents, primarily viruses, can break immunologic tolerance and are implicated in autoimmune diseases. discussed here are mechanisms by which this occurs and speculation on the use of such findings for understanding and treating human autoimmune disease. newly forming autoimmune responses or those already present are enhanced after infection by a wide variety of human dna and rna viruses [ ] [ ] [ ] [ ] [ ] . in fact, patients with immune responses to nucleic acids, cytoskeletal proteins, myosin and lymphocytes, etc. was publicized over years ago by the great swedish immunologist, asterid fagraeus. additionally, experimental acute and persistent infections with dna or rna viruses have induced, accelerated or enhanced autoimmune responses and caused autoimmune disease [ ] . the new zealand mouse family is a genetically defined group in which certain strains spontaneously develop autoimmune disease. for example, among their several typical autoimmune responses, nzb mice develop antibodies to dna and red blood cells, whereas nzb × nzw (f mice) develop antibodies to dna and other nuclear antigens, closely resembling the picture of humans with systemic lupus erythematosus. when these mice are persistently infected with either a dna (polyoma) or an rna (lymphocytic choriomeningitis, lcmv) virus, their autoimmune responses occur earlier, reach higher titres and lead to disease sooner than in their uninfected counterparts. more interestingly, nzw mice, which normally do not develop autoimmune responses but contain the necessary gene(s) for autoimmune disease, develop these autoimmune responses after infection by polyoma virus or lcmv. other viruses, including retroviruses, cause a similar phenomenon. in human autoimmune diseases like multiple sclerosis (ms), insulin dependent diabetes mellitus (iddm) or ankylosing spondylitis, the incidence of disease varies in monozygotic twins suggesting that factors other than genetic and likely environmental also play a role [ ] [ ] [ ] . it has been observed that infectious agents [ ] [ ] [ ] or cytokines [ ] [ ] [ ] [ ] released in the presence and/or absence of infections can break tolerance in potentially autoreactive cd þ t or cd + t cells. others have reported on epidemiologic and serologic correlations between certain viruses and autoimmune diseases like ms and iddm. for example, coxsackie b virus and rubella virus have been linked with iddm [ , [ ] [ ] [ ] . in a few instances, coxsackie b virus has been directly isolated from pancreatic tissues of individuals with acute iddm. inoculation of this virus into mice then produced iddm, fulfilling koch's postulates [ ] . a clue for a novel mechanism of virally elicited autoimmunity was found in the early s. observations at that time proved that monoclonal antibodies directed against a specific viral protein also cross-reacted with host self proteins [ ] [ ] [ ] . for example, cross-reactivity was clear between measles virus phosphoprotein ( kd molecular weight) and cytoskeletal keratin ( kd molecular weight), and between herpes simplex virus glycoprotein ( kd) and another epitope on keratin [ ] . these observations received increased significance when the laboratories of hilary koprowski, abner notkins and ours established that roughly % of monoclonal antibodies made against different kinds of viruses also reacted with host self determinants [ , ] . these experiments analysed over monoclonal antibodies against such commonly found representatives of dna and rna viruses as herpes simplex, cytomegalovirus, epstein-barr virus, vaccinia virus, myxoviruses, paramyxoviruses, arenaviruses, flaviviruses, orthoviruses, rhabdoviruses, coronaviruses and human retroviruses. these results led to the hypothesis that molecules from dissimilar genes or their protein products shared structural similarities enabling them to mimic one another [ , ] . the idea behind molecular mimicry is that the molecules' linear amino acid sequences or their conformational fits are alike even though their origins are separate, for example, between a virus or a normal host self determinant. cross-reactivity of this type from dissimilar proteins have been identified by assay of both humoral and cellular immune responses. computer searches have also uncovered mimicry between host and viral proteins. coupling this information with data on motifs of proteins (peptides) bound to mhc class i or class ii proteins with outcomes from x-ray crystal-structure analysis has fine-tuned this hypothesis. for example, wucherpfennig & strominger [ ] evaluated the cross-reactivity of known myelin basic protein (mbp)-reactive t cell clones derived from ms patients by using computer predicted peptides from a variety of viruses and bacteria. the results showed that peptides could be selected by predicting that their primary and secondary structures would fit into the hla dr groove of major histocompatibility antigen complex (mhc) molecules (the unique dr allele associated with ms). these authors then showed that the selected microbial peptides bound to and caused proliferation of clonally derived mbp reactive t cells with affinities either greater than or equivalent to the known mbp peptide. this outcome meant that a single t cell receptor could be activated by peptides from several different viruses including herpes simplex virus, epstein-barr virus, adenovirus and influenza a virus as well as one bacteria, pseudomonas aeruginosa. others showed that t cell lines established from ms patients reacted to mbp and also to the sequence from human respiratory coronavirus e [ ] . furthermore, molecular mimicry occurred between human transaldolase expressed selectively in oligodendrocytes, and human t cell lymphotropic virus, human immunodeficiency virus type , gag proteins [ , ] . in other studies, cellular proliferative responses to determinants common to glutamic decarboxylase and coxsackie b were noted [ ] ; a similarly shared proliferative response marked % of newly diagnosed iddm patients but none of healthy matched control subjects [ ] . these combined data indicate that molecular mimicry is not uncommon, is not restricted to any specific class or group of viruses, and clearly accompanies several autoimmune disease. an important addendum to these observations is the possibility that an immune response elicited against an infecting pathogen could or would eliminate it but could also cross-react with any self antigen that shares determinants with that pathogen. in this way, the immunopathologic process could continue chronically or be reinitiated by multiple viral infections. if so, disease continues after the determining agent has been eliminated, so its presence is no longer detectable, a ''hit-andrun'' phenomenon [ , ] . several rules concerning the structure of peptides and their binding to mhc as well as the peptide-mhc complex binding the tcr are now established. mutational and crystallographic studies of mhc molecules complexed with viral peptide show that the molecule's flexible conformations allow peptides to bind within the mhc groove once their anchoring residues are fixed [ , , ] . analysis of residues flanking the anchoring residue(s) indicates the critical importance of minor pockets of mhc-binding clefts in peptide selectivity, leading to the concept that these structural factors are likely responsible for the preferential selection of specific peptides so often observed in interactions with mhc molecules [ ] [ ] [ ] . this flexibility is biologically shown when a single tcr can cross-react with multiple ligands [ , , ] including super antigens [ ] and related self peptides [ , , , ] . a major component for the argument for the biological importance of molecular mimicry emanates from experiments with mbp and experimental allergic encephalomyelitis (eae) in living animals [ ] . for these experiments, mbp was selected as the host self component to test because its encephalitogenic site of - amino acids has been mapped in several animal species. computer-assisted analysis showed significant homology between the encephalitogenic site of mbp and several viral proteins, but the best fit occurred between the mbp encephalitogenic site in the rabbit and hepatitis b virus polymerase. when this viral peptide was injected into rabbits, the histopathologic and immunologic hallmarks of eae appeared (perivascular infiltration localized to the central nervous system; generation of lymphocytes that reacted (proliferated) to both rabbit mbp and the viral peptide). other studies witnessed a similar scenario when the principle players were coxsackie b or mouse cytomegalovirus infections, cardiac myosin and virus-induced myocardial disease [ , , ] . these experimental models confirmed that molecular mimicry caused not only autoimmune responses but also autoimmune disease. to better understand the molecules and events involved in virusinduced autoimmune disease, we and others have designed transgenic mouse models [ , , , [ ] [ ] [ ] . a cartoon of the model used for virus-induced iddm and virus-induced oligodendrocyte autoimmune (demyelinating) disease is shown in fig. . our results from several studies based on the iddm model [ , , , , ] are reproduced in fig. . for this model we used the rat insulin promotor (rip) to express a viral gene in b cells of the islets of langerhans. diabetes does not occur spontaneously (incidence < % in these mice) unless tolerance is broken to the self (viral) antigen [ , , ] , in this situation the incidence of iddm is - %. when the transgene is expressed only in b cells, potentially autoreactive t cell clones of high viruses and autoimmune diseases affinity pass through the thymus by positive selection and reside in the periphery [ , , ] . these t cells are of high affinity. upon challenge with the virus, iddm follows within - days. however, the picture changes when the transgene is expressed in the target cell (b cells) and also in the thymus. in this instance, high affinity antiviral (self) t cells are removed by negative selection [ , , ] . passing to the periphery are low affinity t cells that are unresponsive (anergic). in the absence of viral infection, iddm can be induced when such anergic ctl clones (of high or low affinity) in the periphery are activated as they pass into an islet environment where interferon-g or b . are expressed [ , ] . in addition, activation of low affinity but not high affinity ctl clones requires cd help [ ] . for iddm to occur in both the high affinity and low affinity models, perforin [ , ] and g-interferon [ ] are required. experimentally disallowing g-interferon expression [ ] and/or establishing il- expression in the islets [ ] aborts the iddm. expression of g-interferon [ ] or tnf-a [ ] in the islets quickens the kinetics and/or enhances the severity and incidence of iddm. these observations allowed us (von herrath, gairin, horwitz, sarvetnick & oldstone) to design therapeutic approaches to halt the virus-induced iddm [ ] [ ] [ ] [ ] [ ] [ ] . when the cytokine profile in the islet of langerhans milieu was changed from a th to a th phenotype (g-interferon to il- , il- , tgfb), iddm was blocked [ , , , ] . one interesting way this occurred was through the oral administration of porcine insulin [ , , ] [ , , , , , , , [ ] [ ] [ ] [ ] [ ] . data generated for this model were obtained primarily with drs matthias von herrath and nora sarvetnick. the figure is modified from one proposed by dr matthias von herrath. of effector cd þ ctl. however, when a single or double amino acid change was made in the b chain of the insulin molecule that ordinarily protected against iddm, the protective effect was lost and in some preparations iddm was enhanced [ , ] . in the latter instances, transgenic mice got iddm within week after lcmv challenge [ , ] . this outcome documents how little we know about what controls immunity vs. tolerance with respect to oral therapy. a second approach was to design a peptide that bound at high affinity to the mhc allele involved in iddm [ , ] . by this means iddm did not occur, cd þ and cd þ t cells were not placed in the islets but were found around them (peri-islets). a third approach was to abort expression of the mhc class i molecule by expressing the e transcription complex of adenovirus in b cells [ ] . the focal reduction of mhc class i expression in the islets was associated with a normal precursor frequency of cd þ ctls. effector t cells were localized not in the islets but in the peri-islet positions and iddm did not develop. to examine whether molecular mimicry between a virus and a protein expressed in oligodendrocytes could lead to a central nervous system (cns) autoimmune disease much like the demyelinating disease, multiple sclerosis, transgenic mice were generated whose oligodendrocytes expressed either the nucleoprotein or glycoprotein of a virus [ ] . again, since the viral transgene integrated into the germline and passed to progeny mice, it becomes a ''self'' antigen. peripheral infection (initiated via intraperitoneal or intravenous routes) with a virus that encoded the same gene led to an antiviral immune response (predominantly cd þ ctl) that cleared the infection within days, with entry and retention of activated antiviral (''self'') t cells (both cd þ and cd þ t lymphocytes) in the cns and their retention along oligodendrocyte-produced myelinated tracts. activation of microglia and enhancement of mhc class i and ii expression paralleled the findings with t cells in the cns [ ] (fig. ) . a more lasting effect, however, was chronic inflammatory disease of the cns with activated t cells (both cd þ and cd þ ) found in the white matter for over year. a second infection with the initiating virus or an unrelated virus enhanced the cns inflammation followed by loss of myelin associated with a clinical disorder of motor dysfunction (incoordination, weakness) (fig. ) [ ] . hence, a cns autoimmune disease with myelin loss can be induced by infection with viruses that share epitopes with proteins expressed in oligodendrocytes, and this disease worsens after a second or multiple infections with the same or an unrelated virus. however, infection with the unrelated virus is, itself, unable to initiate the disease. herein may lie an explanation for the clinical observations in ms patients [ , , , ] of, first, the association of several different viruses with this autoimmune disease; second, the finding in their cerebral spinal fluids of antibodies to multiple viruses; and third, the epidemiologic pattern that exposure to an ''environmental factor'' early in life often predicts the later occurrence of such disease. the concept of molecular mimicry is a viable hypothesis for framing questions and approaches in the investigation of the aetiology, the pathogenesis, treatment and prevention of autoimmune disorders. useful tools in this query are computer data banks, links between specific mhc alleles and particular autoimmune disease, identification of anchoring amino acids, and important flanking sequences that bind to the mhc allele or face to the t cell receptor within viral (microbial) peptides and information on the conformational fit. these tools now allow us to evaluate suspected microbial causes of autoimmune disorders and also self determinants that are likely contributors to the disease process. transgenic models designed to evaluate molecular mimicry and virus-induced diseases reveal that unresponsive but potential autoimmune inducing t lymphocytes exist in the periphery. such t lymphocytes, when activated by cytokines like interferon-g or viral infection can act to break immune tolerance. once activated, these lymphocytes release additional cytokines, the balance of which within the local milieu may determine whether autoimmune disease occurs rapidly, slowly, or not at all. after tissue damage begins, an autocatalytic reaction may follow during which other self antigens that crossreact with the infecting virus may participate. specific therapies designed to inhibit viral replication, inactivate effector antiviral cd þ ctl, or change the cytokine profile from a th to th have proven successful for treatment in these animal models and will likely, in the future, be adaptable to human autoimmune diseases. overview: infectious agents as etiologic triggers of autoimmune disease molecular mimicry as a mechanism for the cause and as a probe uncovering etiologic agent(s) of autoimmune disease virus induced autoimmunity using transgenic mouse models to dissect the pathogenesis of virusinduced autoimmune disorders of the islets of langerhans and the central nervous system virus-induced autoimmune disease virus infection triggers insulin-dependent diabetes mellitus in a transgenic model: role of anti-self (virus) immune response ablation of tolerance and induction of diabetes by virus infection in viral antigen transgenic mice infection breaks t-cell tolerance sensitization to self antigens by in situ expression of interferon-g co-expression of b . and viral (self) transgenes in pancreatic bcells can break peripheral ignorance and lead to spontaneous autoimmune diabetes induction of diabetes is influenced by the infectious virus and local expression of mhc class i and tnf-a interferon-g is essential for destruction of b cells and development of insulin-dependent diabetes mellitus coxsackie viruses and diabetes mellitus high frequency of diabetes mellitus in young adults with congenital rubella molecular mimicry virus-induced diabetes mellitus sv large t shares an antigenic determinant with a cellular protein of molecular weight , molecular mimicry in virus infection: cross-reaction of measles virus phosphoprotein or of herpes simplex virus protein with human intermediate filaments infection with vaccinia favors the selection of hybridomas synthesizing auto-antibodies against intermediate filaments, among them one cross-reacting with the virus hemagglutinin molecular mimicry: frequency of reactivity of monoclonal antiviral antibodies with normal tissues molecular mimicry and autoimmune disease molecular mimicry in t-cell mediated autoimmunity: viral peptides activate human t-cell clones specific for myelin basic protein myelin basic protein and human coronavirus e: cross-reactive t cells in multiple sclerosis oligodendrocyte-specific expression and autoantigenicity in transaldolase in multiple sclerosis comparative analysis of antibody and cell-mediated autoimmunity to transaldolase and myelin basic protein in patients with multiple sclerosis t cell cross-reactivity between coxsackievirus and glutamate decarboxylase is associated with a murine diabetes susceptibility allele cellular immunity to a determinant common to glutamate decarboxylase and coxsackie virus in insulin-dependent diabetes structure of the human class i histocompatibility antigen, hla-a the alphabeta t cell receptor structure at . Å and its orientation in the tcr-mhc complex binding of viral antigens to major histocompatibility complex class i h- d b molecules is controlled by dominant negative elements at peptide non-anchor residues: implications for peptide selection and presentation antigen analogs/mhc complexes as specific t cell receptor antagonists essential flexibility in the t-cell recognition of antigen a single t cell receptor recognizes structurally distinct mhc/peptide complexes with high specificity superantigens: mechanism of t-cell stimulation and role in immune responses specific t cell recognition of minimally homologous peptides: evidence for multiple endogenous ligands amino acid homology between the encephalitogenic site of myelin basic protein and virus: mechanism for autoimmunity cardiac myosin induces myocarditis in genetically predisposed mice autoantibodies to cardiac myosin in mouse cytomegalovirus myocarditis how virus induces a rapid or slow onset insulin-dependent diabetes mellitus in a transgenic model expression of il- in b cells of the islets of langerhans aborts insulin dependent diabetes mellitus in a transgenic model viral infection of transgenic mice expressing a viral protein in oligodendrocytes leads to chronic central nervous system autoimmune disease thymic selection and adaptability of cytotoxic t lymphocyte responses in transgenic mice expressing a viral protein in the thymus development of insulitis without diabetes in transgenic mice lacking perforin-dependent cytotoxicity molecules involved in b cell destruction induction of diabetes is influenced by the infectious virus and local expression of mhc class i and tumor necrosis factor-alpha oral insulin treatment suppresses virus-induced antigen-specific destruction of b cells and prevents autoimmune diabetes in transgenic mice a specific mhc class i restricted ''blocking peptide'' prevent activation of virus-induced ctl and the development of virusinduced autoimmune diabetes expression of adenoviral e transgenes in b-cells prevents autoimmune diabetes a single amino acid change in the b-chain of insulin completely abrogates the efficacy of this compound to induce ''oral tolerance'' and prevent autoimmune diabetes a single amino acid change in the b-chain of insulin completely abrogates the efficacy of this compound to induce ''oral tolerance'' and prevent autoimmune diabetes design of high-affinity major histocompatibility complex-specific antagonist peptides that inhibit cytotoxic t-lymphocyte activity: implications for control of viral disease multiple sclerosis and other demyelinating diseases epidemiologic contributions to multiple sclerosis: an overview this is publication number -np from the division of virology, department of neuropharmacology, the scripps research institute, la jolla, ca, usa. this work was supported in part by usphs grants ag , ai , and a grant from the juvenile diabetes foundation (jdfi ). the author thanks matthias von herrath for input and assistance in the design of figs and . key: cord- -yqhzyqs authors: umemoto, eric y.; brokaw, james j.; dupuis, marc; mcdonald, donald m. title: rapid changes in shape and number of mhc class ii expressing cells in rat airways after mycoplasma pulmonis infection date: - - journal: cell immunol doi: . /s - ( ) - sha: doc_id: cord_uid: yqhzyqs mycoplasma pulmonis infection in rodents causes a chronic inflammatory airway disease with a strong immunological component, leading to mucosal remodeling and angiogenesis. we sought to determine the effect of this infection on the shape and number of dendritic cells and other major histocompatibility complex (mhc) class ii expressing cells in the airway mucosa of wistar rats. changes in the shape of subepithelial ox (anti-mhc class ii)-immunoreactive cells were evident in the tracheal mucosa days after intranasal inoculation with m. pulmonis. by week, the shape of the cells had changed from stellate to rounded (mean shape index increased from . to . ). the number of ox -positive cells was increased -fold at week and -fold at weeks. coincident with these changes, many columnar epithelial cells developed ox immunoreactivity, which was still present at weeks. we conclude that m. pulmonis infection creates a potent immunologic stimulus that augments and transforms the ox -immunoreactive cell population in the airways by changing the functional state of airway dendritic cells, initiating an influx of mhc class ii expressing cells, and activating expression of mhc class ii molecules by airway epithelial cells. antigen presentation by cells expressing major histocompatibility complex class ii (mhc class ii) molecules on their surface is an important step in the initiation of the primary immune response [ ] . dendritic cells (dc) are the principal mhc class ii expressing cells in the normal airway mucosa. these cells play a major role in the processing of inhaled antigens and may participate in the pathogenesis of infectious and allergic airway diseases [ ] [ ] [ ] . during steady-state conditions, dc turnover every - h [ ] . however, the dc network rapidly expands after exposure to a variety of pathogens and antigens [ ] . for example, exposure to aerosolized heat-inactivated moraxella catarrhalis causes dc influx into the airways which peaks within h and remains elevated for h [ ] . inoculation of viable bordatella pertussis results in dc recruitment with similar kinetics but with a maximal cell density at h [ ] . even at the peak of the inflammatory response, dc are the principal mhc class ii expressing cells-greatly outnumbering macrophages and b lymphocytes [ ] . such stimuli can also evoke transient expression of mhc class ii molecules by airway epithelial cells [ ] [ ] [ ] . less is known about the involvement of dc and other mhc class ii expressing cells in chronic inflammatory airway disease, in part because most disease models have focused on transient conditions. the longlasting consequences of mycoplasma pulmonis infection make it useful for studying changes in chronic disease cellular immunology ( ) - www.elsevier.com/locate/ycimm [ ] [ ] [ ] . the organisms attach to the luminal surface of the airway epithelium of rats and mice [ ] and are not cleared from the airways despite strong cellular and humoral immune responses [ , [ ] [ ] [ ] . the ongoing stimulus causes an influx of mononuclear cells, including dc, macrophages, and lymphocytes [ ] [ ] [ ] [ ] . the development of mucosal lymphoid tissue is a prominent part of the remodeling of the airway mucosa, and is accompanied by epithelial cell and mucous gland hyperplasia, fibrosis, angiogenesis, and increased sensitivity of the newly formed blood vessels to the neuropeptide substance p [ , , , ] . although some of these changes may occur after viral infection [ , ] , m. pulmonis infection is unusual in that it causes life-long disease and, if untreated, can result in severe remodeling of the airway mucosa [ ] . the role of dc and other mhc class ii expressing cells in these changes is unknown [ , ] , but is of interest because of the rapid cellular response after infection and the strong immunological component of mycoplasmal airway disease. in the present study, we used m. pulmonis infection as a model of chronic inflammation to determine the time course of changes in shape, number, and distribution of mhc class ii expressing cells in the airway mucosa, with a focus on the region beneath the airway epithelium where m. pulmonis organisms are attached. we also determined whether epithelial cells express mhc class ii molecules after m. pulmonis infection. mhc class ii expressing cells in the tracheal mucosa, stained immunohistochemically with the ox monoclonal antibody [ , , ] , were examined in rats infected with m. pulmonis for days to weeks. tracheal whole mounts were used to determine the -dimensional shape and number of ox -immunoreactive cells within and near the airway epithelium, and tracheal cross-sections were used to determine the distribution of these cells within the thickness of the airway wall. male pathogen-free wistar rats were purchased from charles river breeding laboratories (hollister, ca) and housed under barrier conditions in autoclaved microisolator units, three animals per cage. charles river documented the pathogen-free status of the animals as evidenced by serological assays for multiple pathogens, including m. pulmonis, parainfluenza type i (sendai) virus, coronavirus (rat coronavirus/sialodacryoadenitis virus), and cilia-associated respiratory bacillus. animals were weeks old and - g upon arrival. all experiments were approved by the committee on animal research at the university of california, san francisco. m. pulmonis strain c was grown in mycoplasma broth, harvested in the late log phase of growth, and frozen at ) °c in ml aliquots [ , ] . the frozen aliquots contained :  colony forming units of m. pulmonis per milliliter, as determined by quantitative culture [ , ] . after anesthesia (intramuscular injection of . - . ml of a mixture of ketamine, . mg/ml, parke-davis, morris plains, nj, and xylazine, . mg/ ml, the butler, columbus, oh), rats were inoculated intranasally with ll aliquots of m. pulmonis medium or sterile culture medium into each nostril daily, on three consecutive days [ ] . at or days or , , or weeks after the first inoculation, rats (n ¼ per time-point) were anesthetized by intraperitoneal injection of sodium pentobarbital ( mg/kg, abbott laboratories, north chicago, il) and then perfused via the ascending aorta for min with % paraformaldehyde in phosphate-buffered . % nacl (pbs, ph . ) at - mmhg. pathogen-free controls included five uninoculated rats and six rats inoculated with sterile culture medium (three rats at days and three rats at week). all animals had ml of blood drawn from an external jugular vein for measurement of serological antibody titers to m. pulmonis, sendai virus, and rat coronavirus/sialodacryoadenitis virus. after the vascular perfusion of fixative, tracheas were removed and fixed in % paraformaldehyde for - days at °c before being processing for immunohistochemistry. tracheas, washed and permeabilized in six -h changes of pbs containing . % triton x- (pbs/ triton at room temperature, sigma), were cut transversely into three segments, then were incised longitudinally and pinned flat, mucosal surface up, on sylgard slabs (dow corning, midland, mi). alternatively, -lm cross-sections were cut with a vibratome (series , technical products international, st. louis, mo). tracheal whole mounts or cross-sections were processed for immunoperoxidase histochemistry using techniques described previously [ , ] . briefly, specimens were incubated for h in pbs containing % normal goat serum (jackson immunoresearch laboratories, west grove, pa) and then for h in pbs containing % normal goat serum and mrc ox anti-mhc class ii monoclonal antibody (pharmingen, san diego, ca) at a : dilution [ , ] . the tissues were washed again for h in pbs followed by a -h incubation in peroxidase-conjugated goat anti-mouse igg (jackson) at a : dilution. the solutions contained . % thimerosal (sigma) to prevent microbial growth. another wash in pbs was followed by min in . m trizma base-trizma hcl buffer (sigma), ph . , containing . % diaminobenzidine and . % hydrogen peroxide to localize the peroxidase-labeled secondary antibody. all steps were done at room temperature. finally, the specimens were dehydrated in ethanol, cleared in toluene, and mounted in permount (fisher scientific, fair lawn, nj). whole mounts were mounted mucosal surface up. tracheas were examined with a zeiss axiophot microscope (carl zeiss, thornwood, ny) equipped with differential interference contrast optics or with an edge r microscope (edge scientific instrument, los angeles, ca), which uses oblique specimen illumination to produce -dimensional images. color photographs taken with kodak ektachrome film were scanned (polaroid sprintscan , cambridge, ma) and printed on a digital image printer (fujix pictography , fuji film, tokyo, japan). the shape of ox -immunoreactive (ox -positive) cells was estimated using a shape-sensitive parameter (shape index ¼ pa=p , where a is the projected cell area and p the projected perimeter) that expresses the ratio of area to perimeter relative to that of a circle [ ] . circular cells have a shape index of , whereas irregularly shaped cells have smaller shape indices, which decrease toward zero as the perimeter increases with respect to the area. dc with many processes have smaller shape indices than more rounded cells. the projected area and perimeter of ox -positive cells were measured in the rostral third of each trachea. measurements were made with a digitizing tablet on real-time color video images magnified with a zeiss axiophot microscope (projected magnification approximately Â) [ ] . ox -positive cells, located within or beneath the epithelium to a depth of about lm, were counted in consecutive regions of mucosa, each measuring . mm , in tracheal whole mounts. these particular cells were selected so the measurements would reflect the cells near the m. pulmonis organisms in the airway lumen, and the values would be comparable to published data on subepithelial dc [ , ] . measurements were made on regions of mucosa over cartilage rings - . the number of ox -positive cells was expressed per square millimeter of mucosal surface. specimens were viewed at a projected magnification of Â. epithelial basal cells, which had faint immunoreactivity, and co-lumnar epithelial cells, which had variable immunoreactivity after m. pulmonis infection, were readily identified and excluded from the counts of dc. mucosal thickness was measured in tracheal cross-sections. serological antibody titers to m. pulmonis, sendai virus, and rat coronavirus/sialodacryoadenitis virus were measured by enzyme-linked immunosorbent assays (elisa; bioreliance, rockville, md). values are expressed as means ae se (n ¼ rats per group, unless specified otherwise). the significance of differences between groups was evaluated by analysis of variance and fisherÕs test or scheff e eÕs f test for multiple comparisons or, for values that were not normally distributed, by the mann-whitney test. differences were considered significant when p < : . in pathogen-free rats, a network of mhc class ii expressing cells, identified by their ox immunoreactivity, occupied a thin layer just beneath the epithelium of the tracheal mucosa (fig. a) . in rats infected with m. pulmonis, the mucosa became progressively thicker and much more densely populated with ox -positive cells over the -week period of the study (figs. b and c). mucosal thickness increased from about lm in pathogen-free rats to lm at week after infection, and lm at weeks after infection. most of the ox -positive cells in the tracheas of pathogen-free rats had the characteristic branched morphology of dc [ , , ] . cells with - branched cytoplasmic processes formed a thin network roughly in the plane of epithelial basal cells (fig. d) . consistent with their stellate shape, ox -positive dc in pathogenfree rats had a comparatively low shape index (mean ¼ . ; fig. ). after m. pulmonis infection, the shape, number, and distribution of ox -positive cells in the tracheal mucosa underwent conspicuous changes. ox -positive cells became progressively rounder during the first week (fig. e) , as reflected by an increase in shape index from . in pathogen-free rats to . at days after infection and to . at week (fig. ) . at and days, the shape of many of the cells was intermediate between stellate and round. at , , and weeks, almost all of the ox positive cells had a rounded phenotype. ox immunoreactivity in tracheas of rats inoculated with sterile culture medium was indistinguishable from that in uninoculated rats. the staining was abolished by omission of the ox primary antibody. the numerical density of ox -positive cells in the most superficial lm of tracheal mucosa increased % during the first week after infection, from ae cells=mm in pathogen-free controls to ae cells= mm in infected rats (p < : ; fig. ). increasing ox immunoreactivity of mucosal cells made cell-counting more difficult in tracheal whole mounts after the first week; however, an analysis of cross-sections showed that ox -positive cells were uniformly abundant throughout the mucosa at and weeks after infection (figs. b and c). when mucosal thickening was taken into ac- count, the number of ox -positive cells was increased -fold at week and -fold at weeks compared to the pathogen-free value. in pathogen-free rats, columnar epithelial cells of the tracheal mucosa had no ox immunoreactivity, but processes of ox -positive cells in the lamina propria penetrated the epithelium (fig. a) . some epithelial basal cells had faint immunoreactivity but not a dendritic shape. scattered columnar epithelial cells had ox immunoreactivity at days after infection (fig. b) , and the number increased progressively (fig. c) . by week, most of the epithelium had granular ox immunoreactivity, which appeared to be associated with intracellular organelles (fig. c) . no epithelial cells were stained when the ox primary antibody was omitted. rats that were pathogen-free or infected for week or less did not have significant serological antibody ti-ters to m. pulmonis, but at weeks the infected rats had detectable titers ( : ae : elisa units), and at weeks the titers were significantly higher ( : ae : elisa units). none of the rats had significant antibody titers to sendai virus or rat coronavirus/sialodacryoadenitis virus. in the present study, m. pulmonis infection resulted in conspicuous changes in the shape, number, and distribution of mhc class ii expressing cells in the tracheal mucosa. during the first week after infection, ox -positive cells changed in shape from dendritic to rounded, increased in number, and changed in distribution from a concentration at the base of the epithelium to scattered throughout the mucosa. by weeks, the mucosa had times the normal thickness and an even larger increase in number of ox -positive cells. in addition, many columnar epithelial cells acquired ox immunoreactivity. changes in the shape and number of ox -positive cells were assessed after immunohistochemical staining of tracheal whole mounts. in these preparations, we could see the -dimensional shape as well as determine the distribution of cells in the mucosa. the -lm vibratome sections complemented the whole mounts by highlighting the increase in mucosal thickness and the distribution of ox -positive cells within the mucosa. only the most superficial ox -positive cells were counted in whole mounts so the values could be related to data from previous studies [ ] . nonetheless, the number of these cells found in pathogen-free rats was - % larger than corresponding values obtained from tangential lm sections of mucosa just beneath the epithelium [ ] . this difference could be explained by the greater thickness of the region analyzed in the present study. the combination of whole mounts and cross-sections produced a picture of ox -positive cells that neither method could give independently and could not be readily obtained from conventional histological sections. tracheal whole mounts provided a clear view of the number and -dimensional structure of ox -positive cells in pathogen-free rats and during the first week after m. pulmonis infection. however, as the mucosa became thicker, only the most superficial cells of whole mounts were stained, probably due to limited penetration of reagents and increasing immunoreactivity of the epithelium. by comparison, cross-sections, which did not have the limitation of reagent penetration, clearly revealed the thickening of the mucosa and showed abundant ox positive cells throughout the mucosa. together, the two types of preparations revealed that the total number of ox -positive cells increased -fold over the first week after infection and -fold over weeks. pathogen-free animals. most ox -immunoreactive cells in the airways of pathogen-free rats are considered to be dc because they express mhc class ii (ia) determinants and have the characteristic dendriform morphology [ , , ] . holt and co-workers [ ] reported that virtually all of the cells that stain for mhc class ii in the airways of pathogen-free rats are dc. in addition, ox -positive cells, which are most abundant at the base of the epithelium, can be distinguished from ed -positive tissue macrophages, which are most numerous deeper in the mucosa [ ] . although tissue macrophages in pathogen-free rats may have cytoplasmic processes, they typically do not express mhc class ii determinants in the absence of antigenic stimulation [ ] . m. pulmonis-infected animals. the phenotypic distinction between dc and other cell types blurs after mycoplasmal infection, when multiple cell types express mhc class ii molecules [ , ] . the characteristic branched morphology of dc in pathogen-free rats was no longer present in the ox -positive cell population week after infection and could, therefore, not be used to distinguish dc from other mhc class ii expressing cells. accordingly, some ox -positive cells observed after infection may represent activated macrophages or b lymphocytes that have infiltrated the airway mucosa [ , ] . the exact proportion of dc relative to other mhc class ii expressing cells cannot be definitively determined without using multiple cell markers. for example, activated b-cells could be identified by using ox as a marker [ ] . however, it is unlikely that activated macrophages make a major contribution to the ox -positive cell population, because in infected ani-mals they acquire a distinctive distribution around angiogenic blood vessels, quite different from the pattern of ox immunoreactivity [ ] . in rats exposed to heat-killed m. catarrhalis, ''small, round, intensely class ii positive cells'' migrate to the airway epithelium and reach a maximal density of approximately three times normal [ ] . in sections of airway epithelium stained for ox immunoreactivity, class ii-positive cells become pleomorphic about h after challenge with m. catarrhalis and by h develop into mature dc with highly branched processes [ ] . rounded dc precursors are not seen in control animals, and their transformation from rounded to stellate phenotype is most likely explained by local factors affecting dc maturation [ ] . in our experiments, ox -positive cells with a rounded phenotype accumulated in the airways during the first week after m. pulmonis infection. perhaps the transformation from stellate to rounded phenotype reflects changes in dc maturation, differentiation, or motility. alternatively, the apparent change in dc phenotype may represent the gradual replacement of stellate dc by rounded mhc class ii expressing cells such as b-cells. the number of ox -positive cells remained fairly constant during the first days after m. pulmonis infection and then increased significantly. the number continued to increase along with the -fold increase in thickness of the tracheal mucosa evident at weeks. the accumulation of ox -positive cells in the airway mucosa after m. pulmonis infection was accompanied by the accumulation of airway-associated lymphoid tissue [ , ] . changes in the number of ox -positive cells after intranasal inoculation of m. pulmonis differed both in onset and duration from what has been found after exposure to other bacteria or to viruses. exposure to aerosolized bacteria can increase the number of ox positive dc within h [ , ] . by contrast, the kinetics and time course of ox -positive cell recruitment seen with m. pulmonis infection is closer to the response demonstrated by sendai virus infection, which peaks at - days and remains elevated for weeks [ ] . similarly, in rats exposed to heat-killed bacillus calmette-gu e erin, numerous ox -positive dc accumulate at the borders of pulmonary granulomas, peaking at weeks and then gradually decreasing [ ] . the strong ox immunoreactivity of columnar epithelial cells that developed after m. pulmonis infection is consistent with the establishment of persistent infection and the corresponding immune response. in pathogenfree rats, some basal cells had faint staining but other epithelial cells had none. after infection, immunoreactivity was detectable in scattered columnar epithelial cells at days, was strong and fairly uniform at week, and was still strong and uniform through weeks. the upregulation of mhc class ii determinants by airway epithelial cells has also been observed during sendai virus infection [ ] . additionally, ox immunoreactivity has been reported in other types of epithelial cells after exposure to endotoxin or ifn-c [ , , ] . it is not clear whether the m. pulmonis organisms themselves or perhaps their ability to stimulate ifn-c production is driving the mhc class ii expression by epithelial cells. the granular immunoreactivity of epithelial cells resembles mhc-rich vacuoles in dc [ ] [ ] [ ] , where internalized antigen associates with mhc molecules. m. pulmonis infection provides a potent immunologic stimulus that augments and transforms the mhc class ii-expressing cell population of the airway mucosa. mhc class ii-expressing cells become much more abundant in the mucosa during the first four weeks after infection, and concurrently, columnar epithelial cells begin to express mhc class ii molecules. the rounding of ox -positive cells after infection suggests that existing dc change shape during maturation or differentiation or are replaced by ox -positive cells with a rounded phenotype. changes in the ox -positive cell population probably reflect the activation of an adaptive immune response and may play a role in the progression to chronic disease by persistently stimulating t lymphocyte responses. the dendritic cell system and its role in immunogenicity mhc class ii antigenbearing dendritic cells in pulmonary tissues of the rat. regulation of antigen presentation activity by endogenous macrophage populations studies on the density, distribution, and surface phenotype of intraepithelial class ii major histocompatibility complex antigen (ia)-bearing dendritic cells (dc) in the conducting airways a contiguous network of dendritic antigen-presenting cells within the respiratory epithelium rapid dendritic cell recruitment is a hallmark of the acute inflammatory response at mucosal surfaces dendritic cells are recruited into the airway epithelium during the inflammatory response to a broad spectrum of stimuli acute laryngitis in the rat induced by moraxella catarrhalis and bordetella pertussis: number of neutrophils, dendritic cells, and t and b lymphocytes accumulating during infection in the laryngeal mucosa strongly differs in adjacent locations inflammatory infiltration of the upper airway epithelium during sendai virus infection: involvement of epithelial dendritic cells expression of class ii antigen in endotoxin induced uveitis induction of rat thyroid cell mhc class ii antigen by thyrotropin and c-interferon significance as a research complication and experimental production with mycoplasma pulmonis the pathogenic potential of mycoplasmas: mycoplasma pulmonis as a model mycoplasma pulmonis infections cause long-lasting potentiation of neurogenic inflammation in the respiratory tract of the rat changes in epithelial secretory cells and potentiation of neurogenic inflammation in the trachea of rats with respiratory tract infections interactions of mycoplasmas with b cells: antibody production and nonspecific effects the development of extranodal lymphoid follicles in experimental bronchopneumonia non-lymphoid cells of bronchus-associated lymphoid tissue of the rat in situ and in suspension. with special reference to interdigitating and follicular dendritic cells tissue macrophages associated with angiogenesis in chronic airway inflammation in rats upregulation of substance p receptors in angiogenesis associated with chronic airway inflammation in rats exacerbation of murine respiratory mycoplasmosis in gnotobiotic f /n rats by sendai virus infection exacerbation of murine respiratory mycoplasmosis by sialodacryoadenitis virus infection in gnotobiotic f rats dexamethasone and oxytetracycline reverse the potentiation of neurogenic inflammation in airways of rats with mycoplasma pulmonis infection accessory cells of the lung. ii. ia+ pulmonary dendritic cells display cell surface antigen heterogeneity intracage ammonia promotes growth of mycoplasma pulmonis in the respiratory tract of rats clearance of different strains of mycoplasma pulmonis from the respiratory tract of c h/hen mice calcitonin gene-related peptide is a marker of secretory cells of the rat tracheal epithelium glucocorticoid-induced apoptosis of dendritic cells in the rat tracheal mucosa endothelial gaps and permeability of venules in rat tracheas exposed to inflammatory stimuli studies on the surface phenotype and functions of dendritic cells in parenchymal lung tissue of the rat mitogenic activity of mycoplasma pulmonis. i. stimulation of rat b and t lymphocytes comparison of mononuclear cell subpopulations in bronchoalveolar lavage fluid in acute rejection after lung transplantation and mycoplasma infection in rats the biology of airway dendritic cells nakamura, dendritic cell involvement in pulmonary granuloma formation elicited by bacillus calmette-gu e erin in rats up-regulation of surface antigens on epiplexus cells in postnatal rats following intraperitoneal injections of lipopolysaccharide identification of a novel cell type in peripheral lymphoid organs of mice. ii. functional properties in vitro identification of a novel cell type in peripheral lymphoid organs of mice. i. morphology, quantitation, tissue distribution a comparison of murine epidermal langerhans cells with spleen dendritic cells the authors thank dr. j. russell lindsey and ms. julie erwin of the university of alabama, birmingham for supplying the m. pulmonis organisms and for helpful suggestions, dr. peter baluk for assistance with the immunohistochemistry, dr. gary anderson for insightful comments regarding the manuscript, and dr. gavin thurston, dr. john mclean, and ms. evelyn clausnitzer for help throughout the project. this study was supported in part by nih grants hl and hl from the national heart, lung, and blood institute and novartis pharmaceuticals, basel, switzerland. key: cord- -r os kn authors: lu, dan; liu, kefang; zhang, di; yue, can; lu, qiong; cheng, hao; wang, liang; chai, yan; qi, jianxun; wang, lin-fa; gao, george f.; liu, william j. title: peptide presentation by bat mhc class i provides new insight into the antiviral immunity of bats date: - - journal: plos biol doi: . /journal.pbio. sha: doc_id: cord_uid: r os kn bats harbor many zoonotic viruses, including highly pathogenic viruses of humans and other mammals, but they are typically asymptomatic in bats. to further understand the antiviral immunity of bats, we screened and identified a series of bat major histocompatibility complex (mhc) i ptal-n* : –binding peptides derived from four different bat-borne viruses, i.e., hendra virus (hev), ebola virus (ebov), middle east respiratory syndrome coronavirus (mers-cov), and h n influenza-like virus. the structures of ptal-n* : display unusual peptide presentation features in that the bat-specific –amino acid (aa) insertion enables the tight “surface anchoring” of the p -asp in pocket a of bat mhc i. as the classical primary anchoring positions, the b and f pockets of ptal-n* : also show unconventional conformations, which contribute to unusual peptide motifs and distinct peptide presentation. notably, the features of bat mhc i may be shared by mhc i from various marsupials. our study sheds light on bat adaptive immunity and may benefit future vaccine development against bat-borne viruses of high impact on humans. in recent years, emerging and re-emerging viral diseases with high mortality have continuously posed serious threats to human health [ ] [ ] [ ] . in etiologic studies with convincing evidence, a series of such fatal diseases in humans have been confirmed or hypothesized to be caused by bat-borne viruses such as hendra virus (hev), ebola virus (ebov), marburg virus, and coronaviruses, including severe acute respiratory syndrome coronavirus (sars-cov) and middle east respiratory syndrome coronavirus (mers-cov) [ ] [ ] [ ] [ ] [ ] [ ] [ ] . similarly, fetal diseases a a a a a in livestock have also been associated with emerging viruses of bat origin, such as the fatal disease outbreak of pigs in china, which was found to be caused by a novel coronavirus, swine acute diarrhea syndrome coronavirus (sads-cov), from bats [ , ] . furthermore, it was shown that influenza-like viruses, termed h n and h n circulating among bats in central america, may act as an ancient influenza reservoir [ , ] . it is well accepted now that bats harbor an exceptionally high proportion of zoonotic viruses with interspecies transmission potential [ ] that have the potential to become virulent pathogens for humans. however, studies from wild or experimental bats indicate that most of these lethal viruses in humans and other mammals cause only asymptomatic infection in bats, suggesting a potentially special immune system in bats that is different from most other mammals [ , ] . studies of comparative genomics and transcriptomics confirm that the critical components of the innate and adaptive immune system are conserved and functional in bats [ , ] . however, a large number of bat-unique characteristics related to immunity and antiviral responses have recently been identified. the most notable immune features involve the absence of key natural killer (nk) cell receptors and dampened cell signaling of type i interferons (ifns) [ ] [ ] [ ] . meanwhile, stimulator of interferon genes (sting), an essential adaptor protein in multiple dna sensing pathways, has a substitution in bats, compared with other mammals, that leads to decreased ifn activation [ ] . meanwhile, bats also possess special features to maintain an effective immune state. the genome analysis of rousettus aegyptiacus revealed a dramatic difference from their functional gene counterparts in other mammals [ ] . indeed, bat ifns and some ifn-stimulated genes are constitutively transcribed or maintain detectable expression levels in the absence of stimulation [ ] [ ] [ ] . these characteristics may allow bats to fine-tune innate defense responses against insults by viral, bacterial, or host cytosolic dna while avoiding excessive inflammation. however, the adaptive immune system in bats is less well studied. major histocompatibility complex (mhc) class i molecules (mhc i) present antigen peptides to the surface of antigen presenting cells (apcs) to active t cells through interaction with t-cell receptors (tcrs), which play pivotal roles in antiviral defense [ , ] . epitopes or peptides are accommodated in the six pockets (a-f) of peptides/mhc i complexes, which were initially defined in humans [ ] . pocket a anchors the amine group of the amino terminal residue of the bound peptide, pocket b binds the side chain of peptide residue two, and pocket f accommodates the side chain of the carboxyl terminal residue [ ] . in bats, the partial map of the pteropus alecto mhc i region shows that mhc i genes are highly condensed and present within only one of the three highly conserved class i duplication blocks [ ] . genomic analyses of r. aegyptiacus demonstrate an expanded and diversified set of mhc i genes, with mhc i genes found outside the canonical region [ ] . sequences analyses, based on bat mhc i genes identified thus far, show that many of these bat mhc i molecules have a -or -amino acid (aa) insertion in the α domain compared with other mammals [ , ] . in the r. aegyptiacus genome, it is shown that of the mhc class i loci identified display the -aa insertion and only one locus without the insertion. interestingly, one of the mhc class i loci with -aa insertion is located in the canonical mhc alpha loci, indicating that both the mhc class i molecules with and without insertion can present the canonical binding surface [ ] . furthermore, the binding peptide motif of mhc i ptal-n � : derived from p. alecto has been identified. it displays a preference for peptides with pro at their c terminus, which has never been seen in mhc i proteins of any other vertebrates [ ] . however, the molecular basis for the peptide binding and presentation by bat mhc i remains unclear. in this study, we screened bat mhc i ptal-n � : -binding peptides from different batborne viruses (hev, ebov, mers-cov, and h n ) and determined the structures of bat mhc class i complexed with these viral peptides. unusual peptide presentation by bat mhc i with was demonstrated, which may help to understand the greater capacity of bats to coexist with a variety of viruses, from the perspective of adaptive immunity. previous work identified bat mhc i genes from more than seven different species of bats on different continents. each of these bat mhc i genes has the typical mhc i domains as in other mammals. however, examination of the retrieved set of bat mhc i sequences from gen-bank revealed several unusual features. first, bat mhc i genes contain a -or -aa insertion within their peptide binding groove (pbg) compared with those from a variety of other mammals (i.e., between trp and ile of human hla-a � ) (fig a and b) . within the bat mhc i genes, . % possess a -aa insertion and . % have the -aa insertion (fig a, s fig and s table) . the -aa insertion is unique to bat sequences, while the -aa insertion is present in both bats and some marsupials ( . % of the opossum mhc i, % of the koala mhc i, and % of both tammar wallaby and tasmanian devil mhc i contain the -aa insertion). all of the higher mammals, such as humans, nonhuman primates (nhps), mouse, and horse, lack any insertion at this site ( fig a and s a fig) . another feature of bat mhc i is the higher prevalence of a negatively charged residue at position and a positively charged residue at position , as well as their pairing (according to the ptal-n � : residue code) (fig b and s a fig) . for the mhc i molecules of the higher mammals, the residue at the position corresponding to (position of human, nhp, mouse, and horse mhc i) is a highly conserved gly. however, . % of bat mhc is have an asp or glu ( d/e) at this position ( fig c) . the pairing of asp and arg (d +r ) also occurs at a high fraction ( . %) in bat mhc i compared with the mhc i of humans ( %), nhp ( %), mouse ( %), and horse ( %). actually, the pairing of the charged residues at these two positions include different types: the negatively charged residues asp/glu at paired with positively charged residues arg/lys at , and even positively charged residue arg/lys at paired with asp/glu at (as gene epq . in myotis brandtii). also, the pairing of the charged residues, termed as charge matching at the two positions (+/−), occurs at an even higher fraction for bat mhc i ( . %). interestingly, these features of bat mhc i are also prevalent in marsupials ( fig c) . to determine whether the insertion has any correlation with the unusual substitutions at positions / , we analyzed the fractions of d/e, d +r , and the +/− charge matching in the bat mhc i genes with the -aa insertion. we found a significantly higher fraction of d/e ( %), d +r ( . %), and the +/− charge matching ( . %) in the bat mhc i genes with the -aa insertion compared with the corresponding fractions of other bat mhc i genes (no insertion and -aa insertion) ( % for d/e, . % for d +r , and . % for the +/− charge matching) ( fig d) . collectively, these distinct features suggested an unusual pbg of bat mhc i, which may affect peptide binding and presentation. to verify the peptide binding motif of ptal-n � : and to screen the potential bat mhc i tcell epitopes from recently emerging and re-emerging viruses with bats as their potential reservoir, we predicted ptal-n � : -binding peptides from ebov, mers-cov, and h n / h n (s table) . the previously determined ptal-n � : -binding peptides derived from hev were also included as a positive control [ ] . the binding capacity of these peptides were evaluated by their ability to facilitate the in vitro renaturation of ptal-n � : . generally, the peptides with higher binding capability to mhcs would have a higher production of the heterotrimer mhc complexes but lower production of β -microglobulin (β m). seven peptides from mers-cov, five from ebov, one from h n , and the two hev-derived peptides helped ptal-n � : naturally refold (fig ) . the lengths of the ptal-n � : -binding peptides cover a range from octamers to tridecamer. these peptides possess an asp at the p position (with gln in one peptide: mers-cov-s ), an aromatic aa (phe or tyr) at the p position, and a pro or leu at the po position (c terminus of the peptide) (s table) . the proportions of mhc class i alleles with -or -aa insertions in bats, marsupials (opossum, koala, tammar wallaby, and tasmanian devil), and higher mammals (human, nhp, mouse, and horse). the proportions of mhc class i alleles with the -aa insertion, -aa insertion, and no insertion are represented with orange, yellow, and cyan columns, respectively. the deletions (mainly including a -aa deletion) or insertions (mainly -aa insertions) other than the -and -aa insertions are termed as "others" in gray columns. the numerical data are included in s data. (b) structure-based sequence alignment of ptal-n � : and other representative (bats, marsupials, and higher mammals) mhc i molecules covering the residues from positions to (as in ptal-n � : ). the full information of the mhc i molecules of these species was listed in s fig and s table. coils above the sequences indicated α-helices. residues highlighted in red are completely conserved, and residues in blue boxes are highly ( %) conserved, with consensus amino acids in red. the residues at position and are shown in yellow. special insertion positions in ptal-n � : are marked with red arrows above the sequences. the sequence alignment was generated with clustalx and espript. to the right of the sequences, mhc class i alleles with the -aa insertion, -aa insertion, no insertion, and others are labeled with orange, yellow, cyan, and gray boxes, respectively. (c) the proportions of negatively charged residue asp or glu (" d/e," cyan columns), the pairing of the asp and arg ("d +r ," yellow columns), and the pairing of the negative-positive charged residues, termed as charge-matching at the two positions ("+/−," purple columns) at the corresponding locations of mhc i in bats, marsupials, and higher mammals. (d) statistical analysis of the -aa insertion and no -aa insertion alleles (no insertion and -aa insertion) in bats, respectively. fisher exact test or the chi-squared test was used for the statistical analyses. �� p < . . aa, amino acid; mhc, major histocompatibility complex; nhp, nonhuman primate. table ) . , and h n influenza-like virus (h n ) (d) with ptal-n � : were evaluated by co-refolding. co-refolding without any peptide was termed as the negative control (no pep), as curves in gray color. after properly refolding, the high-absorbance peaks of the correctly refolded mhc i with the expected molecular mass of kda were eluted at the estimated volume of ml on a superdex increase / gl column. the profile is marked with the approximate positions of the molecular mass standards of . , . , and . kda. inset, reduced sds-page gel ( %) of ptal-n � : /hev complex for peak (p ), peak (p ), and peak (p ). lane m contains molecular-mass markers (labeled in kda). p , p , and p represent the aggregated heavy chain, the correctly refolded heterotrimer ptal-n � : complex ( kda) , and the extra β m, respectively. β m, β -microglobulin; hev, hendra virus; ebov, ebola virus; mers-cov, middle east respiratory syndrome coronavirus; mhc, major histocompatibility complex; p , peak ; p , peak ; p , peak . the overall structures of ptal-n � : display the common characteristics of classical mhc i molecules in other mammals, with the extracellular region of the heavy chain folding into three different domains. the α and α domains construct a typical pbg that contains two α -helices and eight β-sheets, and the α domain and β m display typical immunoglobulin values in parentheses refer to statistics in the outermost resolution shell. b data completeness = (number of independent reflections)/(total theoretical number). where i i is the observed intensity, and hii is the average intensity of multiple observations of symmetry related reflections. (ig) domains and underpin the peptide binding domain ( fig a) . the all-atoms superimposition of ptal-n � : /hev onto the other five structures demonstrates a similar overall conformation, with root mean square deviations (rmsds) of . - . Å (fig b) . the superimposition of ptal-n � : /hev onto human mhc i hla-a and mouse mhc i h- k d generated rmsds of . and . Å, respectively ( fig c) . the most distinct differences between ptal-n � : and the mhc i from other vertebrates are located in the n terminus of the pbg, with an extension of the α -helix in ptal-n � : ( fig d) . to elucidate whether the peptide-presenting features of a bat mhc i molecule can be influenced by binding to human β m, we solved the structure of the ptal-n � : heavy chain complexed with human β m (ptal-n � : -h) at a resolution of . Å (table ) . comparing hev / ptal-n � : renatured with bat β m and human β m, the structural conformations of both hev peptides in the two structures are also similar, with an rmsd of . Å in the two binding grooves (fig e) . in addition, the overall structures are quite similar, with the rmsd of . Å of all atoms ( fig f) . and the key residues binding to α α domains and α domain further analysis shows trp in bat β m binds to gln and asp in ptal-n � : when forming a complex, which is also conserved in human β m when binding to ptal-n � : ( fig g) . it indicates that the structure of the peptide loaded in the groove of ptal-n � : was not affected by the substitution of the β m subunit. we also determined the structure of monomer bat β m without mhc i ( table ). the structure alignment of bat β m monomer with β m subunit in mhc complexes indicates a minor conformational shift of the loop resides ser to tyr in bat β m after forming a complex (fig h) . although having different lengths, the -mer peptide hev , -mers hev and h n -np, -mer ebov-np , and -mer ebov-np in the pbg of ptal-n � : all display an m-shaped conformation, with p and po residues as the primary anchors and the p or p residue as the secondary middle anchors (fig a- f and s a-s e fig) . the p asp of all of the peptides adopts a rigid conformation upward to the α -helix of the heavy chain. in the structure of the ptal-n � : /mers-cov-s complex, the conformation of the p -p residues of the peptide could not be determined due to their poor electron densities (s f fig), revealing a flexible conformation in the middle region of this mers-cov-s peptide. however, the other three residues (p -asp, p -phe, and po-pro) with electron densities available adopt similar conformations as in the other five structurally determined peptides. as a -mer peptide, mers-cov-s possesses a glu at p , corresponding to the p -asn in -mer hev , which is the secondary anchor residue. the larger glu may not be able to locate in the pbg, which leads to a flexible conformation of the mers-cov-s . to further validate the role of the unusual binding peptide motif of bat mhc i ptal-n � : , ala mutations at residues p , p , po, and the middle (p or p ) positions of peptides hev and hev were evaluated by refolding assays (fig g and i) . the thermal stability of the resulting heterotrimers was monitored by circular dichroism (cd) spectroscopy (fig h and j , s table) . substitution of ala at p (hev -d a), p (hev -y a), and p (hev -p a) of peptide hev led to nearly no refolding for ptal-n � : , whereas ala at p (hev -d a), p (hev -f a), and p (hev -p a) of peptide hev still supported refolding but with significantly lower stability than the original peptide. in contrast, substitution of ala at p of hev (hev -t a) and at p of hev (hev -n a) led to similar yield of refolded heterotrimers, indicating a similar stability compared with the original peptides. thus, through these analyses, the p anchor of ptal-n � : -binding peptides seems to be as significant as the p and po anchors. bat ptal-n � : possesses the met-asp-leu insertion within the n terminus of its α -helix (between residues and of hla-a � ). compared with the structures available for the mhc i of other mammals, such as humans and mouse, the ptal-n � : structure displays an extension of the α -helix of the pbg (fig a) . the -aa insertion pushes residue asp closer to the n terminus of the binding peptide, which leads to the extension of the negatively charged side chain of asp into the pbg (fig b) . the bat mhc i residue asp participates in the formation of the a pocket (s fig) . detailed analysis indicated that asp , arg , and the p -asp of the peptides in all six structures of ptal-n � : form a triangular network of hydrogen bonds (fig c and s fig) . to further investigate the role of the -aa insertion in the peptide binding and presentation of ptal-n � : , we constructed the ptal-n � : (- aa) mutant, which deleted the -aa insertion ( fig g) . the structure of ptal-n � : (- aa)/hev displayed a shortened α -helix that is similar to the human hla-a � ( fig d) . meanwhile, although the salt bridge was still observed between arg of ptal-n � : and the p -asp, both the arg and p -asp displayed a conformational shift (fig e) . the triangular network of the hydrogen bonds between asp (residue asp in the mutant), arg (residue arg in the mutant), and the p -asp of hev within the structure of the wild-type ptal-n � : was broken (fig f) . one of the hydrogen bonds between the two residues arg (residue arg in the mutant) and the p -asp of hev we also investigated whether the -aa deletion of ptal-n � : influenced binding ability to the peptides. ptal-n � : (- aa) was still renatured in the presence of peptide hev but with a much lower yield of the heterotrimer complex ( fig h) . cd spectroscopy also indicated a weaker binding of ptal-n � : (- aa) with the peptide (fig i) . to further verify the unusual peptide presentation and preference for peptides with a p -asp in bat ptal-n � : , we constructed the hla-a m mutant, which has a -aa insertion and the charge matching residues at positions / based on hla-a � : (fig j) . both the refolding assay and cd spectroscopy indicated a stronger binding of dl (g d mutant at p of peptide gl ) with the hla-a m compared with hla-a � : , and the hla-a � : has a higher binding capacity to gl than dl (fig k and l ). although ptal-n � : can bind peptides with leu as the po anchor, the binding peptides can also possess pro at this position. this is uncommon in the peptides bound by mhc i molecules from other mammals. the structures of ptal-n � : with the -mer peptides hev (dyintnvlp) and h n -np (dfekegysl) showed that both of the peptides adopt similar overall conformations as the hla-a � -presented -mer peptide gl (gilgfvftl) with a leu at the po site ( fig a) . however, to the best of our knowledge, no peptide motif with a pro at the po position has been reported for hla-a � . detailed comparative analysis of the f pockets of ptal-n � : and hla-a � revealed that the f pocket of ptal-n � : is shallow but with a wide opening (fig b- e to verify the allele-specific preference of ptal-n � : for peptides with pro at the po site, we examined the binding ability of hla-a � to a mutated gl peptide, gl -l p, with a pro at p (fig f and g) . we found gl -l p has a weaker capacity to help the hla-a � refold, and the generated heterotrimer complex has lower midpoint transition temperature (t m ) ( . ˚c) compared with the wild-type peptide gl ( . ˚c) with a leu at po. thus, although pro at po may also act as a suboptimal anchor for hla-a � , ptal-n � : uses pro as one of its optimal po anchors. the analysis of the different component amino acids for the proteins from the mers-cov and sars-cov indicated that pro possesses a relatively low mutation rate compared with the other amino acids (fig h) . the mutation rate of pro is only higher than trp and gly, which are the largest and smallest residues, respectively. it indicates that, as special residues trp and gly, the residue pro may also keep the natural conformation and function of viral proteins. in other words, structural constraints favoring conservation of pro in certain positions of proteins may operate to preserve viral protein conformation and function. thus, its mutation rate is restricted. the selection of pro as the anchor residue of bat mhc i ptal-n � : presented peptides may restrict the viral mutation pushed by t-cell immunity and accelerate virus clearance. the p positions of ptal-n � : -binding peptides are predominantly aromatic amino acids such as tyr and phe as the anchor, which is common in human hla-a � or mouse h- k d . however, when we superimposed the structures of ptal-n � : onto the previously determined structures of hla-a � and h- k d , we found that the p anchors of ptal-n � : protrude in a different direction (fig a) . the hla-a � -and h- k d -presented peptides have a tyr or phe pointing to the c terminus of the pbg, while the tyr or phe of ptal-n � : -binding peptides swing toward the n terminus of the pbg (fig c and d) . comparison of the amino acids lining the b pockets of these mhc i molecules from different mammals demonstrated that ptal-n � : possesses a tyr (compared with the small residues ser in hla-a � or val in h- k d ), which takes up space and can push a p -tyr or -phe to the other direction ( fig b) . furthermore, compared with the residues met in hla-a � or phe in h- k d , the ptal-n � : -specific ala leaves a large space to accommodate the p -tyr or -phe. indeed, detailed analyses showed that the p -tyr of ptal-n � : -binding peptides form hydrogen bonds directly with the main chain of the β-sheet on the floor of the pbg. in contrast, the p -tyr of hla-a � -restricted peptides bind to the side chain of his on the α -helix (fig b) . sequence superimposition of the bat mhc i with other mammal mhc is indicated that ala is prevalent ( %) among bat mhc i molecules but is never seen in human and mouse mhc i (s a fig). previously, it was indicated that ptal-n � : has a special preference for the binding of long peptides, together with the common - -mer peptides in other mammal mhc i molecules. to further elucidate whether the -aa insertion of ptal-n � : has an impact on the preference for long peptides through an n-terminal extension manner, we synthesized long peptides ( -mer to -mer) that were previously eluted from ptal-n � : -expressing cells (s table) bat mhc class i presenting viral peptides peptides, we also synthesized naturally n-terminally extended peptides based on the ptal-n � : -binding peptides from hev , mers-cov-s , and ebov-np (s table) . although these three peptides have a typical motif of ptal-n � : -binding peptides, the n-terminal extension led to a failure in peptide binding (s b fig and s table) . these data indicate that the -aa insertion into ptal-n � : leads to a more restrictive binding peptide selection for ptal-n � : but not an extension to longer peptides via the n terminus. the identification of bats as natural reservoirs of several highly pathogenic viruses that impact human and animal health and the fact that these viruses are harmless to bats have resulted in an increasing interest in the investigation of the specificities of the bat immune system. herein, we screened and identified a series of bat mhc i ptal-n � : -binding peptides derived from four different bat-borne viruses: hev, ebov, mers-cov, and h n . the subsequent determination of the structures of ptal-n � : complexed with peptides from these viruses revealed unusual peptide presentation features of bat mhc i. interestingly, this uncommon feature of pocket a of bat mhc i may be shared by the mhc is from different marsupials. in addition, as the traditional primary anchoring positions for peptides, the b and f pockets of ptal-n � : also display unconventional conformations that contribute to the distinct peptide presentation and special peptide motif compared with other higher mammals. the sequence combination of the -aa insertion at the n terminus of the α -helix and the charge matching residues at positions / enable an unusually tight anchoring of the p -asp in pocket a of ptal-n � : . but more significantly, this insertion site is located at a position called helix (residues - ); newly synthesized mhc i molecules complexed with β m are poised in the endoplasmic reticulum in a peptide-receptive (pr) form, ready to bind and be stabilized in the mature peptide-loaded (pl) form by peptides destined for display at the cell surface. the movement of a hinged unit containing a conserved helix promotes the pr transition state to a pl mature molecule [ ] . chaperone-mediated loading of high-affinity peptides onto mhc i is a key step in the mhc i antigen presentation pathway. tap binding protein (tapbpr; related) remodels the peptide-binding groove of mhc i, resulting in the release of low-affinity peptide [ ] . in the absence of tapbpr, y (y in ptal-n � : ) plays a role in closing of the α - -helix "latch" by associating with the c terminus of bound peptides in the f pocket, the release of nonoptimal peptides induced by tapbpr [ ] . however, the hydrogen bond network of the a pocket could stabilize peptides with p -asp during peptide processing and exchange and high-affinity peptides are guided by initial contacts spanning both the a-and f pockets to form a prolonged interaction within the groove, resulting in a closure of the α - helix latch, which triggers tapbpr release from the peptide/mhc i complex. being highly polymorphic, subtle substitutions or insertions in the pbgs of mhc i molecules may dramatically affect the binding peptide pool and also the peptide presentation to t cells [ , ] . comparative genomic and transcriptomic analysis demonstrated that a series of bat mhc i molecules have an insertion in the n terminus of the pbg compared with other higher mammals [ ] . herein, our structural study visually shows that the -aa insertion in the bat mhc i leads to an extension of the α -helix. this conformational change causes the protrusion of the bat-specific residue asp into the a pocket of the pbg, which forms a hydrogen bond network with the arg and the peptide p -asp. study of the peptide repertoire of key human and mouse mhc i alleles demonstrates that anchor asp is disfavored at the p position of the peptides [ ] . in contrast, recently reported data on the peptide elution from bat mhc i molecules [ ] , together with viral peptide screening results in this study, demonstrate that bat mhc i ptal-n � : prefers peptides with a p -asp. either depletion of the three inserted residues (met asp leu ) or substitution of the p -asp with ala impaired the mhc/peptide binding. indeed, the electrostatic nature of the contacts between asp , arg , and p -asp and the fact that it is involved in solvent-exposed elements in pocket a of the mhc complex suggest that the p -asp acts as a "surface anchor residue." this surface anchor residue was also previously defined in a phosphopeptide-mhc complex [ ] in which the solvent-exposed p phosphate moiety can enhance the stability of the peptide-mhc association. the additional surface anchor residue for the bat mhc i binding peptides may have at least two advantages for antiviral t-cell immunity. first, the peptides tightly bind to ptal-n � : and present a special peptide-mhc landscape at the n terminus of the peptides for the t-cell recognition. second, negatively charged residues such as asp/glu in the peptides may also act as key residues in the original viral proteins for virus replication, which will have lower mutation rates to escape t-cell recognition. in addition, the insertion of met asp leu also leads to a special exposed landscape of the α -helix of ptal-n � : . thus, heterozygous bats with mhc i alleles of both -aa insertion and no insertion may possess a t-cell repertoire with broader diversity, which need more work to investigate. bats are one of the most ancient extant lineages of eutherian mammals, believed to be located in distinct mammalian lineages different from marsupials and other higher eutherians [ ] . the evolution of the mhc i gene family is closely tied to the evolution of the vertebrates genome [ ] . however, the -aa insertion and the charge-matching residues at positions / of mhc is are also prevalent among different marsupials: opossum, koala, tammar wallaby, and tasmanian devil. this may be a phenomenon of convergent evolution under the pressure of related pathogens. to comparatively investigate the peptide presentation of mhc i from marsupials, we have synthesized a marsupial mhc i gene (trvu-ub � ), which has similar characteristics to -aa insertion and charge-matching residues at positions / to ptal-n � : , and also the small residue g at position (s a fig). however, the preference of the trvu-ub � protein for peptides may not be similar to that of ptal-n � : (s b fig) . this result indicates that although some alleles of mhc i from the marsupials possess the same key residues in the pbg of ptal-n � : , the preferred peptide motifs are only partially similar between them (pro as po), which may reflect the contribution of some other adjacent residues. thus, the detailed peptide motifs and the presentation features of mhc is from these lower mammals still require further laboratory investigation. meanwhile, whether the -aa insertion in the bat mhc i impacts the peptide presentation in the same manner as -aa insertion or with a new molecular mechanism needs more structural studies. in this context, it is also worth mentioning that we also tried two additional online mhc-peptide binding predicting servers, netmhcpan and rosetta flexpepdock, to verify the peptide-binding experiments in the study. however, neither prediction server was able to match the experimental results. this may indicate that current mhc binding peptide predictions were not suitable for non-mouse and nonhuman mammals such as bats, which may have a different manner of peptide binding. like the human and murine mhc i [ , ] , ptal-n � : has b and f pockets in the pbg to accommodate the primary anchors of the binding peptides, but uncommon conformational features of ptal-n � : -loaded b and f pockets were identified through our structural investigations. the b pocket of ptal-n � : , featuring the bat-specific residue ala , has a novel position in the pbg for the p anchor. in this position, the p -tyr of the peptides form hydrogen bonds directly with the main chain of the β-sheet on the floor of the pbg of ptal-n � : , which leads to a stable anchoring of the p -tyr. meanwhile, ptal-n � : , with a relatively unique gly at position , has an unusually shallow f pocket with a wide entrance, like a large bowl, which can accommodate the uncommon po-pro anchor. to the best of our knowledge, although the po-pro anchor is not observed in the previously reported peptides from mammalian mhc i, the precedents for the p -pro anchor have been reported in the context of murine h- l d and human b � and b � [ ] [ ] [ ] . requirements for pro in the p position as an anchor residue are also observed in the murine h- d d -and macaque mamu-a � -restricted epitopes [ , ] . a previous study indicates that the antiviral effect of t cells is sufficiently strong to force the virus to adopt a relatively unfavorable mutation, which reduces viral replication [ ] . the proline anchor may be a result of special antigen processing in bats. for humans, most products of ornithine decarboxylase degraded in vitro by the s atp-dependent proteasome, which contained one or two pro residues, implied that the pro residue has a role in the escape from random cleavage by proteasomes [ ] . in addition, pro residue(s) within epitopic sequences presumably contribute to efficient production of mhc class i ligands through prevention of their random cleavage by proteasomes [ ] . thus, the peptides with proline as a c terminus are still seldom in human and other common mammals. however, the current research on the proteasome of bats is still blank. our data also showed that among the different component amino acids for the proteins from the bat-related viruses, the residue pro possesses a relatively low mutation rate compared with the other amino acids (fig h) . pro, with a unique conformation, may act as a key residue for the structure and function of viral proteins, and thus its mutation rate is low. therefore, the usage of pro as the po anchor of the t-cell epitopes in ptal-n � : -carrying bats may also restrict the formation of escape mutations. however, based on the currently limited amount of bat mhc i sequences available, gly in the f pocket does not seem prevalent in bat mhc i. more sequencing of bat genomes and especially mhc i genes are needed to verify whether the accommodation of po-pro as a peptide anchor is common in bat mhc i or a specific feature of ptal-n � : . in conclusion, through a series of structural and functional investigation, we demonstrated several novel features of bat mhc class i molecules presenting virus-derived peptides. our results provide new insight into the adaptive immune system of bats, which may contribute to the unique virus-host interactions in these important mammals. due to the high containment nature of the viruses and the difficulty in conducting live bat infection studies, our current study lacks in vivo functional characterization, which we hope to conduct in the future with international collaborations. the sequences of mhc class i genes (including predicted genes) from bats were retrieved from the ncbi database (s table) . higher mammal mhc i heavy chain sequences were retrieved from the immuno polymorphism database (ipd) (www.ebi.ac.uk/ipd/mhc) and the uniprot database (www.uniprot.org). previously deposited marsupial (opossum, tammar wallaby, koala, tasmanian devil) and platypus mhc i transcripts were included in these analyses (s table) . sequence alignments were generated with clustalx [ ] and espript [ ] . similarities were calculated using dnaman (https://www.lynnon.com/). the proteomes of , mers-cov genomes and , sars-cov genomes were retrieved from genbank, respectively. after sequence alignment with mafft, the dominant amino acid for each site was elected as a reference sequence. the mutation frequency = the number of overall mutations for each amino acid/(the number of occurrences of the amino acid in the reference sequence×total number of sequences). to screen potential peptides for binding to ptal-n � : , the proteomes of the bat-related viruses ebov (np: genbank no. af . ; gp: genbank no. akg . ), mers-cov (genbank no. axn . ), h n influenza-like virus (a/little yellow-shouldered bat/ guatemala/ / (h n )), and h n influenza-like virus (a/flat-faced bat/peru/ / (h n )) were utilized to predict the candidate peptides. the candidate peptides were predicted and selected according to the recently reported motif, by which the two ptal-n � : -binding peptides, hev and hev , derived from hev were also synthesized (s table) [ ] . the potential binding scores of the selected peptides were also predicted through the online netmhcpan . server (http://www.cbs.dtu.dk/services/netmhcpan/) [ ] and rosetta flexpepdock, which is based on structure modeling [ , ] , so that we prefer choose peptides that conform to the motif of ptal-n � : [ ] . the peptide purity was determined to be > % by analytical hplc and mass spectrometry. the peptides were stored at − ˚c as freeze-dried powders and were dissolved in dmso before use. the cdnas for the heavy chain of p. alecto mhc i ptal-n � : (genbank no. kt ) [ ] and bat β m (genbank no. xp_ . ) were synthesized (genewiz, beijing, china). ptal-n � : sequence was deposited to genbank by wynne and colleagues, and ptal-n � : -binding peptides hev and hev were identified in their study [ ] . although ng and colleagues reported the first ptal-n � : [ ] , the sequence is not available online. to investigate the function of met asp leu in ptal-n � : , a mutant termed ptal-n � : (- aa) with a deletion of these three amino acids was constructed. the amplified products expressing the extracellular domain (residues - ) of ptal-n � : and bat β m (residues - ) were cloned into a pet a vector (novagen). the expression plasmid for human β m (residues - ) was previously constructed in our laboratory [ ] . molecular replacement with the program phaser mr in ccp [ ]. the model used was the structure coordinates with protein data bank (pdb) code f i [ ] , and restrained refinement was performed using refmac from ccp . extensive model building was performed by hand using coot [ ] . the stereochemical quality of the final model was assessed with the program refine in phenix or ccp (table ) . structure-related figures were generated using pymol (http://www.pymol.org/) and coot. the thermostabilities of ptal-n � : with two group key peptides were tested by cd spectroscopy. all complexes were refolded, purified, and measured at . mg/ml in a solution of mm tris (ph ) and mm nacl. cd spectra at nm were measured on a chirascan spectrometer (applied photophysics) using a thermostatically controlled cuvette at temperature intervals of . ˚c at an ascending rate of ˚c/minute between and ˚c. the unfolded fraction (%) is expressed as (θ−θ a )/(θ a −θ b ), where θ a and θ b are the mean residue ellipticity values in the fully folded and fully unfolded states, respectively. the denaturation curves were generated by nonlinear fitting with originpro . (originlab) [ ] . the t m was calculated by fitting data to the denaturation curves and using inflection-determining derivatives. -mers) to ptal-n � : elucidated by in vitro refolding. twenty long peptides (peptide bat to bat ) that were previously eluted from ptal-n � : -expressing cells were synthesized (s table) [ ] . the gray curve is a negative control without any peptide in the refolding reaction. renaturation and purification of ptal-n � : assembled with peptides were performed as previously described heavy chain and bat β m were overexpressed as inclusion bodies in the bl (de ) strain of escherichia coli, and the purified inclusion bodies of the proteins were solubilized in m guanidine-hcl buffer with a concentration of mg/ml. then, injection and dilution of mhc heavy chain, β m, and peptide occurred at a molar ratio of : : in refolding buffer ( mm tris-hcl the ptal-n � : /peptide complexes were screened through crystal screen kit i/ii, index screen kit, pegion kit i/ii, and the pegrx kit (hampton research) % (w/v) polyethylene glycol , , and % (v/v) glycerol at a protein concentration of mg/ml. single crystals of ptal-n � : /ebov-np were grown in . m succinic acid (ph . ) and % (w/v) polyethylene glycol , . single crystals of ptal-n � : /ebov-np were grown in . m ammonium acetate, . m tris (ph . ), and % (w/v) polyethylene glycol , . single crystals of ptal-n � : (- aa)/hev were grown in . m sodium formate and % (w/v) polyethylene glycol , human t-cell immunity against the emerging and re-emerging viruses protective t cell responses featured by concordant recognition of middle east respiratory syndrome coronavirus-derived cd + t cell epitopes and host mhc the triphibious warfare against viruses. sci china life sci fruit bats as reservoirs of ebola virus infection of humans and horses by a newly described morbillivirus bats and their virome: an important source of emerging viruses capable of infecting humans bats are natural reservoirs of sars-like coronaviruses severe acute respiratory syndrome coronavirus-like virus in chinese horseshoe bats a"iv to "z"ikv: attacks from emerging and re-emerging pathogens intra-host ebola viral adaption during human infection fatal swine acute diarrhoea syndrome caused by an hku -related coronavirus of bat origin the persistent prevalence and evolution of cross-family recombinant coronavirus gccdc among a bat population: a two-year follow-up bat-derived influenza-like viruses h n and h n a distinct lineage of influenza a virus from bats host and viral traits predict zoonotic spillover from mammals going to bat(s) for studies of disease tolerance studying immunity to zoonotic diseases in the natural host-keeping it real comparative analysis of bat genomes provides insight into the evolution of flight and immunity the egyptian rousette genome reveals unexpected features of bat antiviral immunity the immune gene repertoire of an important viral reservoir, the australian black flying fox dampened sting-dependent interferon activation in bats contraction of the type i ifn locus and unusual constitutive expression of ifn-alpha in bats type iii ifns in pteropid bats: differential expression patterns provide evidence for distinct roles in antiviral immunity a potential robust antiviral defense state in the common vampire bat: expression, induction and molecular characterization of the three interferon-stimulated genes -oas , adar and pkr generation of murine ctl by a hepatitis b virus-specific peptide and evaluation of the adjuvant effect of heat shock protein glycoprotein and its terminal fragments lack of prominent peptide-major histocompatibility complex features limits repertoire diversity in virus-specific cd + t cell populations specificity pockets for the side chains of peptide antigens in hla-aw refined structure of the human histocompatibility antigen hla-a at . a resolution evolution and comparative analysis of the bat mhc-i region characterization of the antigen processing machinery and endogenous peptide presentation of a bat mhc class i molecule the peptide-receptive transition state of mhc class i molecules: insight from structure and molecular dynamics crystal structure of a tapbpr-mhc i complex reveals the mechanism of peptide editing in antigen presentation peptide exchange on mhc-i by tapbpr is driven by a negative allostery release cycle an invariant arginine in common with mhc class ii allows extension at the c-terminal end of peptides bound to chicken mhc class i diversified anchoring features the peptide presentation of dla- * : first structural insight into domestic dog mhc class i major histocompatibility complex class i (flae* ) molecular structure in domestic cats demonstrates species-specific characteristics in presenting viral antigen peptides phosphorylationdependent interaction between antigenic peptides and mhc class i: a molecular basis for the presentation of transformed self marsupials and monotremes possess a novel family of mhc class i genes that is lost from the eutherian lineage cross-immunity against avian influenza a(h n ) virus in the healthy population is affected by antigenicity-dependent substitutions an altered position of the alpha helix of mhc class i is revealed by the crystal structure of hla-b* bound water structure and polymorphic amino acids act together to allow the binding of different peptides to mhc class i hla-b the three-dimensional structure of an h- ld-peptide complex explains the unique interaction of ld with beta- microglobulin and peptide the crystal structure of h- dd mhc class i complexed with the hiv- -derived peptide p -i at . a resolution: implications for t cell and nk cell recognition first glimpse of the peptide presentation by rhesus macaque mhc class i: crystal structures of mamu-a* complexed with two immunogenic siv epitopes and insights into ctl escape escape from hla-b* -restricted cd t cells by hepatitis c virus is associated with fitness costs atp-and antizyme-dependent endoproteolysis of ornithine decarboxylase to oligopeptides by the s proteasome contribution of proline residue for efficient production of mhc class i ligands by proteasomes we thank dr. jianhui li (institute of biophysics, chinese academy of sciences) for instruction in circular dichroism spectroscopy. the collected intensities were subsequently processed and scaled using the denzo program and the hkl software package (hkl research). the structures were determined using key: cord- -wuve tjz authors: anderson, robert title: manipulation of cell surface macromolecules by flaviviruses date: - - journal: adv virus res doi: . /s - ( ) - sha: doc_id: cord_uid: wuve tjz cell surface macromolecules play a crucial role in the biology and pathobiology of flaviviruses, both as receptors for virus entry and as signaling molecules for cell–cell interactions in the processes of vascular permeability and inflammation. this review examines the cell tropism and pathogenesis of flaviviruses from the standpoint of cell surface molecules, which have been implicated as receptors in both virus–cell as well as cell–cell interactions. the emerging picture is one that encompasses extensive regulation and interplay among the invading virus, viral immune complexes, fc receptors, major histocompatibility complex antigens, and adhesion molecules. flaviviruses comprise a rich and diverse family of agents that infect a variety of hosts and cause a wide spectrum of disease. three disease types are recognized for flaviviruses, namely encephalitis, hemorrhagic fever, and fever-arthralgia-rash. disease distinctions are not absolute, and overlapping pathologies among various flavivirus members are often observed. the ability of flaviviruses to cause such divergent clinical syndromes, associated with virus replication in a number of different organs, has profound implications for the types of cell surface molecules the virus recognizes as receptors. mutational analyses of the flaviviral e protein have demonstrated a striking ability of flaviviruses to adapt to different cells and receptors. given the considerable homologies among them, flaviviruses show a remarkable capacity to cause vastly different diseases with a minimum of alterations in the e protein. the cell surface molecules, which act as receptors for flaviviruses, are only starting to be identified. in addition to providing the molecules involved in virus attachment and penetration, the host cell erects a battery of surface structures that mediate communication with other cells and trigger host defense and pathological processes. many of these are modulated by flavivirus infection and contribute to the overall picture of pathogenesis. the flavivirus e protein is a multifunctional protein involved in cell receptor binding (anderson et al., ; chen et al., ; he et al., ) and virus entry via fusion with a host cell membrane (rice, ) . some of the functional activities of the e protein, notably membrane fusion, are regulated by interaction with a second viral protein, prm. it is believed that the association of prm with e stabilizes certain ph-sensitive epitopes on the e protein, thereby preventing the conformational changes that normally occur at acidic ph and activate the fusogenic activity of the e protein guirakhoo et al., ; heinz et al., ) . in addition to its normal role in flavivirus assembly, the prm protein has also been included in novel recombinant formulations in which it is generally coexpressed with the e protein; the resultant e/prm complexes have been shown to be immunogenic and protective as vaccines against challenge with several flaviviruses, including japanese encephalitis virus (mason et al., ) , yellow fever virus (pincus et al., ) , dengue virus (fonseca et al., ) , and tick-borne encephalitis (tbe) virus . in tbe virus, the majority of extracellular virus is largely free of prm protein due to a late intracellular processing event that generates a carboxy-terminal fragment designated m and which together with the e and c proteins are believed to constitute the protein components of the mature virus particle (heinz et al., ) . cleavage of prm to m enhances low ph-dependent virus-cell fusion (guirakhoo et al., ) and infectivity (guirakhoo et al., ; heinz et al., ; randolph et al., ; shapiro et al., ; wengler, ) . dengue virions containing prm are still infectious (randolph et al., ) and bind to permissive cells in a manner that can be blocked using e-specific antibodies wang et al., ) . virus particles containing mainly e and prm also show antibody-enhanced binding to fc receptor-bearing k cells as well as to platelets . thus, in addition to being requisite precursors to mature virus particles, virus particles containing prm possess many properties associated with mature virus particles. flaviviruses appear to gain entry to the cell by the endocytic pathway (rice, ) . at low ph, the e protein undergoes a conformational change involving dissociation of the e dimer (stiasny et al., ) , thereby exposing a hidden fusion peptide, followed by reorganization of e into a trimer , in which the fusion peptide is brought close to the membrane-anchoring carboxy terminus (ferlenghi et al., ) . remarkably similar structural features and conformational rearrangements have been noted between the flavivirus e protein and the alphavirus e lescar et al., ; pletnev et al., ; strauss and strauss, ) , suggesting a common evolutionary origin for these two virion surface proteins. considerable homology exists among flaviviral e proteins, raising the possibility that different flaviviruses may have similar receptorbinding motifs. for example, many mosquito-borne flaviviruses contain an rgd sequence (e.g., residues - of the murray valley encephalitis virus e protein), which has been implicated in virulence (lobigs et al., ) and receptor binding by analogy with integrin-binding motifs (rey et al., ) . mutagenesis studies of the yellow fever virus (van der most et al., ) and murray valley encephalitis virus (hurrelbrink and mcminn, ) rgd motifs, however, have cast doubt on the role of integrins in flavivirus attachment or entry. studies with tbe virus have identified important determinants for pathogenicity within the suspected receptor-binding site on the upperlateral surface of domain iii (mandl et al., ) . acquisition of heparan sulfate-binding mutations by passaging tbe in cell culture has also implicated amino acids in this region in receptor binding (mandl et al., ) . the selection of virus mutants on the basis of weak binding to brain membranes has been used with several neurotropic flaviviruses (holbrook et al., ; ni and barrett, ; ni et al., ) and has identified a variety of mutations within domain iii as well as other regions of e. for dengue virus, blocking of virus cell binding correlates more closely to virus neutralization for mab h than for mab b . this may suggest that mab h neutralizes dengue virus predominantly by blocking virus-cell attachment, whereas mab b neutralizes dengue virus largely by a postattachment mechanism. the mab h -binding site on the dengue viral e protein has been partly characterized (hiramatsu et al., ; megret et al., ; trirawatanapong et al., ) and probably encompasses, at a minimum, residues - (hiramatsu et al., ) within domain iii. more recent data involving a larger number of monoclonal antibodies indicate that mabs that interact with domain iii are in fact the most effective blockers of virus-cell attachment (crill and roehrig, ) . a putative heparan sulfate-binding site on the dengue- e protein is also located within this region , and comparative sequencing of dengue type genomes has implicated amino acid of the e protein as a major determinant of pathogenicity (leitmeyer et al., ) . the ph-dependent conformational ''hinge'' region (between domains i and ii) of the e protein has also been implicated in virulence, receptor interaction, and/or membrane fusion (hurrelbrink and mcminn, ; lee et al., ; monath et al., ) . further mutagenesis studies will undoubtedly help define the sites of the e protein involved in flavivirus-cell macromolecule recognition. transmission of flaviviruses to humans generally occurs via the bite of an infected mosquito or tick. in the case of dengue, inoculated virus is thought to first replicate in skin langerhans (dendritic) cells (palucka, ; taweechaisupapong et al., a taweechaisupapong et al., , b wu et al., ) . dendritic cells have also been shown to be involved in the transport of intradermally inoculated west nile virus to local draining lymph nodes, with a subsequent accumulation of leukocytes . it is likely that dendritic cells will prove to be efficient carriers of a wide number of flaviviruses from their cutaneous site of infection to lymphoid and possibly other tissues. given the importance of dendritic cells in initiating immune responses (banchereau et al., ) , they probably play a pivotal role in stimulating host defense against invading flaviviruses. dengue virus infection of immature myeloid dendritic cells has been shown to induce their maturation accompanied by the expression of major histocompatibility complex (mhc) class i and ii antigens; the costimulatory molecules cd , cd , and cd ; and the dendritic cell marker cd (libraty et al., ) . such changes were seen in both dengue-infected and bystander cells, indicating that upregulation of cell surface molecules could be a consequence of virus infection as well as virusinduced cytokine expression. similarly, langerhans cells infected with west nile virus, as well as an alphavirus, semliki forest virus, express increased cell surface mhc class ii and appear to undergo maturation to a cell type similar to lymphoid dendritic cells (johnston et al., ) . the efficient presentation of both mhc class i-and ii-associated viral peptides on the surface of dendritic cells permits the generation of potent cytotoxic and helper t cell responses (see also section v,a). monocytes and macrophages have long been recognized as major targets of flavivirus replication in the human host (halstead, ; scott et al., ) . they are also important host cells for the antibody-enhanced replication of certain flaviviruses (see section iv,c). because of their presence in the circulation, blood monocytes may be particularly important to the pathogenesis of hemorrhagic viruses, such as dengue. because most of the pathological changes associated with dengue virus are hemostatic in nature, it is suspected that blood cells, particularly virus-infected blood monocytes, orchestrate many of these effects. dengue virus-infected human monocytes have been shown to be potent sources of vasoactive cytokines such as tumor necrosis factor (tnf)- and interleukin (il)- (chang and shaio, ) . monocytes are also known producers of several other vasoactive mediators, including il- , platelet-activating factor (paf), prostaglandins, thromboxanes, leukotrienes, and nitric oxide (bulger and maier, ; funk, ; lefer, ; maruo et al., ; montrucchio et al., ; szabo and billiar, ) , any of which could have powerful effects on endothelial cell physiology. a crucial aspect in understanding dengue pathogenesis will be the identification of additional vasoactive mediators, which trigger the key dysfunctional events in vascular integrity. various tissue macrophages are undoubtedly important in the pathogenesis of flaviviral diseases but have, to date, not received much attention. skin mononuclear cells, pulmonary, splenic, and thymic macrophages and liver kupffer cells have been recognized carriers of viral antigen (halstead, ) . in the liver, virus or viral antigen has been found in kupffer cells and hepatocytes in infections with yellow fever (monath et al., ) and dengue (bhamarapravati et al., ; hall et al., ; halstead, ; rosen and khin, ) . destruction of kupffer cells, possibly by apoptosis, has been reported in the liver of some patients with fatal dengue (huerre et al., ) . primary cultures of kupffer cells apparently undergo an abortive infection with dengue virus in which viral antigen but no progeny virus is produced (marianneau et al., ) . many flaviviruses invade either visceral or central nervous system tissues following initial replication in dendritic cells, monocytes, or macrophages. often this necessitates a transfer of virus across blood vessel endothelial layers. for neurotropic flaviviruses, endothelial cells of the cerebral microvasculature constitute a barrier that must be overcome in order to gain access to the central nervous system. how this occurs remains uncertain. transendothelial passage of virus may direct infection of cerebral microvascular endothelial cells, may transport across the endothelial layer, or both (dropulic and masters, ) . japanese encephalitis virus has been observed electron microscopically to traverse mouse cerebral endothelial cells by transcytosis (liou and hsu, ) . alternatively, virus may spread from blood vessels to the olfactory neuroepithelium and from there to olfactory neurons (mcminn et al., ; monath et al., ) . even normally nonneurotropic flaviviruses may occasionally invade the central nervous system under certain conditions. modulation of the blood-brain barrier by anesthetics (ben-nathan et al., ) or lipopolysaccharide (lustig et al., ) has been reported to facilitate neuroinvasion by a normally noninvasive strain of west nile virus. flaviviruses may also trigger the production of soluble factors that perturb the integrity of the blood-brain barrier, leading to increased leakage of proteins and cells into the central nervous system (chaturvedi et al., ) . these studies indicate that even nonneurotropic flaviviruses may infect tissues of the central nervous system or otherwise affect the integrity of the blood-brain barrier under special circumstances. transendothelial migration of individual leukocytes (e.g., lymphocytes, monocytes, neutrophils, eosinophils) is regulated in a highly specific manner by the differential expression of selected adhesion molecules on endothelial cells (reviewed in crockett, ; lowell and berton, ) . flaviviruses, including dengue and west nile (shen et al., ) viruses, activate endothelial cell adhesion molecule expression by either direct (virus-mediated) or indirect (cytokine-mediated) mechanisms (see section v,c). in the presence of leukocyte-attracting chemokines, such virus-triggered activation of the vascular endothelium may contribute toward the migration of leukocytes into extravascular tissues. in addition to being a mechanism for virus dissemination, this process may also be a factor in phenomena such as leukopenia and particularly neutropenia (loss of circulating leukocytes, neutrophils) often observed in flavivirus, particularly dengue, infection (reviewed in halstead, ) . due to the lack of suitable animal models for severe dengue disease, i.e., dengue hemorrhagic fever (dhf) or dengue shock syndrome (dss), there are difficulties in assessing the roles of such events, particularly the identification of adhesion molecules mediating the transendothelial migration of neutrophils using blocking antibodies against specific integrins, as has been performed for other disease states (doerschuk et al., ; gao et al., ; issekutz and issekutz, ; laberge et al., ; springer, ) . the hallmark feature of increased vascular permeability in hemorrhagic flavivirus (e.g., dengue) infection suggests that vascular endothelial cells may mediate the fluid leakage and hemorrhaging that occur in dhf/dss. endothelial cells line the inner surface of blood vessels and play essential roles in maintaining an antithrombogenic surface and regulating vascular permeability. increased vascular permeability can arise from a variety of mediators associated with acute inflammation and shock (bulger and maier, ; funk, ; lefer, ; michel, ; montrucchio et al., ; schnittler et al., ) . it is thought that vascular permeability is largely controlled by changes in endothelial cell-cell contact, which result in gap formation, thus allowing for fluid exchange between blood and interstitial tissue fluid (michel, ). an electron microscopic study of endothelium from dhf biopsy samples revealed the occasional presence of gaps (sahaphong et al., ) , thus providing evidence that endothelial cell features may indeed be perturbed during dhf/dss. although dengue virus infects endothelial cells in vitro (andrews et al., ; avirutnan et al., ; killen and o'sullivan, ) , there is no evidence that endothelial cell infection occurs clinically, as neither virus particles nor viral antigen has been detected in the endothelium of tissue specimens (halstead, (halstead, , sahaphong et al., ) , in contrast to that seen in cases of ebola (zaki et al., ) or hantaan hemorrhagic fever (gavrilovskaya et al., ; wang et al., ) . it is likely that dengue virus mediates endothelial cell activation via an indirect route, involving blood monocytes, which are a major cell target for dengue virus infection (halstead et al., b; scott et al., ) . a major candidate event in such a route is the activation of endothelial cell adhesion molecules by a factor(s) (particularly tnf-) produced by dengue virus-infected blood monocytes . tnf is a key cytokine in a variety of normal and pathological immune responses, including immunoregulation, regulation of cell proliferation, cytotoxicity, and in the mediation of endotoxic shock (fiers, ; tartaglia and goeddel, ; tracey and cerami, ; vassalli, ) . monocyte-derived tnf-appears to play a pivotal role in dengue-associated endothelial cell activation and may be an important effector in the manifestation of dhf/dss. support for the clinical significance of this observation comes from observations of elevated tnf levels in the sera of patients with severe dengue disease (green et al., b; hober et al., ; vitarana et al., ; yadav et al., ) . taken together, current evidence indicates that dengue virus represents a rather unique group of viruses that target monocytes, thereby triggering the production of factors such as tnf-, which in turn affect other cell targets, including endothelial cells. while the overall picture of endothelial cell dysfunction in dhf/dss is obviously more complex than can be explained by any single factor, the role of tnf in dengue pathogenesis would seem to merit particular attention. current knowledge of endothelial cell responses observed in endotoxic shock may be instructive for the understanding of vascular leakage in dhf/dss. plasma leakage induced by endotoxin (lipopolysaccharide, lps) from gram-negative bacteria encompasses a complex cascade of processes, including activation and functional alteration of endothelial cells. major mediators of endothelial cell perturbation in endotoxic shock are lps itself, as well as cytokines such as tnf-and il- (bevilacqua, ) . these factors can modulate endothelial cell function to varying degrees by activating cytokine and vasoactive factor release (rink and kirchner, ; shanley et al., ) , upregulating adhesion molecule expression (bevilacqua, ; luscinskas et al., ; moser et al., ; smith et al., ) , and mediating transendothelial migration of specific leukocytes luscinskas et al., ; morzycki et al., ; moser et al., ; smith et al., ) . additional factors, particularly lipid mediators such as paf, leukotrienes, thromboxanes, and prostaglandins, may contribute to further endothelial cell dysfunction, including vascular leakage (bulger and maier, ; funk, ; lefer, ; montrucchio et al., ) . while the involvement of these vasoactive mediators is recognized in endotoxic shock, more needs to be learned of their role in the vascular dysfunction that occurs in severe dengue disease. although lymphocytes are potently involved in the host response and immunopathology of flavivirus (especially dengue) diseases, their role as virus-permissive host cells is unclear. dengue virus has been identified in circulating b cells from acutely ill dengue patients by immunocytochemistry and by recovery of infectious virus after passage in mosquitoes . in vitro studies showed that cells and cultured cell lines of both b and t cell derivation could be infected with dengue virus (bielefeldt-ohmann et al., ; kurane et al., ; marchette and halstead, ; mentor and kurane, ; sung et al., ; takasaki et al., ; theofilopoulos et al., ) . continued passage of dengue virus in lymphoblastoid (raji) cells can give rise to dengue virus variants capable of replication in human lymphocytes (brandt et al., ) . interestingly, lymphocytes do not appear to undergo antibody-enhanced dengue virus infection (brandt et al., ; kurane et al., ) , even though b cells do have fc receptors (dijstelbloem et al., ; see section iv,c). the initial stages of pathogenesis for neurotropic flaviviruses appear to be common for flaviviruses in general in that the virus progresses from the subcutaneous site of inoculation to lymph nodes, followed by viremia and replication in extraneural tissues. invasion into the central nervous system is marked by high virus titers in the brain and detectable virus or viral antigen in neurons (albrecht, ) . cell destruction in tick-borne encephalitis may be less extensive than that seen in herpes simplex type encephalitis (studahl et al., ) , although this is variable and may involve considerable inflammation (chu et al., ; matthews et al., ; suzuki et al., ) . susceptible cell types include both neurons and glial cells (chu et al., ; ramos et al., ; steele et al., ) . as notorious producers of vasoactive mediators, mast cells have been a source of controversial speculation for years in dengue pathogenesis. cells resembling degranulated mast cells have been reported in skin perivascular infiltrates from dhf/dss cases (bhamarapravati et al., ) . dengue patients showed elevated levels of urinary histamine (a major granule product of mast cells), which correlated with disease severity (tuchinda et al., ) , suggesting that mast cells may have a contributory role in the pathogenesis of dengue. although antihistamine treatment does not resolve shock in severely dengue-diseased patients (halstead, ) , histamine is only one of several potent vasoactive factors produced by mast cells (benyon et al., ; bradding et al., ; galli et al., ; grabbe et al., ; marshall and bienenstock, ; moller et al., moller et al., , moller et al., , nilsson et al., ; schwartz and austen, ) , some of which could cause vascular dysfunction in dengue infection. dhf/dss patients have been reported to have elevated serum levels of ige (pavri et al., ) , which has been speculated to relate to ige-triggered histamine release in the manifestation of shock (pavri and prasad, ) . mast cells reside mainly in the tissues, often closely associated with blood vessels (alving, ; anton et al., ; pesci et al., ; pulimood et al., ; selye, ; selye et al., ) . they are present in large numbers in the skin (marshall et al., ) , where transmission of insect-borne flaviviruses occurs. basophils, however, comprise about % of total circulating cells and would be accessible to virus in the blood. dengue virus infects basophil/mast cell-like ku cells in an antibody-enhanced manner, coupled with the release of vasoactive cytokines, il- and il- (king et al., , . this cell line, which can be differentiated easily toward either a basophil or mast cell phenotype (saito et al., ) , may provide further insights into potential roles for basophils and mast cells in dengue disease. dengue patients show increased serum levels of anaphylatoxins c a and c a (malasit, ) , which can attract (nilsson et al., ) and activate (kownatzki, ) mast cells. among the expected mast cell secretion products would be vasoactive factors, including histamine, which has been detected in elevated amounts in the urine of dengue patients (tuchinda et al., ) . evidence for platelet involvement in dengue pathogenesis comes from at least two (probably related) sources. first, thrombocytopenia (loss of circulating platelets) is one of the most consistent clinical features of severe dengue infection (halstead, ) . second, viral immune complexes have been detected on platelets from dengue patients phanichyakarn et al., a) . functional studies on platelets in dengue-diseased individuals have been sparse, but include a markedly reduced half-life (mitrakul et al., ) , deficient adp release (mitrakul et al., ) , increased adhesiveness (doury et al., ) , increased tagging by complement fragments (malasit, ) , and increased release of -thromboglobulin and platelet factor (srichaikul et al., ) . there is also evidence for platelet activation in dengue patients (doury et al., ; krishnamurti et al., ; srichaikul et al., ) . although these results relate to a variety of platelet functions, they do indicate a general alteration in platelet physiology, which is consistent with platelet involvement and triggering of thrombocytopenia in dengue disease. dengue virus has been recovered from washed patient platelets (scott et al., ) , and virus has been reported to bind to platelets in the absence of antibody as assayed using immunofluorescence and immunoperoxidase techniques (funahara et al., ) . however, the levels of antibody-independent bound virus are very low compared to the levels of virus bound in the presence of dengue-specific antibodies . as noted earlier, dengue immune complexes have been demonstrated on platelets from dengue patients phanichyakarn et al., a) . weiss and halstead ( ) originally proposed the possibility that dengue virus interactions with platelets might be involved in the thrombocytopenia observed in severe dengue disease. the finding that dengue virus binding to platelets is dependent on a virus-specific antibody is consistent with epidemiological and experimental data linking preexisting host antibodies to an increased risk of dhf/dss (reviewed in halstead, ) . several other viruses have been shown to bind directly to platelets (bik et al., ; danon et al., ; forghani and schmidt, ; larke and wheelock, ; lee et al., ; zucker-franklin et al., ) . platelet association may stabilize or protect blood-borne viruses (larke and wheelock, ) and may function as a mechanism of hematogenous dissemination (forghani and schmidt, ) . virus binding to platelets has been suggested to be a contributing mechanism to thromobocytopenia arising from infections with vaccinia (bik et al., ) , chikungunya (larke and wheelock, ) , and rubella (bayer et al., ) . thrombocytopenia in these virus infections is generally much milder than that observed in severe dengue disease. levels of dengue virus in the blood can exceed infectious units/ml (gubler, ; monath, ) . such high viremic titers are likely necessary to ensure infection and transmission of the obligate mosquito intermediary host (monath, ) . assuming a reasonable particle:infectivity ratio of : , virus particle titers in blood may rival normal platelet counts (  /ml). such parity between numbers of virus particles and platelets suggests that antibodyenhanced binding of virus to platelets may have a profound effect on platelets. circulating virus-immune complexes are detected in dhf/ dss, and levels of immune complexes have been correlated with severity of disease (ruangjirachuporn et al., ) and some of these are platelet associated phanichyakarn et al., a) . these observations suggest that sufficient binding of virus immune complexes to platelets may occur to tag the majority of circulating platelets. such an event could lead to immune clearance by the reticuloendothelial system, thereby precipitating the thrombocytopenia frequently associated with severe dengue disease. it is likely that molecules other than fc receptors on the platelet surface may mediate antibody-enhanced binding of dengue virus . drug-induced thrombocytopenias provide interesting examples in this regard. it is known that given the appropriate accessory ligand (i.e., drug), igg can bind to platelets through either the fc receptor or other surface proteins. a variety of clinical thrombocytopenias are known that involve an immune component in pathogenesis. many of these reflect activities of host antibodies, which react with proteins on the surface of platelets. these antibodies may be autoimmune in nature (i.e., antibodies that bind to platelet surface molecules) or dependent on a third party ligand (drug or protein), which then induces binding of the antibody-ligand complex to either the platelet fc receptor or to another surface protein. for example, a number of individuals are susceptible to drug-dependent thrombocytopenia when administered drugs such as heparin or quinine/quinidine (aster, ; hackett et al., ) . while heparindependent antibodies bind to the platelet fc receptor (adelman et al., ; chong et al., a chong et al., , b kelton et al., ) , quinine/quinidine-dependent antibodies bind to platelet protein heterodimers gpiib/iiia and gpia/ix (berndt et al., ; chong et al., ; christie et al., ; devine and rosse, ) . this latter category of immune-mediated thrombocytopenia may be relevant to the understanding of dengue-associated thrombocytopenia, as patient antibodies mediate dengue virus binding to platelets via a platelet surface protein other than the fc receptor . communication between platelets and endothelial cells is a frequent intermediate step in certain events such as platelet adhesion, aggregation, and regulation of vascular permeability. how this occurs in dengue infection and what the effects are on endothelial cell function are unknown. binding of viruses to platelets can have potentially profound immunological effects [e.g., the stimulation of tgf-release by platelets bound by epstein-barr virus (ahmad and menezes, ) ]. in light of reports of altered platelet function in dengue patients, discussed earlier, there is a tantalizing need to determine the immunological consequences of antibody-enhanced dengue virus binding to platelets in terms of platelet as well as endothelial cell physiological responses. many products of complement activation can also be deposited on platelets (devine, ) . in view of evidence for complement activation in severe dengue disease (halstead, ; malasit, ) , binding of complement products might play a role in the immune destruction of platelets leading to thrombocytopenia. platelets display surface receptors, e.g., c q receptor ghebrehiwet, , ) , membrane cofactor protein (seya et al., ) , and decay-accelerating factor (devine et al., ) , for specific components of complement activation. in addition, the platelet surface can act as a substrate for the deposition of c dg and c b- (devine, ) . fragments of c have been detected on the platelets of dhf/dss patients (malasit, ) . in addition to immune complex deposition on platelets, thrombocytopenia associated with dhf/dss might also arise by the immune destruction of platelets through antiplatelet autoantibodies. antiplatelet autoantibodies have been reported in the sera of dengue patients (lin et al., ) , although they have also been detected in patients recovering from a variety of viral infections (imbach, ) . antiplatelet antibodies are strongly linked to the pathogenesis of immunemediated thrombocytopenias, such as idiopathic thrombocytopenic purpura (winkelstein and kiss, ) . while this brief discussion of cell targets for flaviviruses is by no means complete, it highlights some of the major interactions as they relate to pathogenesis. because pathogenesis is probably best understood for dengue, fig. illustrates the interactions of hemorrhagic flavivirus (e.g., dengue) with cell targets both within and outside the vascular system. glycosaminoglycans and proteoglycans (i.e., proteins bearing glycosaminoglycans) are important cell surface molecules involved in a variety of ligand recognition and cell signaling processes (gallo, ) . because glycosaminoglycans are widely distributed on cells, they are attractive candidates as virus receptors. some degree of specificity (i.e., virus tropism) may arise from the compositional heterogeneity of glycosaminoglycans, as well as quantitative differences in the degree of expression on various cell types. flaviviruses seem to share, with a large number of virus families, the ability to bind glycosaminoglycans (birkmann et al., ; dechecchi et al., dechecchi et al., , duisit et al., ; feldman et al., feldman et al., , giroglou et al., ; goodfellow et al., ; heil et al., ; hsiao et al., ; hulst et al., hulst et al., , lin et al., ; liu and thorp, ; patel et al., ; rue and ryan, ; shukla et al., ; shukla and spear, ) . glycosaminoglycans such as heparin and its structural analogues have been investigated for their ability to bind dengue virus and thereby to gain insights as to the structural requirements for dengue receptors. potential glycosaminoglycan-binding motifs have been identified on the dengue viral e protein at two sites, the best characterized of which appears to be composed of amino acids , - , and - and which may also play a role in virus-cell attachment . heparin (minimum of carbohydrates) and an uncharacterized highly sulfated heparin sulfate isolated from bovine liver were found to show the best binding to dengue e protein . attachment of dengue virus to human hepatoma cells has also been reported to be inhibited by heparin (hilgard and stockert, ) . a further study involving a panel of natural and synthetic polyanionic, sulfated compounds suggested that binding of the dengue e protein required a highly sulfated (and highly charged) oligosaccharide with a minimum size of Å and a high degree of structural flexibility (marks et al., ) . the role of glycosaminoglycans in natural (i.e., nontissue cultureadapted) strains of flaviviruses needs to be studied further. it has long been recognized that dengue virus passaged in various host cell types can give rise to virus variants with altered cell specificity (brandt et al., ; halstead et al., a halstead et al., , b halstead et al., , c . passage-dependent mutations of the dengue virus e protein at a number of different amino acid residues have been documented (lee et al., ) . following passage of tbe virus in cultured bhk- cells, virus mutants were selected that contained more positively charged amino acids in the putative receptor-binding region of the e protein, resulting in dependence on cell surface heparan sulfate (mandl et al., ) . such mutants were diminished in their neurovirulence in mice as well as in their replication in primary chicken cells and plaque formation in porcine kidney cells (mandl et al., ) . a large number of other viruses have also been shown to undergo loss of virulence upon adaptation to cell culture associated with heparan sulfate utilization (bernard et al., ; byrnes and griffin, ; klimstra et al., klimstra et al., , lee and lobigs, ; neff et al., ; sa-carvalho et al., ) . cd and the toll-like receptor (tlr) pattern recognition receptors are involved in the innate response to lipopolysaccharide and other microbial products (diamond et al., ; imler and hoffmann, ) . a role for cd and tlr has been found for respiratory syncytial virus (rsv) (kurt-jones et al., ) , suggesting that these receptors may have a broader involvement in host response than previously thought. a possible role for cd in dengue infection has been postulated on the basis of inhibition of dengue virus infection of human monocytes with bacterial lipopolysaccharide (chen et al., ) . however, this has been disputed (bielefeldt-ohmann et al., ) and requires further investigation. as indicated earlier, flaviviruses are capable of initiating infection of appropriate host cells through as yet largely unidentified primary receptors. in addition, a number of flaviviruses are capable of using subneutralizing levels of virus-specific antibodies to attach to and gain entry to cells bearing fc and/or complement receptors (cardosa et al., ; halstead, ; halstead and o'rourke, a; schlesinger and brandriss, a ) by a process known as antibody-dependent enhancement (ade) of infection (table i) . ade has been documented for dengue , west nile (peiris and porterfield, ) , yellow fever (schlesinger and brandriss, b) , tick-borne encephalitis (phillpotts et al., ) and japanese encephalitis (cecilia and ghosh, ) viruses. early work with dengue virus and monocytes differentiated between trypsin-sensitive and trypsinresistant cell surface molecules as the putative receptors for antibody-independent and antibody-dependent infection, respectively (daughaday et al., ) . to date, dengue virus appears to be the only flavivirus in which strong evidence exists for antibody-dependent enhancement as a major contributing factor to severe disease (halstead, ; thein et al., ) . severe dengue disease, encompasing conditions known as dengue hemorrhagic fever/dengue shock syndrome, involves several well-defined hemostatic abnormalities, including the leakage of plasma into interstitial spaces, as well as thrombocytopenia and bleeding (halstead, ; kurane et al., ) . the potential to cause severe hemorrhagic disease is a general property of dengue viruses and is not limited to any one viral serotype (gubler, ; rigau-perez et al., ) . although different strains of dengue may influence the severity of hemorrhagic symptoms (leitmeyer et al., ; rico-hesse et al., ) , it is also generally accepted that pathogenesis depends on immunopathological processes (rothman and ennis, ) . thus the roles of prior immunity, antibody-enhanced virus infection, and immune-mediated pathologic effects on the vascular system are key points in understanding the pathogenesis of dengue hemorrhagic disease. while the pathogenesis of severe dengue disease is not completely understood, it is clear from laboratory and epidemiological studies that a considerable risk factor is prior immunity. severe dengue disease, dhf/dss, rarely occurs in seronegative individuals suffering their first dengue infection, but instead occurs in individuals who have preexisting dengue viral antibodies, either from a previous infection or from passive antibody transfer, e.g., following maternal transmission of antibodies to the fetus (kliks et al., (kliks et al., , . estimates suggest that % of children suffering from dhf/dss have preexisting immunity from a prior dengue virus infection (halstead, ) . consequently, from this and other studies, it has been calculated that prior exposure to dengue increases the risk for hemorrhagic disease in a okayama et al. ( ) , anselmino et al. ( ) , and tuijnman et al. ( ) . b from . c from wu et al. ( ) and libraty et al. ( ) . d from king et al. ( ) . e abortive infection, but expressing viral antigen (marianneau et al., ) . f from littaua et al. ( ) and kontny et al. ( ). second dengue infection by at least -fold (halstead, ; thein et al., ) . preexisting serum antibodies can potentiate virus infection by the mechanism of antibody-dependent enhancement, giving rise to amplified virus replication and to an increased potential for the development of hemorrhagic symptoms (halstead, ) . viremic titers are higher in secondary dengue infections in both humans (gubler et al., ) and experimental monkeys (halstead et al., ) . antibody-enhanced dengue virus infection of human blood monocytes is necessary for the production of endothelial cell activators , thereby providing a link between antibody-dependent enhancement and alteration of endothelial cell properties, which might contribute to vascular permeability in dengue infection. for certain other viruses, e.g., influenza (tamura et al., ) and hiv (takeda et al., (takeda et al., , , distinct ''neutralizing'' and ''antibodyenhancing'' epitopes have been identified on the respective viral attachment proteins. surprisingly, no systematic approach has yet been undertaken to identify regions on the e protein that are essential for ade, even though this issue was raised as a challenge to research on dengue many years ago (halstead, ) . human fc receptors are currently categorized into three classes: fcri (cd ), fcrii (cd ), and fcriii (cd ). while fcri shows high affinity for monomeric igg, fcrii and fcriii bind monomeric igg poorly and are more likely involved in binding immune complexes (dijstelbloem et al., ) . fcrii is the most widely distributed, being expressed on most circulating leukocytes (van de winkel and anderson, ) . monocytes express all three fcrs to varying degrees (van de winkel and anderson, ) , although fcri and fcrii predominate, whereas fcriii appears to be limited to a subpopulation ($ %) of monocytes (anderson et al., ; passlick et al., ) . fcriii constitutes the major fcr on macrophages (fanger et al., ) , although fcri and fcrii are also present (tuijnman et al., ; van de winkel and anderson, ) . it is also important to recognize that fcr expression on cells, including macrophages, can vary depending on the microenvironment (tomita et al., ) . although strong evidence exists for fcr involvement in ade of dengue virus, the participating fcrs in vivo have not yet been identified rigorously. in cultured cell lines (monocytic u or erythroleukemic k cells), fcri (kontny et al., ) and fcrii (littaua et al., ) have been shown to mediate ade of dengue virus infection. that fcri has the ability to mediate ade of dengue has been demonstrated using cos cells transfected with fcri (schlesinger and chapman, ) . dengue and dhf patients show elevated serum levels of interferon (ifn)- (kurane et al., ) . because ifn-can upregulate both mhc class i and ii molecules as well as fc r (particularly fc ri) expression in monocytes (erbe et al., ; perussia et al., ) , the chances for ade may be increased, thereby creating a vicious cycle involving positive cytokine feedback and virus amplification (kurane and ennis, ) . ifn-has been shown to enhance ade of dengue virus infection of human monocytic u cells (kontny et al., ) , although any enhancing effect on dengue infection of peripheral blood monocytes may be negated by the antiviral properties of ifn- (sittisombut et al., ) . mast cells and basophils express mainly fc rii (anselmino et al., ; okayama et al., a; wedi et al., ) and some (ifn--inducible) fc ri (okayama et al., (okayama et al., , b as well as the highaffinity fc eri for ige (guo et al., ; sperr et al., ) . as noted previously, the basophil/mast cell ku cell line exhibits antibodyenhanced dengue virus infection and produces vasoactive cytokines . although fc r-mediated ade of flaviviruses has been examined extensively as a mechanism for virus amplification, the biological consequences for the participating host cell are not well understood. because fc r-mediated cell signaling is complex, the functional effects of virus-antibody interactions with cell surface fc rs need to be investigated. monocytes infected with dengue virus in the presence of antibody release cytokines such as tnf- . induction of tnf-requires infectious virus , suggesting that virus replication (or perhaps expression of one or more crucial viral genes) is responsible for the stimulation of tnf-release. therefore, in this case, the fc r is likely facilitating antibody-enhanced virus replication rather than providing a signal triggered by virus binding to the fc r. similarly, antibody-enhanced dengue virus infection of ku basophil/mast cells produces il- , il- (king et al., , , and selected chemokines (king et al., ) . suppressive effects of antibody-enhanced flavivirus or alphavirus infection on monocyte cytokine secretion have also been reported (lidbury and mahalingam, ; yang et al., ) . both activating (fcri, fcriia, and fcriiia) and inhibitory (fcriib) forms of fcrs exist, which mediate signal transduction via a cytoplasmic immunoreceptor tyrosine-based activation motif (itam) or inhibitory (itim) motif, respectively (dijstelbloem et al., ) . the itam and associated molecules are necessary for the endocytosis of fcr-bound immune complexes (amigorena and bonnerot, ) and therefore play a likely role in the initiating events of antibody-enhanced flavivirus infection. although not necessary for fcrii, an accessory subunit (homo-or heterodimeric or chains) is required for signaling through fcri and fcriiia (ravetch, ) . a further fcr (fcriiib) lacks transmembrane and cytoplasmic domains and is instead anchored to the cell surface membrane via a glycosylphosphatidylinositol (gpi) linkage (selvaraj et al., ; simmons and seed, ) . it apparently does not participate in signal transduction and has been speculated to sequester and accumulate immune complexes at specific sites on the cell surface (huizinga et al., ; selvaraj et al., ) . the roles of activating and inhibitory fcrs in viral ade have not yet been ascertained. activating fcrs are expressed on monocytes, macrophages, granulocytes, natural killer (nk) cells, and platelets but not on most lymphocytes (dijstelbloem et al., ) . inhibitory fcrs, however, are found on b cells, dendritic cells, and macrophages (dijstelbloem et al., ) . interestingly, ade of dengue virus is best documented for monocytes/macrophages and related cell lines (halstead, ) . in contrast, lymphocytic cells (brandt et al., ; kurane et al., ) and dendritic cells (wu et al., ) do not appear to support antibody-enhanced dengue virus infection. whether this is due to differential expression of activating versus inhibitory fcrs remains to be investigated. fcrs for ige (primarily the high-affinity fceri) are expressed on cells such as monocytes, macrophages, mast cells, basophils, and dendritic cells and are structurally related to fcrs (ravetch, ) . their role in binding ige and/or immune-complexed flaviviruses, such as dengue, remains unexplored. similarly unexplored is the potential role of the neonatal fc igg receptor (fcrn), structurally related to mhc class i and involved in igg transport across cells (ghetie and ward, ) . in addition to being expressed on certain epithelial and endothelial cells, fcrn is also expressed functionally on monocytes, macrophages, and dendritic cells (zhu et al., ) . in addition to the fcr, the antibody-complexed flavivirus has been shown to be taken up by a macrophage cell line using the complement receptor- (cardosa et al., ) . in the case studied-west nile virus infection of mouse p d macrophages-ade was mediated by the presence of antiviral igm and was inhibited with a cr -blocking antibody. this mode of ade was, however, found to be quantitatively less productive than the more commonly studied route of ade, i.e., involving fcr-mediated uptake route of igg-virus complexes (cardosa et al., ) . the recent demonstration of dc-sign as a functional dengue virus receptor on human dendritic cells represents an important advance in the definitive identification of flavivirus receptors (navarro-sanchez et al., ; tassaneetrithep et al., ) . several studies have identified cell surface proteins that bind flaviviruses, generally assayed by virus overlay blots of sds-page-resolved cell proteins (table ii) . further work is required to confirm the involvement of these and other proteins as receptors in flavivirus infection. a number of flaviviruses are able to stimulate the expression of cell surface molecules. notable among these are adhesion molecules and major histocompatibility antigens. multiple mechanisms appear to be involved, including virus-and cytokine-dependent pathways. flavivirus infection of a number of cell types causes an increase in cell surface mhc class i expression (king and kesson, ; king et al., ; libraty et al., ; liu et al., ; lobigs et al., ; shen et al., a shen et al., , . evidence for both virus-dependent (lobigs et al., ) and cytokine-dependent (libraty et al., ; shen et al., ) mechanisms has been reported. one process appears to be driven by the amount of flaviviral peptides generated by proteolysis and imported into the transporter associated with antigen processing (tap), which results in increased cell surface expression of peptideloaded mhc class i (momburg et al., ) . the upregulation of mhc class i molecules by flaviviruses is perhaps reminiscent of that observed in infections by coronaviruses (suzumura et al., ) but stands in contrast to the virus-manipulated downregulation of mhc class i by viruses such as herpesviruses (jennings et al., ; ploegh, ) , adenoviruses (sparer and gooding, ) , poxviruses (boshkov et al., ) , and hiv (scheppler et al., ) . although enhanced chu and ng ( ) mhc class i expression would be expected to lead to greater cytotoxic t (tc) cell-mediated cytolysis, it would render cells less susceptible to recognition by nk cells. evidence has been presented that flavivirusinfected cells in fact show reduced susceptibility to nk cells at the cost of enhanced tc cell-mediated lysis (lobigs et al., ) . it has been suggested that such a response may permit flaviviruses to evade an early nk cell response and thereby allow for substantial amplification of virus during the viremic phase of infection (momburg et al., ). nevertheless, evidence shows that nk cells are activated during dengue infection (green et al., a) , and nk cell-mediated cytotoxicity has been reported to correlate with the severity of disease (homchampa et al., ) . dendritic cells also undergo upregulation of mhc class i molecules following infection with dengue virus (libraty et al., ) . compared to other antigen-presenting cells, dendritic cells have superior t cellstimulating activities (mckinney and streilein, ; timares et al., ) . because antigen presentation via dendritic cell mhc class i can provoke exceptionally strong proliferation in cd -bearing t cells (bhardwaj et al., ; elbe et al., ; mckinney and streilein, ) , much of the overall cytotoxic t cell response arising in flavivirus infection may be dictated at the level of the dendritic cell. west nile virus infection induces mhc class ii expression in mouse macrophages (shen et al., a) , mouse astrocytes (liu et al., ) , rat schwann cells (argall et al., ) , and human myoblasts (bao et al., ) . upregulation of dendritic cell mhc class ii occurs in response to dengue (libraty et al., ) and west nile (johnston et al., ) virus infection. given the potent ability of dendritic cells to activate t cells (banchereau et al., ) , the communication between dendritic cell mhc class ii-peptide complexes and recognition molecules on cd -expressing t cells should provide insights into some of the molecular processes underlying t cell activation. adhesion molecules are expressed on a variety of cells and mediate a spectrum of processes (ley, ; roebuck and finnegan, ; springer, ) . from the standpoint of flaviviruses, the most significant processes likely concern adhesion molecules on vascular endothelial cells, as these cells regulate permeability as well as transendothelial migration of leukocytes (springer, ) . of particular importance are intercellular adhesion molecule (icam- ; cd ), vascular cell adhesion molecule- (vcam- ; cd ), and e-selectin (cd e), which are upregulated on the surface of the endothelium by inflammatory cytokines, cellular stress, and virus infection (roebuck and finnegan, ) . in the case of dengue, activation of endothelial cells occurs in vitro via tnf-released from antibody-enhanced dengue virus infection of monocytes . such activation involves upregulation of adhesion molecules e-selectin, icam- , and vcam- . evidence that similar activation processes occur in vivo comes from clinical studies showing elevated serum levels of tnf- (green et al., b; hober et al., ; vitarana et al., ; yadav et al., ) and soluble vcam- (murgue et al., ) in dengue-and dhf/dss-infected patients. surprisingly, serum levels of soluble icam- were actually found to be lower than those of control subjects, although this may reflect plasma protein loss through leakage (bethell et al., ) . moreover, the function of soluble forms of icam- remains unclear, and their expression appeazrs to be regulated differently from that of membranebound icam- (komatsu et al., ; van den engel et al., ) . two phases of icam- upregulation have been noted in west nile and kunjin virus infection of human embryonic fibroblasts, namely an early ($ h postinfection) virus-dependent process and a later ($ h postinfection) event that is mediated by type interferon (shen et al., b) . for neurotropic flaviviruses, such as west nile virus in the mouse, the development of encephalitis has been correlated with viremia (weiner et al., ) , suggesting virus penetration of the blood-brain barrier. the endothelium of the brain microvasculature normally represents a block between circulating virus and the central nervous system. expression of endothelial cell adhesion molecules, thereby facilitating leukocyte adherence and diapedesis through the endothelium, may be an important mode of dissemination of virus-infected monocytes or other leukocytes into the brain. west nile virus infection of human endothelial cells causes the upregulation of e-selectin, icam- , and vcam- (shen et al., ) , which could mediate the transendothelial migration of leukocytes. upregulation of these adhesion molecules was observed to occur early ( - h) in infection and appeared to be triggered by the virus rather than by cytokines (shen et al., ) . further studies are required to clarify the role of endothelial cell adhesion molecule expression in the neuroinvasion of certain flaviviruses. assuming such a role is confirmed, it will be incumbent to identify the mechanisms by which either free or cell-borne flaviviruses are stimulated to cross the vascular endothelial layer. for virusinfected leukocytes, such stimulation likely arises, at least in part, from chemokines produced by cells of the central nervous system. astrocytes infected with je virus have been reported to release chemokines (rantes and mcp- ), which may play a role in the transendothelial migration of leukocytes (including those possibly carrying virus) across the blood-brain barrier (chen et al., ) . thus, once neural infection is initiated, the process could be amplified by the production of leukocyte-attracting chemokines at the site of infection. complement activation is well documented in dengue disease (nishioka, ; phanichyakarn et al., b; russell et al., ) , with peak activation and the production of c a and c a occurring at the time of vascular leakage and/or shock (malasit, ) . complement activation is likely to be largely mediated by immune complexes consisting of igg and virus (bokisch et al., a (bokisch et al., , b shaio et al., ; sobel et al., ) , although the low levels of circulating immune complexes detected in patients have stimulated thought as to other possible mechanisms (malasit, ) . receptors for c a and c a are found on a wide variety of cells, including many human peripheral blood leukocytes (chenoweth and hugli, ; fureder et al., ; kretzschmar et al., ; nilsson et al., ; van epps and chenoweth, ) . c a receptors have been reported on endothelial cells, although at lower levels than myeloid cells (zwirner et al., ) . although endothelial cells do not appear to be major targets for dengue virus in vivo (halstead, (halstead, , sahaphong et al., ) , endothelial cells infected with dengue virus in vitro can become a substrate for deposition of c dg and c b- , provided the dengue antibody is present (avirutnan et al., ) . the presence of complement activation products on the endothelial cell surface could be a contributing factor to vascular permeability . furthermore, anaphylotoxins and/or deposition of sublytic c b- on the endothelial cell surface has the potential to activate the expression of adhesion molecules (foreman et al., ) , cytokines (saadi et al., ) , chemokines (selvan et al., ) , cyclooxygenase- (bustos et al., ) , tissue factor , heparan sulfate proteoglycan proteinases (ihrcke and platt, ) , and even functional or morphological changes such as permeability loss and gap formation . thus, in addition to being activated by leukocyte-derived cytokines , endothelial cells may also be coaxed toward a more permeability-enhancing state by virus infection and virusmediated complement deposition. at present, the lack of evidence for in vivo infection of endothelial cells by dengue virus would suggest that the cytokine-mediated pathway is dominant. figure shows a model illustrating the potential role of endothelial cell perturbation by monocyte-derived cytokines and complement activation products in initiating vascular permeability and leukocyte extravasation in severe hemorrhagic flavivirus disease. or by deposition of c b- and other products of complement activation (avirutnan et al., ) . c b- is represented as a membrane attack complex pore structure, although the deposition of c b- on dengueinfected cells appears associated with sublytic, rather than lytic, responses (avirutnan et al., ) . increased adhesion molecule expression, along with uncharacterized vasoactive factors, can lead to endothelial leakage and can mediate rolling, adhesion, and transendothelial migration of leukocytes into extravascular tissues. similar processes may also contribute to the invasion of cell-borne neurotropic flaviviruses through the endothelial blood-brain barrier. much remains to be learned about the primary receptors for flaviviruses, though much knowledge has been gained about the initial interactions of flaviviruses with cell surface structures. the ability of flaviviruses to affect cell entry through heparan sulfate-type proteoglycans, as well as their dexterity to adjust mutationally to different receptors, depending on host cell type, illustrates the plasticity of the viral e protein to adapt to 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disorders human skin langerhans cells are targets of dengue virus infection dengue haemorrhagic fever and dengue shock syndrome: are they tumour necrosis factor-mediated disorders? antibodydependent enhancement of heterotypic dengue infections involved in suppression of ifn-gamma production a novel immunohistochemical assay for the detection of ebola virus in skin: implications for diagnosis, spread, and surveillance of ebola hemorrhagic fever mhc class i-related neonatal fc receptor for igg is functionally expressed in monocytes, intestinal macrophages, and dendritic cells internalization of human immunodeficiency virus type and other retroviruses by megakaryocytes and platelets expression of the anaphylatoxin c a receptor in non-myeloid cells key: cord- -er sm u authors: terry, frances e; moise, leonard; martin, rebecca f; torres, melissa; pilotte, nils; williams, steven a; de groot, anne s title: time for t? immunoinformatics addresses vaccine design for neglected tropical and emerging infectious diseases date: - - journal: expert rev vaccines doi: . / . . sha: doc_id: cord_uid: er sm u vaccines have been invaluable for global health, saving lives and reducing healthcare costs, while also raising the quality of human life. however, newly emerging infectious diseases (eid) and more well-established tropical disease pathogens present complex challenges to vaccine developers; in particular, neglected tropical diseases, which are most prevalent among the world’s poorest, include many pathogens with large sizes, multistage life cycles and a variety of nonhuman vectors. eid such as mers-cov and h n are highly pathogenic for humans. for many of these pathogens, while their genomes are available, immune correlates of protection are currently unknown. these complexities make developing vaccines for eid and neglected tropical diseases all the more difficult. in this review, we describe the implementation of an immunoinformatics-driven approach to systematically search for key determinants of immunity in newly available genome sequence data and design vaccines. this approach holds promise for the development of st century vaccines, improving human health everywhere. vaccines have been invaluable for global health, saving lives and reducing healthcare costs, while also raising the quality of human life. however, newly emerging infectious diseases (eid) and more well-established tropical disease pathogens present complex challenges to vaccine developers; in particular, neglected tropical diseases, which are most prevalent among the world's poorest, include many pathogens with large sizes, multistage life cycles and a variety of nonhuman vectors. eid such as mers-cov and h n are highly pathogenic for humans. for many of these pathogens, while their genomes are available, immune correlates of protection are currently unknown. these complexities make developing vaccines for eid and neglected tropical diseases all the more difficult. in this review, we describe the implementation of an immunoinformatics-driven approach to systematically search for key determinants of immunity in newly available genome sequence data and design vaccines. this approach holds promise for the development of st century vaccines, improving human health everywhere. neglected tropical & emerging infectious diseases: new challenges climate change and international travel have had a dramatic impact on the geographic distribution of pathogens infecting humans and animals. old world pathogens such as dengue and chikungunya virus, previously restricted to the middle east, africa and asia have now appeared in the americas [ ] . newer pathogens such as middle east respiratory syndrome coronavirus (mers-cov), an entirely new coronavirus affecting humans, have been spreading beyond the region of the world from which they derive their names [ ] . meanwhile, human populations in developing areas of the world continue to be threatened by neglected tropical diseases (ntd). more than two billion people -nearly % of the world's population -suffer from one or more ntd [ ] , which include leishmaniasis, lymphatic filariasis, onchocerciasis, schistosomiasis and soil-transmitted helminthiasis, among others (table ). in addition to climate change and airline travel, economic conditions leading to transmigration contribute to the spread of ntd. recent examples include the reemergence of leishmania in spain [ ] and chagas disease in texas [ ] . even while ntd expand their reach, vaccine development for these diseases lags behind. in contrast, vaccines for important emerging infectious diseases (eid, table ) are being developed, to a certain extent, by large biotechnology companies, particularly when these companies receive guaranteed purchase agreements or other incentives to accelerate vaccine development. examples include the development of a vaccine for h n (an emerging avian influenza) by novartis and novavax in [ , ] and work toward the development of a new mers-cov vaccine in [ ] . however, the standard approach to develop new vaccines for emerging (and reemerging) infectious disease threats, which is to implement previously existing vaccine design methodologies such as cloning and expressing the dominant surface antigen [ ] , frequently results in the development of vaccines that are only effective when given with strong adjuvants [ ] . this approach is particularly unlikely to work for pathogens that have complex lifecycles (such as parasites) or are highly mutable (such as rna viruses). this article will discuss new, computational approaches that may accelerate and improve the design of vaccines for ntd and eid. truly effective vaccines do not exist for the majority of ntd. although vaccines are in development for several ntd pathogens [ ] , lack of financial incentive to invest in research and development programs for diseases concentrated in lowincome countries has mired the progress of vaccine efforts among large pharmaceutical companies [ ] . preventative chemotherapy mass drug administration (mda) programs employing donated or extremely low-cost generic drugs are currently in progress to control lymphatic filariasis, onchocerciasis, leprosy, trachoma and helminthiases in many areas of the world [ ] . a complication inherent in this strategy is that because the regions affected by different ntd overlap, coinfections can be difficult to manage with antiparasitic agents [ ] . the success of these long-term efforts and off-target effects of mass-eradication campaigns at the individual and population levels remain to be determined [ ] . as has been observed for polio, geopolitical upheaval may hamper global efforts to eradicate ntd. thus, effective ntd vaccines are still needed [ ] . new cost-effective design methodologies can help to bridge the gap between the great need for these vaccines and return on investment for pharmaceutical companies. fortunately, genomes for many newly emerging pathogens and neglected tropical disease-associated pathogens are becoming available due to research efforts worldwide [ ] . the availability of these genomes now makes it possible to apply computational vaccinology tools to these diseases of global health importance. host immune response to pathogens is mediated by the innate and adaptive arms of the immune system. innate immune cells such as macrophages, neutrophils and natural killer cells are responsible for the first line of defense, while adaptive immunity provides a more targeted response to pathogens that establishes immune memory for more rapid responses upon repeated exposures. b cells produce antibodies which are capable of recognizing and neutralizing pathogenic antigens. t cells support antibody production, activation and memory development, and are capable of lysing infected cells. the potent response from t and b cells, however, comes at a cost. whereas innate immune cells can respond within - h, the primary adaptive response normally takes - days to mature. secondary adaptive responses driven by immune memory are much faster and much stronger. this is the principle behind vaccination: pre-exposure to pathogen-derived antigens can induce pathogen-specific immune memory. the discovery of critical antigens that drive protective memory is facilitated by new computational tools. indeed, the general principle that immune cells develop memory to specific pathogen components -has driven the development of genome-derived vaccines over the past two decades. since t cells play a critical role in adaptive immunity and the development of immune memory required for an efficacious vaccine, computational tools have been used to search for small linear peptides (t-cell epitopes) derived from protein antigens that drive class i and class ii t-cell responses. these peptides are displayed on the surface of apc by multiple alleles of the mhc. as human beings express multiple alleles of class i and class ii mhc molecules, called human leukocyte antigens (hla), computational vaccinologists now search for t-cell epitopes that can bind to the most common hla alleles in the human population, reasoning that broad hla coverage will contribute to the development of effective genome-derived vaccines. computational tools can also be used to select epitoperich surface proteins that are better immunogens to drive b-cell response. fortunately, while b cells and antibodies generally recognize surface proteins, t cells recognize epitopes derived from a broader range of proteins, giving the computational vaccinologist many possible sources (internal and external proteins as well as secreted proteins) for the selection of t-cell epitopes for vaccines. exposure to a given pathogen generates memory t-cell clones capable of rapid and efficient response upon subsequent reinfection [ ] . this response may include t cell help for induction of higher antibody titers, t-cell-mediated lysis of infected cells and the expression of cytokines to coordinate other cell-mediated immune processes such as activation of apc. breadth of t-cell response (responding to many different epitopes) appears to be correlated with protection from severe disease for many pathogens that affect humans. more specifically, for hiv, hbv, hcv, lymphocytic choriomeningitis virus and malaria, protection from disease has been correlated with broad t-cell epitope response to both 'immunodominant' and subdominant t-cell epitopes [ ] [ ] [ ] [ ] [ ] . these discoveries have contributed to the concept that vaccines can be made directly from genomes by selecting sets of epitopes that will stimulate immune responses and protect against diseases. based on the observation that broad t-cell response may be protective, computational vaccinologists have worked to define collections of t-cell epitopes that can recreate the requisite features of this response. t-cell-driven, epitope-based strategies for developing vaccines against eid and ntd are currently the focus of several ntd research laboratories. proof of principle exists for a number of disease models: cellular immunity elicited by epitope immunization provided complete protection against respiratory syncytial virus challenge, partial protection of balb/c mice against sporozoite challenge, elimination of malaria-infected hepatocytes in vitro, partial protection of balb/c and cba against encephalitis following intracerebral challenge with a lethal dose of measles virus, complete protection from intraperitoneal hsv challenge, protection against infection with malaria or influenza a virus and full protection of sheep against bovine leukemia virus (these examples are reviewed in [ ] ). we have demonstrated complete protection against lethal vaccinia challenge [ ] and successful clearance of a chronic bacterial infection (helicobacter pylori) following t-cell epitope-driven vaccination [ ] . in earlier studies, we achieved partial protection against an aerosolized bacterial pathogen (tularemia [ ]) using a vaccine that contained only epitopes. while mice are not humans, growing evidence that t-cell epitope-driven vaccines can be effective in humans has led to the establishment of a number of biotech startups and venture-backed companies focused entirely on t-cell epitopebased vaccines. t-cell epitopes as ' payload' as described in the following sections, computational tools are being used to identify proteins or antigens of interest directly from the genomes of pathogens. in theory, a minimal set of antigens or epitopes that induce a competent immune response to a pathogen can be discovered using the new tools. adjuvant triggers innate immunity, which is an essential component of the protective immune response, directing it toward inflammation rather than tolerance. when combined with the minimum antigenic components that comprise the 'payload' of a genomederived vaccine, delivered in the right vehicle, may trigger protective immune response. the fundamental principle of the genome-derived epitope-driven vaccine approach is illustrated tthus: the importance of epitopes as key determinants of protective immune responses is reflected by the flurry of immunoinformatics activity over the past decades. a number of t-cell epitope mapping tools have been developed to accelerate the identification of these critical components of the immune response. using methods such as frequency analysis, support vector machines, hidden markov models and neural networks, researchers have developed highly accurate tools for modeling the mhc-peptide interface and predicting t-cell epitopes. computational vaccinologists have been unable to successfully develop accurate tools for b-cell epitope prediction, even though one of the most commonly measured outcomes of vaccination and accepted determinant of protection is antibody generation [ , ] . thus, current computational vaccinology approaches to vaccine development must take b-cell response into consideration and develop approaches that include means of stimulating effective humoral immunity where it is required for protection against challenge. given t-cell dependence for essential features of an effective antibody response, including bcell affinity maturation, class switch recombination, plasma cell differentiation and memory b-cell differentiation [ ] , t-cell epitope analysis and quantification have been used by our group as a proxy for identifying good b-cell immunogens, linking in silico sequence analysis to desired putative b-cell responses [ ] . the ivax approach to design genome-derived epitope-driven vaccines de groot and colleagues have integrated epitope-mapping tools with a wider array of vaccine design algorithms into the webbased ivax toolkit, which will be described in some detail in the following sections. the tools were initially used by epivax and collaborators [ , [ ] [ ] [ ] [ ] [ ] , and then expanded and refined for projects that have been in progress at the institute of immunology and informatics (icubed) [ ] [ ] [ ] . the ivax toolkit is currently in use for ntd research at the icubed and with academic collaborators under an agreement established between epivax and uri in . ivax tools are being used to evaluate the protective potential of existing ntd and eid vaccines [ , ] , to predict immune response to newly emerging pathogens [ , ] and to design novel ntd vaccines composed of t-cell epitopes (for chagas disease, brugia malayi and several different species of leishmania [ ] ). in the following few sections, we describe the ivax approach to design genomederived epitope-driven vaccines for ntd and eid. one of the first questions facing computational vaccinologists is how to prioritize their search for antigenic proteins and epitope subunits. the entire set of proteins derived from a pathogen's genome is an unlikely point of departure for epitope mapping, since many of these proteins may not be part of the 'core genome' for a set of bacterial or viral strains of the same pathogen. others may be proteins that serve as 'housekeeping' genes that are also well conserved in harmless commensal organisms. on the other hand, proteins that are highly conserved across variant strains, that are pathogen-specific and those that are upregulated during interactions with the host, particularly those that are secreted by a pathogen (presumably in an attempt to alter the host environment), are excellent targets for vaccine development. in addition to targeting upregulated, secreted and pathogen-specific antigens, other means of selecting antigens for epitope screening include identifying proteins that are more common in virulent as compared to avirulent strains, selection of genes differentially expressed in immunopathogenesis, prioritizing proteins exposed on the surface of the pathogen and focusing on proteins that are expressed early in the course of natural infection. the expert protein analysis system (expasy) proteomics server of the swiss institute of bioinformatics offers a wide variety of proteomics tools that can be used for this purpose, including tools related to protein identification and characterization. our groups have adapted an approach first described by gennaro et al. for mycobacterium tuberculosis (mtb) [ ] , employing a series of expasy tools (signalp, tmpred and prosite scan [ ] ) to triage pathogen genomes, reducing the number of potential targets from thousands of proteins to several dozen candidate antigens. in our first test of this approach, we found that a subset of epitopes derived from the mtb genome elicited ifn-g response from mtb-exposed human samples, and prototype epitope-based tb vaccines were shown to be robustly immunogenic in murine studies [ ] . reflect more epitope content than expected, while negative scores reflect less epitope content than expected [ ] . large numbers of protein sequences derived directly from the genome of selected pathogens can be ordered by potential class i (ctl), class ii (t helper) or both class i and class ii epitope content and placed on an immunogenicity scale (figure ). this tool allows researchers to quickly rank a given set of proteins both in relative (i.e., relative to each other) and absolute (i.e., relative to a panel of known immunogens and nonimmunogenic proteins) terms [ ] . in our experience, epitope-rich proteins are good vaccine targets and elicit strong antibody responses -thus, as previously stated, t-cell epitope content is a useful proxy for overall immunogenic potential. antigen selection is particularly complicated when targeting parasitic organisms due to their comparatively massive genomes and multistaged life cycles, and ranking of these antigens may assist with the selection of better targets. for example, in figure , we show two candidate antigens derived from b. malayi, a causative agent of lymphatic filariasis, whose life cycle is divided into multiple larval stages including a microfilarial stage [ ] . in this case, tpx- , a protein that has been identified as a potential vaccine target [ ] , is shown to contain minimal t-cell epitope content, with an immunogenicity score of - . , and thus it may be less successful as a vaccine candidate. in contrast, juv-p , a b. malayi ortholog of a litomosoides sigmodontis antigen implicated in conferring protection against microfilarial infection [ ] carries substantially more t-cell epitope content, scoring + . on the immunogenicity scale, in the same range as other well-known immunogens. furthermore, as is illustrated here in the case of b. malayi, we frequently find evidence that pathogens appear to reduce t-cell epitope content in key proteins to avoid human immune responses. epitope deletion is an established means of immune evasion in hiv and hcv [ , ] ; thus, the mechanism may also be relevant in the context of infections that are associated with chronic infection caused by filaria, leishmania and other chronic ntd, particularly in stages associated with chronic parasitism and parasite persistence in the face of immune pressure. we will discuss additional means of immune evasion that can be uncovered by computational tools below. epimatrix: t-cell epitope mapping of selected antigens t-cell epitopes are short linear peptides that can bind to mhc molecules and engage t cells through their receptors (tcr), activating specific populations of cd + and/or cd + lymphocytes. these epitopes are key to forming the immunological synapse between antigen-presenting cells and t cells. because tcrs are produced in a myriad of possible conformations (much like antibodies, to which they are related), mhc binding is the dominant event in immune recognition. in other words, most mhc ligands are also t-cell epitopes, and t-cell epitopes are by definition, mhc ligands. the mhc-peptide interaction is well characterized [ , ] . based on these characterizations, pattern-matching algorithms such as epimatrix have been developed to screen protein sequences for peptides that will bind mhc. the human mhc molecules, or hla, are among the most variable proteins in the human genome. this variation ensures that the surveillance capabilities of the human immune system are both broad and deeply redundant, making immune escape through mutation more difficult for pathogenic organisms. fortunately, some alleles are much more common than others in the human population and the binding repertoire of many alleles significantly overlap. by focusing on alleles that are both common (in the human population) and significantly different from each other (representative of human diversity), hla alleles can be grouped into all other factors being equal, the more hla ligands (i.e., putative t-cell epitopes) contained in a given protein, the more likely that protein is to induce an immune response. to capture this concept, the epimatrix immunogenicity scale presents proteins by the epimatrix protein score, and compares them to other known immunogens. the epimatrix protein score is the difference between the number of predicted t-cell epitopes expected in a protein of a given size and the number of putative epitopes predicted by the epimatrix. the epimatrix protein scores are 'normalized' and can be plotted on a standardized scale. 'average' proteins score near zero. protein scores above zero indicate the presence of excess mhc ligands and denote a higher potential for immunogenicity, while scores below zero indicate the presence of fewer potential mhc ligands than expected and a lower potential for immunogenicity. the epimatrix protein score is correlated with observed immunogenicity in vitro and in vivo. as shown here, proteins scoring above + , such as brugia malayi antigen juv-p , are considered to have a significant immunogenic potential. proteins scoring below - , such as tpx- above, are less likely to be immunogenic in vivo. 'supertypes,' which can reduce the search space to a manageable number of evaluations. six of these class i super-type alleles that 'cover' the genetic backgrounds of most humans worldwide have been used to define ctl epitopes: a* , a* , a* , a* , b* and b* [ ] . for class ii t helper epitopes, mapping for a panel of eight common alleles: drb * , * , * , * , * , * , * and * , gives broad t helper epitope coverage [ ] . the concept of supertype alleles is generally accepted and widely applied to vaccine design in the field of computational vaccinology [ , ] . using the set of selected protein antigens as a starting point, ivax uses epimatrix to parse each into overlapping -mer frames where each -mer overlaps the last by eight amino acids. each -mer is then scored for predicted binding affinity to a panel of class i or class ii hla alleles. the epimatrix algorithm compares the amino acid sequence of each given -mer peptide to the coefficients contained in stored probability matrices and produces a raw score. in order to compare potential epitopes across multiple hla alleles, epimatrix raw scores are converted to a normalized 'z' scale. peptides scoring above . on the epimatrix 'z' scale (typically the top % of any given sample) are likely to be mhc ligands [ ] . evidence from animal studies suggests that the number of epitopes required for full protection is a small and definable subset (~ ) [ , ] ; thus, epitope-driven vaccines developed by our group generally contain a payload of - epitopes that provide broad coverage of human genetic backgrounds. with a combination of promiscuous class ii epitopes and class i supertype epitopes, it is possible to attain > % coverage of the hla of most human populations [ , ] . eliminating regulatory or suppressor epitopes using janusmatrix a recent development in vaccine design includes the consideration of epitopes that induce regulatory or suppressive immune responses [ ] . our group has been investigating epitope crossconservation with the human genome and its association with diminished or regulatory immune responses. using a recently developed tool called janusmatrix we first determined that published effector t-cell epitopes can be distinguished from reported regulatory t-cell epitopes on the basis of tcr-specific cross-reactive potential with the human genome and human microbiome [ ] . janusmatrix differs from whole-sequence alignment tools such as blast [ ] in its basis upon t-cell receptor homology. pathogenic peptides whose tcr-facing residues are identical to the epitopes contained in multiple self may be recognized by t cells specific to those human proteins. of course, even though the mhc-facing residues may differ, these peptides must still have the capacity to bind to the same mhc as the pathogen sequence, provided that binding is preserved. taking this into account, janusmatrix compares the tcr-facing contour of pathogen ligands to other genomes of interest, identifying matches therein that are predicted to bind the same mhc. tcr-homologous epitopes shared between pathogens and humans, or pathogens and other microbes, can be uncovered with remarkable speed using the janusmatrix tool. exploring further, we have uncovered a high degree of host (human) homology in viruses that tend to establish chronic infections in humans such as ebv and cmv [ ] . furthermore, 'commensal' viruses can be shown to contain significantly more human genome-homologous epitopes relative to those causing acute infection (e.g., ebola, marburg) [ ] . the limited clinical efficacy of some vaccines against selected microbial pathogens may, in fact, have been due to their extensive crossconservation with the human genome [ ] . the janusmatrix tool is currently being used by our team and collaborators to identify significant homology between candidate payload epitopes and proteins contained within the human genome and the human microbiome. using the tool, we find that not only viruses but also bacteria that establish chronic infections in humans 'deimmunize' (remove t-cell epitopes) and 'tolerize' (modify epitopes to be more cross-reactive to human t-cell epitopes). comprehensive studies of ntd genomes (and stageby-stage analysis of parasite antigens) will be performed using the janusmatrix tool in the near future. it follows that careful selection of t-cell epitopes, and redesign of whole antigens, to avoid the inclusion of t-cell epitopes that may be highly cross-reactive with the human genome could improve the efficacy of whole-antigen and epitope-based vaccines. janusmatrix complements recent research [ ] on the development of adaptive immunity and supports the hypothesis that adaptive t-cell responses are reinforced by cross-reactivity with the human microbiome [ ] [ ] [ ] . cytoscape is an online tool that is usually used by bioinformaticians to illustrate the relatedness between proteins, for example, all of the intracellular proteins that might be involved in the stimulation of a cell through toll-like receptors. we have repurposed cytoscape to describe the relationship between epitopes across proteins in groups of sequences (the human genome, the human microbiome, pathogen genomes [ ] ). using cytoscape [ ] , the results of janusmatrix analysis (e.g., comparing a pathogen epitope to the human genome) can be visualized as networks where each epitope derived from a pathogen is linked to its tcr-matched counterparts in the search database, which themselves are linked to their source proteins. for example, an influenza t-cell epitope previously identified by mark davis and colleagues [ ] that stimulates t cells in subjects never exposed to influenza can be shown to have an extensive network of cross-reactive tcr-facing epitopes in the human microbiome. in contrast, an epitope from vaccinia virus synthesized and tested by larry stern's group is shown to have extensive cross-reactivity with the human genome by janusmatrix. this epitope was nonimmunogenic in vitro (by ifn-g elispot) even though it was shown to bind to the correct class ii mhc [ ] . this epitope fits the emerging in silico definition of a treg epitope. due to their commensal nature and need to avoid human immune responses over many years of coexistence, it is even ntd/eid: time for t? review informahealthcare.com more likely for selected human parasites to share putative t-cell epitope content with their human hosts. in figure , we offer two example peptides from b. malayi antigens tpx- and juv-p , compared to published treg epitopes from human immunoglobulin (tregitopes) and effector epitopes, the ceft pool (a set of peptides used as 'control positive' peptides in eli-spots [ ] ). the potential cross-reactivity network differential is evident between the tpx- epitope, with many related epitopes derived from human sequences, and the juv-p sequence, whose related human epitopes are few. this finding underscores the importance of validating the response phenotype of t cells stimulated by epitopes identified in silico prior to their inclusion in vaccine constructs, and also illustrates the importance of this type of analysis for the selection of candidate epitopes for ntd. promiscuous hla binding potential is a feature of class ii-restricted t-cell epitopes particularly exploitable for vaccine design purposes. it has been shown that putative epitopes for hla class ii are not often distributed evenly across protein sequences, but instead tend to cluster in specific regions, where it is not uncommon to observe several reactive -mer frames in close proximity [ ] . these 'clusters' of unusually high predicted epitope density can be identified in silico using the clustimer algorithm. in general, t-cell epitope clusters identified by the clustimer algorithm tend to be promiscuous mhc binders and are frequently t-cell epitopes [ ] . due to overlapping peptide-binding preferences among hla-dr alleles, it is also possible to identify single -mers capable of binding four or more hla alleles [ ] . these sequences have been dubbed 'epibars' due to their horizontal, band-like signature in readout from epimatrix (figure ) . t-cell epitope clusters can be very powerful, and epibars may be a characteristic feature of highly immunogenic, promiscuous class ii epitopes. these compact, highly reactive peptides are relatively easy to deliver and show great promise as vaccine components when cross-reactivity with the human genome is limited (see above). we have used these clusters extensively in our own work [ , , ] . promiscuous t-cell epitopes also exist, to a certain degree, for class i alleles; however, this is much less common than for class ii. some laboratories have demonstrated cross-presentation of peptides within hla 'superfamilies,' such as the a superfamily: a , a , a , a and a [ ] . cross-mhc binding and presentation to t cells has been confirmed in hiv vaccine studies [ ] . however, we have found that weighting toward the selection of highly promiscuous class i epitopes may lead to identification of candidate epitopes that have lower binding affinities overall. higher binding affinities appear to be a critical aspect of ctl epitope efficacy [ ] , thus our group prefers to select a small set of the best-scoring putative epitopes for each of the six class i hla superfamilies from a given protein or set of conserved peptides (figure ) . potential lf t eff epitope (juv-p ) potential t eff epitope ceft pool published t reg epitope human igg a b c d figure . janusmatrix analysis. this tool considers identity of tcr-facing residues to target proteins or genomes independently from residues that contribute to mhc binding. peptides that have similar tcr-facing residues and are presented in the context of the same hla can be identified. extensive homology is easy to identify using the cytoscape network visualization tool. the extent of the network can be used to distinguish potential regulatory t-cell epitopes (a) from potential effector t-cell epitopes (b). a published treg epitope example is shown in (c), and several published teff epitope examples are shown in (d). for these illustrations, yellow hexagons identify the source antigens, turquoise diamonds identify the source t-cell epitope clusters, gray squares indicate the source -mers, dark blue triangles indicate matched human -mers and light blue circles indicate human antigens in which matched -mers are found. data for c, d taken from [ ] . selecting epitopes that are broadly reactive across circulating strains can enhance broad applicability of new vaccines. the problem of pathogen variability significantly complicates the selection of epitopes for vaccine design. to address this problem, epivax has developed epiassembler [ ] to identify sets of overlapping, conserved and immunogenic epitopes and to assemble them into extended immunogenic consensus sequences (ics, figure ). the theory behind developing ics is that processing and presentation of these sequences would allow for presentation of the highly conserved class ii-restricted epitopes contained in the ics in the context of more than one mhc. the resulting peptide is not a 'pseudo-sequence' as such, since each constituent epitope occurs in its corresponding position in the native protein; adjacent epitopes may be similarly conserved but not in the same variant of the pathogen. the ics approach has been useful for identifying highly immunogenic epitopes for hiv vaccine design [ ] . using hiv as an example, while the full composite ics peptides happen to be exactly conserved in a few individual strains of hiv, each peptide represents a significant percentage of circulating strains because every constituent overlapping epitope is conserved in a large number (range - ) of individual hiv- strains [ ] . by extending the approach described above, it is possible to develop completely synthetic antigens whose sequences are optimized for t helper potential. with an eye to structural considerations, even recombinant protein-only vaccines could be optimized in this way, enabling primary cognate t help to be maximized and b-cell memory to be elicited. an ideal vaccine might include whole proteins in addition to some epitopes; some or all of these antigens could be optimized using the ics approach. linking ics epitopes to a carrier protein (such as a surface protein target of b-cell response) would further maximize primary cognate t help, since b cells that capture the recombinant proteins would be able to process and present t helper epitopes derived from more variable proteins. as compared with ics, randomly selected counterparts, on average, contain half as many binding motifs and cover one-third fewer isolates [ ] . to develop vaccines of equivalent antigenic 'payload' using conventional methods would be prohibitively expensive, as it would require use of multiple variants of each antigen. we believe that this and similar approaches that harness conserved t help have tremendous potential and deserves careful consideration in vaccine design. after generating a preliminary list of candidate vaccine components, the next step in the ivax approach is to review the putative epitopes produced by the epimatrix system, adding qualitative and quantitative annotations wherever possible, leading to an investigator-driven down-selection process. putative epitopes derived from known antigens or from proteins overexpressed during early stages of infection or proteins known to be exposed to immune surveillance as reported in the literature may be prioritized. furthermore, putative epitopes with the in silico profile of potential regulatory t-cell epitopes (based on janusmatrix analysis) are removed from further consideration. figure . example of an epibar: epimatrix analysis of candidate lymphatic filariasis epitope. in addition to providing an overall immunogenicity score, epimatrix can be used to analyze epitopes at the local level. a brugia malayi juv-p peptide is shown above, parsed into -mer frames and analyzed for predicted immunogenicity. epimatrix assessments above . constitute the top % of predicted hla binders and are shaded medium blue, while scores above . fall in the top % and are shaded dark blue. this juv-p peptide registers significant scores for all eight alleles in epimatrix in a single -mer frame, and based on the epimatrix method, has a cluster score of . (reflecting the number of predicted binders per amino acid length). cluster scores higher than are considered to be significant based on retrospective and prospective studies carried out by the epivax group. the band-like pattern illustrated in frame is called an epibar and is characteristic of promiscuous epitopes. algorithms can also be helpful to interpret vaccine component responses in preclinical and clinical studies. in studies of immune response to therapeutic proteins and vaccines, the authors have observed that subject-to-subject variation in t-cell response closely relates to subject hla type and the number of motifs or peptides that match the subject's hla haplotype. to describe this relationship, epivax researchers have developed a metric that may be useful in clinical assessment of immune response to vaccines, called the 'individualized t-cell epitope measure' or item. for a given t-cell epitope, an individual's item score can be calculated by weighting and summing the epitope's epimatrix z-scores for each hla allele in a given subject's haplotype. this calculated score allows for individualized immunogenic potential to be predicted based on the number of putative epitopes contained in a protein and a given individual's hla haplotype. using this score, it is possible to analyze the contribution of haplotype to the corresponding t-cell response. in prospective and retrospective evaluations, significant correlations were found between the ifn-g response to a given antigen and the item scores for individual subjects [ ] . in addition, correlations between the item score and patient hla have been observed for antibody titers [ , , ] , reflecting the importance of hla-restricted t-cell responses to the genesis of a robust antidrug antibody response. a number of methods for enhancing epitope-based vaccines have been described and implemented [ , ] . one approach is to align the individual epitopes in a protein or dna vaccine construct as a 'string of beads' without any intervening figure . class i epitope 'staircase' ranking. in the process of generating a selection of predicted high-affinity class i epitopes for inclusion in t-cell-driven vaccines, parsed -mers from any antigen are ranked by potential to bind supertype hla alleles and collated in a 'staircase' report. in this example, the top five highest-scoring peptides from a given antigen are shown. in general, prioritizing class i epitopes by score for each of the individual alleles is preferred to define epitopes that bind across alleles. sequences or spacers between the payload epitopes [ ] . however, the lack of spacers between the payload epitopes has raised concern that these sequences may contain junctional epitopes. vaccinecad, an algorithm that iteratively analyzes epitope assemblies and minimizes the potential for junctional immunogenicity in any string-of-beads construct, has been developed to address this concern [ ] . peptide sequences contained in the junctional regions between the target epitopes are evaluated for potential immunogenicity. the highest scoring junction is identified and the algorithm optimizes the order of epitopes by evaluating potential alternative sequences. the process is repeated until no additional reductions in junctional immunogenicity can be achieved or until all junctional immunogenic potential has been eliminated. when the potential for junctional immunogenicity cannot be sufficiently reduced, a cleavage promoting spacer sequence, typically 'aay' for class i restricted constructs [ ] or a binding inhibiting 'breaker' sequence such as 'gpgpg' for class ii restricted constructs [ ] is placed between the two offending epitopes. the ability to minimize junctional immunogenicity while simultaneously minimizing the presence of transmembrane domains or highly hydrophobic peptide segments which may be difficult to express would be a logical extension of this tool's capabilities. the integration of computational tools for epitope discovery has enabled the development of genome-derived vaccines [ , , ] . compared to conventional strategies, this approach has the potential to create more effective and safer next-generation vaccines, as carefully selected epitopes focus immune responses on the minimal, essential pathogen-specific antigenic elements; epitopes directed against conserved 'self' (host) antigens are eliminated. this approach is also well suited for highly variable pathogens, as selection of epitopes that are conserved across multiple strains or subtypes enables the development of a broadly applicable, multipathogen vaccine. the genome-derived vaccine strategy has been applied by our team to a wide range of pathogens, including f. tularensis, variola, hiv, mtb, h. pylori and influenza. these studies demonstrate that immunoinformatic-predicted epitopes are immunoreactive in vaccinees and survivors of infection, and stimulate de novo, protective immune responses in vivo in hla transgenic mice (e.g., [ ] [ ] [ ] ] ). epitope-driven vaccines offer distinct advantages over traditional subunit vaccines. multiple epitopes derived from several antigens can be packaged together. thus, a broad-based immune response directed against several different antigenic proteins can be elicited without manufacturing and administering the entire protein, much of which will be immunologically irrelevant. this may reduce formulation challenges, cost and safety risk. the use of epitopes also mitigates safety concerns arising from the use of intact recombinant proteins that may have undesired biological activity. this review of vaccine design tools developed by the epivax team is by no means comprehensive, and has mainly focused on antigen selection and design. topics not covered in this review include formulation of epitope-driven vaccines, route of delivery (mucosal, intradermal, etc.), adjuvanting, selection of delivery vehicles and preclinical and clinical testing. a major caveat concerning the use of the ivax toolkit is that none of the vaccines designed using these tools have advanced to the clinic. given the cycle of vaccine development, this is not surprising (it may take up to years to develop a vaccine with full industry support). retrospective and prospective studies have provided extensive validation of the tools described here [ , , , ] . nonetheless, algorithms developed and applied by this group to a wide range of pathogens have met with significant preclinical success and are currently in use for the development of vaccines against ntd parasites, eid viruses and bioterror pathogens. access to nearly all of the tools described in this article is freely available to trained users through the ivax toolkit [ ] . the website was developed with funding from the national institutes of health in . access to the ivax toolkit and training on the tools is available for interested researchers under collaborative agreements with the university of rhode island (primarily for ntd, but other arrangements are possible). commercial users are directed to epivax [ ] , which provides a secure-access version of the ivax website for commercial users. in general, the field of vaccine research has been slow to adopt new vaccine design tools, and even fewer ntd researchers are familiar with the use of the tools, despite proof of principle for the genome-derived vaccine approach and the fact that it significantly reduces time and effort to make vaccines. for eid, 'tried and true' approaches often win out over newer strain: a b c d e f g h conserved epitope figure . epiassembler construction of immunogenic consensus sequences. this figure illustrates the process of assembling highly conserved t-cell epitopes into a single molecule. first, a highly conserved, promiscuous epitope is identified to form the -mer core of the ics peptide (red bar). overlapping conserved epitopes (pink, orange, green and blue bars) are then added to the n-and c-termini of the peptide until a suitable length is reached for binding in the class ii hla binding groove. this economical approach allows for targeting of multiple strains of a given pathogen using a single peptide, as illustrated by the blended bar at the bottom of the figure. ics: immunogenic consensus sequences. a similar approach was used during the emergence of sars and completely failed to protect against rapidly evolving sars viruses in animal challenge models [ , ] . application of advanced immunoinformatics tools to ntd vaccines has also lagged for a number of reasons. ntd researchers do not use the tools because they lack access to and familiarity with them, and there are no widely publicized examples focusing on diseases that impact the developing world. a series of technical challenges for ntd vaccines have been described recently, including antigen discovery, process development, preclinical development, clinical trials in resource-poor settings and the immune response to ntd infection, including what is commonly referred to as the ige trap, through which certain individuals, perhaps especially those in endemic regions, may have elevated preexisting ige antibodies for potential ntd vaccine antigens, leading to increased risk with vaccination [ ] . computational vaccinology cannot currently address all of these challenges; however, the approach described here offers a unique opportunity to address certain hurdles early in the developmental process. early in the pipeline, antigen discovery using t-cell epitope prediction and ranking, along with candidate epitope triage using cluster analysis and crossreactivity prediction provide valuable leads. selected peptide candidates can be screened ex vivo in order to verify the phenotype of the immune response prior to inclusion in a final vaccine product. finally, t-cell epitope-based strategies are exceptionally platform-flexible, adaptable to synthetic peptide formulations deliverable in saline, emulsion or microparticle, or encoding into plasmid vectors for dna vaccination or recombinant protein production, thus allowing for novel distribution strategies necessary to reach the world's poorest. this flexibility extends to the antigen discovery approach as well, in that many kinds of targets may be explored using immunoinformatics tools. a pertinent example for ntd and eid applies to vector-based targets. vaccine components based upon the salivary proteins of arthropod vectors are already under investigation [ ] . however, vector salivary antigens also have known immunomodulatory properties allowing for extended host tolerance [ , ] . the same discovery and evaluation strategy described for pathogenic antigens could be applied to such proteins, potentially providing a mechanism through which to stimulate robust immune response in the absence of the immunomodulatory properties of the complete salivary antigens. the amount of data generated through new technologies, such as next-generation sequencing, continues to expand exponentially. by applying these technologies to the study of eid and ntd causative agents, and expanding our genomic knowledge of these organisms, the feasibility of using high-throughput, informatics-based tools for the identification of putative protein and peptide targets increases. vaccine efficacy may also improve, as the selection of targets can be refined by comparing the antigen to other genome sequences, such as the human genome and the human microbiome. the in silico-based approach to vaccine design may also alleviate many of the funding-associated challenges common to traditional vaccine design, by reducing the number of assays that need to be performed to select vaccine targets. reduced cost should allow for the reallocation of critical funding to the testing of in silico-predicted targets and constructs. and finally, improved safety, by eliminating human genome cross-conserved epitopes, may reduce unwanted adverse effects. looking further into the future, we are confident that the evolution of the tools described here will eventually contribute to the development of personalized, on-demand vaccines [ ] . considering the importance of controlling infectious diseases to global economic stability, the integration of computational vaccinology tools and their application to the design of vaccines for ntd and eid is of paramount importance. delay is no longer acceptable. vaccine developers must implement computational vaccinology tools if they wish to contribute to improve world health in the st century. the authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed. no writing assistance was utilized in the production of this manuscript. • new immunoinformatics tools have been developed that address critical problems in vaccine design. • these tools have been extensively validated in preclinical models. • the design of vaccines for neglected tropical diseases would benefit from expanded use of these tools. chikungunya fever diagnosed among international travelers-united states the emergence of the middle east respiratory syndrome coronavirus control of neglected tropical diseases needs a long-term commitment re-emergence of leishmaniasis in spain neglected parasitic infections in the united states: chagas disease bringing influenza vaccines into the st century a recombinant viruslike particle influenza a (h n ) vaccine current advancements and potential strategies in the development of mers-cov vaccines how the sars vaccine effort can learn from hiv-speeding towards the future, learning from the past cross-conservation of t-cell epitopes: now even more relevant to (h n ) influenza vaccine design this study connects the dots between low t-cell epitope content and low immunogenicity of h n vaccines in humans, and points out important changes to t-cell epitopes that might further reduce immune response through the induction of tregs innovation for the 'bottom million': eliminating neglected tropical diseases in the americas preventive chemotherapy as a strategy for elimination of neglected tropical parasitic diseases: endgame challenges neglected tropical diseases and the millennium development goals: why the "other diseases" matter: reality versus rhetoric the contribution of mass drug administration to global health: past, present and future the human hookworm vaccine genomics of emerging infectious disease: a plos collection evolution of the t-cell repertoire during primary, memory, and recall responses to viral infection cytotoxic t-lymphocytes in asymptomatic long term nonprogressing hiv- infection. breadth and specificity of the response and relation to in vivo viral quasispecies in a person with prolonged infection and low viral load degenerate cytotoxic t-cell epitopes from p. falciparum restricted by multiple hla-a and hla-b supertype alleles human memory ctl response specific for influenza a virus is broad and multispecific vaccines: correlates of vaccine-induced immunity conformational b-cell epitope prediction on antigen protein structures: a review of current algorithms and comparison with common binding site prediction methods follicular helper cd t cells (tfh) • a study that links innate, adaptive and humoral immunity at the follicular dendritic cell level confirmation of immunogenic consensus sequence hiv- t-cell epitopes in bamako diversity of francisella tularensis schu antigens recognized by t lymphocytes after natural infections in humans: identification of candidate epitopes for inclusion in a rationally designed tularemia vaccine hiv vaccine development by computer assisted design: the gaia vaccine developing an epitope-driven tuberculosis (tb) vaccine coupling sensitive in vitro and in silico techniques to assess cross-reactive cd (+) t cells against the swine-origin h n influenza virus careful prospective validation of epitope predictions demonstrating that h n 'seasonal' vaccination or exposure might protect against h n 'pandemic' disease at the t-cell level. it also validates the individualized t-cell epitope measure tool for hla-specific immunogenicity prediction human immune responses to h. pylori hla class ii epitopes identified by immunoinformatic methods peptide-pulsed dendritic cells induce the hepatitis c viral epitope-specific responses of naïve human t cells immunogenic consensus sequence t helper epitopes for a pan-burkholderia biodefense vaccine analysis of chimerivax japanese encephalitis (je) virus sequence for t cell epitopes and comparison to circulating wild type je virus strains immunoinformatic comparison of t-cell epitopes contained in novel swine-origin influenza a (h n ) virus with epitopes in - conventional influenza vaccine a study demonstrating that analysis of hemagglutinin prior to production of vaccine might reveal important insights: in this case, that seasonal influenza might protect against pandemic influenza, a prediction that was later corroborated by other researchers in vitro and in vivo. prospective use of this method for evaluating influenza antigens might be cost-saving in the context of vaccine development and world health programs immunogenicity and immune modulatory effects of in silico predicted l. donovani candidate peptide vaccines identification of secreted proteins of mycobacterium tuberculosis by a bioinformatic approach expasy bioinformatics resource portal epitope-driven tb vaccine development: a streamlined approach using immuno-informatics, elispot assays, and hla transgenic mice immunomics: discovering new targets for vaccines and therapeutics low immunogenicity predicted for emerging avian-origin h n : implication for influenza vaccine design a fearless prediction of h n immunogenicity that was later validated (see cross-conservation publication in the same journal apple trees productions, llc novel phage display-based subtractive screening to identify vaccine candidates of brugia malayi juvenile female litomosoides sigmodontis produce an excretory/secretory antigen (juv-p ) highly modified with dimethylaminoethanol mutational escape from cd + t cell immunity: hcv evolution, from chimpanzees to man mechanisms of hiv- escape from immune responses and antiretroviral drugs allele-specific motifs revealed by sequencing of self-peptides eluted from mhc molecules • one of the first mhc-binding motif descriptions exact prediction of natural t cell epitope nine major hla class i supertypes account for the vast preponderance of hla-a and -b polymorphism one of the several studies describing hla class i 'supertypes', that is, families of hlas that group together based on their hla binding preferences. this one describes supertypes for hla-a and -b (class i). these papers made it possible to design vaccines in silico several common hla-dr types share largely overlapping peptide binding repertoires hlas that group together based on their hla binding preferences. this one describes supertypes for hla-dr (class ii). these papers made it possible to design vaccines in silico a comparison of two methods for t cell epitope mapping: "cell free" in vitro versus immunoinformatics a consensus epitope prediction approach identifies the breadth of murine t(cd +)-cell responses to vaccinia virus putting immunoinformatics to the test elimination of il- -inducing t-helper epitopes from an igfbp- vaccine ensures potent antitumor activity the two-faced t cell epitope: examining the host-microbe interface with janusmatrix basic local alignment search tool integrated assessment of predicted mhc binding and cross-conservation with self reveals patterns of viral camouflage a mathematical exploration of the cross-conservation between human pathogen (viruses) and human host that demonstrated a significant increase in the number of cross-conserved epitopes in viruses that 'hit and stay' (commensals) as compared to 'hit and run' viruses such as ebola virus-specific cd (+) memory-phenotype t cells are abundant in unexposed adults further evidence that cross-reactive t-cell recognition is discernable gut immune maturation depends on colonization with a host-specific microbiota has the microbiota played a critical role in the evolution of the adaptive immune system? peripheral education of the immune system by colonic commensal microbiota a travel guide to cytoscape plugins human cd + t cell epitopes from vaccinia virus induced by vaccination or infection t cell epitope: friend or foe ? immunogenicity of biologics in context hla supertypes and supermotifs: a functional perspective on hla polymorphism from genome to vaccine: in silico predictions, ex vivo verification identification of subdominant cytotoxic t lymphocyte epitopes encoded by autologous hiv type sequences, using dendritic cell stimulation and computer-driven algorithm engineering immunogenic consensus t helper epitopes for a cross-clade hiv vaccine clinical validation of the "in silico" prediction of immunogenicity of a human recombinant therapeutic protein the first double-blinded prediction of therapeutic protein immunogenicity using in silico tools a method for individualizing the prediction of immunogenicity of protein vaccines and biologic therapeutics: individualized t cell epitope measure (item) targeting a polyepitope protein incorporating multiple class ii-restricted viral epitopes to the secretory/endocytic pathway facilitates immune recognition by cd + cytotoxic t lymphocytes: a novel approach to vaccine design enhancing dna immunization defined flanking spacers and enhanced proteolysis is essential for eradication of established tumors by an epitope string dna vaccine optimization of epitope processing enhances immunogenicity of multiepitope dna vaccines evasion of antibody neutralization in emerging severe acute respiratory syndrome coronaviruses vaccines to combat the neglected tropical diseases a listeria-based vaccine that secretes the sand fly salivary protein ljm confers long-term protection against vector-transmitted leishmania major sand-fly saliva-leishmania-man: the trigger trio tick salivary compounds: their role in modulation of host defences and pathogen transmission making vaccines "on demand": a potential solution for emerging pathogens and biodefense? disability-adjusted life years (dalys) for diseases and injuries in regions, - : a systematic analysis for the global burden of disease study key: cord- -ae x wpg authors: vieira, g. f.; chies, j.a.b. title: immunodominant viral peptides as determinants of cross-reactivity in the immune system – can we develop wide spectrum viral vaccines? date: - - journal: medical hypotheses doi: . /j.mehy. . . sha: doc_id: cord_uid: ae x wpg summary when we look back to edward jenner vaccination of a young man in , we cannot help thinking that he was both lucky and crazy. crazy because he decided to test in a human being a hypothesis based mainly in the traditional belief that people who had acquired cowpox from the udders of a cow were thereafter resistant to smallpox, a quite devastating disease, and lucky because (even considering that he did not know this at that time) he succeeded to induce protection against a pathogen through the induction of an immune response directed against a different agent. not only was he able to protect the young man but he took the first step towards the development of a vast new field, vaccination. it is acceptable to say that jenner was lucky because he succeeded in promoting protection against smallpox using a cowpox virus and this induction of protection in a cross-reactive way is believed to be quite rare. nevertheless, more and more examples of cross-reactive immune responses are being described and we are beginning to admit that cross-reactivity is far more common and important than we used to think. here we review cross-reactivity in the immune system and the plasticity of t cell recognition. based on the existence of t cell receptor promiscuous recognition and cross-recognition of conserved viral immunodominant epitopes, we propose two approaches to develop wide spectrum viral vaccines. the first one is based on the identification, characterization, and cloning of immunodominant viral epitopes able to stimulate responses against different viruses. the produced peptides could then be purified and serve as a basis for vaccine therapies. a second strategy is based on the identification of conserved patterns in immunodominant viral peptides and the production of synthetic peptides containing the amino acid residues necessary for mhc anchoring and tcr contact. although we are still far from a complete knowledge of the cross-reactivity phenomenon in the immune system, the analysis of immunodominant viral epitopes and the identification of particular “viral patterns” seems to be important steps towards the development of wide spectrum viral vaccines. immunodominant viral peptides as determinants of cross-reactivity in the immune system -can we develop wide spectrum viral vaccines? q g.f. vieira, j.a.b. chies * introduction the human immune system has developed, throughout its evolution, different mechanisms to eliminate pathogenic organisms. pathogens, in turn, have developed strategies to evade our defense mechanisms. some of the interaction mechanisms between the immune system and pathogens are already well understood. for viral systems, however, the subject is not fully elucidated, mainly due to the highly mutable nature of viruses, which requires the development of specialized and complex defense mechanisms. many cells of the immune system are involved in the eradication of viral infections. we will give special attention to the interactions between cells that express major histocompatibility complex (mhc) class i molecules (all the nucleated cells) and cytotoxic cd + t lymphocytes (ctls). when an mhci-expressing cell is infected by a virus, it starts producing proteins necessary to viral assembly. part of these proteins is, however, proteolytically degraded in the proteasomes, along with other cytosolic self-proteins or even proteins provenient from intracellular bacteria that have been also ubiquitinated. this degradation process generates peptides - amino acids (aa) long that may, potentially, bind to the mhc class i molecule peptidic cleft, in the endoplasmic reticulum, being transported to the cell surface. these peptides have little variation in size, presenting normally - amino acids. the peptide size restriction is due to the nature of the mhc i peptidic cleft, which presents closed extremities, contrasting to the peptidic cleft of mhc class ii molecules which can accommodate longer sequences (up to aa). imagine that a given viral protein possesses amino acids and that its proteolysis generates, in average, peptides with aa (it must be emphasized that fission does not occur, necessarily, at aa from the initial extremity, but that random cuts in any part of the protein generate peptides of different sizes). the number of sequences yielded, in such case, would reach some hundreds. considering that the exemplified protein size is relatively small and considering the classical idea that each cd + t cell recognizes only one peptide, the repertoire of memory t cells would have to be larger that , the total number of lymphocytes in human beings, to be efficient [ ] . therefore, the recognition of peptidic sequences must involve mechanisms with a certain plasticity and adaptability, without, however, loss of specificity. this would allow the immune cell repertoire to be shaped according to ongoing experiences leading to the establishment of memory, but at the same time, it would allow the maintenance of a source of naive, inexperienced cells. we will discuss here some mechanisms that could indicate events that bypass the problem of the size limitation of the immune system and, at the same time, could confer it a powerful resource for viral recognition. most of these mechanisms involve cross-reactivity. the capacity of a t lymphocyte to recognize nonrelated peptides derived from the same virus, or even peptides from heterologous viruses, will be defined here as cross-reactivity [ , ] . this phenomenon is mainly observed in cytotoxic t lymphocytes, even though it also occurs in t helper cells [ ] . several examples of cross-reactivity between heterologous viruses have already been described and some of them will be presented throughout the text. brehm et al. [ ] demonstrated that the sensitization with a subdominant epitope of the lymphocytic choriomeningitis virus (lcmv) elicits strong immune response against an heterologous subdominant epitope of the pichinde virus (pv). it is important to note that the pv peptide which elicited the cross-reactivity shared six out of eight amino acids with the one used to sensitize the cells. in that case, infection with an heterologous virus stimulated a strong t cell immunodominant response to an epitope that was previously weak and subdominant, indicating that not only the hierarchy of virus-specific t cells can be more malleable than formerly imagined, but also that the hierarchy of immunodominance can be deeply affected by previous encounters with heterologous pathogens. another interesting work involved a panel of four heterologous viruses: lcmv, pv, vaccinia virus (vv) and murine cytomegalovirus (mcmv). this study evidenced that memory t cells primed by a given virus may alter the host immune response to a second unrelated virus. thus, cross-reactivity was observed between those viruses, mainly between lcmv and pv, and between lcmv and vv. it was also demonstrated that previous immunization with one of those viruses, in some cases, enhanced the clearance of a second unrelated virus, early in infection, although the sequence of viral infection was important and cross-protection was not necessarily reciprocal [ ] . in the work of wedemeyer et al. [ ] , the presence of specific cd + t cells directed to one immunodominant epitope of the hepatitis c virus (hcv) among blood donors that did not present any history of infection by hcv or hepatitis b virus (hbv), led to an inquiry that revealed that those patients had been cross-sensitized by a previous encounter with influenza a virus (iva). the crossresponse occurred against one endogenously pro-cessed epitope of the iv neuraminidase (iv na- ), which is conserved among influenza viruses, being normally included in vaccines, and an hcv immunodominant viral determinant called hcv ns - , which is an hla-a determinant frequently recognized during acute hcv infection. in the previous example, the degree of similarity between the sequences of both epitopes, which share seven out of nine amino acids, seems to be quite important. additionally, both peptides present conserved aa in residues and , the critical residues for binding to the hla-a molecule. although they differ in two aa, these non-identical amino acids belong to the same chemical group and share certain physicochemical characteristics. several examples of cross-reactivity involving the influenza virus have already been reported. this is, to a certain extent, an expected fact since any given human immune system is challenged by this virus several times throughout life. consequently, memory cells for many epitopes, from different influenza strains, must be abundant in the t cell repertoire of any human being. in , shimojo et al. [ ] observed that a rotavirus-derived peptide could sensitize hla-a . + targets, inducing their lysis by ctls specific to a iv-derived matrix peptide. one variant of this iv peptide, the flu-m : - , presented cross-reactivity with an hiv-i epitope. cells stimulated in vitro with flu-m were capable of lyse not only cells marked with the iv epitope but also cells presenting the hiv epitope [ ] . it is interesting to point out that this observation was done in peripheral blood mononuclear cells from both hiv-infected and uninfected individuals, suggesting that, in individuals vaccinated against influenza, the response generated against the iv matrix protein could direct a specific immune response to an hiv epitope. it is known that the iv matrix protein and the hiv capsid and matrix proteins present notable structural similarities, mainly in the three-dimensional structure of the proteins rather than homology in the amino acid sequence. interestingly, both virus proteins have similar functions in their assembly, mediating the encapsidation of the ribonucleoprotein complex by the viral membrane [ ] . another work demonstrated the existence of cross-reactivity in the immune responses directed to japanese encephalitis and dengue viruses [ ] . among phylogenetically related viruses there are several examples of cross-recognition, such as the occurrence of cross-recognition to different peptides from the same subtype of influenza virus a (iva) [ ] , or among epitopes from different subtypes of iva [ ] , although cross-reaction between viruses with widely divergent primary amino acid sequences (hiv- and hiv- , for instance) seems also to be common [ ] . much of the cross-reactivity phenomenon involves the degenerated capacity for antigen recognition of the t cell receptor (tcr). the tcr consists of an heterodimeric structure formed by an a chain and a b chain or, alternatively, a c and a d chain. within each one of these chains, there are three hypervariable sites known as complementary determining regions (cdrs), which protrude as loops from the tcr and directly contact sites on the peptide and mhc molecule [ ] . the classical view of a monogamous relationship between the tcr of a given lymphocyte and the corresponding mhc-peptide (mhc-pep) complex has been wearing. as previously mentioned, the number of mhc-pep complexes that could be generated exceeds that of t cells in the repertoire of an individual. specifically, this limitation is due to spatial and/or numerical restrictions on the mature t cell pool, rather than the number of possible tcr rearrangements. the size of the potential tcr repertoire is estimated in , considering all gene segment rearrangements and the imprecise junctions generated by insertions and deletions in the n terminal regions. this number is greater than the theoretical number for different nonamer peptides that could be generated, which equals [ ] . it is logical that the repertoire full combinatorial potential cannot be exhausted in humans or other animals, even if there were a mechanism ensuring that any tcr is produced only once in a given individual. thus, cross-reactivity is necessary for t cell receptors. such wide spectrum cells can be activated by a primary peptide and also by closely related peptides, or even by peptides that present a certain homology with the required sequence. the tcr flexibility is exhibited at different levels, influencing the positive selection of immature thymocytes as well as the immune response to heterologous antigens, in peripheral mature t cells [ ] . considering that, during an immune response, it is expected that multiple t cell clones recognize the same mhc-pep complex and considering that the number of potential antigens exceeds that of available t cells, it can be suggested that a high degree of cross-reactivity is an intrinsic and necessary property for an efficient immune system. a mathematical model supports this idea, predicting that a single tcr may be able to interact with over one million different peptides [ ] . the cdr loops of the tcr are subject to significant conformational alterations to accommodate the three-dimensional structure of the mhc-pep complex surface. the natural tcr flexibility allows it to be promiscuous in peptide recognition, making it inherently able for cross-recognition [ ] . it can be argued that not all possible peptides will naturally occur and that, among them, some will associate with mhc molecules while others will not. even so, the number of mhc-pep complexes effectively formed is extremely high. nevertheless, we must consider that not all the mhc-pep complexes formed will trigger clonal expansion, memory ctls reactivation and infected cell lysis. another level in the antigen recognition concerns the mhc. although the classical view dictates that the majority of antigenic peptides could be recognized in the context of only one or a few alleles of the mhc, nowadays it has been observed that t cell determinants can be recognized in the context of a variety of class ii alleles [ ] , although the structural basis for this promiscuous recognition is not yet understood. brehm et al. [ ] , through the infection of mice, obtained specific ctls for lcmv-derived peptides which also recognized allogeneic antigens. these allospecific cells generated in response to the viral infection have been kept in high frequencies in the memory cell compartment, indicating that allospecific cd + memory t cells can develop as a consequence of viral infection. in fact, mice previously infected with lcmv were refractory to the tolerance induction to skin allogeneic graft. the susceptibility to tolerance induction is influenced by the immunological history of the individual, i.e., individuals that have suffered multiple viral infections tend to be more refractory to tolerance induction than those who have had less infections. such observation can have some implications in the transplant research field [ ] . also, previous immunity to a given virus can significantly improve the clearance of a second, nonrelated virus, in initial stages of the infection, when specific high affinity t cells directed against this second virus (generated by the stimulation of naïve t cells) were not yet available. thus, the task of reducing infection spreading can be delegated to cross-reactive cells and the organism will gain time to assemble a more specific artillery. the idea that cross-reactive t cells lead to a low affinity response to heterologous epitopes generated the argument that this could cause deleterious effects to the host, who would lack an acute, virus-specific immune response for some types of viral infections. in fact, some works have verified that such situation is possible and can be used by viruses as an extra evasion mechanism. it was observed that human papillomavirus type (hpv )-infected individuals that had developed cervical cancer presented an almost complete absence of specific t lymphocytes directed to the oncoprotein (hpv ) e - ( ) epitope. cross-reactivity between this epitope and a coronavirus epitope called ns - was identified. it was proposed that frequent contacts with a virus could lead to an ''exhaustion'' of the t cell repertoire, which could lead to an inefficient ctl response. moreover, this same hpv epitope presented considerable sequence homology with peptides from diverse organisms, including some sequences from self-peptides, providing support for the suggestion of toleration of the immune system [ ] . another strategy used by viruses to evade the immune system is exemplified by the murine cytomegalovirus (mcmv), which codes for an evasion immune protein (m /gp ). this protein prevents the presentation of an immunodominant peptide, provenient from an antiapoptotic protein (m ), in pathologically relevant tissues. thus, this peptide stimulates a cd + long-term response, but the target is not present in the tissues of interest [ ] . a possible role for the plasticity of t cell recognition in the development of human immunity is supported by de silva-udawatta et al. [ ] . human t cell clones were generated against two autoantigens which are frequent targets of autoantibodies and t cells in the same lupus patient. interestingly, the tcrs from all isolated clones had substantial sequence homology in their cdr region . cloning of the tcr a and b chains from these cells, in a tcr-negative human cell line, and a subsequent analysis of interaction between the tcr and the antigenic peptides revealed tcr stimulation by both peptides, evidencing a degeneration or plasticity in the recognition of these lupus autoantigens by the t cell receptor. memory, in the immune system, is also affected by cross-reactivity. the immunological history of an individual will constantly shape the t cell repertoire and, consequently, affect the induction of cells in future viral infections. memory cells are not isolated in the immune system but participate in an interactive network, which is continuously evolving in such a way that the any immune response modifies the frequency, distribution and activity of all other components of the immune system [ ] . finally, if it was previously thought that memory t cells were present in relatively low frequency and were essentially resting cells, recent studies have demonstrated that virus-specific ctls were kept in high frequencies during the lifespan of a mice that had been infected by lcmv, pv or vv. in addition, a subpopulation of those memory t cells consisted of cytolytically active circulating cells, expressing il- receptors and high levels of adhesion molecules, which is in agreement with a plastic and interconnected immune system [ ] . considering the previous discussion, and focusing specifically in viral immunodeterminants, some questions arise. if the same peptide can, potentially, trigger either a strong immune response or a barely detectable response, how can we talk about immunodominant peptides? in other words, how can the immune system detect a virus? beyond the need for costimulation, it seems that viral immunodominant peptides share some characteristics. joshi et al. [ ] studied cross-reactivity of three promiscuous epitopes of t helper cells (linked to mhc class ii), which did not show strong sequence homology and were of distinct origins. the sequences seemed totally not related. a more careful analysis, however, revealed some structural similarities, such as the presence of positively charged residues in the middle of their sequences. the same work comments that few lateral chains from the amino acids of the peptide concentrate the focus of the tcr action. moreover, the same tcr can accommodate peptide sequences with different lateral chains, depending on the size and chemical properties of the tcr contact surface. in addition, a study with an epstein-barr virus immunodominant epitope that represents the main target for ctls identified that only out of amino acids of the peptide possessed crucial function for t cell recognition. two of those amino acids were anchorage residues and occupied positions and , and the residue in position was responsible for making contact with the tcr. polyalanine analogues that share these amino acids were capable of inducing reactivation and clonal expansion of specific ctls for the wild-type epitope, indicating that a simple but specific amino acid residue is sufficient to productively interact with the tcr. not only was the original amino acid (aspartic acid) at position capable of inducing reactivation of specific ctls, but also the glutamic acid or glutamine, all of them presenting a carbonylic group on the side chain. such peptides, however, have failed to trigger the cytolytic mechanisms, demonstrating the existence of different requirements in tcr stimulation. thus, different interactions must induce reactivation of memory ctls and unchain the cytotoxic mechanisms [ ] . we can suggest that, even though there is a need for co-stimulation, some factors really characterize some epitopes as viral sequences and that these ''sequences'' will probably not be represented in higher organisms, otherwise we would observe a high incidence of autoimmune diseases. however, in some cases, viral epitopes present considerable similarities to self peptides and can be involved in the pathogenesis of autoimmune diseases. epidemiologic evidences alert that infections frequently precede autoimmune reactions, moreover, variant epitopes of viral origin may act as antagonists of their own ctls, by possessing the anchor residues but not the contact residue. how can we use cross-reactivity to develop wide spectrum viral vaccines? considering the phenomena previously discussed, we can propose at least two major strategies to develop wide spectrum viral vaccines. the first one is based on the identification, characterization, and cloning of immunodominant viral epitopes able to stimulate responses against different virus involved in frequent human diseases. the produced peptides could then be purified and serve as a basis for vaccine therapies. as previously mentioned, a number of peptide candidates fits this initial criteria of cross-reactivity induction, making this first step easy and feasible. after the identification of this potentially useful peptides, several admixtures could be elaborated, aiming at inducing protection to different virus-induced diseases. certainly, the administration of such a peptide pool should be associated to adequate immune stimuli (meaning the inflammatory/infectious context necessary to induce immune responses) in order to enhance the probability of immune response stimulation and long term protection. a second strategy is potentially more powerful than the first one, but also demands more time and studies to become a reality. it starts from the identification of patterns (in the amino acid sequence or in the biochemical properties) in immunodominant viral peptides (such as conserved amino acids in specific residues) that are capable to induce immune response against a broad range of different organisms. the deduced conserved amino acid sequences (or biochemical properties) should be used for the production of synthetic peptides containing the amino acid residues necessary for mhc anchoring and tcr contact. except for these conserved residues, these synthetic peptides could be filled with amino acids with few or none lateral chains (that could interfere with the mhc/ tcr contact). thus, a priori, a limited number of highly conserved and immunogenic peptides could be enough to induce protection, by means of cross-reactivity, against a variety of virus. nevertheless, a series of technical problems surround the identification of conserved viral immunodominant peptides. obviously, the existence of a ''golden sequence'' containing the elements necessary to stimulate immune responses against all kinds of virus is highly improbable, although the identification of consensus sequences representing different viral groups is a concrete possibility. also, different hla alleles require different peptides as anchor motifs and this variability should also be considered during peptide screenings. it is important to point out that sometimes viral peptides are associated to the development of autoimmunity and, consequently, it will always be necessary to take into account the cross-reactive phenomenon as a whole and not only as a characteristic of immune responses against pathogens. conversely, a potential application to conserved viral epitopes includes tolerance induction against peptides involved in autoimmune diseases. as discussed, these features do not invalidate the potential of immunodominant viral peptides as inducers of long term immune responses but points out the need of a deep knowledge of cross-reactivity phenomena. both vaccination approaches previously proposed have as an advantage, the use of single peptides instead of complex viral particles, when compared to conventional vaccine strategies. this is extremely relevant when organisms such as hcv or hiv are the therapy targets. besides, if relevant conserved features in immunodominant peptides could be identified and associated to specific viral groups (a pool of peptides that confer protection against different iv lineages, for instance), the vaccine could be directed to this viral group, with minimal interference in the remaining t cell pool. interestingly, as immunodominance hierarchy is affected by previous encounter with other epitopes, also subdominant epitopes could be envisaged as putative candidates to vaccine development. thus, the analysis of immunodominant viral epitopes in search of particular features and the subsequent use of these features to the development of synthetic peptides with a ''viral pattern'' seems to be interesting steps towards the development of wide spectrum viral vaccines. cross-reactivity between hepatitis c virus and influenza a virus determinant-specific cytotoxic t cells analysis of cd (+) t-cell responses to human papillomavirus (hpv) type l in healthy adults reveals a high degree of responsiveness and cross-reactivity with other hpv types protection against lethal vaccinia virus challenge in hla-a transgenic mice by immunization with a single cd + t-cell peptide epitope of vaccinia and variola viruses cross-reactivities in memory cytotoxic t lymphocyte recognition of heterologous viruses t cell immunodominance and maintenance of memory regulated by unexpectedly cross-reactive pathogens protective heterologous antiviral immunity and enhanced immunopathogenesis mediated by memory t cell populations specificity of peptide binding by the hla-a . molecule cross-reactivity between hla-a -restricted flu-mi: - and hiv p gag: - epitopes in hiv-infected and uninfected individuals structural similarities between influenza virus matrix protein m and human immunodeficiency virus matrix and capsid proteins: an evolutionary link between negative-stranded rna viruses and retroviruses antibody responses determined for japanese dengue fever patients by neutralization and hemagglutination inhibition assays demonstrate cross-reactivity between dengue and japanese encephalitis viruses diversity of epitope and citokine profiles for primary and secondary influenza a virus-specific cd + t cell responses selective expansion of cross-reactive cd + memory t cells by viral variants cytotoxic t cells from human immunodeficiency virus type -infected patients frequently cross-react with different human immunodeficiency type i clades cd + t cell responses to viral infections in sequence cross-reactivity in t-cell antigen recognition a very high level of crossreactivity is an essential feature of the t-cell receptor flexibility in mhc and tcr recognition: degenerate specificity at the t cell level in the recognition of promiscuous th epitopes exhibiting no primary sequence homology direct visualization of cross-reactive effector and memory allo-specific cd t cells generated in response to viral infections heterologous immunity provides a potent barrier to transplantation tolerance human papillomavirus type e peptide-directed cd + t cells from patients with cervical cancer are cross-reactive with the coronavirus ns protein cytomegalovirus misleads its host by priming of cd t cells specific for an epitope not presented in infected tissues cloned human tcr from patients with autoimmune disease can respond to two structurally distinct autoantigens plasticity of t cell memory responses to viruses a single specific amino acid residue in peptide antigens is sufficient to activate memory ctl: potential role of cross-reactive peptides in memory t cell maintenance we thank andréia escostesguy vargas for critical review of this manuscript. key: cord- - i v ecg authors: kopitar-jerala, nataša title: the role of cysteine proteinases and their inhibitors in the host-pathogen cross talk date: - - journal: curr protein pept sci doi: . / sha: doc_id: cord_uid: i v ecg proteinases and their inhibitors play essential functional roles in basic biological processes in both hosts and pathogens. endo/lysosomal cathepsins participate in immune response in pathogen recognition and elimination. they are essential for both antigen processing and presentation (host adaptive immune response) and activation of endosomal toll like receptors (innate immune response). pathogens can produce proteases and also natural inhibitors to subvert the host immune response. several pathogens are sensed through the intracellular pathogen recognition receptors, but only some of them use the host proteolytic system to escape into the cytosol. in this review, i provide an update on the most recent developments regarding the role of proteinases and their inhibitors in the initiation and regulation of immune responses. multicellular organisms have continuous interactions with both pathogenic and non-pathogenic microbes and have developed a set of antimicrobial recognition and defense systems, which enable them to survive. the innate immune system detects molecular structures, denoted as pathogen associated molecular patterns (pamps) that are distinct from host molecular pattern and are frequently found in bacteria, fungi, virus, and some protozoans. these pamps are detected using an array of pattern-recognition receptors (prrs) [ ] [ ] [ ] , which are expressed at the first line of defense against infection by cells like macrophages, monocytes, dendritic cells, neutrophils and epithelial cells, as well as cells of the adaptive immune system [ ] . prrs include the membranebound toll-like receptors (tlrs), the cytosolic nod like receptors (nlrs) and the rna-sensing rig-like helicases (rlhs) [ ] . the outcome of pamp recognition by prrs leads to signal transduction from these receptors which converges on a common set of signaling modules, often including the activation of the nf-b and ap- transcription factors that drive proinflammatory cytokine/chemokine production [ , ] . ligand recognition by tlrs is mediated by the extracellular or ectodomains that contain to leucine-rich repeat (lrr) motifs, a transmembrane domain and a cytoplasmic tir domain [ ] [ ] [ ] . structural and biochemical studies revealed that all tlrs form either hetero or homodimers (e.g., tlr /tlr , tlr /tlr , tlr /tlr , and tlr /tlr ) [ , ] . in mammals, tlr family members have been described ( tlr in mice and tlr in humans) [ ] . while tlr , , , and are primarily expressed on the cell sur-*address correspondence to this author at the department of biochemistry, molecular and structural biology, »jo ef stefan« institute, jamova , ljubljana, slovenia; tel: + ; fax: + ; e-mail: natasa.kopitar@ijs.si face and recognize pamps derived from bacteria, fungi and protozoa, tlr recognizes lipopolysaccharide (lps), a major cell wall component of gram-negative bacteria [ ] [ ] [ ] [ ] . an essential component of gram-positive bacteria, peptidoglycan is sensed by tlr [ ] , which also detects lipoarabinomannan (lam) of mycobacteria [ ] . tlr could form also heterodimers (in conjugation with tlr or tlr ) and as a heterodimer recognizes diacyl or triacyl lipopeptides on bacteria, mycobacteria and mycoplasma. tlr senses the flagellin protein expressed by flagellated bacteria [ ] . tlr participates in the recognition of macrophage-activating lipoprotein kd (malp- ) derived from mycoplasma [ ] . the intracellular tlr , tlr , tlr , and tlr are localized on the er membrane and only upon stimulation with pamps, they are targeted into the endosomes [ ] . the intracellular localization of tlr , tlr , tlr , and tlr is regulated by the er membrane protein unc b, which directly interacts with the intracellular tlrs [ ] . tlr recognizes genomic dna from dna viruses such as hsv- , hsv- , or mcmv [ ] . viral singlestranded rnas (ssrnas) derived from hiv or influenza virus are recognized by tlr [ ] . tlr recognizes dsrna derived from reoviruses and a synthetic double-stranded rna (dsrna) analog, polyinosinic-polycytidylic acid (poly i:c) [ ] . signaling through tlr , tlr , tlr , tlr and tlr primarily induces the production of inflammatory cytokines, whereas tlr and tlr induce type i interferons (ifn) [ ] . recently, several excellent reviews have described the signaling of innate immune receptors, therefore those aspect will not be discussed in detail [ ] [ ] [ ] ] . proteases play significant roles in innate as well as adaptive immune response. they are classified by their gene sequence homology and according to their catalytic mechanism as cysteine, serine, threonine, aspartate and metalloproteases, or unknown proteases [ ] (http://www.merops. sanger.ac.uk). the activity of different proteases is tightly controlled at the level of expression, zymogene activation, localization in different cellular compartments, or by protease inhibitors. many pathogenic organisms synthesize proteases that resemble host proteases. indeed, amino-acid sequences typical of pathogen proteases exist in key molecules involved in host immune response regulation including immunoglobulins (ig), cytokines and chemokines. here, i will discuss the functional role of proteinase and inhibitors from metazoan hosts and microbial pathogens, at the cross talk of host pathogen interactions and the contribution of these interactions to host protection and susceptibility to infections. cysteine cathepsins were long known to be involved in protein degradation in lysosomes [ ] . they are papain-like cysteine proteinases belonging to clan ca, family c [ ] . eleven human cathepsins are known (b, h, l, s, c, k, o, f, v, x and w). with the exception of cathepsins s, v, k and w, they are widely expressed in a number of different cells and tissues. despite similarities in sequence and structure, cysteine cathepsins differ among each other in specificity. most of the cathepsins are endopeptidases, although cathepsin b and x are also carboxydipeptidases, and cathepsin h and c are aminopeptidases [ , ] . cysteine cathepsins exhibit a broad variety of functions [ ] [ ] [ ] . the human genome encodes for two cathepsin l-like proteases, namely the human cathepsin l and cathepsin v (cathepsin l ), whereas in mouse only cathepsin l is present [ ] . cathepsin v expression is restricted to thymus, testis and corneal epithelium, while cathepsin l is ubiquitously expressed [ , ] . cathepsins are synthesised as preproproteins, which are activated either by other proteinases or self-activated (in the case of endopeptidases). cathepsins are optimally active in the acidic environment in endolysosomes. however, they are still active in the extracellular space and in the nucleus despite a neutral ph [ ] . seminal study by goulet et al. showed that nuclear procathepsin l processed the transcriptional factor cux into a form with enhanced dna binding and that promotes cell cycle progression [ ] . cathepsin l was targeted into the nucleus through translation initiation at alternative start codons downstream of the normal signal sequence [ ] . recently, also cathepsin b and f were reported to be localized in the nucleus [ ] [ ] [ ] . our recent work demonstrated that the activity of cathepsin l in the nucleus is regulated by a nuclear cystatin, denoted as stefin b [ ] . the regulation of nuclear cathepsin f activity by stefin b in hepatic stellate cells was involved in the transcriptional regulation of two activation markers and implies the role of stefin b in transcriptional regulation [ ] . the activity of cathepsins is regulated by interaction with their endogenous protein inhibitors: the cystatins [ ] [ ] [ ] , thyropins [ ] and some of the serpins [ ] . thyropins are a superfamily of inhibitors homologous to the thyroglobulin type- domains [ ] . the best characterized human representative so far is the mhc-class ii associated invariant chain (ii) fragment, which strongly inhibits cathepsin l and cruzipain [ ] [ ] [ ] . cystatins are reversible and tight-binding inhibitors of papain (c ) and legumain (c ) families of cysteine proteases and are characterized by a strong sequence and structure conservation [ ] . the tertiary structures of cystatins are conserved and exhibit the so called "cystatin fold", which is formed by a five stranded anti-parallelsheet wrapped around a five-turn -helix [ , ] . the cystatin family i contains three subfamilies: i a, b and c, as defined in the merops database of protease and protease inhibitor information (http://merops.sanger.ac.uk/) [ ] . cystatins are found in plants, fungi and animals as well as in viruses. type cystatins, denoted as stefins, are predominantly present in the cytosol and the nuclei, while type cystatins are mainly extracellular, secreted proteins. these latter are synthesized with - residue long signal peptides, most of them found in physiologically relevant concentrations in body fluids. type cystatins are multidomain proteins of high molecular mass ( - kda) and present three tandemly repeated type -like cystatin domains [ ] . the mammalian cystatins belonging to this type are called kininogens [ ] , which were first known as kinin precursor proteins. the serpins are essentially serine proteinase inhibitors [ , ] , only some of them inhibit both serine and cysteine proteases [ ] . the mechanism by which cysteine proteases are inhibited involves the cleavage of the serpin, in some cases involving a stable covalent complex [ ] [ ] [ ] and in other cases not [ ] . macrophages play a critical role in host defense against pathogens and are present in virtually all tissues [ ] . they can change their physiology in response to microenvironmental stimuli. classically activated macrophages or m , primed with ifn-and stimulated with lps, are involved in inflammatory responses to bacterial and viral infection [ ] . stimulation of macrophages with the cytokines interleukin (il- ) or il- induces alternatively activated (called m ) macrophages [ ] [ ] [ ] . the m macrophages include several types of activated macrophages, not only wound healing macrophages, but also regulatory macrophages and tumor-associated macrophages. regulatory macrophages can secrete large amounts of interleukin- (il- ) in response to fc receptor-ligation [ , ] . m macrophages produce high amounts of proinflammatory cytokines, such as tumor necrosis factor alpha (tnf-), upon recognition of invading pathogens by a set of prrs including tlrs, rlrs and nlrs. m macrophages are known to produce nitric oxide (no) by expressing inducible no synthase (inos) and are critical for clearing bacterial, viral and fungal infections. early studies reported that macrophages activated with ifn-and stimulated with ifnchicken cystatin generated increased amounts of no and the cytokines tnf-and interleukin (il- ), in comparison with macrophages activated only with ifn- [ , ] . experiments on macrophages prepared from cystatin cdeficient mice revealed that ifn--primed cystatin cdeficient macrophages exhibit significantly higher il- but lower tnf-expression, compared to similarly primed wild type macrophages [ ] . upon the classical activation mechanism, macrophages up-regulate a variety of proteinases that can degrade endocytosed pathogens and play a critical role in antigen processing and presentation. the role of endosomal cathepsins has been described in several extensive reviews [ , ] . only some of the endogenous inhibitors (cystatin f and spia g) are also up regulated, while cystatin c is down regulated [ , ] . ser-pina g (spia g) is highly induced in macrophages during bacillus calmette-guérin infection as well as in infection with salmonella typhimurium and listeria monocytogenes [ ] . it was demonstrated that a ubiquitin homolog, ifnstimulated gene of -kda (isg ) is conjugated to spia g in activated macrophages. it was reported that the stimulation of murine macrophages with ifn-resulted in increased cathepsin s activity and down-regulation of intracellular cathepsin l activity, despite the persistence of high levels of mature cathepsin l protein [ ] . the reason for the lack of cathepsin l enzyme activity is still not clear. beers et al. showed that inhibitors of cysteine proteinases cystatin c and p form of major histocompatibility complex invariant chain did not inhibit cathepsin l and the authors suggested that cystatin f might be the inhibitor that selectively regulated cathepsin l activity in macrophages [ ] . recently, colbert et al. found equivalent loss of cathepsin l activity in ifn-stimulated wild type and cystatin f-deficient macrophages, indicating that cystatin f did not inhibit cathepsin l activity in activated macrophages [ ] . we showed that cathepsin l is targeted to the nucleolus of classically activated (m ), but not un-stimulated and alternatively activated (m ) macrophages [ ] . therefore, we proposed that lack of activity of cathepsin l in classically activated macrophages could be, at least in part, due to different nucleolar localization of cathepsin l and co-localization with spia g [ ] . since only the pro-inflammatory stimuli (ifn-and lps) and not the anti-inflammatory stimuli (il- ) induce increased nucleolar localization of spia g, it is possible that spia g functions in the nucleolus are important for the host defense against pathogens. spia g is a mouse specific serpin and a human homologue has not been described so far, therefore it is not clear which protein compensates for spia g deficiency in human macrophages. inflammasomes are multiprotein complexes that activate caspase- , an event which leads to maturation of the proinflammatory cytokines interleukin (il- ) and il- [ ] . cytosolic nlr (nlrp , nlrp , and nlrp ) are involved in assembly of inflammasome and the nlrp can be activated not only by bacteria, bacterial pore forming toxins or viruses [ ] , but also by a number of molecules like crystals silica, asbestos, alum and -amyloid [ ] [ ] [ ] [ ] [ ] . it was shown that cathepsin b (and possibly other cathepsins) leaking from the lysosomes had an important role in a direct nlrp activation by crystals and -amyloid [ , ] . in addition, it was reported that live intracellular mycobacterium m. kansasii triggered the activation of the nlrp inflammasome and cathepsin b release from the endosomes and that the production of reactive oxygen species was essential in this process [ ] . therefore cysteine cathepins participate in the defense against pathogens in cytosol. the cytosolic cysteine proteinase inhibitors could regulate cathepsin activity in this process and prevent excessive inflammation. dcs are antigen presenting cells characterized by their efficient processing of internalized antigens and the presentation of peptide bound to major histocompatibility (mhc) complexes to the t cells [ , ] . immature dcs reside in tissues and actively uptake antigens. maturation of dcs can be achieved by tlrs [ ] [ ] [ ] . the expression of a unique set of tlrs renders each type of dc susceptible to particular subsets of pathogens and the outcome of stimulation with tlr ligands can result in increased antigen uptake and presentation [ ] . the population of dcs can be divided into major sub populations: conventional dcs (cdcs) and plasmacytoid dcs (pdcs) [ ] . while the macrophages contain high levels of endolysosomal proteases and rapidly degrade internalized antigens, cdcs express low levels of endolysosmal proteases and in vivo degrade internalized antigens slowly. however , the resulting limited lysosomal proteolysis in cdcs is favourable to the antigen presentation [ ] . subtle differences exist also among cdc; only cdcs isolated from peripheral blood or derived in vitro from cd + hematopoietic progenitor cells are protease poor, whereas cdcs differentiated in vitro from monocytes exhibit protease expression and activity similar to macrophages [ ] . t lymphocytes recognize proteolytic fragments of antigens that are presented to them on mhc molecules. mhc class i molecules present primarily products of proteasomal proteolysis to cd + t cells, while mhc class ii molecules display mainly degradation products of lysosomes for stimulation of cd + t cells [ ] . mhc class ii molecules are assembled in the endoplasmic reticulum with the assistance of chaperone invariant chain (ii). a portion of ii, termed clip, binds in the peptide groove of mhc class ii molecules, thereby preventing premature loading of peptides [ ] . gene targeting studies showed a critical role for the lysosomal cysteine protease cathepsin s in the late stages of ii degradation in b cells, dcs and macrophages [ ] [ ] [ ] and cathepsin l (v in humans) in thymic cortical epithelium [ ] . it has been proposed that cystatin c regulates the cleavage and removal of the mhc class ii invariant chain (ii) by regulating the activity of cathepsin s, and hence in the formation of mhc class ii-peptide complexes [ ] . experiments on dc isolated from cystatin c-deficient mice showed that the lack of cystatin c did not change the formation of peptide-loaded mhc class ii complexes in any of the dc types, nor the efficiency of antigen presentation [ ] . plasmacytoid dcs constitute a minor population of dcs; in the endolysosomes they express tlr and tlr and produce large amounts of ifnin upon tlr and tlr stimulation by viral nucleic acids [ ] . activated pdcs behave differently than conventional dcs in antigen presentation following activation via tlr ligands such as cpg dna. in models of influenza infection, conventional dcs undergo maturation and present antigens in complex with mhc class ii, with a parallel downregulation of mhc ii synthesis. although pdcs also undergo maturation and present antigens, mhc class ii molecules synthesis is not down-regulated upon stimulation with tlr ligands, indicating that the pdcs have the ability to continuously present viral antigens in activated state [ ] . tlr recognition of viruses leads to ifn-production, which positively feedbacks via interferon receptor to drive further type i ifn production by pdcs [ ] . in the steady state, tlr , tlr and tlr are mostly sequestered in the endoplasmic reticulum [ ] and are transported into endosomes upon the activation with ligands [ , ] . an additional level of control is achieved by the proteolysis full length tlr and tlr by endosomal cysteine cathepsins and aep.tlr can bind its ligand cpg dna, but it cannot trigger activation signals without first being processed by endolysosomal proteases, which remove n-terminal region [ ] [ ] [ ] [ ] . cells derived from cathepsin b, l, k and s deficient mice show that no single protease is responsible for tlr or tlr processing, indicating that there is redundancy in this reaction [ , ] . the role of endogenous inhibitors in this process remains to be fully elucidated. many pathogens also synthesize cysteine proteases that act on target proteins in the host and thereby modulate host immune response. pathogen-derived proteases range from nonspecific proteases that degrade multiple proteins involved in the immune response to enzymes that are very specific in their mode of action. staphylococcus aureus is the most frequently isolated pathogen in gram-positive sepsis. among others, s. aureus secretes papain-like cysteine proteases: staphopain a (scpa) and staphopain b (sspb). it was reported that enzymatically active staphopains degraded collagen and fibrinogen in the host, and the authors suggested that these activities could contribute in the clotting impairment and tissue destruction caused by staphylococcal infection [ ] . chemerin is a proinflammatory plasma protein that binds to the serpentine receptor cmklr on macrophages and plasmacytoid dendritic cells and promotes chemotaxis [ ] . it is secreted as a precursor protein and activated upon proteolytic cleavage of its c-terminus by serine proteases of the coagulation, fibrinolytic, and inflammatory cascades [ ] , as well as human cathepsins k and l [ ] . sspb was reported to cleave and activate chemerin [ ] . it was proposed that sspb may help to recruit pdc and macrophages at the site of infection and contribute to the ability of bacteria to elicit and maintain a chronic inflammation [ ] . in silico studies revealed the presence of cystatin superfamily representatives in bacterial genomes and only some of them were pathogens of humans (v. cholerae and v. vulnificu) [ ] . since bacterial cystatins are homologues of eukaryotic ones, the authors of the analysis suggested that they might inhibit the cysteine proteases of their eukaryotic hosts [ ] . bacterial pathogen streptococcus pyogenes secretes a highly specific protease, called ides, which cleaves only immunoglobulin igg [ , ] . contrary to the expectations, ides activity is not inhibited by host protease inhibitors cystatin c, instead the protease activity was markedly stimulated. kinetic studies revealed that the human cystatin c efficiently accelerated the enzymatic velocity of the pathogen cysteine protease ides and thus functions as a facultative cofactor for this bacterial protease [ ] . therefore more experimental data will be needed before we can make conclusions regarding the role of bacterial cystatins. recently, it was reported that gram negative pathogen anaplasma phagocytophilum, which causes human granulocytic anaplasmosis and is harboured within neutrophils, upregulates cathepsin l expression in the nucleus and leads to enhanced cux cleavage and the repression of cux regulated essential genes for effective neutrophil function [ ] . the ways in which viruses entry the host cells are largely defined by the interactions between virus particles and their receptors at the host cell surface [ ] . enveloped viruses, such as orthomyxoviruses [ , ] , paramyxoviruses [ ] and retroviruses [ , ] encode proteins that mediate fusion of the viral envelope with target cell membranes, thus facilitating viral cell entry. the majority of viruses are internalized by endocytosis and delivered to the endosomes. in addition to host cell receptors, at least in some cases, endolysosomal cysteine cathepsins are required for their transport into the cytosol [ ] . filoviruses are enveloped, single-stranded, negativesense rna viruses. infections by the ebola and marburg filoviruses cause a fatal haemorrhagic fever in humans and non human primates, for which no approved antivirals are available [ ] . it has been shown that the endolysosomal processing by cathepsins b and l of the ebola virus glycoprotein is essential for the delivery of viral material into the cytosol [ ] . ebola and marburg entry into the host cell is mediated by the viral spike glycoprotein (gp), which attaches viral particles to the cell surface [ , ] . ebola virus gp is synthesized as a single polypeptide chain that is cleaved in the golgi into its receptor-binding (gp ) and fusion (gp ) subunits, which remain together through noncovalent interactions and through a disulphide bond. three gp -s-s-gp units come together to form the homotrimer that protrudes from the virion surface [ , ] . it was reported that the gp cleavage is a two-step event: first cathepsin l cleaved gp into kda fragment and after the cleavage with cathepsin b kda fragment is generated [ ] . the translocation of pseudovirions bearing kda gp into cytoplasm was strongly inhibited by cathepsin b inhibitors, while the entry of pseudovirions bearing kda gp was not [ , ] . a recent study confirmed that gp cleavage by endosomal cathepsins was essential to reveal a putative binding domain for the endolysosomal cholesterol transporter niemann-pick c (npc ) [ ] . lack of npc on target cells prevented ebola virus glycoprotein-dependent fusion [ ] . furthermore, it was reported that the virulence of only some of the ebola virus species is strongly dependent on cathepsin b [ ] . ebola and marburg viruses are highly pathogenic with mortality in humans up to % within days of exposure. while there are no fda-approved vaccines or post-exposure treatment modalities available for preventing or managing ebola virus or marburg virus infections, several promising vaccines have been tested on nonhuman primates [ , ] . considering the aggressive nature of ebola infections, in particular the rapid and overwhelming viral burdens, early treatment with cathepsin inhibitors alone or in combination with neutralizing antibodies could help to bring down the number of death cases after infection. not only filoviruses, but also some of coronaviruses, positive sense rna viruses, were reported to entry the host cell by proteases dependent manner [ ] . human coronavirus e, a causative agent of the human common cold, en-ters cells via endosomes in which cathepsins are involved in the fusogenic activation of e s protein [ ] . in addition, cathepsin l was reported to cleave spike (s)-protein of the severe acute respiratory syndrome (sars) coronavirus [ ] . the cleavage and the activation of the viral s protein by cathepsin l induced a conformational change in the sprotein required for the binding to the cellular receptor, angiotensin-converting enzyme (ace ) [ ] . the activation of s protein involves at least two consecutive cleavages by host cell proteases and is essential for viral infectivity [ ] . although ace is a cellular receptor for two divergent coronaviruses, sars coronavirus and human coronavirus nl , it was reported that the inhibitors of cathepsin l blocked the infection only by sars, but not by nl virus [ ] . in addition, expression of exogenous cathepsin l significantly enhanced infection mediated by the sars s protein, but not by the nl s protein or the vesicular stomatitis virus g protein [ ] . sars infections emerged in affecting > persons and resulting in death in % of cases [ ] . coronaviruses still represent a leading source of novel viruses for emergence into the human population [ ] . recently, several neutralizing antibodies have been developed and some of them showed promising results in the therapy of non-human primates [ ] . however, the combinational therapy with antibodies and inhibitors could have several advantages. paramyxoviruses are enveloped, single-stranded, negative-sense rna viruses which include a number of major human pathogens, such as measles virus, mumps virus, human respiratory syncytial virus, and hendra and nipah virus [ ] . proteolytic activation of the fusion protein of the nipah virus is a prerequisite for the production of infectious particles and for virus spread via cell-to-cell fusion [ ] . recently cathepsins b and l were reported to be required for the cleavage and productive replication of pathogenic nipah virus, but not hendra virus [ ] . reoviruses form non-enveloped, double-stranded rna viruses. the virus entry into cells is initiated by the attachment of virions to cell surface receptors [ ] and by receptor-mediated endocytosis [ ] . in the host cell endolysosomes, virions undergo stepwise disassembly, forming discrete intermediates, the first of which is the infectious subvirion particle [ ] . a recent study examined a contribution of individual cathepsins b, l and s to the virus spread in newborn mice [ ] . in was shown that the survival rate of cathepsin b-deficient mice was enhanced in comparison to that of wild type mice, whereas the survival rates of cathepsin l and cathepsin s deficient mice were weakened. virus titers at sites of secondary replication in all strains of cathepsin-deficient mice were lower than those in wild type mice, indicating that all cathepsins could participate in the spread of the virus. clearance of the virus was delayed in cathepsin l-and cathepsin s-deficient mice in comparison to the levels for wild type and cathepsin b-deficient mice, as a consequence of the important functions of the two cathepsins in immune response [ ] . the study shows that the functions of proteinases in the virus entry into the cell as well as in host immune response are relevant for the possible therapy with inhibitors. during the last decade, our 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endosome-recycling compartment protein is the reovirus cell attachment protein evidence for a second step in the intracellular uncoating and transcriptase activation process genetic and pharmacologic alteration of cathepsin expression influences reovirus pathogenesis this work is supported by grants from slovenian research agency: grant j - (to n. kopitar-jerala), grant p- (to b. turk) and grant j - . key: cord- -jqy lcbk authors: gupta, vandana; tabiin, tani m.; sun, kai; chandrasekaran, ananth; anwar, azlinda; yang, kun; chikhlikar, priya; salmon, jerome; brusic, vladimir; marques, ernesto t.a.; kellathur, srinivasan n.; august, thomas j. title: sars coronavirus nucleocapsid immunodominant t-cell epitope cluster is common to both exogenous recombinant and endogenous dna-encoded immunogens date: - - journal: virology doi: . /j.virol. . . sha: doc_id: cord_uid: jqy lcbk correspondence between the t-cell epitope responses of vaccine immunogens and those of pathogen antigens is critical to vaccine efficacy. in the present study, we analyzed the spectrum of immune responses of mice to three different forms of the sars coronavirus nucleocapsid (n): ( ) exogenous recombinant protein (n-gst) with freund's adjuvant; ( ) dna encoding unmodified n as an endogenous cytoplasmic protein (pn); and ( ) dna encoding n as a lamp- chimera targeted to the lysosomal mhc ii compartment (p-lamp-n). lysosomal trafficking of the lamp/n chimera in transfected cells was documented by both confocal and immunoelectron microscopy. the responses of the immunized mice differed markedly. the strongest t-cell ifn-γ and ctl responses were to the lamp-n chimera followed by the pn immunogen. in contrast, n-gst elicited strong t cell il- but minimal ifn-γ responses and a much greater antibody response. despite these differences, however, the immunodominant t-cell elispot responses to each of the three immunogens were elicited by the same n peptides, with the greatest responses being generated by a cluster of five overlapping peptides, n( – ), each of which contained nonameric h (d) binding domains with high binding scores for both class i and, except for n( – ), class ii alleles. these results demonstrate that processing and presentation of n, whether exogenously or endogenously derived, resulted in common immunodominant epitopes, supporting the usefulness of modified antigen delivery and trafficking forms and, in particular, lamp chimeras as vaccine candidates. nevertheless, the profiles of t-cell responses were distinctly different. the pronounced th- and humoral response to n protein plus adjuvant are in contrast to the balanced ifn-γ and il- responses and strong memory ctl responses to the lamp-n chimera. sars coronavirus nucleocapsid immunodominant t-cell epitope cluster is common to both exogenous recombinant and endogenous dna-encoded immunogens vaccines designed to prevent viral infection must promote the adaptive expansion of t cells that are associated with band t-cell long-term memory of epitopes identical to or crossreactive with those of the natural pathogen (welsh et al., ; crotty and ahmed, ) . the processing and presentation of antigen epitopes of vaccine immunogens must therefore mimic that of pathogens. the conventional understanding has been that epitopes presented by mhc class i are derived from endogenous cellular proteins that have been cleaved by proteasomal and post-proteasomal peptidases (villadangos et al., ; yewdell and bennink, ; kloetzel ) and that the epitope loading of mhc class ii molecules generally involves exogenous proteins that are taken into antigenpresenting cells (apcs) by specialized endocytic receptors and processed by proteases of the endosomal/lysosomal compartments (moreno et al., ; bryant and ploegh, ) . it has been widely reported that various antigen-processing pathways may affect immune epitope formation either positively or negatively and may influence the repertoire of t-cell epitope-specific immune responses either quantitatively or qualitatively (pamer and cresswell, ; kessler et al., ; lautwein et al., ; kloetzel, ) . such specificity in antigen processing would suggest that the form and delivery of an immunogen can be critical to the generation of biologically effective antigen epitopes and thus to vaccine efficacy. there is, however, abundant evidence for the presentation of exogenous antigens by mhc class i and of endogenous proteins by mhc class ii through a variety of alternative antigen trafficking and processing mechanisms, including specialized processing pathways (reimann and schirmbeck, ; norbury et al., ; bonifaz et al., ; imai et al., ) , cross priming and presentation (brode and macary, ; tewari et al., ) , alternative endolysosomal pathways (brooks et al., ; lich et al., ; chen and jondal, ) , vesicular translocation (reis e sousa and germain, ; ackerman and cresswell, ) , and phagocytic or autophagic mechanisms for antigen delivery to both the mhc class i and ii compartments (houde et al., ; guermonprez et al., ; nimmerjahn et al., ; rocha and tanchot, ; paludan et al., ) . nevertheless, little is known about the consequences of these alternative antigen trafficking and processing pathways for the production of pathogen-specific antigen epitopes. thus, an examination of t-cell epitopes is a critical early step in the development of a new generation of vaccines, including dna vaccines, chimeras with other protein sequences, or epitopebased antigens, all of which involve antigens as non-viral structures that may access processing pathways that differ from those followed by the native pathogens. these issues regarding the pathways used for antigen delivery and processing are particularly relevant to mhc class ii epitopes. presentation of antigen epitopes and activation of cd t cells by mhc class ii molecules are critical steps in generating memory b cell and cd + t-cell phenotypes (wang and livingstone, ; , and failure to elicit immune memory is one of the recognized problems of conventional genetic vaccines (maecker et al., ) . this defect may reflect an inadequate level of antigen expression or restricted access to the mhc ii processing and presentation compartments by dna-encoded endogenous antigens that lack a dedicated trafficking pathway (martins et al., ; maecker et al., ; oehen et al., ; rush et al., ) . in response to this problem, we have characterized dna vaccine constructs synthesized in the form of chimeras that link the antigen sequences to the trafficking/targeting signals of the lysosome-associated membrane protein- (lamp- ) (chen et al., ) . lamp molecules are known to traffic to lysosomes and to specialized multilaminar vesicular compartments of immature apcs, termed miic, where mhc ii antigen processing and the formation of antigenic peptide -mhc ii complexes occur (kleijmeer et al., ; geuze ; murk et al., ) . studies of several antigens (including hiv- env and gag, hpv e , cytomegalovirus pp , carcinoembryonic antigen, dengue prem/e and yellow fever env, and the catalytic subunit of the telomerase reverse transcriptase (htert)) expressed from viral vectors and naked dna or rna have shown that lamp-targeted antigens generated greater antigenspecific lymphoproliferative activity, antibody titers, and cytotoxic t-lymphocyte activities than do dna constructs encoding wild-type antigens (rowell et al., ; wu et al., ; ruff et al., ; nair et al., ; raviprakash et al., ; bonini et al., ; su et al., ; lu et al., ; marques et al., ; chikhlikar et al., ; barros de arruda et al., ; anwar et al., ; su et al., ) . most recently, in a clinical study, patients who received dendritic cells transfected with rna encoding an htert/lamp chimera developed higher frequencies of htert-specific t-cell responses than did subjects receiving dendritic cells transfected with the unmodified htert template (su et al., ) . despite this abundance of evidence of increased immune responses to lamp chimeras of antigen proteins, the biological consequences of the enhanced immune responses in relation to protective immunity have not been defined, and there remains a critical question of the identity of t-cell epitopes of lamp-targeted antigens as compared to those of natural pathogens. lamp trafficking is not a normal mechanism for antigen access to the mhc class ii compartment of antigen-presenting cells, and the epitope specificity of antigen processing and presentation in vivo in comparison to other antigen delivery systems remains to be demonstrated. in the present study, we have used the severe acute respiratory syndrome coronavirus (sars cov) nucleocapsid (n) as a model antigen to address this issue of immune response and t-cell epitope presentation as a function of antigen formulation. the n protein of amino acids is abundantly expressed and highly immunogenic (lau et al., ; huang et al., a) . the primary function of this structural protein in virus assembly is to bind to sars cov rna during the formation of the ribonucleoprotein complex (huang et al., b) . previous studies have shown that both the n protein (lin et al., ; zhao et al., ) and dna encoded n (kim et al., ; zhu et al., ; yang et al., ) elicit antibody and cytotoxic t-cell responses in mice. in this analysis of the effect of immunogen formulation and delivery on immune responses in vivo, we have utilized three forms of n delivery: ( ) recombinant n-gst protein combined with freund's adjuvant as a classical exogenous antigen under conditions of profound activation of the innate immune response; ( ) n encoded in a dna plasmid as an unmodified endogenous cytoplasmic/nuclear antigen; and ( ) a dna construct with n inserted into the luminal domain of the entire lamp- molecule as an unnatural endogenous antigen targeted to the luminal compartment of endosomal/lysosomal vesicles (guarnieri et al., ) . validation of n expression by p-n and p-lamp-n dna constructs was carried out by analyzing cells transfected in vitro. cdna encoding the n protein was prepared by rt-pcr with n-specific primers and sars rna purified from a singapore clinical isolate (genbank accession no. ay ). the corresponding -bp n sequence was inserted into the p vector (kessler et al., ) to form the wild-type p-n dna expression vector and into the p -hlamp- construct in a position v-proximal to the lamp transmembrane domain to make the p-hlamp-n vector (fig. a) . western blot analysis of n in cell lysates and culture supernatants of transfected cos- cells showed the presence of the¨ kda n protein at comparable levels in cell lysates and culture supernatants of p-n and p-hlamp-n-transfected cos- cells and of various forms of the higher molecular weight lamp-n chimera in the cell lysate and culture supernatant of p-hlamp-n-transfected cells (fig. b) . the n-like protein in the cell lysate of p-hlamp-n-transfected cells is attributed to the cleavage of n from the protease-resistant lamp at some stage in the cellular trafficking of the molecule. intact or near-intact hiv- gag protein was also observed with cells transfected with the corresponding lamp/gag dna vaccine construct . several forms of the larger lamp-n molecule were also present in both the cell lysate and culture supernatants of the transfected cells. the intact lamp-n is found at about kda, and the other bands are attributed to various degradation products. however, we are cautious about the presumed cell trafficking pathway for the newly synthesized n, which lacks an endoplasmic reticulum translocation (signal) sequence, is to the cell cytoplasm. in contrast, the lamp-n chimera is expected to traffic to endosomal/ lysosomal vesicles because of the lamp vesicular trafficking sequences. we used confocal microscopy to validate these trafficking destinations for the immunoreactive unmodified n antigens and n of the lamp-n chimera expressed in transfected mouse lb . b cells and human melanoma mel-juso cells, comparing their cellular distribution to that of the endogenously expressed lamp- and mhc class ii proteins (fig. ) . the colocalization of endogenous lamp and mhc class ii proteins in vesicular compartments of apcs has been extensively documented (kleijmeer et al., ; geuze, ; murk et al., ) . trafficking of the lamp-n chimera protein to the lamp and mhc ii compartments of the transfected cells was validated by the colocalization of the lamp-n chimera (labeled by anti-n antibody) with endogenous lamp- and mhc class ii in both mouse b ( figs greater definition of the localization of n in transfected cells was obtained by analyzing of immunolabeled ultrathin cryosections of mouse lb . b cells. the endocytic compartments of these b cells are morphologically similar to those of other reported mouse b-cell lines ( h .dm and a .a b ), with well-defined multivesicular and multilaminar miics and end organelles (geuze, ) . double immunogold labeling of the transfected b cells confirmed that n of the lamp-n chimera was present with both endogenous lamp and mhc class ii molecules in the miic multilaminar vesicles (figs. a, b). in contrast, unmodified n was predominantly localized in the cytoplasm or in vacuolated structures with no evident colocalization with lamp or mhc class ii (figs. c-e). remarkably, lower power magnification showed that some of the cytoplasmic vesicles contained large amounts of n. these vesicles may correspond to the strongly labeled vesicular structures shown by immunofluorescent microscopy, and the presence of n in these structures suggests a possible route for the secretion of the protein (fig. e ). n antigen in three forms, representing differences in antigen delivery, adjuvants, and peptide processing pathways, were administered according to the following protocols: ( ) n as an exogenous n-gst protein was administered three times, on day one and and weeks later, initially with cfa and then with ifa. ( ) n as an unmodified endogenous, dna-encoded, cytoplasmic protein, and ( ) n as an artificial chimera encoded in the luminal domain of lamp and targeted to cellular lysosomal and mhc ii compartments were administered five times, on day , and , , , and weeks later, without added adjuvant but subject to the molecular adjuvant effects of the dna cpg motifs and the targeting of the lamp-n chimera to the mhc ii compartment. dna encoding p-hlamp was used as the negative control. t-cell responses were assayed by ifn-g elispot and elisa assays using spleen cells collected days after the final dna immunization. for these assays, the t cells were stimulated with n-his protein as a positive control or with a library of overlapping n peptides, to amino acids in length. the results were confirmed in three experiments and in multiple assays with three independent sets of peptides from different sources. the ifn-g responses were also assayed with an elisa protocol, with similar results (data not shown). the strongest ifn-g responders were cells from mice immunized with p-hlamp-n, with comparable but weaker responses by the p-n-immunized mice, and minimal responses by cells of the n-gst-immunized mice (table ; fig. ). the major grouping of ifn-g responses was elicited by peptides of amino acid sequence n - , encompassing of the overlapping peptides. more recently, these results have also been confirmed in a new experimental protocol currently being conducted that also show that all of the peptides that encompass n - elicit both cd -and cd -specific elispot responses (data not shown). other dominant responses were to peptides n - and n - and the overlapping peptides n - and n - . all significant epitope-specific responses of the pn or n-gst-immunized mice were shared by the p-hlamp-nimmunized mice. the weak ifn-g responses of the n-gst protein immunized mice were observed only with peptides that elicited the strongest responses with splenocytes of the dnaplasmid-immunized mice (figs. a -c). this comparatively weak ifn-g response of n-gst-immunized mice was in marked contrast to the strong il- responses as shown below and to the exceptional strong antibody responses. the n-specific antibody titers to n-gst immunization on day (after the first boost) was : , and at day (after the second boost) was : , , as compared to titers increasing to a maximum of : on days and in response to immunization with the p-n and p-hlamp-n dna constructs. memory immune responses (total igg antibody titers, ctl activity, and elispot ifn-g and il- responses) were examined by use of a separate experimental protocol with groups of mice treated with the same three n antigen preparations but receiving only three inoculations over weeks. after an interval of weeks, the memory immune response was activated by a single injection at week of the p-n construct as a surrogate of virus infection. antibody and tcell elispot and ctl responses were measured immediately before p-n injection at weeks and three times at week intervals thereafter (figs. and ). the t-cell assays were conducted with the selected peptides that had elicited immunodominant responses in pilot experiments for and ctl, and n - . the residual t-cell ifn-g and il- responses of mice sacrificed on day before p-n memory activation were very low and were significant only in mice initially immunized with p-hlamp-n (fig. ) . following the recall p-n injection, increased ifn-g and il- responses were observed on day , reaching an apparent maximum on days to . the strongest memory t-cell responses in all assays were those of mice initially immunized with p-hlamp-n, with strong ifn-g and il- responses to both peptides. n-gst plus freund's adjuvant elicited strong il- responses but minimal ifn-g responses, as seen for the primary response. the responses of mice initially immunized with p-n were uniformly lower than those seen for the lamp chimera and the il- responses to n-gst. however, the ifn-g responses were greater for p-n than for n-gst. memory ctl responses were strongest in p-hlamp-n-immunized mice, with a much weaker response to p-n dna immunization and little if any after immunization with n-gst with adjuvant (fig. ) . memory antibody responses followed the pattern of the primary response, with a much stronger igg titer of about : , on days and following activation of the n-gst-immunized mice as compared to the responses of about : by the p-hlamp-n-immunized mice. there was relatively little response ( : ) by mice initially immunized with p-n. peptide epitope activation of t cells relies on receptor (tcr) recognition of complexes of antigen peptide sequences bound to the nine amino acids of the binding clefts of mhc class i and ii molecules (benacerraf, ) . it is postulated that the kinetic stability of the mhc-peptide complexes is a key parameter that dictates immunodominance (lazarski et al., ) . in the further analysis of the t-cell epitope specificity of n, the predicted h d class i (h -k d , -l d , -d d ) and class ii (i-e d , i-a d ) binding motifs of the sars n overlapping peptides were examined by a murine immunoinformatics system based on quantitative matrices, pred balb/c (zhang et al., ) . each of the nine-amino-acid sequences of n was analyzed for binding to the core sequence of the h d alleles. all of the nine ifn-g elispot-positive peptides scored for a high probability of binding to class i and/or class ii alleles (table ) , and, in ongoing experiments, all of a new set of peptides encompassing n - elicit both cd -and cd specific responses (data not shown). in the present study, we have demonstrated that the dominant t-cell immune responses of mice immunized with the sars cov n protein plus adjuvant or with dna encoding two different cellular trafficking forms of n are directed to the same peptide epitopes of all three immunogens. this finding points to the existence of versatile antigen-processing mechanisms that are not defined by the initial site of delivery of the protein, whether exogenous or endogenous, or the state of the innate responses to the immunogens. the data are consistent with multiple in vivo routes for antigen access to proteolytic processing compartments that produce the same or comparable mhc i and ii peptide epitopes. this versatility would not all elispot responses were to a group of dominant peptides, shown by peptide number, amino acid region in n, sequence, and number of ifn-g-positive cells of mice immunized with the three immunogens. a cluster (n - ) of peptides of to aa, overlapping by to , contains of the strongest dominant epitopes. preclude quantitative variation in epitope presentation as a result of differences in the levels of antigen, the degree of processing, or access to cellular compartments for antigen presentation. for example, in this study, the greater response to the lamp-n chimera could be related to the quantitatively greater delivery of n to the mhc class ii compartment of apcs. there can also be major differences in the repertoire or functionality of t cells responding to any given epitope, such as the differences in the ifn-g and il- responses of mice immunized with dna or with n-gst plus cfa. others have reported similar findings that the same ctl-defined epitope peptide is generated by an exogenous protein and by plasmid dna-encoded antigens (schirmbeck et al., ) and that the efficiency of mhc class i cross-presentation of peptides derived from exogenous antigens is comparable to that for presentation by the classical mhc class ii pathway (storni and bachmann, ) . additionally, ria et al. ( ) have compared the response to hen egg-white lysozyme (hel) and its reduced and carboxymethylated form (rcm-hel) and have found that rcm-hel induces an in vivo t-cell response that is focused on the same immunodominant determinant that characterizes the response to native hel but that the rcm-hel response is skewed toward the th pathway. no difference in the efficiency of processing was observed between hel and rcm-hel. we conclude that the makeup of the t-cell epitopes of an antigen is primarily determined by the amino acid composition of the protein in the context of the peptide binding sites of mhc molecules, and not by the mechanism of delivery of the protein. these findings support the usefulness of novel immunogens, such as antigen chimeras and dna vaccines encoding antigens with modified cell trafficking signals, as vaccine candidates, despite their differences from normal pathogen proteins in terms of antigen structure, delivery, and processing mechanisms. the differences in the results we obtained for the three antigen preparations were largely related to the character and magnitude of the immune responses. mice immunized with exogenous n protein plus freund's adjuvant responded with a greatly enhanced antibody response and a high ratio of t-cell il- to ifn-g responses to the antigen peptides. in contrast, the same peptides elicited a balanced ifn-g and il- response and lower antibody responses in mice immunized with dna vaccines. freund's adjuvant has been associated with the preferential induction of t-cell th responses (shibaki and katz, ; yip et al., ; billiau and matthys, ) , and different t-cell cytokine responses to the same epitope (varga et al., ) have been attributed to variable expression patterns of single cells and populations that reflect transient rather than heritable differences in the expression profile (kelso and groves, ; kelso et al., ) . another difference between the vaccines was the comparatively poor memory response to the n-gst protein and p-n dna construct as compared to the p-hlamp-n chimera construct. the limited efficacy of the conventional p-n dna construct is consistent with many previous studies of our and other laboratories (martins et al., ; maecker et al., ; oehen et al., ; rush et al., ; marques et al., ; barros de arruda et al., ) . although it is likely that there are multiple factors involved in memory t-cell differentiation (masopust et al., ) , we attribute the enhanced t-cell and memory responses to p-lamp-n, at least in part, to the increased efficiency of trafficking of the lamp-n chimera to the mhc ii compartments of apcs. the localization of the dominant t-cell responses to a cluster of overlapping peptides epitopes within n - is similar to findings from other ongoing studies of hiv- gag and yellow fever virus envelope proteins where the major t-cell responses fig. . primary t-cell ifn-g elispot responses of (a) p-hlamp-n, (b) p-n, and (c) n-gst-immunized mice. as described in materials and methods, mice were immunized five times with the dna immunogens and three times with n-gst plus adjuvant, and the mice were sacrificed for elispot assay of antigenspecific splenocyte ifn-g secretion days after the final immunization. the splenocytes were stimulated with recombinant n-his protein and with n peptides from panels of overlapping peptides spanning the entire molecule, with mice immunized by hlamp plasmid as the negative control. all significant t-cell peptide-specific ifn-g responses elicited by each immunogen were stimulated by the same peptides, with the strongest responses to p-hlamp-n and the weakest to n-gst. also involve clustered epitopes (unpublished observations). regions of overlapping t-cell epitopes are also reported in studies of hiv- proteins (shankar et al., ; surman et al., ; brown et al., ; berzofsky et al., ) , the outer membrane protein of chlamydia trachomatis (kim and demars, ) , and in predictions of hla t-cell epitopes (srinivasan et al., ; zhang et al., ) . clustered t-cell epitopes appear to be a common, perhaps ubiquitous occurrence and, as such, may facilitate the development of epitope-based vaccines if a single or few hot spots elicit all of the required tcell functions. the basis for such hot spots is unknown but is likely to be related in some measure to the binding affinities of the peptide sequences to mhc molecules as well as to other functional parameters that affect mhc -peptide binding to tcell receptors during t-cell activation (chen et al., ) . analysis of the nonameric mhc binding motifs of sars n by a newly developed pred balb/c computational system for predicting the probability of binding of each nine-amino-acid sequence of an antigen to the h d class i (h -k d , -l d , -d d ) and class ii (i-e d , i-a d ) binding motifs (zhang et al., ) showed that each of the sars n peptides that elicited elispot-positive ifn-g t-cell responses contained highprobability binding motifs for mhc class i and/or ii alleles. there may also be protein structural effects in the selection of epitope hot spots. the three-dimensional structure of the n amino-terminal domain, n - , of a truncated model lacking n - and n - has been determined by nmr spectroscopy (huang et al., b) . two of the elispot-positive sequences, n - and n - , were present on the exposed surface of this structure. the n - t-cell hot spot sequence also contains the n - sequence reported to comprise a b cell epitope (huang et al., a ). an association between tand b-cell epitopes has also been found for the hiv envelope; these are seen to be present in exposed loops or strands when mapped onto the crystal structure of the protein (brown et al., ; del porto et al., ; shirai et al., ) . mouse b lymphoma and monkey kidney cell lines (lb . and cos- ) were obtained from atcc (rockville, md). human melanoma cell line (mel-juso) was obtained from german collection of microorganisms and cell cultures (braunschweig germany). insect cell lines sf and high were obtained from invitrogen. all the cell lines were maintained according to the supplier's protocols. the sars cov nucleocapsid (n) cdna was prepared from a singapore clinical isolate and purified total rna. the bp n gene sequence was obtained by pcr with primers v-cggctagcatgtctgataatggaccccaatc- v and v-gcggtaccttatgcctgagttgaatcagcag- v, having nhei and kpni sites. the pcr product was cloned into the p expression vector containing aav-itr sequences flanking the expression elements and a cmv promoter (kessler et al., ) to make the wild-type plasmid p-n. this n-sequence (without the stop codon) was amplified, and xholi and ecori sites were introduced by using primers v-cgctcgagatgtctga-taatggaccccaatc- v and v-cggaattctgcct-gagttgaatcagcagaa- v. the p-hlamp-n chimera vector was constructed by substitution of the hiv-gag sequence into the p-hlamp-gag vector . due to the presence of internal nhei or xhoi sites in the n sequence, the p-n and p-hlamp-n plasmids were constructed by sequential cloning of the two fragments of the n gene. sars-cov n was amplified from the rt-pcr sequence using forward primer v-acgtcagaattcatgtctga-taatggaccccaa with an ecori site and reverse primer v-agtttagcggccgctgcctgagttgaatcagcag with a noti site. the cdna sequence was cloned into the ecori and noti sites of the pgex p- (invitrogen, a kind gift from dr. roland of the institute of molecular and cell biology, singapore) and pfastbac ht-a (invitrogen, carlsbad, ca) vectors. all clones were verified by sequencing. the n-specific h d binding motifs were analyzed by the pred balb/c system. each of the ifn-g elispot peptides contained multiple nine amino acids binding motifs that were within the top % of all binding scores to the five h d alleles. all of the ifn-g elispot peptides except n - scored were predicted to contain class i binding motifs. similarly, class ii binding motifs were predicted in all except n - and n - . expression of the n protein from the vaccine plasmids p-n and p-lamp-n in the cos- cell line was analyzed by western blot analysis. cells were transfected with the sars plasmids using polyfect transfection reagent (qiagen gmbh). cell culture and polyacrylamide gel electrophoresis were carried out as described . the n protein was detected by incubation with polyclonal serum from mice immunized with n-gst protein followed by horseradishperoxidase-conjugated goat anti-mouse igg monoclonal antibody (pierce biotechnology). binding of secondary antibody was visualized with the supersignal west pico chemiluminescent kit or dab (pierce biotechnology). recombinant n protein was generated by use of two dna plasmid expression systems, n-gst in e. coli for use as an antigen for mouse immunization and n-his in a baculovirus system as an antigen for elisa and elispot assays. recombinant n protein was expressed as a gst fusion in top e. coli cells (invitrogen) after iptg induction. the protein was purified by affinity chromatography with glutathione s -sepharose beads (amersham pharmacia biotech ab), and the preparation was validated by western blot analysis using mouse monoclonal anti-gst (santa cruz biotechnology) and rabbit polyclonal anti-n (imgenex) antibodies. the bac to bac baculovirus expression system version c (invitrogen) was used for n protein expression in insect cells. pfastbacht-a-n was constructed by cloning the ecori-and noti-digested sars cov n fragment into the pfastbacht-a vector. n protein was purified from lysates of infected high cells using ni-nta agarose beads (invitrogen), and the protein preparation was verified by western blot analysis with mouse monoclonal penta-his-antibody (qiagen, gmbh) and anti-n (imgenex) antibodies. purification of the culture products of each expression system yielded fractions enriched for the -kda n-gst and -kda n-his proteins. the cellular localization of the expressed viral proteins in transfected mouse b lymphoma cells was studied by confocal and immunoelectron microscopy and confirmed by confocal microscopy of transfected human mel-juso cells. cells were transfected with the sars cov plasmids (p-hlamp-n and p-n) using polyfect (qiagen, gmbh). the expression of the recombinant viral proteins was visualized by the use of rabbit polyclonal anti-n (imgenex) or mouse monoclonal anti-human lamp antibody h a (obtained from supernatant of hybridoma cultures prepared in our laboratory). localization of the transgenic proteins in mhc-class-ii-containing compartments and lysosomes of b cells was determined by the use of anti-mouse mhc class ii antibody m / . . (bd biosciences) and anti-mouse lamp (mlamp ) id b antibody (prepared in our laboratory). localization of the viral proteins in the mhc class ii-containing compartments of mel-juso cells was analyzed with anti-hla-dr, dp, and dq antibodies (bd pharmingen) and rabbit anti-n antibody (img- ; imgenex). three immunization protocols were carried out: ( ) three groups (p-hlamp control, p-n, and p-hlamp-n immunogens) of female ( to weeks old) balb/c mice per group were immunized subcutaneously at the base of the tail with ag of specified endotoxin-free dna plasmid diluted in pbs on days , , , , and . blood samples were collected by phlebotomy from the tail vein on days , , , , , , and , and the animals were sacrificed on day , days after the last immunization. ( ) another group was immunized intraperitoneally with ag of n-gst protein emulsified with cfa followed by two booster immunizations weeks apart with n-gst protein emulsified with ifa. blood samples were collected as outlined above on days , , and , and the animals were sacrificed on day , days after the last immunization. protocols and were carried out twice with five mice per group each time. ( ) an additional protocol was carried out for analysis of memory responses: four groups of mice each (p-hlamp control, p-n, p-hlamp-n, and n-gst immunogens) were immunized as outlined above, but with only three injections over weeks. at week , after an interval of weeks, the memory immune response was activated by a single injection of the p-n construct with p-hlamp as the negative control. three mice of each group were sacrificed day before the final p-n injection and at weekly intervals for weeks thereafter. these experiments were performed as approved by the johns hopkins university animal care and use committee under protocol number mo m . anti-sars cov n igg antibodies in the serum from each mouse were assayed by elisa for seroconversion at a dilution of : , and the total igg titers of -fold serial dilutions, starting from : , were determined by standard quantitative elisa. in brief, elisa plates ( -well, maxisorb f ; nunc inc.) were coated overnight at -c with ag/ml recombinant n-his protein in pbs followed by blocking with elisa buffer (pbs containing . % milk and . % tween ). after washing, appropriately diluted mouse serum was added and incubated for h at room temperature. total igg was detected with the mouse extravidin alkaline phosphatase staining kit (sigma). the response of antigen-specific t cells from immunized mice was measured by the use of ifn-g and il- elispot sets (bd pharmingen) according to the manufacturer's protocol. splenocytes were stimulated with ag/ml of recombinant n-his (baculovirus-produced) or ag/ml of the overlapping sars cov synthetic peptides from three sources (nih aids research and reference reagent program, rockville, md; synpep corp, dublin, ca; the johns hopkins biochemistry core facility). negative control stimulation produced less than five spots per well in > % of experiments. the average number of spots in the negative control wells was . t . . each experiment was repeated at least twice on different groups of mice from two immunizations. cytotoxic t-lymphocyte assay ctl assays were performed using the cytotox nonradioactive cytotoxicity assay kit (promega corporation, usa). the pooled splenocytes (from two mice in the n-gst group and three mice in each of the other groups) obtained at each time point (days , , , and ) after memory recall with pn were stimulated in vitro with each of the two peptides, n - and n - , at a final concentration of ag/ml for days. sp /o target cells were pulsed overnight separately with ag/ ml of each of the two peptides, washed two times, resuspended in rpmi supplemented with % fcs, and seeded onto a well u-bottom plate (  cells/well in al of culture medium) with an equal volume of pooled, in-vitro-stimulated splenocytes from recalled mice (effector cells) at different effector/target ratios, : , : , and : . all incubations were done in quadruplicate. the negative control contained an equal concentration of unrelated peptide, and controls for effector cell spontaneous release, target spontaneous release, and target maximum release were included in the assays. all the values were computed as the mean t standard deviations of the four replicate assays after subtracting the average of the values obtained for the negative controls. the use of overlapping peptides for antiviral cd and cd t-cell responses has been described (maecker et al., ; draenert et al., ) . balb/c mouse h d t-cell binding motifs of the overlapping n peptides were predicted by use of the pred balb/c class i (h -k d , -l d , -d d ) and class ii (i-e d , -a d ) models (zhang et al., ) . the pred balb/c system scores the binding index of all immunogen nine-amino-acid sequences to h d molecules, based on quantitative matrices. cellular mechanisms governing crosspresentation of exogenous antigens west nile premembrane-envelope genetic vaccine encoded as a chimera containing the transmembrane and cytoplasmic domains of a lysosome associated membrane protein: increased cellular concentration of the transgene product, targeting to the mhc ii compartment and enhanced neutralizing antibody response dna vaccine encoding human immunodeficiency virus- gag, targeted to the major histocompatibility complex ii compartment by lysosomal-associated membrane protein, elicits enhanced long-term memory response a hypothesis to relate the specificity of t lymphocytes and the activity of i region-specific ir genes in macrophages and b lymphocytes construction of peptides encompassing multideterminant clusters of human immunodeficiency virus envelope to induce in vitro t cell responses in mice and humans of multiple mhc types modes of action of freund's adjuvants in experimental models of autoimmune diseases efficient targeting of protein antigen to the dendritic cell receptor dec- in the steady state leads to antigen presentation on major histocompatibility complex class i products and peripheral cd + t cell tolerance targeting antigen in mature dendritic cells for simultaneous stimulation of cd + and cd + t cells cross-presentation: dendritic cells and macrophages bite off more than they can chew class ii-restricted presentation of an endogenously derived immunodominant t-cell determinant of hen egg lysozyme clustering of th cell epitopes on exposed regions of hiv envelope despite defects in antibody activity class ii mhc peptide loading by the professionals endolysosomal processing of exogenous antigen into major histocompatibility complex class i-binding peptides identification of two lysosomal membrane glycoproteins structural and kinetic basis for heightened immunogenicity of t cell vaccines inverted terminal repeat sequences of adeno-associated virus enhance the antibody and cd (+) responses to a hiv- p gag/lamp dna vaccine chimera immunological memory in humans high prevalence of hypervariable region -specific and-cross-reactive cd (+) t cells in hcv-infected individuals responsive to ifn-alpha treatment comparison of overlapping peptide sets for detection of antiviral cd and cd t cell responses the role of endosomes and lysosomes in mhc class ii functioning the motif tyr -x -x-hydrophobic residue mediates lysosomal membrane targeting of lysosome-associated membrane protein er-phagosome fusion defines an mhc class i crosspresentation compartment in dendritic cells phagosomes are competent organelles for antigen cross-presentation evaluation of antibody responses against sars coronaviral nucleocapsid or spike proteins by immunoblotting or elisa structure of the n-terminal rna-binding domain of the sars cov nucleocapsid protein exogenous antigens are processed through the endoplasmic reticulum-associated degradation (erad) in cross-presentation by dendritic cells a single peripheral cd + t cell can give rise to progeny expressing type and/or type cytokine genes and can retain its multipotentiality through many cell divisions the genes for perforin, granzymes a -c and ifn-gamma are differentially expressed in single cd (+) t cells during primary activation gene delivery to skeletal muscle results in sustained expression and systemic delivery of a therapeutic protein mhc class i antigen processing regulated by cytosolic proteolysis-short cuts that alter peptide generation epitope clusters in the major outer membrane protein of chlamydia trachomatis generation and characterization of dna vaccines targeting the nucleocapsid protein of severe acute respiratory syndrome coronavirus characterization of mhc class ii compartments by immunoelectron microscopy major histocompatibility complex class ii compartments in human and mouse b lymphoblasts represent conventional endocytic compartments generation of major histocompatibility complex class i antigens: functional interplay between proteasomes and tppii detection of severe acute respiratory syndrome (sars) coronavirus nucleocapsid protein in sars patients by enzyme-linked immunosorbent assay human b lymphoblastoid cells contain distinct patterns of cathepsin activity in endocytic compartments and regulate mhc class ii transport in a cathepsin s-independent manner the kinetic stability of mhc class ii: peptide complexes is a key parameter that dictates immunodominance cytoplasmic processing is a prerequisite for presentation of an endogenous antigen by major histocompatibility complex class ii proteins identification of an epitope of sars-coronavirus nucleocapsid protein dengue prem-e/lamp chimera targeted to the mhc class ii compartment elicits long-lasting neutralizing antibodies cytotoxic t cell responses to dna vaccination: dependence on antigen presentation via class ii mhc use of overlapping peptide mixtures as antigens for cytokine flow cytometry hiv- p gag encoded in the lysosome-associated membrane protein- as a dna plasmid vaccine chimera is highly expressed, traffics to the major histocompatibility class ii compartment, and elicits enhanced immune responses dna vaccination against persistent viral infection the role of programming in memory t-cell development processing of an endogenous protein can generate mhc class iirestricted t cell determinants distinct from those derived from exogenous antigen -d structure of multilaminar lysosomes in antigen presenting cells reveals trapping of mhc ii on the internal membranes induction of primary carcinoembryonic antigen (cea)-specific cytotoxic t lymphocytes in vitro using human dendritic cells transfected with rna major histocompatibility complex class ii-restricted presentation of a cytosolic antigen by autophagy multiple antigen-specific processing pathways for activating naive cd + t cells in vivo antiviral protection after dna vaccination is short lived and not enhanced by cpg dna endogenous mhc class ii processing of a viral nuclear antigen after autophagy mechanisms of mhc class i-restricted antigen processing synergistic neutralizing antibody response to a dengue virus type dna vaccine by incorporation of lysosome-associated membrane protein sequences and use of plasmid expressing gm-csf alternative pathways for processing exogenous and endogenous antigens that can generate peptides for mhc class i-restricted presentation major histocompatibility complex class i presentation of peptides derived from soluble exogenous antigen by a subset of cells engaged in phagocytosis selection of similar naive t cell repertoires but induction of distinct t cell responses by native and modified antigen towards a cellular definition of cd + t-cell memory: the role of cd + t-cell help in cd + t-cell responses lysosome-associated membrane protein- -mediated targeting of the hiv- envelope protein to an endosomal/lysosomal compartment enhances its presentation to mhc class ii-restricted t cells the enhanced immune response to the hiv gp /lamp chimeric gene product targeted to the lysosome membrane protein trafficking pathway efficient priming of cd + and cd + t cells by dna vaccination depends on appropriate targeting of sufficient levels of immunologically relevant antigen to appropriate processing pathways similar as well as distinct mhc class i-binding peptides are generated by exogenous and endogenous processing of hepatitis b virus surface antigen three regions of hiv- gp contain clusters of immunodominant ctl epitopes induction of skewed th /th t-cell differentiation via subcutaneous immunization with freund's adjuvant t cell recognition of hypervariable region- from hepatitis c virus envelope protein with multiple class ii mhc molecules in mice and humans: preferential help for induction of antibodies to the hypervariable region prediction of class i t-cell epitopes: evidence of presence of immunological hot spots inside antigens loading of mhc class i and ii presentation pathways by exogenous antigens: a quantitative in vivo comparison enhanced induction of telomerase-specific cd (+) t cells using dendritic cells transfected with rna encoding a chimeric gene product cd + t cells are required for the maintenance, not programming, of memory cd + t cells after acute infection localization of cd + t cell epitope hotspots to exposed strands of hiv envelope glycoprotein suggests structural influences on antigen processing a cytosolic pathway for mhc class ii-restricted antigen processing that is proteasome and tap dependent the attachment (g) glycoprotein of respiratory syncytial virus contains a single immunodominant epitope that elicits both th and th cd + t cell responses proteases involved in mhc class ii antigen presentation cutting edge: cd + t cell help can be essential for primary cd + t cell responses in vivo immunological memory to viral infections engineering an intracellular pathway for major histocompatibility complex class ii presentation of antigens a dna vaccine induces sars coronavirus neutralization and protective immunity in mice cut and trim: generating mhc class i peptide ligands adjuvant-guided type- and type- immunity: infectious/noninfectious dichotomy defines the class of response pred balb/c : a system for prediction of peptide binding to the h d molecules, a haplotype of the balb/c mouse immune responses against sars-coronavirus nucleocapsid protein induced by dna vaccine induction of sars-nucleoprotein-specific immune response by use of dna vaccine this research has been funded in part by the national institute of allergy and infectious diseases, national institutes of health, department of health and human services, contract no. hhsn c, and by contracts from the agency for science, technology and research, singapore to the division of biomedical sciences, johns hopkins in singapore, and institute for infocomm research, singapore. we thank a/p ng mah lee and a/p charanjit kaur (national university of singapore) for critical analysis of the iem images and j.l. koh (institute for infocomm research) for computational analysis of the balb/c proteome. the support of the nih aids research and reference reagent program in providing the sars n peptides is gratefully acknowledged. key: cord- -bu pzbnv authors: miller, craig s. title: pleiotropic mechanisms of virus survival and persistence date: - - journal: oral surg oral med oral pathol oral radiol endod doi: . /j.tripleo. . . sha: doc_id: cord_uid: bu pzbnv viruses are enormously efficient infectious agents that have been implicated in causing human disease for centuries. transmission of these pathogens continues to be from one life form to another in the form of isolated cases, epidemics, and pandemics. each infection requires entry into a susceptible host, replication, and evasion of the immune system. viruses are successful pathogens because they target specific cells for their attack, exploit the cellular machinery, and are efficient in circumventing and/or inhibiting key cellular events required of survival. this article reviews some of the advances that have taken place in human virology in the past years, emphasizing mechanisms that contribute to, and are involved with, virus survival and persistence. the field of virology was barely half a century old in when dr thomas francis wrote articles, ''viruses as agents of disease'' and ''the prevention of virus disease,'' that were published in this journal. early leadership by mayer, ivanovsky, loeffler, frosch, walter reed and others, allowed progress from ''contagium vivum fluidum'' and a simple understanding of the existence and predatory nature of viruses to the characterization of viruses with regard to size, resistance to chemical and physical agents, host and tissue selectivity, and pathogenic and immunologic effects. these investigations made it clear that viruses were a very diverse group of pathogens. however, our knowledge of viral-cell interactions and the effect of viruses on the immune system was rudimentary. we held the understanding that a recovered individual is not susceptible to reinfection with the same virus, and that serum contained components that when mixed with virus and injected into a susceptible animal that animal was protected. these basic concepts served as the basis for classic studies of active and passive immunization. but several important advances that occurred between and jump-started the field of modern virology. these included the development of cultures of single animal cells, , watson and crick's identification of dna and the genetic code, the development of optimal medium for growing cells, and the development of the viral plaque assay. by the early s, max theiler and jonas salk (killed virus) had developed vaccines for yellow fever and polio, respectively, and through the benefits of growing the viruses in cell culture, shortly thereafter sabin developed the oral (live attenuated virus) vaccine. introduction of these vaccines into the human masses remains to this day one of the greatest accomplishments of preventive medicine. in the s, the transition from basic virology to molecular biology began. viruses and components of viral infections were analyzed using gel electrophoresis, protein-antibody interactions, and biochemical assays to answer basic biologic questions. as a result, greater knowledge of virus replication, viral and cellular receptors, and immunologic interactions was achieved. specifically, research during this time led to an understanding of the regulation of gene expression including transcription factors, enhancer elements, promoters, aspects of rna polymerase, and reverse transcriptase, as well as the discovery of proto-oncogenes and tumor suppressor proteins. scientists tagged viruses to identify intracellular locations of viral proteins, understand nuclear and cytoplasmic shuttling, and map neural circuitry. complete genomic sequences of viruses have been recorded and entered into public databases. the benefits of databases such as fasta and blast have led to searches in homology between motifs characteristic for specific gene products and the identification of novel viral genes and their functions. our knowledge also has advanced from the use of positive and negative selection procedures. positive selection being the method whereby genomic fragments or single candidate genes are expressed in a suitable cell system and tested for functionality (ie, infection phenotype). in contrast, negative selection is based on the construction of viral mutants that lack specific genes and the implementation of studies that identify a change in phenotype when the viral mutant infects a particular this work was supported in part by nih grant de . cell or animal. use of these techniques has led to the identification of virulence factors, novel mechanisms of regulation of cell surface receptors, and signal transduction pathways. within the last decade, the emerging fields of genomics and proteomics have allowed for the functional analysis of a large number of transcripts and protein sequences that are expressed during viral infection that provide new clues as to the regulation of acute, chronic, and persistent viral infections, as well as reactivation and malignant transformation. excitingly, the last decade has demonstrated that scientists have the knowledge and skill to harness unique features of viruses (eg, adenoviruses, retroviruses, herpesviruses) in implementing gene therapy and targeting processes important in chronic disease and cancer. however, mastery of this field remains to be seen. despite the exponential growth in virology during the last years, mankind still suffers from transmission and disease when humans serve as hosts to viruses. moreover, the outcomes are often severe when humans serve as novel hosts to emerging virus infection (eg, avian flu virus, ebola virus, equine hemorrhagic fever viruses, hanta virus, human immunodeficiency virus (hiv), hendra virus, nipah virus, sudden acute respiratory syndrome (sars) coronavirus, and west nile virus). of great importance is the fact that the oral cavity continues to be the source of transmission of many viruses, the site of replication and asymptomatic shedding of viruses, and a site where persistent viral infections exist, the latter being a prerequisite for virally induced malignant transformation. clearly, the field of virology has grown to the extent that a ''state-of-the-art'' paper would be exhaustive in length. accordingly, this review focuses on specific viral cell interactions that allow the virus to survive the cellular attack and evade the immune system, establish persistent infections, and cause chronic disease. additional topics will be covered in future reviews. viruses have developed numerous strategies for subverting the host defenses that are launched during infection. the first innate defense encountered is cellular selectivity during the entry process. viral attachment proteins bind to specific cell receptors (proteins, carbohydrates, or glycolipids) and coreceptors. the absence of a specific receptor shields the cell from attack. if this level of defense is foiled, upon binding receptors can sense microbial infection and trigger a multitude of antimicrobial and inflammatory responses. the toll-like receptor (tlr) family which consists of to members are well characterized in their ability to detect bacterial components (ie, lipoproteins and lipoteichoic acids, flagellin) as well as unmethylated cpg motif dna of bacteria and viruses (detected by tlr ), double-stranded rna (detected by tlr ) and single-stranded viral rna (detected by tlr ). in particular, tlrs , , , and specialize in viral detection and recognition of nucleic acids within the intracellular compartments which results in defensive signaling. after receptor binding, entry is modulated by either direct fusion with the plasma membrane or clathrin-or nonclathrin-mediated endocytosis. viruses that gain entry uncoat and deliver their genetic material and undergo a permissive or nonpermissive infection. a permissive cell permits virus replication and ultimate lysis of the host cell. in contrast, a nonpermissive cell downregulates virus replication and lytic gene expression resulting in little to no viral progeny. nonpermissive infections can be abortive or persistent, and persistent infections can be active or latent. latent infections are characterized by silencing of gene transcription, intermittent reactivation, or rarely oncogenic transformation. at the onset of infection, for a virus to survive within a cell, the virus must balance its own growth with death of the host and circumvention of the immune response. strategies for survival involve regulating apoptosis, inhibiting interferon production, modulating the major histocompatibility complex (mhc) class i function that ultimately affects the cytotoxic lymphocyte (ctl) and natural killer (nk) response, and limiting cytokine and chemokine production/function. long-term survival (ie, latency) requires downregulation of lytic gene expression, inhibition of apoptosis, and minimizing the inflammatory response. apoptosis, or programmed cell death, is a highly regulated and conserved series of sequential cellular events that results from receptor-or mitochondrialmediated pathways in response to a variety of stimuli, including viral infection and the appearance of doublestranded rna. the process is regulated (fig ) by a family of aspartate-specific cysteinyl proteases, or caspases, that converge at a number of downstream points resulting in proteolytic cleavage and enzyme activation. caspases are segregated into distinct subfamilies. the ''apoptotic'' caspases ( , , , , , , and ) are involved in the cascade that results in protease production, chromatin condensation, and cellular degradation. the ''inflammatory'' caspases ( , , and ) provide a second round of defense against viral infection. the inflammatory caspases are involved in the proteolytic maturation of key cytokines (ie, interleukin (il)- b and il- ). cytokine il- , also known as interferon (ifn)-inducing factor, directs the production of ifn-c. in turn, ifn-c induces expression of proteolytic active subunits that lead to proteolysis and antigenic processing by tap proteins. tap proteins are critical for displaying viral antigens on the cell surface (see cellular immunity, below). clearly, apoptosis is an important target of virus defense, because early destruction of an infected cell could greatly reduce replication and the number of viral progeny produced. interestingly, viruses have evolved several methods for suppressing or delaying apoptosis as well as encoding proteins that function as inducers of apoptosis. this apparent yin-yang relationship with apoptosis is important to prolong the life of the cell yet facilitate the release and spread of viral progeny at the appropriate time. , viruses regulate apoptosis by several mechanisms including the targeting of the tumor suppressor gene product p , the fas death receptor, and by producing caspase inhibitors and viral bcl- homologs. adenovirus, for example, encodes several gene products that influence apoptosis. the e a gene product stabilizes p and induces p -dependent apoptosis. , in contrast, the adenovirus e gene product promotes degradation of fas, and the adenovirus e b proteins antagonize p function. viral homologs of bcl- , an apoptosis suppressor that binds with bax, are produced by adenovirus, epstein-barr virus (ebv, bhrf protein), and other viruses. , there are several classes of caspase inhibitors encoded by viruses. these include the serine proteinase inhibitors (serpins: crma/spi- ), viral inhibitors of apoptosis (viaps), p , and inhibitors of procaspase protease (also known as flice). crma and p block caspase , previously termed il- beconverting enzyme (ice). caspase functions primarily as an activator of proinflammatory cytokines, but also has apoptosis-inducing ability in select mammalian cells, such as neurons. the viaps appear to inhibit bax-mediated apoptosis in human cells rather than directly inhibiting caspases. several human herpesviruses encode fliceinhibitory proteins (flips) that block trail-mediated cell death by interfering with procaspase protease (flice) activation. for example, the b-herpesviruses (cytomegalovirus (cmv)) encode a viral inhibitor of caspase activation (vica) which inhibits caspase (flice) activation, and c-herpesviruses encode vflips (eg, k ) which inhibit activation of caspases by molecular mimicry. cmv also encodes a viral mitochondrial inhibitor of apoptosis (vmia) (encoded by the u l gene) which inhibits activation of mitochondrial pores in a manner similar to members of the antiapoptotic bcl family. , the alpha herpesvirus hsv- encodes several antiapoptotic gene products (ie, icp , icp , c . , u s , gj) [ ] [ ] [ ] [ ] [ ] that modulate apoptosis at several levels, including antagonism of double-stranded rna-activated protein kinase (pkr), a downstream induction molecule of the interferon signaling pathway , of note, all c-herpesviruses express viral homologues of cellular antiapoptotic genes, including or bcl- homologues. interferon, discovered in the late s when scientists observed that virus-infected cells secrete a factor that mediates the transfer of a viral-resistant state, is a family of regulatory glycoprotein cytokines that modulate both innate and adaptive antimicrobial immunity. they are products of an infected cell genome and one of the key factors in the host response against viral infection. ifns serve as an early defense system that precedes the onset of the immune response and are triggered by envelope glycoproteins, cpg dna, or double-stranded rna. in recombinant formulations, they have been used in medicine and dentistry to combat various viral infections. , human ifns are classified based on the sequence of amino acids into main groups -a, b, and c -and that are less extensively studied (x, j, and s, not discussed further in this review). ifn-a and -b are produced rapidly when viral factors interact with cellular patternrecognition receptors such as tlrs and cytosolic receptors. historically, synthesis of ifn-a has been attributed to macrophages and b cells, and ifn-b has been considered to be produced by fibroblasts. more recently, plasmacytoid dendritic cells have been shown to produce ifn-a preferentially to ifn-b. both ifn-a and -b prevent the replication of viruses by inducing formation of secondary messengers which include ifn regulatory factor (irf) , irf- , irf- , c-jun/atf- , and nf-jb. ifn-c is synthesized by activated t lymphocytes and natural killer (nk) cells following receptor-mediated stimulation or in response to cytokines produced by macrophages or antigen-presenting cells (ie, primarily il- , il- , and ifn-a/b) or by stimulation through t cell receptors (tcrs) or nk cell receptors. it is a powerful activator of mononuclear phagocytes, thus enhancing their ability to destroy intracellular microorganisms and tumor cells. ifns mediate their antiviral action through ifn-stimulated genes (isg), which number in hundreds. ifns also regulate the cell cycle and have antiproliferative effects. viral evasion of ifn occurs by several strategies. in the majority of infections, viruses encode products that antagonize either the ifn signal transduction pathway or cellular proteins induced by ifn that are responsible for inhibiting virus replication (fig ) . adenovirus, ebv, papillomavirus, and members of the paramyxovirinae subfamily encode proteins that inhibit the jak-stat (janus kinaseesignal transducer and activator of transcription) signaling pathways that are required for ifn production. specifically, adenovirus encodes the oncoprotein e a which inhibits the activation of isg factor (isgf ). , paramyxovirinae reduce the effectiveness of the ifn response by targeting stat for degradation or by interference with stat phosphorylation or stability. , kaposi's sarcomaeassociated herpesvirus (kshv) encodes the pleiotropic gene product latency-associated nuclear antigen (lana) that acts downstream of isgf and inhibits p . , in an alternate approach, hpv encodes proteins, e and e , that bind to irf- and irf- , respectively, both of which inhibit the transactivation functions of the bound irf. , viruses also encode proteins that mimic cellular components of the ifn signal transduction pathway, including homologs of the ifn receptors, a viral isrelike promoter element, and viral homolog of irf (virf). for example, poxviruses antagonize ifn signals by encoding soluble ifn receptor homologs. , ebv encodes a viral isre and hhv encodes virf from the k orf that functions as a repressor of transcriptional activation induced by ifn-a, -b, and -c. in addition, several viruses have developed strategies to inhibit ifn-inducible, rna-dependent protein kinase (pkr). pkr, when antagonized, leads to phosphorylation of eif- a which results in inhibition of the ifn-induced antiviral response of the host. adenovirus, herpesviruses, influenza, and sv antagonize pkr by different mechanisms involving degradation of pkr, prevention of pkr activation, and resistance to downstream kinase activation. [ ] [ ] [ ] the mhc class ierestricted t cell response can result in a lethal hit before virus replication. thus, many viruses have developed strategies for interfering with antigen presentation to mhc class i molecules and intracellular trafficking of mhc molecules. viruses target the mhc-i at almost all steps of its trafficking: in the endoplasmic reticulum (er), in the cytoplasm on its way to the surface, and after the mhc reaches the cell surface (fig ) . one key target in the viral defense against the cellular arm of the immune system is attack of the transporter protein associated with antigen processing (tap). tap loads short antigenic peptides to the mhc which stabilize the class i complexes and allows their migration to the cell surface. without the peptide cargo, mhc class i molecules are unstable and dissociate. hsv- and hsv- encode infected cell polypeptide (icp)- , an immediate early gene product, that interacts with the tap protein in the cytosol to prevent peptide binding to tap. human cytomegalovirus (hcmv) encodes u s , a eamino acid glycoprotein, that blocks peptide transport by binding to tap in the endoplasmic reticulum. although efficient at both retaining mhc-i molecules and preventing ctl recognition, hcmv also uses additional viral proteins (u s , u s , u s , and u s ) to evade the immune system. a different approach is taken by ebv. this human c-herpesvirus encodes a glycine-alanine repeat (gar) domain on ebv-encoded nuclear antigen (ebna) that inhibits ubiquitin/proteasome-dependent proteolysis of ebv antigens. thus, processing (ie, degradation) of viral proteins into antigenic peptides is restricted. kshv, a lymphotropic c-herpesvirus, interferes with mhc-i antigen presentation by ubiquitinating the cytosolic domain of the mhc-i. herpesviruses also produce proteins that target mhc class i molecules for degradation in lysosomal compartments and downregulate expression of major histocompatibility complex molecules by shutting off host cell protein synthesis by the gene known as virion host shutoff (vhs, u l ). in contrast, hiv with its simple genome encodes fewer proteins but accomplishes similar immune evasion by pluripotent accessory proteins. for example, nef, of the regulatory proteins encoded by hiv, has multiple functions. in addition to enhancing virion infectivity, nef binds to and inhibits the surface expression of the major mhc-i, downregulates the cell surface expression of cd (the main hiv receptor), and facilitates cd receptor endocytosis. through the function of nef in redirecting the trafficking of immune receptors, infected t lymphocytes are able to hide from the immune system allowing for viral spread. hpv utilizes early proteins (e and e ) to persist undetected within epithelial cells. the early gene product e of the oncogenic strains hpv- and - downregulates mhc-i expression at the transcriptional level by inhibiting the promoters of the mhc-i heavy chain, tap- , and lmp- . e decreases mhc-i expression at the transcriptional level and causes retention of mhc-i in the golgi apparatus. within the golgi, hpv e inactivates the atpase proton pump system. as a result, acidification is blocked, local ph rises, and mhc-i trafficking is perturbed. thus, it is clear that viruses have achieved ingenious methods for interference with mhc-i antigen presentation and inhibition of the cellular immune response. viruses persist in cells because they are able to downregulate key processes that if left unattended would result in cell death. originally attributed in part to the immune response, increasing evidence suggests regulation of key genes plays an important role in the process. specifically, regulation of viral transcription and genomic replication allows for long-term viral stability and survival. many viruses, including those that cause persistent infections and chronic disease (ie, hepatitis c virus, hepatitis b virus, hiv, human herpesviruses, hpv, and jc virus), are successful because of their cell tropism and ability to autoregulate their replication efficiently within specific cells. common features of autoregulation include sensors to the external environment, negative feedback loops, transcriptional enhancers specific for cells that host the persistent infection, and transcriptional silencers. in some cases autoregulation results in steady-state levels of virus replication; in other infections, the virus enters latency only to reactivate intermittently. the importance of autoregulation is apparent from both in vivo and in vitro studies. for example, during lentivirus (hiv, simian immunodeficiency virus, and feline immunodeficiency virus) infection, viremia peaks early after infection then declines to a steady-state level. the effect is not altered by the presence of steroidinduced immunosuppression, and clearance of infected white blood cells is not associated with an earlier presence of antibody, cytokine response, or cytotoxic lymphocyte activity. in another common clinical example, successful antiviral therapy results in dramatic drops in viremia, often to undetectable levels. however, replication of virus often rebounds rapidly to pretreatment levels upon drug withdrawal, and in vitro studies indicate that cellular and immune functions are not contributory to the observed outcome. even when antiviral therapy achieves a sustained virologic response (ie, absence of viremia months after the end of treatment), highly sensitive assays (ie, polymerase chain reaction) detect residual viral genomes in most patients, indicating the ability of viruses to persist and autoregulate based on their environment. , herpesviruses are well known to establish latency in a variety of cell types, and this family of viruses has the ability to autoregulate. this is illustrated by hsv- and hsv- , which can undergo dichotomous life cycles: a lytic infection in epithelium and a latent infection in neurons. in fact, when neuronal cells are infected with hsv- or hsv- at low multiplicity of infection in vitro, the majority of cells can survive for many days even in the absence of immune cells and without the addition of antiviral drugs to the culture medium. similarly, if the rare neuronal cells supporting replication are eliminated using acycloguanosine in the above mentioned system, over % of the remaining population harbors a quiescent infection for weeks after the antiviral drug is removed, again in the absence of immune cells. , viruses regulate replication of their genome in a complex manner, but achieve the outcome through use of viral sensors, repressors, and effectors. viral sensors ''sense'' perturbations in the viral equilibrium within the cell and signal change at the appropriate time. with many viruses (ie, hiv, hepatitis c virus, hepatitis b virus), envelope proteins play the role of sensors, because envelope proteins can influence virus replication in both a positive and a negative manner. [ ] [ ] [ ] [ ] for hpv, regulation is through cellular factors that bind to the promoters of e and e . for hsv- , transcription of the immediate-early (ie) genes during the lytic infection is regulated by the binding of a tegument protein (vp ) with the cellular protein host cell factor (hcf) and oct- . thus, vp would seem to be a logical choice for a sensor. however, latency can be established in the presence of vp and reactivation occurs without the transactivating domain of vp . thus, downstream factors of vp (eg, icp , icp , or other unknown factors) serve as sensors of the environment and regulate the balance between latency and reactivation. the ''effectors'' (transactivators and replicative enzymes such as rna polymerase) modulate virus replication and are the targets of the sensors. effectors are tightly regulated (ie, repressed at certain times) but dynamically modifiable, typically by proteins bound to critical regions of the genome. these proteins afford protection by limiting changes in conformation of, or enzymatic action on, the restricted gene. histones are the most notable guardians of the effectors. histones permit access of dna to specific activators or repressors, general transcription factors, and rna polymerase by posttranslational modification (acetylation, methylation and phosphorylation) of their amino terminal tails. , for example, hyperacetylation of histones is associated with an ''open chromatin'' conformation and transcriptional activation, whereas hypoacetylation of the histone complex is associated with condensed (hetero-) chromatin and gene silencing. several human herpesviruses - utilize these mechanisms for regulating latency and reactivation. in addition, the active regions of the latent a-herpesvirus genome appear to be segregated from the repressed gene regions by boundary or insulator elements, similar to that found on cellular chromosomes (d bloom, personal communication). these chromatin insulators appear to be able to protect genes in one region from the regulatory influence of adjacent regions through conserved ctcf motifs. herpesviruses also encode proteins such as lana and latency-associated transcripts (lat) that appear to regulate viral transcription during latency. , integration, and the site of integration, into the host chromatin is another mechanism that can regulate viral gene transcription. for example, viruses that integrate (ie, hiv) preferentially select chromosomal sites where high-level transcription of key transactivators is maintained. this is accomplished by viruses preferentially integrating in chromatin regions characterized by an open structure (a hallmark of actively transcribed genes). the process by which this is regulated is not completely clear, but it has been suggested that cellular proteins may interact with integrase, the viral protein that catalyzes the integration reaction, in a manner that is site specific. viral persistence increases the likelihood of chronic infection and replication, but under certain circumstances also contributes to increased risk of oncogenic transformation. this can occur through chromosomal instability and virus integration, and the ability of several specific viral proteins to bind and inactivate p or less frequently prb (fig ) . p is a checkpoint protein that interacts with cdk/cyclin inhibitors and p , p , and p to arrest the cell cycle in the g phase and can send signals for apoptosis through the regulatory proteins bax, bcl- , and c-myc. the clinical importance of p inactivation is exemplified in that persistent hpv infection is associated with an increased risk of developing cervical cancer in young women, and recent findings suggest that the persistence of hpv dna in treated tissue after cancer therapy is highly predictive of local recurrence. in this brief review, examples of mechanisms that contribute to survival and persistence of viruses within their host were presented. emphasis was placed on mechanisms that permit survival of host defenses, evasion of the immune system, and establishment of chronic infections. detailed knowledge of these processes has led to many therapeutic successes. however, additional knowledge is required for us to make strides in eliminating human suffering caused by these intracellular pathogens. it is hoped that years from now when another review may appear in this journal on this topic, we will have a better understanding of how to eliminate persistent viral infections and identify patients at risk for virally induced complications of acute and chronic infections and will have harnessed the power of viruses to undergo selective lytic replication in tumor cells and modulate chronic disease. the origins of virology further studies on the proliferation in vitro of single isolated tissue cells the infection of cells in tissue culture with rous sarcoma virus the structure of dna a plaque assay for foot-and-mouth disease virus and kinetics of virus reproduction 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virus latency insulators: many functions, many mechanisms a lat-associated function reduces productive-cycle gene expression during acute infection of murine sensory neurons with herpes simplex virus type integration site selection by retroviruses viral and host factors in human papillomavirus persistence and progression perspectives in studies of human tumor viruses human tumor suppressor p and dna viruses persistence of human papillomavirus infection as a predictor for recurrence in carcinoma of the cervix after radiotherapy key: cord- -h knm y authors: hussey, séamus; travassos, leonardo h.; jones, nicola l. title: autophagy as an emerging dimension to adaptive and innate immunity date: - - journal: seminars in immunology doi: . /j.smim. . . sha: doc_id: cord_uid: h knm y abstract autophagy is an evolutionary conserved cellular process during which cytoplasmic material is engulfed in double membrane vacuoles that then fuse with lysosomes, ultimately degrading their cargo. emerging evidence, however, now suggests that autophagy can form part of our innate and adaptive immune defense programs. recent studies have identified pattern recognition molecules as mediators of this process and shown that intracellular pathogens can interact with and even manipulate autophagy. recent translational evidence has also implicated autophagy in the pathogenesis of several immune-mediated diseases, including crohn disease. in this review, we present autophagy in the context of its role as an immune system component and effector and speculate on imminent and future research directions in this field. throughout evolution, facets of our cellular biology have been conserved and adapted. one process at the forefront of homeostasis and environmental interaction is autophagy. this complex process in eukaryotic cells involves the trafficking of cellular elements from the cytosol to the lysosome wherein they are degraded and processed. ongoing developments in this field point to an inextricable link between autophagy and the innate and adaptive immune system. in this review, we summarize the pertinent research findings to date and suggest future research directions in this dynamic field, especially with respect to pattern recognition receptor interaction. three sub-types of autophagy have been described-chaperonemediated autophagy, microautophagy and macroautophagy (hereafter called autophagy). the term 'autophagy' was first suggested by de duve over years ago [ ] . lamellar vesicles that encapsulated portions of the cytosol and organelle remnants had been described in early electron microscopy studies as vacuoles and lysosomes, and were speculated to arise from focal cytoplasmic degradation [ ] [ ] [ ] . such vesicles bore the hallmarks of what are now termed 'autophagosomes', the characteristic vacuoles synonymous with autophagy. metabolic manipulation was shown to affect autophagy induction, demonstrating that autophagy was a malleable rather than a static process. the catabolic hormone glucagon and deprivation of amino acids and nutrients were shown to induce autophagy while insulin and certain exogenous amino acids impaired autophagy and proteolysis, defining a role for autophagy in adaptation to cellular stresses [ , [ ] [ ] [ ] [ ] [ ] [ ] [ ] . the complexity of signaling molecules that influence autophagy is an ongoing focus of research and will be discussed later. the stepwise process of autophagosome biogenesis is a cornerstone of autophagy. over thirty governing autophagy genes (atg) and their proteins (atg) have been identified in elegant studies in yeast species [ ] [ ] [ ] . while not all mammalian orthologs have been identified, some have numerous mammalian paralogs with striking similarities in structure and/or function to their yeast counterparts [ , ] . ultra-structural studies of autophagosome membranes have shown that they harbor relatively few transmembrane proteins and are thinner than other cellular membranes e.g. the plasma membrane [ ] . the earliest identifiable structure in the sequence of autophagosome formation is the diskshaped, isolation membrane or phagophore (fig. ) . once formed, this membrane progressively elongates, encircling its cytosolic target, e.g. bacterium, within a portion of the cytosol, eventually sealing to complete the autophagosome. speculation continues whether the foundation template for the isolation membrane originates from the endoplasmic reticulum, golgi, mitochondria, a pre-formed organelle membrane or even de novo [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . the molecular mechanisms that lead to isolation membrane appearance continue to be elucidated in both yeast and mammalian cell systems. two ubiquitin-like conjugation systems are pivotal to autophagosome formation and completion. the first system modifies a core autophagy protein-microtubule-associated protein light chain (lc ). multiple paralogs of atg exist in mammals -lc a, lc b, gate , gabarap -hereafter referred to collectively as lc [ , ] . lc has a diffuse cytosolic distribution. it is cleaved at its c-terminus by the cysteine protease atg and in turn undergoes sequential ubiquitin-like modifications by the e -like enzyme, atg , and the e -like enzyme, atg , to form lc - . the c-terminal carboxyl group of lc -i is ultimately conjugated to the amine of phosphatidylethanolamine, forming lc -ii. this lipidation of lc -i to form lc -ii is notable in that lc -ii is exclusively found on autophagosome membranes. the conjugated yeast ortholog of lc , atg , is known to have membrane tethering properties, which may explain one of its roles in autophagosome formation [ ] . atg also deconjugates lc -ii on the autophagosome membrane, releasing lc , highlighting the plasticity of this process. the multifunctional protein p interacts with both ubiquitinated proteins and lc , whereby it is incorporated into autophagosomes. p accumulates during autophagy inhibition and has been implicated in targeting proteins to autophagosomes, although it is not itself essential for autophagosome formation [ ] . in the second conjugation system, the ubiquitin-like autophagy protein atg is covalently conjugated to atg via its c-terminal glycine, forming the dimeric atg -atg complex, following ubiquitin-like reactions involving atg and atg . the autophagy scaffold protein atg l is then conjugated to atg via its nterminus, forming the atg -atg -atg l complex. the atg l complex self-multimerizes, forming large kda complexes. these are found in the cytosol and on the evolving isolation membrane, and are likely necessary for the ultimate conjugation of lc -i. fujita et al. showed that the atg l complex behaves as an e -like enzyme and targets lc -i to its membrane site of lipid conjugation [ ] . the atg l complexes dissociate from autophagosome membranes as they near completion. other essential groups of autophagy proteins participate in isolation membrane formation. the mammalian autophagy proteins ulk (unc- -like kinase), fip (focal adhesion kinase family interacting protein) and atg were recently identified in a complex which subsequently co-localized at the nascent isolation membrane on autophagy induction, similar to the atg -atg -atg complex in yeast [ ] . the c-terminus of ulk- binds to fip and atg , and ulk- also interacts with lc [ ] . mammalian studies of the trans-membrane protein atg have similarly underscored its essential role early in autophagosome formation. it associates with the trans-golgi network, late endosomes, lc , the rab-gtpase proteins (rab and rab ) and re-distributes following autophagy induction, localizing to the nascent autophagosome [ ] . yeast atg was recently shown to self-multimerise via its c-terminus, facilitating its intra-cellular trafficking, independent of other autophagy proteins. this novel finding suggests a potential further role for such atg complexes in contributing to early isolation membrane formation [ ] . the discovery of the target of rapamycin in yeast (tor) and mammalian cells (mtor) led to significant advances in understanding autophagy regulation, through the family of phosphatidylinositol kinase-related kinases [ ] [ ] [ ] . these signaling networks are involved in broad cellular functions from metabolic responses to growth and proliferation. the key serine/threonine kinase, akt, links the mtor and phosphatidylinositol- kinase (pi k) pathways which are activated by a diverse array of stimuli, including cytokine receptors and toll-like receptors (tlr) [ ] . following receptor activation, class-i pi ks are recruited by receptor adaptor molecules to phosphorylate phosphatidylinositol- , -bisphosphate, which in turn phosphorylates and activates akt [ , ] . the mtor complexes, down-stream positive effectors of akt, integrate multiple cellular signals, including those from growth factors, amino acids and intracellular atp. mtor activation increases cellular anabolic activity and protein translation [ ] [ ] [ ] . autophagy is under negative regulation by activated akt and mtor [ ] . recently, mtor was shown to phosphorylate and therefore inhibit the ulk kinase-complex activity, disrupting autophagosome formation [ , ] . rapamycin inhibition of mtor and amino acid deprivation reversed these effects. mtor may further affect autophagy through its control of autophagy gene transcription [ , ] . the class iii pi k enzyme, vps (vacuolar protein sorting ), solely phosphorylates phosphatidylinositol and is involved in regulating vesicular trafficking, nutrient sensing and autophagy [ , ] . the pharmacological agent -methyadenine ( -ma) inhibits its function in vitro. together with vps (another kinase), beclin- , uvrag (ultraviolet radiation resistance associated gene) and ambra- , vps forms a multiprotein complex that is necessary for early stages of autophagosome biogenesis and can up-regulate autophagy overall [ ] [ ] [ ] . however, its seemingly paradoxical role in signal transduction to the mtor complex following amino acid sensing suggests that its signaling function may depend on the nature of its interacting protein complexes [ , ] . beclin- , a tumor suppressor protein, is itself also involved in modulating autophagy through its interaction with bcl- , an anti-apoptotic protein that inhibits both autophagy and apoptosis. the beclin- /bcl- interaction is an evolutionary conserved phenomenon, the balance of which determines either up-or downregulation of autophagy. silencing or over-expression of bcl- was shown to enhance or suppress starvation-induced autophagy respectively [ ] . these effects were specifically dependent on beclin- /bcl- interaction, suggesting that nutrient sensing affects the equilibrium of the beclin- /bcl- interaction. bcl- dominant interactions with beclin- likely disrupt beclin- /vps complex formation, leading to autophagy suppression, although the mechanism has not been fully elucidated. recently, the toll-like receptor (tlr) signaling molecules myd and trif were shown to modulate the beclin- /bcl- interaction, enhancing their interaction with beclin- to induce autophagy [ ] . a myriad of other signal transduction and effector molecules influence autophagy regulation, directly or indirectly. the akt and jnk pathways have been shown to enhance or reduce expression of lc and beclin- in response to tumor necrosis factor-␣ (tnf-␣) and insulin-like growth factor- respectively [ ] . the mammalian transcription factor, nfb, is a key regulator of gene expression, modulating physiological processes including inflammation, apoptosis and also autophagy. tnf␣-induced nfb activation suppresses autophagy, while nfb suppression enhances starvation-induced autophagy [ , ] . nfb may signal through mtor activation or by affecting enhanced bcl- expression to modulate autophagy. autophagy itself may in turn influence nfb activity since it is involved in degradation of ib kinase, the upstream activator of nfb, through association with the heatshock protein, hsp [ , ] . reactive oxygen species (ros) are highly reactive molecules generated from mitochondrial respiratory activity and the products of oxidase enzymes, including nadph oxidase, and are capable of modulating autophagy [ , ] . atg is redox-regulated via a conserved cysteine residue and, furthermore, starvation-induced autophagy depends on h o signaling [ ] . starvation lead to local h o formation, partly dependent on class iii pi k activity, and anti-oxidant treatment in vitro attenuated autophagy induction. recently, a transgenic mouse model harboring a mutant form of super-oxide dismutase, a key anti-oxidant enzyme, also showed increased autophagic activity due to ros accumulation [ ] . evidence also suggests that autophagic (type ii) cell death may stem from ros accumulation, as seen following in vitro treatments with tnf␣ and lps [ , ] . microbial invasion of the cytosol presents a serious challenge to our innate defenses, including autophagy. while several agents succumb to autophagic destruction (xenophagy), others have evolved mechanisms of autophagy evasion and manipulation. various gram+ and gram− bacteria, viruses and protazoa are known autophagy targets ( table ). the mechanisms by which microbes are selectively sequestered in autophagosomes remain elusive and a combination of host and microbial factors are likely to be necessary. microbial molecular motifs themselves may solicit autophagosome formation. alternatively, the up-regulation of autophagy through activating multiple pattern recognition receptors could culminate in xenophagy or perhaps organelle or compartmental damage may lead to targeting by autophagic machinery. microbial factors may be of equal importance for autophagy activation. for example, group a streptococcus (gas) is sequestered in autophagosomes following escape from its early endosomal compartment into the cytosol [ ] . lysosomal degradation of bacteria-containing autophagosomes ensues, effects not observed in autophagy deficient cells. strains of gas lacking the streptolysin o toxin remain within endosomes and avoid autophagic destruction indicating a role for streptolysin o in induction of autophagy. the gram− human diarrheal agent shigella flexneri is a highly adapted pathogen harboring a type iii secretion system (ttss) for delivery of its effector proteins to host cells. in epithelial cells, wild-type (wt) strains secreting the effector icsb are capable of evading entrapment in autophagosomes, in comparison to mutant strains lacking icsb [ ] . interestingly, icsb did not appear to confer autophagy protection in a subsequent study in murine marrow derived macrophages, suggesting a cell-type specific phenomenon [ ] . the intracellular bacterium burkholderia pseudomallei, also avoids autophagic destruction in murine macrophages through secretion of its ttss-delivered effector protein bopa, which shares some homology with icsb [ ] . salmonella enterica serovar typhimurium resides within salmonella-containing vacuoles following intracellular invasion. salmonella employs its ttss to disrupt these vacuoles, facilitating cytoplasmic entry. autophagy promptly contributes to subsequent restriction of intracellular proliferation by targeting bacteria from damaged vacuoles-effects that were dependent on a functioning ttss and reversed in autophagy deficient cells [ , ] . listeria monocytogenes, a gram+ bacillus, replicates within the host cytoplasm following phagosome escape, evading autophagic destruction [ , ] . the virulence factors listeriolysin o, acta and phospholipase c were recently shown to be of importance in modulating listeria-containing phagosomal compartments, blocking lysosomal degradation and facilitating replication and survival [ , ] . mycobacterium tuberculosis has adapted to survive within host macrophages by interfering with and blocking phagosome fusion with lysosomes [ ] . autophagy up-regulation with rapamycin or ifn-␥ overcame this evasion, and lead to phagosome degradation [ , ] . secreted bacterial toxins are themselves capable of interacting with the autophagy pathway. the non-invasive pathogen, vibrio cholerae, causes a potentially fatal secretory diarrhea. its secreted exotoxin, vcc, induces vacuole formation consistent with autophagy induction [ ] . furthermore, cell viability was adversely affected following autophagy inhibition, suggesting that in this situation, autophagy may defend against cell toxicity. our group has recently reported autophagy induction following infection with helicobacter pylori, which was dependent on the vacuolating cytotoxin, vaca. autophagy limited the stability of intracellular vaca, again suggesting a cytoprotective function of autophagy in response to secreted toxins [ ] . viruses also interact with autophagy. the herpes virus hsv- evades autophagy in part through beclin- inhibition by its neurovirulence protein icp . [ ] . poliovirus manipulates autophagic machinery following cellular infection, as evidenced by a marked reduction in viral release following pharmacological inhibition of autophagy and sirna silencing of key autophagy proteins [ ] . rotavirus has been suggested to harness the autophagic apparatus to facilitate replication. its enterotoxin, nsp , was found to co-localize with lc + structures on immunofluorescence microscopy, and the study authors speculate that nsp may interfere with autophagosome-lysosome fusion, enabling viral recruitment of autophagosomes as replication niches [ ] . the antiviral protein kinase, pkr, participates in viral induced autophagy, functioning upstream of beclin- . it is possible that other viruses which inhibit pkr function, including influenza and ebstein-barr virus, may in turn inhibit autophagy to enhance their own survival [ , ] . the possibility of a cell-type dependent autophagy response to viral infection was suggested by coronavirus studies, wherein mouse hepatitis virus replication was impaired in atg −/− stem cells, but not in atg −/− embryonic fibroblasts or marrow derived macrophages [ ] [ ] [ ] . these diverse examples of autophagy-microbial interactions underpin the conserved primary innate role of autophagy as an anti-microbial, protective mechanism and how certain pathogenic organisms have evolved to recognize and commandeer this process for their own advantage. the innate immune system is responsible for the early detection and destruction of pathogens. this first line of defense relies mostly on a set of receptors called pattern recognition molecules (prm) that sense molecular motifs that are common to a wide range of pathogens, triggering different signaling cascades that culminate with the elimination of pathogens and the initiation of an adaptive response [ , ] . the findings that autophagy can specifically target cytosolic pathogens immediately prompted the investigation of the role of prm in the autophagic detection and elimination of intracellular microbes. the tlrs are transmembrane proteins, mostly located at the cell surface, with a toll-il- receptor (tir) domain facing the cytosol. this domain is able to recruit four different adapter molecules: the myeloid differentiation primary response protein (myd ), the tir domain-containing adaptor protein (tirap, also called myd adaptor-like-mal), the tir domain-containing adaptor-inducing ifn-␤-trif, also called tir-domain-containing adaptor molecule -ticam- ) and the trif-related adaptor molecule (tram or ticam ) [ , ] . as we will see in this section, recent data suggest that induction of autophagy after tlr engagement requires the recruitment of specific adaptors (fig. ) . eissa and colleagues provided the first evidence that tlrs are able to trigger an autophagic response by showing the formation of numerous autophagosomes in response to lps stimulation in the murine macrophage raw . cell line [ ] . furthermore, silencing tlr using rna interference resulted in significant reduction in autophagosomes. the tlr -induced autophagic response was dependent on p , rip and trif-, but not myd . as tlr can use fig. . autophagosome biogenesis. the earliest identifiable structure in the initiation (nucleation) sequence of autophagosome formation is the crescent-shaped isolation membrane or phagophore. key elements include atg , the ulk -fip -atg complex, lc -ii, the atg -atg -atg l complex. once formed, this membrane progressively elongates (elongation), encircling its cytosolic target, e.g. bacterium, within a portion of the cytosol. the membrane tips fuse and eventually seal, forming the autophagosome (completion). the autophagosome may fuse with the endosomal compartment, forming an amphisome, prior to its ultimate maturation step, whereby its outer membrane fuses with the lysosome to form an autolysosome (also termed autophagolysosome). this facilitates degradation, processing and recycling of the contents of the autophagosome. both myd and trif adapter molecules for downstream signaling, the authors proposed that by recruiting both signaling cascades, tlr could promote both a fast phagocytic response (through myd ) and a slower autophagic response (via trif). other tlr family members have also been implicated in the control of autophagy (fig. ) . deretic and colleagues have recently demonstrated that when raw . macrophages were stimulated with a panel of tlr ligands such as pam csk (tlr ), flagellin (tlr ), cpg dna (tlr ), poly (i:c) (tlr ), lps (tlr ) and ssrna (tlr ), the latter three were able to up regulate autophagy [ ] . in contrast to tlr (that recruits only trif) and tlr (that recruits both myd and trif), tlr recruits only myd , suggesting that myd may trigger autophagy after tlr activation. however, tlr activation by cpg dna also activates myd but did not induce autophagy. therefore, a simple analysis of which downstream adaptor protein is recruited by tlrs does not fully explain the induction of autophagy by some pathogen associated molecular patterns (pamps) and not others. the mechanistic explanation is still elusive and seemingly conflicting evidence remains difficult to reconcile. for example, tlr recruitment of myd also leads to the activation nfb, which is thought to inhibit autophagy [ ] . trif-dependent signaling leads to the induction of type i interferon, which was previously shown not to affect autophagy [ , ] . two recent studies have proposed a mechanism by which tlrs might regulate autophagy. kehrl and shi demonstrated that not only trif, but also myd targets beclin and reduces its binding to bcl- , upon stimulation with an array of tlr ligands [ , ] . alternatively, wagner and colleagues observed that tlr activation leads to the activation of mtor, which in turn interacts with the adaptor proteins myd and interferon-regulatory factors (irfs) and , thus controlling the transcription of cytokines such as tnf-␣, il- , il , type i interferons but, surprisingly, not il- ␤ [ ] . these lines of evidence suggest a more elaborate tlr control of autophagy whereby tlr-adapter molecules interact with proteins from the autophagic pathway rather than by simply activating the classic hierarchical signaling cascades described heretofore. in contrast with the general notion that tlr ligands up regulate autophagy, green and colleagues suggested a model in which some tlrs, when engaged by their cognate ligands, usurp the autophagic pathway, recruiting lc to the phagosome membrane instead of forming classic autophagosomes [ ] . however, as pointed out by the authors, it is not possible to exclude the possibility that the lc recruited to phagosomes has its origin in rapidly forming autophagosomes. if confirmed, these data would have a deep impact on the understanding on the role of autophagy in the enhancement of antigen presentation for example. pamp recognition as an autophagy trigger seems to be an evolutionary conserved feature. in drosophila, peptidoglycan-recognition protein (pgrp) family members sense peptidoglycan (pg) from gram-negative bacteria [ ] . one of the pgrp family members, pgrp-le was recently implicated in pg sensing and induction of autophagy upon infection with l. monocytogenes thereby leading to clearance of bacteria [ ] . cytosolic prms have also been implicated in regulation of autophagy. suzuki and colleagues demonstrated that ipaf, a nod-like protein previously shown to sense flagellin, down regulates autophagy during infection with the non-flagellated bacterium s. flexneri [ ] . the down regulation of autophagy did not involve the asc adapter protein, normally required for the induction of il- ␤ after ipaf activation. one can speculate that nod proteins, which sense pg in mammalian cells, may play a similar role in the regulation of autophagy. up-coming studies addressing this question are eagerly awaited. in summary, the data above suggest a dynamic interaction between receptors from the innate immune system and regulation of autophagy. additional studies with knockout mice are now needed in order to demonstrate a definitive role for tlr-or nlr in autophagy during infection with pathogens known to activate specific prms. fig. . tlr activation triggers autophagy. lps triggers autophagy after recruitment of trif (also rip and p , not shown) and myd . the latter seems to interact with beclin- , reducing its binding to the anti-autophagic molecule bcl- . tlr engagement induces the incorporation of lc to phagosomes (unkown mechanism). viruses are able to induce autophagy through tlr , rig-i (dsrna) or tlr / -myd (ssrna). conventional dcs sense viral ligads through the rig-i/mavs axis to secrete type i interferon, while the conjugate atg / seems to be a down regulator of such response. plasmocytoid dcs deliver tlr ligands from the cytosol to the compartments containing tlr using basal autophagy. ipaf inhibits autophagy through an unclear mechanism. in the last few years, autophagy induction has been frequently reported as a consequence of innate immune system activation. however, there is compelling evidence that the relationship between autophagy and the immune system is reciprocal. cytokines from the innate and adaptive systems regulate autophagy by different mechanisms. two of the prototypical th cytokines, ifn-␥ and tnf-␣, were shown to up-regulate autophagy. gutierrez and colleagues first demonstrated that mouse macrophages harboring mycobacterium within phagosomes were able to clear bacteria after stimulation with ifn-␥ in an autophagy-dependent manner [ ] . follow up studies implicated gtpases in this process. the mouse genome contains different immunity-related gtpases, most of which respond to ifn-␥ stimulation and play a role in the defense against intracellular pathogens via a mechanism which is as yet unclear [ ] . the studies from deretic's group showed that both mouse immunity-related gtpase (irgm ) and its human ortholog irgm are the key molecules driving the induction of autophagy upon ifn-␥ stimulation, leading to the clearance of mycobacterium from infected macrophages [ , ] . the other th cytokine shown to stimulate autophagy is tnf-␣. codogno and colleagues observed that cells stimulated with tnf-␣ are committed to die when nfb is blocked [ , , ] . these findings are of great interest as the activation of autophagy may represent a way to overcome the resistance of cancer cells to anticancer drugs targeting nfb. in contrast to the autophagy enhancing effect of some th cytokines, th cytokines such as il- and il- , seem to counteract starvation and ifn-␥-induced autophagy by different pathways [ ] . while il- and il- block starvation-induced autophagy by activating the akt-mtor axis, these cytokines inhibit ifn-␥induced autophagy in an akt-independent but stat -dependent manner. the regulation of cytokine secretion by autophagy, has also been reported. jounai and colleagues demonstrated that in response to infection with rna viruses or immunostimulatory rna, ifn-␤ levels were increased in atg knockout embryonic fibroblasts [ ] . the authors demonstrated that the atg -atg conjugate negatively regulates the antiviral immune response by interacting with the rig-i-like receptor (s protein retinoic acid-inducible gene i (rig-i) and ifn-␤ promoter stimulator (ips- ) thus, implying autophagy contributes to viral replication. iwasaki and colleagues showed that in autophagy-impaired cells the increased cytokine secretion in response to immune-stimulatory rna is due to the accumulation of defective mitochondria and consequent ips- and ros accumulation, further strengthening the importance of autophagy in the maintenance of cellular homeostasis [ ] . autophagy has also been proposed to regulate cytokine secretion in crohn disease. crohn disease (cd) is a chronic inflammatory intestinal disease with a complex and multifactorial etiology. several recent independent genome wide association studies have implicated a number of heretofore unappreciated biological pathways in cd pathogenesis, including autophagy [ ] [ ] [ ] [ ] . since the identification of a non-synonymous single nucleotide polymorphism in the atg l gene as a causal risk variant for cd, several groups have sought to elucidate its functional impact on development of cd. akira and colleagues generated mice lacking the coiled-coil domain of atg l and observed aberrant il- ␤ secretion upon lps stimulation of fetal derived liver macrophages. in contrast to previous studies, lps did not induce autophagy in control macrophages indicating the enhanced il- ␤ was not due to disruption of lps-mediated autophagy [ ] . chimeric mice with atg l -deficient hematopoietic cells had an unremarkable baseline intestinal phenotype, but displayed increased susceptibility to dss-induced colitis compared with controls. even though this study used mice expressing a truncated form of atg l , rather than the atg l risk allele, the results point to the importance of functional autophagy machinery for normal intestinal function. using an alternative mouse model hypomorphic for atg l protein expression, cadwell and colleagues noted paneth cell-specific abnormalities including degenerating mitochondria, loss of lysozyme granule integrity and absence of apical microvilli [ ] . parallel findings were observed when intestinal atg expression was suppressed. transcriptional profiling analysis revealed that, among other differences, transcripts for the adipocytokines leptin and adiponectin were highly enriched. similar increased expression profiles were observed previously in patients with cd [ , ] . the above findings, while not specific to atg l suppression, underscore the importance of autophagy pathway integrity to normal paneth cell function. interestingly, atg knockout of pancreatic islet cells resulted in abnormal cellular morphology on em, including mitochondrial swelling, distension of the endoplasmic reticulum and a paucity of insulin granules when compared with controls [ ] . it remains unclear why paneth cells, above others, are susceptible to autophagy interference and how autophagy is involved in maintaining integrity of its lysozyme exocytosis pathway. however, the interaction between autophagy and multivesicular body biogenesis may provide a potential explanation for abnormal granule formation and exocytosis. once again, the paneth cell is placed at the convergence of several innate immune pathway aberrations and cd pathogenesis. translational clinical data are keenly awaited. the products of the two main cellular degradation systems -the proteasome and the lysosome -are not merely unwanted material but are, instead, key molecules utilized to instruct the immune system. this instruction step is achieved by the presentation of these products to cells from both innate and adaptive immune systems. cd + t cells monitor mainly cytosolic and nuclear antigens degraded by the proteasome (a large cytosolic enzyme complex) and loaded into mhc class i. in contrast, cd + t cells respond to extracellular or membrane peptides generated by lysosomal degradation and presented in the context of mhc class ii at the cell surface [ , ] . however, this paradigm has been challenged by the demonstration that dendritic cells (dcs) are also capable of presenting extracellular antigens on mhc class i, and not just on mhc class ii as initially thought, through a mechanism called crosspresentation. cross-presentation allows dcs to instruct also cd + t cells, generating a more efficient t-cell response [ ] . functional evidence for the presentation of endogenous antigens on mhc class ii was first provided by long and colleagues, who demonstrated that measles and influenza antigens could be presented in the context of mhc class ii [ , ] . indeed, the affinity purification of mhc class ii from epstein-barr virus (ebv)transformed b lymphoblastoid cells, murine b cell lymphoma and myeloid cells showed that more than % of natural mhc class ii ligands had their origin in intracellular proteins [ ] . together these studies suggested that an alternative and unknown route could deliver antigens from the cytosolic compartment for presentation on mhc class ii. knecht and colleagues were the first to suggest a role for autophagy in this process by showing that glyceraldehyde- -phosphate dehydrogenase, an important source of human mhc class ii ligands, is degraded via chaperone-mediated autophagy [ ] . additionally, peptides from two atg homologues, lc and gabarap, have been isolated from human and mouse mhc class ii molecules, respectively, providing further support to the notion of autophagy as an alternative route for delivery of cytosolic antigens for mhc class ii. more direct evidence came from studies using pharmacological inhibition of macroautophagy with pi k inhibitors (such as -ma and wortmannin), which are thought to block the sequestration step of autophagy. stockinger and colleagues demonstrated that over-expressed c was processed and loaded onto mhc class ii in an autophagy-dependent manner, as the loading was decreased in the presence of -ma [ ] . a similar approach was used to show that an endogenously expressed bacterial peptide, neor (neomycinphosphotransferase ii), was sequestered in autophagosomes and processed in endosomal/lysosomal compartments for loading onto mhc class ii. brossart and colleagues also used pharmacological inhibition to demonstrate that dcs electroporated with rna coding for the tumor-associated antigen muc- requires not only lysosomal antigen degradation and processing, but also autophagy in order to prime cd + t cells [ ] . further evidence that autophagy contributes to mhc class ii presentation came from munz and colleagues, in which they analyzed the endogenous mhc class ii processing of the nuclear antigen (ebna ) from ebv, the dominant ebv-latent antigen for cd + t cell. inhibition of autophagy by atg sirna in ebv-transformed b cells reduced recognition by ebna -specific cd + t cells [ ] . in another study, the same group demonstrated that the fusion of influenza matrix protein (mp ) with atg /lc drives this molecule to autophagosomes in different cell types and enhances recognition by antigen specific cd + t cells [ ] . knockdown of atg confirmed that the localization of the fusion proteins with mhc class ii molecules was dependent of autophagy. importantly, these results represent great potential for vaccine design, since targeting antigens to autophagosomes induces a more robust t cell response. in support of this contention, jagannath et al. demonstrated that induction of autophagy enhances bcg vaccine efficacy in a murine model [ ] . in contrast to model or viral antigens, very little is known about bacterial antigens requiring autophagy for proper presentation on mhc class ii. so far, only the b antigen from m. tuberculosis was shown to be presented more efficiently on mhc class ii upon induction of autophagy [ ] . accordingly, atg silencing dampened this process, while rapamycin treatment enhanced priming of b-specific cd + t cells, strongly suggesting a role for autophagy in mhc class ii presentation of antigens of bacterial origin. even though studies with other bacterial models are lacking, it is possible to speculate a role for autophagy in mhc class ii presentation during infections with bacteria that escape from phagosomes, such as l. monocytogenes autophagy is steadily emerging from its historic 'house-keeping' role as a new dimension in our host defense program. taken together, the data highlighted above suggest that autophagy impacts on the development of both innate and adaptive immune responses to diverse pathogens and that, conversely, components of the immune system themselves also regulate autophagy. this biological process is now a major target for researchers who want to enhance understanding of and develop strategies to modulate immune responses in a variety of 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tnf-induced accumulation of ros that mediate prolonged mapk activation and necrotic cell death reactive oxygen species are essential for autophagy and specifically regulate the activity of atg skeletal muscle is a primary target of sod g a-mediated toxicity autophagy contributes to caspase-independent macrophage cell death autophagy defends cells against invading group a streptococcus escape of intracellular shigella from autophagy differential regulation of caspase- activation, pyroptosis, and autophagy via ipaf and asc in shigella-infected macrophages stimulation of autophagy suppresses the intracellular survival of burkholderia pseudomallei in mammalian cell lines autophagy controls salmonella infection in response to damage to the salmonellacontaining vacuole autophagy recognizes intracellular salmonella enterica serovar typhimurium in damaged vacuoles cytoplasmic bacteria can be targets for autophagy listeria monocytogenes: a multifaceted model listeria monocytogenes evades killing by autophagy during colonization of host cells listeriolysin o allows listeria monocytogenes replication in macrophage vacuoles tuberculosis toxin blocking phagosome maturation inhibits a novel ca +/calmodulin-pi k hvps cascade autophagy is a defense mechanism inhibiting bcg and mycobacterium tuberculosis survival in infected macrophages lysosomal killing of mycobacterium mediated by ubiquitin-derived peptides is enhanced by autophagy protective role of autophagy against vibrio cholerae cytolysin, a pore-forming toxin from v. cholerae effect of helicobacter pylori's vacuolating cytotoxin on the autophagy pathway in gastric epithelial cells hsv- icp , confers neurovirulence by targeting the beclin autophagy protein subversion of cellular autophagosomal machinery by rna viruses rotavirus nsp induces a novel vesicular compartment regulated by calcium and associated with viroplasms identification of a lytic-cycle epstein-barr virus gene product that can regulate pkr activation 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bowel diseases production of adiponectin, an anti-inflammatory protein, in mesenteric adipose tissue in crohn's disease loss of autophagy diminishes pancreatic beta cell mass and function with resultant hyperglycemia syf-peithi: database for mhc ligands and peptide motifs rapid degradation of a large fraction of newly synthesized proteins by proteasomes autophagy in cd + t-cell immunity and tolerance hla class iirestricted presentation of cytoplasmic measles virus antigens to cytotoxic t cells an endogenous processing pathway in vaccinia virus-infected cells for presentation of cytoplasmic antigens to class ii-restricted t cells excessive degradation of intracellular protein in macrophages prevents presentation in the context of major histocompatibility complex class ii molecules processing and presentation of hla class i and ii epitopes by dendritic cells after transfection with in vitro-transcribed muc rna endogenous mhc class ii processing of a viral nuclear antigen after autophagy antigen-loading compartments for major histocompatibility complex class ii molecules continuously receive input from autophagosomes autophagy enhances the efficacy of bcg vaccine by increasing peptide presentation in mouse dendritic cells enhancing immunity through autophagy funding sources: s.h. is supported by a research fellowship award from cihr, canadian association of gastroenterology and crohn's and colitis foundation of canada. l.h.t is supported by a research fellowship award from cihr. n.l.j. is supported by operating grants from cihr and ccfc. key: cord- - dh authors: kumar, ashutosh; harjai, kusum; chhibber, sanjay title: a multiepitopic theoretical fusion construct based on in-silico epitope screening of known vaccine candidates for protection against wide range of enterobacterial pathogens date: - - journal: hum immunol doi: . /j.humimm. . . sha: doc_id: cord_uid: dh enterobacterial pathogens that have acquired antibiotic resistance genes are a leading cause of community and hospital acquired infections. in such a situation vaccination is considered as a better option to prevent such infections. in the current study reverse vaccinology approach has been used to select peptides from already known immunogenic proteins to design a chimeric construct. we selected yersiniabactin receptor of escherichia coli umn and flagellin of stenotrophomonas maltophila. b-cell linear epitopes were predicted using bepipred prediction tool. peptide binding with reference sets of alleles of mhc class i and class ii was also analyzed. the predicted peptides-mhc complexes were further validated using simulation dynamics. the in-silico construction of chimera was done by restriction mapping and codon optimization. chimera was evaluated using the immunoinformatic approach as done for the selected proteins. from the amino acids of fyua protein, a region from to was selected for containing more linear epitopes and the processing scores obtained were significant for mhc class i and class ii binding. similarly, from flagellin, a region between and amino acids was selected and the peptides present in the selected region showed lower percentile ranks for binding with mhc molecules. the simulation studies validated the predictions of peptide-mhc complexes. the selected gene fragments accommodating maximum part of these peptides were used to design a chimaeric construct of bp. from the immunoinformatic analysis, the chimera was found to be more immunogenic in terms of increased number of b-cell and t-cell epitopes along with increased coverage of global populations with allelic variability. communicable diseases caused by members of enterobacteriaceae family put a great burden on the society by affecting humans and their livestocks. these infections become quite severe when they are not controlled on time. some of the examples include pneumonia, pyogenic liver abscess, pyelonephritis and septicemia [ ] . the treatment has become difficult due to the emergence of antibiotic resistance among some of the pathogens. due to the existing challenge of treating these infections with antibiotics, it is inevitable for the research community to look forward to prophylactic means for the prevention of these infections. a large number of vaccine candidates have been proposed by various researchers for specific infections. however, evaluation of individual vaccine candidates under in-vivo infection conditions is an enduring task. in the recent years, "reverse vaccinology" (rv) has come to play an important role in scrutinizing the vaccine candidates by in-silico analysis [ ] thereby reducing the time required for ruling out ineffective candidates. this immunoinformatic approach is being frequently used by researchers to predict the epitopes on viruses. this has led to the finding of epitopes on nucleocapsid protein and ovarian tumour domain of crimean-congo hemorrhagic fever virus [ ] . another study investigated the variability among epitopes of hepatitis c virus (hcv) identified in genotype and also predicted the immunogenicity of their variants from other genotypes against south african human leukocyte antigen (hla) backgrounds [ ] . epitopes of e protein isolated from hcv have also been identified using the similar approach [ ] . effective immunogens of mers-cov have been discovered through immunoinformatics-driven genome-wide screening strategy [ ] . goodswen et al. [ ] have also used this technique for designing protein based vaccines against eukaryotic pathogens. reverse vaccinology has already been used against other bacterial pathogens like group b streptococcus, where genomic analysis has led to the development of a vaccine composed of four proteins giving protection against all serotypes [ ] . another in-silico study has found a protein bam a of acinetobacter baumanii to be a potential immunogen [ ] . rv has also been implied to predict the potential vaccine candidates from the proteome of burkholderia pseudomallei [ ] . the outer membrane proteins (omps) of these gram-negative bacteria are usually considered as potent vaccine candidates as they are exposed to the host immune defenses [ ] . these omps are not always conserved in different genus of bacteria but their lies a probability of presence of some conserved peptide sequence in these omps. in this study we have taken into account the proteins which have proved to be potential vaccine candidates on the basis of invivo research work on animal models. yersiniabactin receptor fyua is highly conserved protein prevalent in various members of enterobacteriaceae. as per the reports, fyua mediates the uptake of ferricyersiniabactin [ , ] confirming its role in the virulence of bacteria, which makes it an important vaccine candidate. moreover researchers have found it as a potential vaccine candidate against pyelonephritis in a murine model of urinary tract infection [ ] . it has also been found to be protective in murine model of pneumonia caused by k. pneumoniae in our laboratory (manuscript under communication). the flagellin protein is another potential vaccine candidate which has been included in this study. various studies have established the role of flagellin in inducing a systemic inflammatory response via intraperitoneal and intravenous administration [ , ] and a local inflammatory response with intraintestinal administration [ , ] . in our lab, flagellin of stenotrophomonas maltophilia has been shown to induce non-specific immune response which protected mice against subsequent bacterial challenge [ ] . in the current study both of these vaccine candidates have been analyzed using iedb server to design a novel insilico vaccine construct harbouring the properties of both these proteins. gene sequence of fyua accession no. nc_ . and flagellin nc_ . were taken from ncbi database [ ] . the obtained gene sequences were translated into the protein sequences using expasy-translate tool (swiss institute of bioinformatics). protein sequences of yersiniabactin receptor of escherichia coli umn and flagellin of stenotrophomonas maltophilia were analyzed for the presence of linear epitopes using bepipred portal of iedb server. both proteins were also analyzed on other algorithms, parker linear epitope prediction from the protein sequences fyua (a) and flagellin (b) using bepipred prediction portal of iedb server. the yellow peaks show the peptide sequences that are potential epitopes whereas the green peaks show the peptides that are not epitopic in nature. the encircled area on the graphs shows the region of protein having higher frequency of epitopic peptides. conformational epitopes on yersiniabactin receptor were predicted using discotope portal of iedb server by analyzing solvent-accessibility. in human population major histocompatibility complexes (mhc-i and mhc-ii) are encoded by human leukocyte antigen (hla) alleles and are required for the presentation of antigen to t cells. the peptides which could bind to mhc i molecules were predicted by mhc class i binding peptide prediction portal on iedb server using the consensus method. interactions were evaluated in terms of percentile ranks. similarly, peptides binding to mhc ii molecules were predicted by mhc class ii binding peptide prediction portal on iedb server using the consensus method. d structures of alleles were retrieved from rcsb pdb database [ ] . predicted peptide sequences and d structure of mhc class i and class ii alleles were submitted to cabs dock server for docking and simulation studies [ ] . secondary structures of peptides were generated from psipred [ ] . the simulation time was set to cycles. the results were clustered according to the distance between the residues of the peptide and mhc molecules. both gene sequences were analyzed on neb cutter for mapping restriction sites. the enzymes which could cut the dna at only single site were identified and the one that was common in both sequences was selected. the chimeric construct could be constructed after ligation of gene fragments excised from fyua and flagellin. briefly, both the genes could be digested with aatii to produce linear sticky ended fragments of . and . kb respectively. aatii will digest fyua at position bp and flagellin at position bp. these fragments could then be digested with xhoi to generate sticky ended fragments of . kb and . kb from fyua and . kb and . kb from flagellin. the . kb fragment from flagellin and . kb fragment from fyua could be ligated with t dna ligase followed by transformation, screening and sequencing. open reading frame for the sequence of chimeric gene was analyzed on expasy-translate tool (swiss institute of bioinformatics). the model structure of the chimera aksc was generated using modeller . [ ] . the structure was energy minimized and validated using molprobity [ ] server for its stereophysical characteristics. protein sequence of aksc was analyzed using bepipred portal of iedb server. the obtained linear epitopes were analyzed for changes in comparison to the epitopes predicted from the individual proteins. the epitopes were also analyzed for the presence of overlapping regions between individual proteins. protein sequence of aksc was analyzed to observe the peptides which could bind to mhc class i molecules. these peptides were predicted using mhc class i binding peptide prediction portal on iedb server. similarly, peptides binding to mhc ii molecules were predicted using mhc class ii binding peptide prediction portal on iedb server. interactions were evaluated in terms of percentile ranks. amino acid sequences of both the proteins were analyzed for the presence of linear epitopes using the bepipred prediction tool of iedb server. for fyua a threshold score of . for a window size of amino acids was generated in the portal. results in fig. (a) show that a large number of linear epitopes are present in fyua and most of the prominent epitopes were in the region from amino acid position to . the maximum score of . was obtained for the amino acids near position . similarly, linear epitopes were predicted for flagellin with the bepipred generated threshold value of . for a window size of amino acids. results in fig. (b) show a large number of linear epitopes on flagellin and most of the prominent epitopes were in the region from amino acid position to . the maximum value of . was obtained for the amino acids near position . the peptides that were significant linear epitopes are shown here in table . results of bepipred prediction as shown in fig. revealed the presence of a wide range of linear epitopes on both fyua and flagellin proteins. these results were further verified using other parameters like hydrophilicity, surface accessibility and antigenicity. similar results were obtained from all the above predictions as both the proteins were found to possess large number of linear epitopes (supplementary information si. .). the region accommodating the maximum number of epitopes was then selected to be taken further for theoretical construction of chimaera. further, evaluation of other parameters was done by keeping the selected regions into consideration. since, there lies a possibility that the chimeric protein may not take up the proper folding when over expressed due to physical constraint of large size. hence, it may be expressed as inclusion bodies when subjected to over expression. misfolding of protein would certainly not affect the t-cell dependent response since the generation of t-cell response depends on the processing and presentation of peptides on mhc molecules but it may affect the antibody response to conformational epitopes. however, results in table show table . therefore, the presence of linear epitopes can be considered as a very significant feature of the vaccine candidate protein as it could help in the generation of antibody response even if the protein is administered in denatured form. conformational epitopes of fyua were predicted using discotope tool on iedb server. results in fig. (a) as depicted by the green peaks is the region from amino acid position to that possess maximum conformational epitopes. these epitopes (yellow) were also shown on the -d structural image created by j-mol-pdb fig. (b) . since most of the conformational epitopes are present on the exposed surface, these epitopes may become a target of antibodies for effectively neutralizing the bacteria during infection. both the protein could prove to be good vaccine candidates if they are able to generate both b cell as well t cell responses. however, both the proteins qualified for the generation of b cell response therefore predictions were made for their ability to generate t-cell responses. this was done by predicting mhc class i and ii binding peptides. mhc class i binding peptides were predicted using the mhc class i binding prediction tool on iedb server. peptides with a percentile rank below were considered significant. results in fig. (a) show that significant interactions were obtained for fyua protein and among these, interactions were from the selected region of amino acid position to . similarly, for flagellin protein results in fig. (a) show about significant interactions and among these, interactions were from the selected region of amino acid position to (supplementary information si. .). therefore the results in fig. (b) show that % of the significant interactions were from the selected region of fyua and % of significant interactions were from the selected region of flagellin fig. (b) . the top peptides from the selected region having lowest percentile rank were shortlisted and are shown in table . data in table show that both of these proteins possessed significant number of peptidic regions that could bind to it is also seen that the peptides of both these proteins bind with different alleles which are present in different parts of world. this difference in the binding will turn out to be very beneficial when aksc will be used as vaccine as more the number of binding alleles more would be the coverage of human population. peptides binding to mhc class ii molecules were predicted using mhc class ii binding peptide prediction tool on iedb server. for mhc class ii binding, iedb recommended method and a reference set of alleles was used. results in fig. (a) show that significant interactions were obtained for fyua protein. out of which interactions with the reference set of alleles were from the selected region. similarly, for flagellin protein results in fig. (a) show that significant peptide allele interactions were predicted by the server and among these, interactions were from the selected region (supplementary information si. .). also the results in fig. (b) show that % of the significant interactions were from the selected region of fyua and % of significant interactions were from the selected region of flgellin fig. (b) . table depict the top peptides from the selected region having lowest percentile rank for fyua for flagellin respectively. alleles hla-drb * : , hla-drb * : and hla-drb * : are present largely in asian and russian populations (allelefrequencies. net). allele hla-dqa * : /dqb * : is found in asian, african, israel and french populations and hla-dqa * : /dqb * : is present in german, african and asian populations. results obtained after simulated -dimensional docking of predicted peptides with the predicted mhc molecules were presented in tables and . the data shows the average rmsd values of the each peptide-mhc complex. the average rmsd values between and are considered of medium accuracy whereas the values below are considered as highly accurate [ ] . along with the rmsd values the distance between the interacting amino acid residues of mhc molecules and that of the interacting peptide was also analyzed. the results in table and fig. show that the mhc class i and class ii binding peptide predictions made using the iedb server, were also found to be significantly accurate using the -d simulations for docking. this could be interpreted as the values of average rmsd were below in the simulated complexes. the interacting residues shown in the table were lying at a distance of < Å. these results of all the in-silico predictions helped us in choosing the regions from both the proteins, so that they could be combined to produce a single chimaeric construct. we therefore propose a strategy which could lead to the formation of the chimaeric construct. for designing the chimaeric gene construct, cloning strategy was adopted using genetic engineering tool for restriction digestion. gene sequences of both the proteins were analyzed on neb cutter tool of new england biolabs. restriction enzyme aatii (supplementary information si. .) was found to be present in both the protein sequences and cleaves fyua at position and flagellin at position and generates sticky ends. fig. shows the graphical representation of the cloning strategy. in-silico created chimeric construct was found to be consisting of base pairs (supplementary information si. .) which was further translated using expasy/translate tool and a protein of amino acids was formed to give a molecular weight of approximately kda (supplementary information si. .). this ruled out the presence of any stop codon within the whole sequence. the main objective of combining large gene fragments from both the proteins was considered so as to provide the cellular protein processing machinery with enough of proteasome cleavage sites. the possibility of a single peptide to act as a vaccine is usually a rare chance. hence a protein can be processed to in different ways to create epitopes whose presentation to t-cells may add to protection during pathogenesis. analysis of ramachandran plot of the chimera aksc generated using the molprobity server suggested that . % residues lied in the favoured and allowed region while only . % residues were in the outlier region (supplementary information si. .). the obtained values suggest that the model is structurally stable. further analysis of aksc showed an increase in the average threshold value in linear epitope prediction this probably resulted by combining the two proteins (fig. ). there were also the peptides in aksc that were epitopic and lied in the region joining both the proteins. this was another advantage of this chimaera as these overlapping peptides could increase the population coverage of vaccine. table show the peptides that are able to bind to mhc class i and class ii and are present in the region joining the two proteins. these peptides are able to bind to different alleles. results in table show the similarity of overlapping peptides which were generated as a result of fusion of the two proteins. the peptide analyzed using blastp showed its identity with peptides present in microorganisms other than the source of peptide. this also gave a hope that the chimaeric vaccine candidate may confer protection upon global populations from the infectious diseases caused by a wide range of pathogens. from the in-silico analysis it is concluded that reverse vaccinology can be used to create novel chimeric constructs from the already known vaccine candidates to make them more effective and to confer protection among diverse populations against a wide range of enterobacterial pathogens. for this research work no specific grant from any funding agency was provided. mr. ashutosh kumar was granted financial support in the form of senior research fellowship from indian council of medical computer-aided biotechnology: from immuno-informatics to reverse vaccinology epitope-based immunoinformatics and molecular docking studies of nucleocapsid protein and ovarian tumor domain of crimean-congo hemorrhagic fever virus sequence-based in silico analysis of well studied hepatitis c virus epitopes and their variants in other genotypes (particularly genotype a) against south african human leukocyte antigen backgrounds structural analysis and epitope prediction of hcv e protein isolated in pakistan: an in-silico approach epitope-based vaccine target screening against highly pathogenic mers-cov: an in silico approach applied to emerging infectious diseases enhancing in silico protein-based vaccine discovery for eukaryotic pathogens using predicted peptide-mhc binding and peptide conservation scores identification of a universal group b streptococcus vaccine by multiple genome screen in silico analysis of acinetobacter baumannii outer membrane protein bama as a potential immunogen in-silico analysis of burkholderia pseudomallei proteome to predict potential vaccine candidate proteins a multiepitope subunit vaccine conveys protection against extraintestinal pathogenic escherichia coli in mice the pesticin receptor of yersinia enterocolitica: a novel virulence factor with dual function reduced synthesis of the ybt siderophore or production of aberrant ybt-like molecules activates transcription of yersiniabactin genes in yersinia pestis immunization with the yersiniabactin receptor, fyua, protects against pyelonephritis in a murine model of urinary tract infection the innate immune response to bacterial flagellin is mediated by toll-like receptor flagellin, a novel mediator of salmonella-induced epithelial activation and systemic inflammation: iκbα degradation, induction of nitric oxide synthase, induction of proinflammatory mediators, and cardiovascular dysfunction role of flagellin in the pathogenesis of shock and acute respiratory distress syndrome: therapeutic opportunities humoral immune response to flagellin requires t cells and activation of innate immunity stenotrophomonas maltophilia flagellin induces a compartmentalized innate immune response in mouse lung induction of hepatitis a virus-neutralizing antibody by a virus-specific synthetic peptide the protein data bank cabs-dock web server for the flexible docking of peptides to proteins without prior knowledge of the binding site protein secondary structure prediction based on position-specific scoring matrices comparative protein modelling by satisfaction of spatial restraints molprobity: all-atom structure validation for macromolecular crystallography modeling of protein-peptide interactions using the cabs-dock web server for binding site search and flexible docking none to declare. supplementary data to this article can be found online at https:// doi.org/ . /j.humimm. . . . key: cord- -fanlvxqs authors: loureiro, joana; ploegh, hidde l. title: antigen presentation and the ubiquitin‐proteasome system in host–pathogen interactions date: - - journal: adv immunol doi: . /s - ( ) - sha: doc_id: cord_uid: fanlvxqs relatively small genomes and high replication rates allow viruses and bacteria to accumulate mutations. this continuously presents the host immune system with new challenges. on the other side of the trenches, an increasingly well‐adjusted host immune response, shaped by coevolutionary history, makes a pathogen's life a rather complicated endeavor. it is, therefore, no surprise that pathogens either escape detection or modulate the host immune response, often by redirecting normal cellular pathways to their advantage. for the purpose of this chapter, we focus mainly on the manipulation of the class i and class ii major histocompatibility complex (mhc) antigen presentation pathways and the ubiquitin (ub)‐proteasome system by both viral and bacterial pathogens. first, we describe the general features of antigen presentation pathways and the ub‐proteasome system and then address how they are manipulated by pathogens. we discuss the many human cytomegalovirus (hcmv)‐encoded immunomodulatory genes that interfere with antigen presentation (immunoevasins) and focus on the hcmv immunoevasins us and us , which induce the degradation of class i mhc heavy chains by the proteasome by catalyzing their export from the endoplasmic reticulum (er)‐membrane into the cytosol, a process termed er dislocation. us ‐ and us ‐mediated subversion of er dislocation ensures proteasomal degradation of class i mhc molecules and presumably allows hcmv to avoid recognition by cytotoxic t cells, whilst providing insight into general aspects of er‐associated degradation (erad) which is used by eukaryotic cells to purge their er of defective proteins. we discuss the similarities and differences between the distinct pathways co‐opted by us and us for dislocation and degradation of human class i mhc molecules and also a putatively distinct pathway utilized by the murine herpes virus (mhv)‐ mk immunoevasin for er dislocation of murine class i mhc. we speculate on the implications of the three pathogen‐exploited dislocation pathways to cellular er quality control. moreover, we discuss the ubiquitin (ub)‐proteasome system and its position at the core of antigen presentation as proteolysis and intracellular trafficking rely heavily on ub‐dependent processes. we add a few examples of manipulation of the ub‐proteasome system by pathogens in the context of the immune system and such diverse aspects of the host–pathogen relationship as virus budding, bacterial chromosome integration, and programmed cell death, to name a few. finally, we speculate on newly found pathogen‐encoded deubiquitinating enzymes (dubs) and their putative roles in modulation of host–pathogen interactions. relatively small genomes and high replication rates allow viruses and bacteria to accumulate mutations. this continuously presents the host immune system with new challenges. on the other side of the trenches, an increasingly welladjusted host immune response, shaped by coevolutionary history, makes a pathogen's life a rather complicated endeavor. it is, therefore, no surprise that pathogens either escape detection or modulate the host immune response, often by redirecting normal cellular pathways to their advantage. for the purpose of this chapter, we focus mainly on the manipulation of the class i and class ii major histocompatibility complex (mhc) antigen presentation pathways and the ubiquitin (ub)-proteasome system by both viral and bacterial pathogens. first, we describe the general features of antigen presentation pathways and the ub-proteasome system and then address how they are manipulated by pathogens. we discuss the many human cytomegalovirus (hcmv)-encoded immunomodulatory genes that interfere with antigen presentation (immunoevasins) and focus on the hcmv immunoevasins us and us , which induce the degradation of class i mhc heavy chains by the proteasome by catalyzing their export from the endoplasmic reticulum (er)-membrane into the cytosol, a process termed er dislocation. us -and us -mediated subversion of er dislocation ensures proteasomal degradation of class i mhc molecules and presumably allows hcmv to avoid recognition by cytotoxic t cells, whilst providing insight into general aspects of er-associated degradation (erad) which is used by eukaryotic cells to purge their er of defective proteins. we discuss the similarities and differences between the distinct pathways coopted by us and us for dislocation and degradation of human class i mhc molecules and also a putatively distinct pathway utilized by the murine herpes virus (mhv)- mk immunoevasin for er dislocation of murine class i mhc. we speculate on the implications of the three pathogen-exploited dislocation pathways to cellular er quality control. moreover, we discuss the ubiquitin (ub)-proteasome system and its position at the core of antigen presentation as proteolysis and intracellular trafficking rely heavily on ub-dependent processes. we add a few examples of manipulation of the ub-proteasome system by pathogens in the context of the immune system and such diverse aspects of the host-pathogen relationship as virus budding, bacterial chromosome integration, and programmed cell death, to name a few. finally, we speculate on newly found pathogen-encoded deubiquitinating enzymes (dubs) and their putative roles in modulation of host-pathogen interactions. the vertebrate immune system is equipped to deal with invading pathogens, whether by means of mechanical barriers such as the skin and other epithelial surfaces or by means of innate immunity. innate immunity comprises the phagocytic and inflammatory systems, with phagocytes like macrophages and neutrophils, dendritic cells (dcs), and natural killer (nk) cells, as well as soluble mediators such as cytokines and complement. phagocytes are the immune system's first line of defense: they recognize, engulf, and clear the pathogen and are the main cellular component of the innate antibacterial response. nk cells can directly recognize and kill pathogen-infected cells that fail to express mhc molecules and secrete cytokines that affect the immune response. nk cells are the main cellular effectors of the innate response against viruses. the complement system can lyse infected cells or simply coat the surface of the pathogen or pathogen-derived material, resulting in its neutralization and opsonization. to counteract pathogen infection, host cells also have extracellular and intracellular pathogen recognition receptors to alert the immune system, such as toll-like receptors (tlrs) at the cell surface, and protein kinase r (pkr) and nucleotidebinding oligomerization domain (nod) proteins in the cytosol (akira et al., ; inohara et al., ) that can detect pathogen-associated molecular patterns (pamps) such as bacterial peptidoglycan or viral dsrna. these ''danger'' signals initiate the synthesis of cytokines like interferons to induce inflammation, a crucial component of the innate defense against pathogens. because innate immunity is not always successful at recognizing or eliminating the infectious agents, a more sophisticated line of defense, adaptive immunity, is also in place. the adaptive immune system includes cells originated in the thymus, the t lymphocytes, and the bone marrow-derived b lymphocytes (b cells), dcs, and macrophages. the two subsets of t lymphocytes, cd þ and cd þ t cells, possess distinct t cell receptors (tcrs), cd and cd , respectively, that interact with their coreceptors on the surface of the target cell, the polymorphic class i and class ii mhc molecules (ploegh, ) . class i mhc molecules are expressed by nearly all nucleated cells, whereas class ii mhc molecules are constitutively expressed only by professional antigen-presenting cells (apcs), such as macrophages, b cells, and dcs. class ii mhc expression however, can be induced in many cells, in particular by ifn-g treatment. apcs can endocytose, process, and display antigen in the context of class ii mhc products at their cell surface to activate cd þ t cells. in the presence of antigen displayed by the apc and the appropriate lymphocyte costimulatory molecules, cd þ t helper (t h ) cells produce cytokines that ''help'' activate other cells: t h (or inflammatory t cells) activate macrophages to kill the phagocytosed pathogens; t h cells (or helper tcells) trigger tand b cell proliferation and activate the b cell differentiation program into antibody-producing plasma cells. furthermore, activation of cd þ t h cells is carefully regulated by a small subset of t cells, the regulatory t cells (tregs). regulatory t cells play an important role in downregulation of the host immune response, limiting the immunopathology resultant from antipathogen reactions, and preventing autoimmune disease (beissert et al., ; mills, ) . in addition to making antibodies, b cells are a special kind of apcs. unlike dcs and macrophages, b cells are not actively phagocytic. however, stimulation of the membrane immunoglobulin (mig) antigen-recognition component of their b cell receptor (bcr) with cognate antigen triggers the b cell to capture and deliver the antigen to class ii mhc compartments, culminating with antigen presentation for activation of t cells. bone marrow-derived professional apcs include macrophages and dcs. macrophages are phagocytic apcs with a low basal antigen-presenting capacity-owing to low surface expression of class ii mhc and costimulatory molecules-that is induced on macrophage activation, for instance, by ifn-g. macrophages reside in (or are recruited to) peripheral tissues, where they phagocytose and clear pathogens. phagocytosis, in turn, induces release of proinflammatory cytokines like ifn-g that turn macrophages into potent apcs, resulting in initiation of cd þ t cell activation. dendritic cells, the consummate professional apcs, travel through the periphery, sampling all tissues for prospective invaders. immature dcs phagocytose pathogens and home to the nearest lymphoid organ to ''educate'' (prime) naïve cd þ t cells by cross-presenting antigen in the context of class i mhc molecules-a process described in more detail later. mature dcs can also prime naïve cd þ t cells. like resting macrophages, immature dcs have very low antigen-presenting capability, and only on exposure to maturation signals [such as lipopolysaccharide (lps) on bacterial surfaces] does internalized antigen get loaded into class ii mhc products and get displayed at the cell surface to cd þ t cells (bryant and ploegh, ; stockwin et al., ) . ''educated'' (antigen-specific) cd þ t lymphocytes survey all cells in the body, ready to destroy any that displays signs of the presence of cellular alterations (such as viral and tumor peptides) within their surface class i mhc molecules (andersen et al., ; castelli et al., ) . antigen-specific cd þ t lymphocytes can coordinate macrophage bactericidal properties, activation of t and b lymphocytes, and antibody production. not only do the cells of the adaptive immune system provide a more elaborate defense, but also an increased level of protection from a subsequent reinfection with the same pathogen, the bedrock principle of vaccine strategies (crotty and ahmed, ; pulendran and ahmed, ) . adding to the complexity of the immune system is the cross talk between innate and adaptive immunity, which is crucial in eliciting an effective immune response (zingoni et al., ) . as mentioned, phagocytes release cytokines that stimulate the adaptive response. conversely, on activation by antigen recognition, t cells synthesize and secrete cytokines that activate macrophages, increasing their ability to kill ingested microbes, an innate immune response (munz et al., ; salazar-mather and hokeness, ) . the vertebrate immune system, therefore, is the appropriate battleground for microbial pathogens, selecting for those that devise successful strategies to avoid detection and elimination (hilleman, ; ploegh, ) . intracellular pathogens have evolved sophisticated mechanisms to subvert host processes to ensure their own replication and transmission. the initial hurdle is entry into the host cell, which poses great challenges for avoiding immune detection before establishing infection. to promote entry into host cells without alerting the immune system, bacteria possess capsular surfaces that have evolved to minimize antibody and complement deposition while in circulation through the body. on the other hand, filamentous adhesins (like fimbriae and pili) that protrude through the bacteria's capsule enable binding to host cell receptors, which enables secretion systems to deliver bacterial effectors to modulate uptake and invasion (finlay and mcfadden, ; galan and collmer, ) . virus particles are very often coated with highly variable capsid (nonenveloped viruses) or envelope (enveloped viruses) proteins to avoid detection and clearance by antibody-mediated responses. these capsids or envelopes can also be studded with immunomodulatory molecules of viral or even host origin and promote attachment to the host cell membrane, fusion and delivery of the virus internal core. alternatively, they may act as signaling devices and induce intracellular cascades required for virus uptake. ultimately, intracellular release of the viral dna or rna occurs (marsh and helenius, ; skehel and wiley, ) . the establishment of an infection critically depends on bacterial and viral genes dedicated to manipulation of host functions. a number of reviews have covered the bacterial and viral genes involved in manipulation of the host immune system, from control of apoptosis, cytokine signaling, to the antibody response, so the reader is referred to alcami ( ) ; alcami and koszinowski ( ) ; bowie et al. ( ) ; finlay and mcfadden ( ) ; hengel et al . ( ); hilleman ( ) ; and tortorella et al. ( ) . for the purpose of this chapter, we will focus on pathogen manipulation of antigen presentation pathways and the ub-proteasome system. antigen presentation involves the conversion of protein antigens into peptide ligands that can bind to mhc products that are displayed at the cell surface for recognition by t cells. in a simplified view of antigen presentation, the class i and class ii mhc pathways have evolved to sample different sources of antigen to which they have access: the class i mhc pathway usually deals with cytosolic antigens and is crucial for activation of cd þ t cells, whereas the class ii mhc pathway deals with exogenous antigens and the activation of cd þ t cells (bryant and ploegh, ; cresswell et al., ; pamer and cresswell, ) . antigen presentation is, of course, not as simple and clearcut, as we shall discuss later. there are, however, common principles that apply to the discrete steps of antigen processing and presentation by class i and class ii mhc molecules: antigen must be acquired, it is subjected to proteolysis, delivered to mhc þ compartments, and properly assembled with the mhc product. the complex is then subject to sorting through the secretory pathway and delivered to the cell surface ( fig. ) . because each of these steps affords a target for interference by pathogens, we shall survey them for each pathway. the class i mhc is a trimeric complex composed of the class i mhc heavy chain (hc), the b -microglobulin (b m) or light chain, and the antigenic peptide. the structure of the fully assembled complex and its interactions with antigen-specific receptors on t cells have been extensively reviewed (alam et al., ; rudolph and wilson, ; von boehmer, ) . the class i mhc hc is inserted into the er membrane and n-glycosylated and binds in its course of synthesis to the membrane-associated chaperone calnexin (cnx), at which point folding and intrachain disulfide bond formation take place. once dissociated from cnx, the hc binds its soluble partner subunit, b m, and enters the peptide-loading complex (plc). the plc is composed of two mhc-encoded components, tap and tapasin, and two ''housekeeping'' er proteins, calreticulin and erp . the transporter associated with antigen presentation (tap) is an atp-dependent pump with two subunits, tap and tap that transports peptides into the er. tapasin, a transmembrane glycoprotein, mediates the interaction between the tap transporter and peptide-free hc/b m dimers. the soluble calreticulin and erp , a chaperone and a thiol oxidoreductase, respectively, normally involved in folding of nascent glycoproteins, promote assembly of the class i mhc complex. the peptide antigen cargo for class i mhc originates from proteasomal proteolysis in the cytosol. the array of proteasome-generated peptides is subject to trimming by cytosolic endopeptidases and delivered to the er lumen by the tap transporter. further trimming by er-resident endopeptidases can also occur to guarantee a custom-fit of the peptide antigens, typically - amino acids long, into the peptide-binding groove on the hc/b m dimer associated with the plc. empty hc molecules are detained in the er by virtue of interaction with tapasin, until assembly with b m and peptide takes place, at which point the hc/b m/peptide trimeric complex is released from the plc and allowed to exit the er and enter the secretory pathway. once displayed at the cell surface, the antigen-loaded class i mhc complex is ready for inspection by the t cell receptor (tcr) on circulating cytotoxic cd þ t cells (cresswell et al., ; heemels and ploegh, ; rammensee, rammensee, , . figure common principles in antigen processing and presentation by class i and class ii mhc molecules. in the class i pathway, endogenous antigens are derived from cytosolic proteolysis and delivered to the er lumen, where loading onto class i mhc products takes place. the assembled complex is then sorted to the cell surface. in the class ii pathway, exogenous material is internalized from the extracellular space and delivered to the lysosome, where processing and loading onto mhc products occur. sorting through the secretory pathway then delivers the class ii complex to the cell surface. class i mhc products on most cells present exclusively ''self'' peptides, derived from the cell's own proteins, the majority of which results from protein synthesis on free ribosomes in the cytoplasm. because of the intrinsic error-prone nature of protein synthesis and folding, a sizable fraction of translation products (estimated at up to %) may never result in a finished product. these defective ribosomal products (drips) are destroyed within min of their synthesis by the cytosolic proteasomal pathway and enter the class i mhc antigen presentation pathway (yewdell et al., ) . in tumor cells or cells infected by a virus, mutated forms of endogenous proteins or viral proteins will compete with the host's own proteins for presentation by class i mhc products. as ''non-self'' (tumor-or virus-derived) peptides displayed in the context of class i mhc products accumulate at the cell surface, their chance of triggering activation of cd þ t cells with a cognate receptor increases. the activated cytotoxic cd þ t lymphocytes will then lyse the target cell by releasing perforin and granzymes or by fas ligand engagement. secretion of ifn-g and tumor necrosis factor-a (tnf-a) also aids in elimination of infected and tumor cells by cytotoxic t lymphocytes (ctls) (andersen et al., ; castelli et al., ) . the selective pressure imposed by immune surveillance has made loss of class i mhc expression a hallmark of some tumors and virus-infected cells, as this allows them to be invisible to ctls. there is, however, a backup system for when lack of class i mhc expression impairs the cd þ t cell cytotoxic response: nk cells. nk cells display both activating and inhibitory receptors at their surface, which recognize different ligands at the surface of target cells. nk cell activity is ultimately determined by the integration of signals that are perceived by the nk cell surface receptors (lanier, ) . all nk cells express at least one inhibitory receptor, which engages class i mhc molecules on the surface of the target cell, resulting in downregulation of nk cell effector functions. low levels or absence of class i mhc products on the surface of the target cell relieve the inhibitory signals and lead to nk cell cytotoxicity, resulting in clearance of the virus-infected or tumor cells. nk cell recognition has been extensively revised and the reader is referred to backstrom et al. ( ) ; kumar and mcnerne y ( ) ; and lanier ( ) . there is an exception to the rule that the class i mhc pathway is devoted to display of peptide antigens from endogenously generated proteins: the so-called professional apcs, dcs, and macrophages can acquire and process exogenous material and present it at the cell surface in class i mhc products, a process called cross-presentation. cross-presentation allows noninfected professional apcs to prime naïve t cells with pathogen-or tumor-derived peptides acquired through endocytosis of infected cells/cell remnants. this ''cross-priming'' is essential for development of cd þ t cell immunity to viruses and tumors in vivo, since only professional apcs can present viral/tumor antigens in the context of class i mhc products without being themselves infected/tumorigenic. there is considerable controversy as to the exact nature of the antigen acquired and modes of antigen acquisition, as well as the intracellular mechanisms leading to cross-presentation and the subsets of apcs endowed with this property (cresswell et al., ; groothuis and neefjes, ; guermonprez and amigorena, ; jutras and desjardins, ; shen and rock, ) . this controversy is, however, beyond the scope of this discussion. the class ii mhc antigen presentation pathway deals with antigens that reside in extracellular space and are internalized into the endolysosomal pathway. all mammalian cells internalize their own cell surface proteins by constitutive endocytosis. in class ii mhc þ cells, this allows class ii mhc access to self-proteins as a source of peptides. professional apcs, such as b cells, macrophages, and dcs, also engage in receptor-mediated endocytosis to acquire extracellular antigen: the antigen from the extracellular milieu is bound by cell surface receptors, internalized, and delivered to the class ii mhc antigen processing machinery. antibodies, complement system factors, and common bacterial or viral components that coat the surface of pathogens or their toxic products bind receptors on apcs that allow them to recognize and internalize this foreign material. of the many receptors used by professional apcs for this purpose, the mannose receptor, which recognizes mannose residues and glycoproteins on viral and bacterial products, and the scavenger receptor, which recognizes very promiscuously many different classes of macromolecules, are among the most important. professional apcs also have complement receptors and receptors for the fc region of antibodies, the fc receptors, which can assist in the acquisition of opsonized antigen and in its delivery to the proper intracellular destination. b cells can also use their surface immunoglobulin or bcr, to acquire antigen (bryant and ploegh, ; cresswell, ; kim et al., c) . class ii mhc loading with antigenic peptides takes place mostly in the endocytic vesicles of professional apcs. class ii mhc ab dimers assemble in the er and associate with the chaperone invariant chain (ii), which inserts its class ii mhc-associated ii peptide (clip) portion in the peptide-binding groove of the ab dimer, preventing its premature (prelysosomal) loading. ii is also important for correct assembly and transport of class ii mhc in the endocytic pathway. further class ii mhc maturation and peptide loading takes place in acidified compartments of the endolysosomal pathway of apcs, since low ph favors an ''open'' conformation of the class ii mhc molecule and hence peptide exchange, as well as the action of specific cysteine proteases that displace ii from the class ii mhc-ii complex, and that of the class ii mhclike molecule hla-dm which facilitates peptide loading. the many hydrolase activities present in the endolysosomal compartments of apcs, such as the ifn-g-inducible lysosomal thiol reductase (gilt), numerous cysteine proteases of the cathepsin (cat) family, like catb, cats, catl, and asparaginyl endopeptidase (aep), produce the peptide ligands that are loaded onto class ii mhc products. peptides bound by class ii mhc molecules are usually - residues long. the end result is a mature class ii mhc-peptide complex at the cell surface, consisting of a class ii mhc ab dimer loaded with peptide, which interacts with the tcr on cd þ t cells. the result of this interaction is dependent on class ii mhc-tcr contacts and also on the context provided by lymphocyte costimulatory molecules at the immunological synapse. the cd þ t cell response may be cytolytic, but generally these antigen-specific cd þ t lymphocytes function as helper cells, releasing cytokines to enhance the overall immune response by inducing macrophage activation, t and b cell proliferation, and b cell differentiation to produce antigen-specific antibodies and different immunoglobulin isotypes with different effector functions (bryant and ploegh, ; chapman, ; cresswell, ; honey and rudensky, ; hsing and rudensky, ; stern et al., ; villadangos et al., ) . all cellular proteins, regardless of their half-life, are subject to turnover. the main pathway for degradation of short-lived proteins in the cytoplasm of eukaryotic cells is the ub-proteasome system (hershko and ciechanover, ) . since the discovery of ub and ub-dependent proteolysis in the late s, it has become increasingly clear that the ub-proteasome system is pivotal to numerous cellular processes: cell cycle control, transcriptional regulation, signal transduction, antigen presentation and induction of the inflammatory response, degradation from the er, membrane trafficking, receptor endocytosis and downregulation, apoptosis, and development (hershko and ciechanover, ; pickart, ) . ubiquitin is a small -amino acid protein, synthesized as a precursor that is processed by deubiquitinating enzymes (dubs) to expose the glycine-glycine sequence at the ub c-terminus, its site of attachment to target molecules. atp-dependent ub activation is catalyzed by the e (ub-activating) enzyme, which adenylates the ub c-terminus, allowing the subsequent formation of a high-energy thioester bond between the glycine residue of ub and the cysteine residue on the e active site. ub is then transferred from the e cysteinyl side chain to a cysteinyl group on one of several e (ub-conjugating) enzymes. finally, one of hundreds of e (ub-ligase) enzymes, binds the ub-e complex and the substrate, thus facilitating the transfer of ub to a lysine residue in the substrate via an amide (isopeptide) bond (hershko and ciechanover, ) . the functions of e ligases, in particular, are tightly regulated by signalinduced mechanisms, such as localization, oligomerization, degradation, and posttranslational modifications, which makes e s the master orchestrators of specificity in the ub conjugation cascade. this multistep mechanism, much like phosphorylation, endows protein ubiquitination with a high degree of specificity and flexibility, which is paramount to its important biological functions (haglund and dikic, ; hershko and ciechanover, ; pickart, pickart, , varshavsky, ) (fig. a) . overview of the ub-proteasome system. (a) ubiquitin-conjugation cascade and how ub chain linkage type and length influence substrate fate. e , ub-activating enzyme; e , ubconjugating enzyme; e , ub-ligase enzyme; and dub, deubiquitinating enzyme. (b) diversity in e ligases. e s play crucial roles in substrate selection and can be regulated by localization, oligomerization, associated e s, posttranslational modifications, and degradation. hrd p and doa p are yeast e ligases that are multispanning membrane proteins of the er and, in the case of doa p, nuclear envelope. the mammalian scf family of e ligases are mainly cytosolic and can recruit substrate adaptor proteins, the f-boxes, with very diverse substrate specificities. we elaborate on ub e ligases to some extent, as they are key players in several aspects of the immune system, including immune evasion (liu, ) and also in er quality control and degradation kostova and wolf, ; romisch, ) , that yields to some of the ligands on class i mhc products. e ligases can be divided into two broad classes: the homologous to e -ap carboxyl terminus (hect)-domain ligases or the really interesting new gene (ring)-like domain ligases. the first hect e described, e -associated protein (e -ap), was shown to be required for ubiquitination and degradation of p , mediated by the human papillomavirus protein e (scheffner et al., ) . in hect e s, ub is transferred from the e to a conserved cysteine residue in the hect domain, followed by attack of this thioester by a lysine on the substrate (pickart, ) . the ring-ch domain is a ring finger motif with a cysteine residue in the fourth zinc-coordinating position and a histidine residue in the fifth. ring-type e s are more abundant and do not form an obligatory thioester intermediate with ub; rather they bring the ub-loaded e s and the substrate into proximity, thus facilitating the ub transfer from the e to the substrate (pickart, ) . ring-type e s can be single subunit e s, which have both a ring-finger domain and substrate recruitment domain(s) on the same protein, like mdm , a key regulator of p . multisubunit e s include the very diverse cullin-ring ligases (crls) (petroski and deshaies, ) . crls are composed of a catalytic core that recruits the ub-loaded e -formed by a nucleating cullin protein and a ring finger protein-as well as a substrate recognition complex. the archetypal crls are the skp- -cullin- -f-box protein complexes or scf e s. the cullin subunit (any one of cullin- , - , - , - a, - b, - , or - ) forms an elongated bent backbone for the multisubunit ligase. the cullin n-terminus binds the s-phase kinase-related protein- (skp- ), an adaptor that recruits any one of a number of substrate-specific adaptor subunits called f-box proteins. the f-box protein is the main determinant in substrate specificity, as it binds the substrate through its particular substrate recognition domain (jin et al., a) , although the ring box protein may participate (jin and harper, ) . the cullin n-terminus binds the catalytic core composed of the ring-box (rbx) protein with its associated ub-loaded e . this arrangement allows the f-box protein to bring its bound substrate close to the ubiquitination machinery of the complex (fig. b) . phosphorylation of the substrate very often regulates the f-box protein-substrate interaction, converting the substrate into a form susceptible to e activity, adding an extra layer of control to the process (joazeiro and weissman, ; schulman et al., ; zheng et al., ) . crls assemble with numerous substrate receptors. cullins and , for example, recruit substrates through suppressor of cytokine signaling/elongin-bc (socs/bc) boxes and form the so-called scf s and scf s complexes. in scf s and scf s, skp- is substituted by the skp- -like protein elongin c which binds the ub-like elongin b that binds the substrate adaptor subunit (petroski and deshaies, ) . many of these e complexes have important roles in the immune system (liu, ; liu et al., ) . the nuclear factor-kb (nf-kb) transcription factor is a master organizer of both innate and adaptive immunity. nf-kb is activated in response to tlr signaling on recognition of pathogen-associated molecules like bacterial peptidoglycan in a process that is crucially dependent on ubiquitination. one of the steps requires the cytosolic scf b-trcp e complex. the scf b-trcp substrate adaptor component is the f-box protein b-transducin repeat-containing protein (b-trcp) that possesses wd repeats that bind to phosphorylated inhibitor of nf-kb (ikb), inducing its ubiquitination and degradation. nf-kb is thus released from the ikb-nf-kb dimer and translocates into the nucleus, activating downstream transcription. the elongin-c-elongin-b-cullin- -socs (ecs) complex uses socs proteins as the substrate adaptors. socs boxes bind janus kinases (jaks), which are recruited and activated in response to ifn and cytokine signaling, promoting ubiquitination and degradation of jaks by the ecs complex. this, in turn, inhibits phosphorylation and activation of the signal transducer and activator of transcription (stat) family of transcription factors that are crucial for the immune response following ifn and cytokine signaling and following viral infections . there are other families of e s with noncanonical ring-domains, like the k homologues and the ufd homologous box (u-box) e s (which we discuss in more detail later). for a more comprehensive review of hect and ring e s and different classifications read (ardley and robinson, ; coscoy and ganem, ; hatakeyama et al., ; petroski and deshaies, ; sharrocks, ) . originally believed to always deliver a ''kiss of death'' and target the substrate for proteasomal degradation, the much more wide-ranging effects of ub conjugation are beginning to be appreciated. chain length and linkage type also influence the outcome of the ub-conjugated substrate. the multiple ub moieties in a polyub chain (chains of or more ub moieties) are linked to one another by an isopeptide bond between a lysine residue on one ub molecule (usually on lys ) and the c-terminal carboxyl group of the next ub on the chain. at times, extension of a polyub chain on a substrate conjugated with - ''initiator'' ub moieties requires a special subclass of e s, the ufd -homology box (u-box) e s (once called e s) (hoppe, ) . targeting of proteins for proteasomal proteolysis generally requires polyubiquitination in a lysine (lys) -type linkage. by contrast to polyub, substrate monoubiquitination or attachment of noncanonical ub chains-ub chains with non-lys linkages such as lys and lys linkages-usually have nonproteolytic functions in dna repair, endocytosis, signal transduction, transcriptional regulation, and ribosoma l function (d' azzo et al., ; pic kart and ed dins, ) . monoubiquitination can occur on a single lysine residue or on several lysine residues in a substrate (multiubiquitination). monoubiquitination is extremely important as a sorting signal in the endocytic pathway. for example, monoub attachment is sufficient to induce endocytosis of growth hormone receptor and sorting to the lysosome for degradation (hicke, ; hicke and dunn, ) . direct modification of the cargo (cis-regulation) or modification of the proteintrafficking machinery (trans-regulation) by monoub could have many consequences for antigen presentation, as these processes rely heavily on events that take place in endolysosomal compartments. noncanonical ubiquitin chains play many diverse roles in signaling pathways, in dna replication and postreplication dna repair, and modulating protein-protein interactions. for instance, activation of nf-kb is tightly regulated by a balance between lys -and lys -mediated ubiquitination of different components of the nf-kb pathway. triggering of many cell-surface receptors leads to assembly of signaling complexes that recruit tumor necrosis factor receptor-associated factor (traf ), an e ligase that binds ubc , promoting lys -linked polyubiquitination of the g subunit of the inhibitor of nf-kb kinase (ikk) complex, ikkg. this leads to activation of the ikk complex, which in turn results in ikb phosphorylation. phosho-ikb then recruits the scf complex that catalyzes lys -linked polyubiquitination of ikb and consequently activates nf-kb (karin and ben-neriah, ) . for comprehensive reviews see pickart and eddins, ; varshavsky, ) . ubiquitin-like molecules or modifiers (ubls) share structural homology with ub and can also be conjugated onto protein substrates, mostly with outcomes other than proteasomal degradation. ubls like the small ubiquitin-like modifier sumo, neuronal precursor cell-expressed developmentally down-regulated (nedd ) or ifn-stimulated gene product of kda (isg ), to name but a few, are implicated in important physiological processes like nuclear transport, maintenance of chromosome integrity, transcriptional regulation, cell cycle control, signaling and regulation of proteolysis (hochstrasser, ; schwartz and hochstrasser, ) . ubls may regulate ub-mediated proteolysis or signaling through comodification of a substrate, thus modulating the effects of ub conjugation (lamsoul et al., ; sobko et al., ) or by regulating the activity, specificity, localization, or stability of enzymes in the ub-conjugating cascade, as is the case for nedd modification of cullin-ring e s (kawakami et al., ; petroski and deshaies, ; wu et al., ) . deubiquitinating enzymes can cleave isopeptide bonds to remove ub from the substrate or from polyubiquitin chains. there are about dubs in the human genome, organized in five classes according to their catalytic domain structure: the ubiquitin-specific proteases (usps), the ubiquitin c-terminal hydrolases (uchs), the machado-joseph disease proteases (mjds), the ovarian tumor proteases (otus), and the jab /mpn/mov proteases (jamms). the first four classes comprise cysteine-type proteases, whereas jamms are metalloproteases. for a more detailed inventory of dubs, read nijman et al. ( ) . dubs have very diverse specificity properties, in terms of the ubiquitin or ubl moiety itself (substrate specificity), in terms of the target protein to which the ub or ubl is attached (target specificity), and possibly in terms of the context provided by target and attached modification. dub specificity in vivo can be further regulated by subcellular localization or association with different binding partners (amerik and hochstrasser, ; li and hochstrasser, ; reyes-turcu et al., ; soboleva and baker, ) . dub functions are therefore also extremely diverse, ranging from regulation of proteasome function, to regulation of chromatin structure, to membrane protein trafficking, and with obvious implications in processes such as cancer and neurodegeneration (amerik and hochstrasser, ; nijman et al., ; soboleva and baker, ) . the proteasome, very abundant in the cytosol, is a multisubunit protease composed of the s and s proteasome complexes. the s proteasome (or central core particle) has the general architecture of a barrel, formed by four stacked rings of seven subunits each, the outer two rings being composed of a subunits and the innermost two rings of b subunits (groll et al., ) . the b subunits, which line the proteasome's inner cavity, carry out the catalytic activity. for mammalian proteasomes, only three of the seven b subunits in each ring are catalytically active. access to this cavity occurs through narrow pores (with a diameter on the order of - Å ) at both ends of the barrel, so it is usually assumed that protein substrates must be unfolded prior to their delivery to the catalytic chamber (groll et al., (groll et al., , kohler et al., ) . also at both ends of the core particle there is the s cap complex, whose functions range from recognition of poly-ub chains on target proteins, to unfolding of the substrate to facilitate entry into the catalytic cavity, to deubiquitination activity (adams, ; heinemeyer et al., ; rivett et al., ; schmidt et al., ; seeger et al., ) . as mentioned earlier, the proteasome plays an instrumental role in class i mhc antigen presentation and activation of peptide-specific cd þ t cell responses. this process requires not only generation of peptides of the right quality-that is, right size and sequence to allow a correct fit into the peptidebinding cleft-but also in the right quantity to trigger a successful response, which is no small endeavor due to many destructive aminopeptidase activities in the cytosol (shastri et al., ; strehl et al., ) . ifn-g, a crucial component of the innate and adaptive antiviral immune responses, affords the immune system a competitive edge. ifn-g induces expression of auxiliary b subunits, b i, b i, and b i [also known as low molecular weight protein (lmp ), lmp and multicatalytic endopeptidase-like complex (mecl ), respectively], as well as synthesis of the proteasome activator pa , and of the proteasome maturation protein (pomp) (strehl et al., ) . the immunosubunits lmp , lmp , and mecl are incorporated into nascent proteasomes, replacing their endogenous counterparts and constituting the so-called immunoproteasome. the proteasome activator pa (or s proteasome) binds to the outer rings of the s proteasome, thereby opening the central gate and facilitating substrate entry. pomp is important for assembly and maturation of the proteasome (strehl et al., ) . this ifn-g-induced proteolytic cascade, mediated by immunoproteasomes and pa , might be induced to respond to a demand for high proteasome activity when the constitutive cascade is no longer sufficient, altering the proteolytic activity of the proteasome for maximal efficiency in production of the class i mhc peptide repertoire. ifn-g treatment also activates a transcriptional program that increases the synthesis of class i mhc molecules themselves and that of components of the peptideloading complex, thus increasing cell surface presentation (kloetzel, ; kloetzel and ossendorp, ; kruger et al., ; rivett and hearn, ; van den eynde and morel, ) . therefore, even though class i mhc presentation is constitutive, it can be modulated in the course of an immune response, with a proposed role in the early stages of a cytotoxic response. it is thus not surprising that viruses have targeted the ifn-g signaling cascade so aggressively (alcami and koszinowski, ; hengel et al., ; salazar-mather and hokeness, ) . although tightly controlled, er protein synthesis is not always successful. proteins may sustain damage or fail to complete their synthesis early during biogenesis, or be trapped in an irreversible nonnative conformation, or a mutation may result in a structural alteration that leads to misfolding, as is the case for the cystic fibrosis conductance regulator (cftr) (jensen et al., ; ward et al., ) , mutant plasma a -antitrypsin (teckman and perlmutter, ) , or tyrosinase (halaban et al., ) . they may also be expressed in the absence of their cognate subunits, as is the case for unassembled subunits of tcra (huppa and ploegh, ; yang et al., ) . er quality control is a homeostatic process that involves an elaborate machinery that recognizes and retains newly synthesized misfolded or misassembled proteins and targets many of them for degradation by the ub-proteasome system (ellgaard and helenius, ) . this mode of degradation therefore samples sets of proteins otherwise targeted to extracellular space, where the degradation products are available as peptide ligands for class ii mhc products. in addition to its role in er quality control, this er-associated degradation (erad) can also be employed in the physiological regulated proteolysis of normal er proteins whose degradation is subject to metabolic cues, such as hydroxymethylglutaryl-coenzyme a reductase (hmgr) (hampton, ; hampton and bhakta, ) . a feature of this er-associated protein degradation is the spatial separation between targeting of substrates and their proteolysis, which requires substrate export from the er lumen or membrane to the cytoplasm by a process termed dislocation (also called retrograde translocation or retrotranslocation) (werner et al., ) . dislocation is a complicated multistep process that involves substrate recognition, targeting for dislocation, removal from the er membrane, deglycosylation, ubiquitination, and finally proteolysis (kostova and wolf, ; meusser et al., ; romisch, ) . the proteasome is usually considered to be a nonselective degradation apparatus, with selection of erad substrates being mediated mostly by the ub ligases. however, the er quality control e enzymes are mostly cytosolic or membrane-associated and thus are separated from their substrates at least by the er membrane. this invokes the existence of mechanisms, present in e ligases themselves or in upstream factors, which facilitate coupling of erad substrate recognition to ubiquitination by e ligases in the cytoplasm. owing to the extremely diverse nature of proteins that must be examined by the er quality control machinery, a unifying model for how recognition of erad substrates takes place remains intractable. nonetheless, misfolded proteins cleared from the er enter the class i mhc-processing pathway, and hence this route of degradation is an important aspect of the generation of class i mhc epitopes. instead, erad is likely to be custom-fitted to the client protein in question. for glycoproteins, a possible mechanism is the recognition of terminally misfolded proteins by the calnexin/calreticulin (cnx/crt) lectin-type chaperones, which retain immature glycoproteins in the er until productive folding takes place (ellgaard and helenius, ; hammond, ; helenius and aebi, ) . terminally misfolded proteins-that is, proteins that after extensive cxn/crt cycle folding attempts still fail to acquire their native conformation-are trimmed by er mannosidase i, leading to recognition by er degradation-enhancing alpha-mannosidase-like protein (edem), which presumably targets them for degradation (eriksson et al., ; jakob et al., ; molinari et al., ) . most er lumenal proteins require the lumenal chaperone bip for degradation, while transmembrane proteins with large cytosolic domains usually rely on cytosolic chaperone systems, like the heat shock protein (hsp) complexes hsp /hsp and hsp /hsp (ellgaard and helenius, ; romisch, ) . yet it appears that a protein's lumenal or er membrane localization matters less than the localization of the folding alteration within the polypeptide itself. cftr whose cytoplasmic domains are recognized first by the hsp /hsp cytoplasmic chaperone system may be targeted for degradation by the cytosolic hsp /hsp -interacting chip e ligase cochaperone (connell et al., ; murata et al., ) , even if the protein also has a misfolded lumenal domain (which might also target it to a bip-dependent degradation pathway). if the cytoplasmic domain is properly folded, then the lumenal domains are inspected, and if then the protein is recognized as misfolded, it is degraded in a process that involves bip (connell et al., ; meacham et al., ; vashist and ng, ) . this suggests that er quality control uses sequential checkpoints to select degradation substrates and target them to the appropriate degradation pathway. in the case of nonglycosylated substrates, protein disulfide isomerase (pdi), one of a large number of er-resident oxidoreductases that catalyze disulfide bond formation and isomerization, can play a role in er quality control by unfolding certain substrates prior to degradation. another oxidoreductase, erp , interacts with cnx and crt to facilitate folding, but in the event of a terminally misfolded protein may aid in transfer of proteins with improper disulfide bonds to the edem pathway . another possibility, at least in yeast, is that misfolded proteins actually escape to the golgi and are then recycled to the er vashist et al., ) . this er-golgi shuttling model was proposed because mutations in several secretory pathway genes (like ufe p, sec p, and erv p) compromise degradation of erad substrates vashist et al., ) , invoking a functional secretory pathway for efficient degradation of misfolded proteins from the er. the golgi apparatus could presumably endow misfolded proteins with a signal for destruction, but such a modification has not been found. since the ufe p and sec p have since been shown to be required for maintenance of proper er structure (prinz et al., ) , the effects on protein degradation may simply be pleiotropic consequences of perturbing the normal arquitecture of the er (hammond et al., ; romisch, ) . ubiquitination at the er membrane is yet another mode of erad substrate selection. most erad e s are at the er membrane, as is the case for the yeast hrd p/ der p and doa p, or can be brought to the er membrane on demand, as is the case for cytosolic scf complexes and chip, for example (kostova and wolf, ; meusser et al., ; romisch, ). an e ligase can recruit distinct e ub-conjugating enzymes and/or distinct adaptor proteins (as we have seen for the scf and ecs e complexes and will discuss later for erad e s), thus conferring specificity in substrate selection. the yeast hrd p/der p e ligase was first discovered in a genetic screen for saccharomyces cerevisiae genes involved in hmg-coa reductase degradation (hrp) (hampton et al., ) and is an er-resident protein with six predicted transmembrane domains and a c-terminal ring-finger motif facing the cytosol. hrd p can act in a complex with ubc p and ubc p to ubiquitinate substrate proteins ( fig. b ). ubc p is a soluble protein that becomes active only when tethered to the er membrane by the ubc p cofactor cue p membrane protein. degradation of transcription factor mata - protein (doa p) is a transmembrane protein of the er/nuclear envelope, which participates in yeast erad (swanson et al., ) . doa p is predicted to span the membrane times and, like hrd p, uses cue p/ubc p and ubc p to ubiquitinate its substrates (fig. b) . however, the two ligases target different sets of substrates for degradation (bays et al., ; swanson et al., ) . hrd p ubiquitination activity can be directed to a specific subset of er degradation substrates, by virtue of its association of hrd p with hrd p and der p. hrd p is a single-spanning er membrane protein with a large er luminal domain that can recognize misfolded proteins, thus possibly functioning as a substrate recruitment factor for the hrd p ligase complex (gauss et al., ) . the degradation from the er- protein (der p) spans the er membrane four times and is required for degradation of some misfolded glycoproteins (knop et al., ) . der p may function as a substrate adaptor protein or even as a channel for ejection of degradation substrates from the er membrane. hrd p can associate with der p, presumably enabling substrate delivery to downstream components. hrd p can even regulate hrd p activity, which is necessary for substrate extraction from the membrane and delivery to the proteasome (gauss et al., ) (fig. ) . these multifunctional protein complexes can therefore function in substrate selection in the er lumen or membrane and even facilitate subsequent steps that lead to proteasomal degradation. similarly, doa p can catalyze ubiquitination of both membrane and soluble proteins, yet the mechanisms of subsequent proteasome targeting differ (ravid et al., ) , presumably due to association with other regulatory proteins. the many layers of e mediated regulation of erad substrate selection are most likely just emerging. in mammals, there are two predicted homologues of hrd p/der p, the hrd and the gp e ligases. like its yeast counterpart, human hrd is involved in degradation from the er (kikkert et al., ) . hrd may associate with ubc to catalyze ubiquitination of a subset of substrates, like tcra and cd d. hrd is not involved in the regulated degradation of the mammalian hmgr (kikkert et al., ) . hrd may also associate with sel l, a homologue of hrd p, as well as other er membrane and er membrane-associated proteins, including cdc p(p )/npl /ufd , forming multisubunit complexes that seem to coordinate steps that range from substrate selection to delivery to the proteasome for at least a subset of er degradation substrates (lilley and ploegh, a; ye et al., ) . gp was identified as the tumor autocrine motility factor receptor (amfr) (nabi et al., ) and later as an e ligase due to its homology to hrd p and involvement in degradation of cd d and apoliprotein b (fang et al., ; liang et al., ) . gp is an er-resident protein, predicted to span the membrane five times, with a c-terminal cytosolic ring-domain and an additional ubc e -binding site, the cue domain, arranged in tandem. while hrd p recruits ubc through the transmembrane cue p protein, it seems that convergent evolution has made the e -and the e -docking protein come together in a single human protein. curiously, gp is involved in sterol-regulated ub-dependent degradation of hmgr (song et al., ) , suggesting it constitutes the true functional homologue of yeast hrd p. homocysteine-induced endoplasmic reticulum protein (herp) is a singlespanning er membrane protein that is induced by the unfolded protein response (upr) and also required for erad. herp is proposed to improve er protein folding and decrease protein load, protecting cells from er-stress-induced apoptosis (kokame et al., ) . furthermore, herp has an n-terminal ubiquitin-like domain (uld) and is required for the degradation of conexin and cd d (hori et al., ; sai et al., ) . it forms a complex with hrd , p , derlin- , and vimp (schulze et al., ) , a vcp (p )-interacting membrane protein, that recruits p to derlin- (ye et al., ) . herp may function as another adaptor protein in these multiprotein complexes at the er membrane, influencing substrate selection. the doa p mammalian homologue, teb (or march-vi), is a multipletransmembrane-domain-containing protein of the er membrane that functions as an e ligase: it has an n-terminal noncanonical ring-domain in the cytosol that catalyzes ub conjugation and teb self-ubiquitination and degradation (hassink et al., ) , but its regulation is poorly characterized. parkin is a cytosolic e ligase with a c-terminal noncanonical double-ring-finger (ring-ibr-ring), and an n-terminal ub-binding domain, believed to mediate proteasomal degradation of aggregation-prone proteins (imai et al., ) , typical of parkinson's disease. both phosphorylation (yamamoto et al., ) and er stress-induced association with c-terminus of hsc -interacting protein (chip), involved in cytosolic chaperone-dependent folding, regulate parkin e ligase activity (imai et al., ; sahara et al., ) , which could be beneficial for reduction of protein aggregates and cellular pathology. how specificity in substrate selection is conferred can be illustrated by e s involved in glycoprotein turnover. the f-box proteins fbs and fbs bind high mannose n-linked glycoproteins (winston et al., ) . by using fbs / fbs as its substrate adaptor(s), the cytosolic scf fbs ,fbs e ligase is rendered specific for glycoproteins that have been dislocated from the er (yoshida et al., (fig. a) . the chip u-box e ligase which is involved in the degradation of cftr (meacham et al., ) and glucocorticoid hormone receptor (meacham et al., ) , can be ''manipulated'' to function in er glycoprotein turnover. chip usually serves as a cochaperone for the cytosolic heat shock protein hsp /hsp chaperone system. the chip n-terminal tpr motif recruits hsp chaperones loaded with misfolded proteins, whereas its c-terminal u-box ring domain recruits e enzymes (murata et al., ) (fig. b) , effectively linking protein folding with ubiquitination. the chip e ligase activity can be directed to er glycoprotein turnover by binding to fbs (nelson et al., ) , through an interaction between its tpr motif and a pest motif in fbs (fig. c ). n-linked glycans can, therefore, function in er quality control not only to regulate er retention in the folding cycle, but also to function in erad substrate selection and ubiquitination, adding an additional layer of complexity and specificity to glycoprotein quality control (nelson et al., ; yoshida, ) . export of proteins through the er membrane most likely takes place via an aqueous channel that allows the passage of polypeptides through the highly hydrophobic er membrane environment while maintaining proper ionic balance between the er and the cytoplasm. the sec channel, the very same channel responsible for protein import into the er, was initially thought to mediate transport in the reverse direction (hence the name retrograde translocation or retrotranslocation also coined for the dislocation process) (pilon et al., ; plemper et al., plemper et al., , wiertz et al., b) , with accessory factors regulating directionality and specificity of the channel. the extent to which sec is involved in er dislocation or the identity of the ''dislocon'' are not without controversy, and the search for a dislocation channel(s) is a subject of intense research (meusser et al., ; romisch, ) . mammalian derlin- , a member of the der p-like (derlin) family of yeast der p homologues, is involved in dislocation from the er (lilley and ploegh, ; ye et al., ) and was proposed to constitute a channel for protein export from the er membrane to the cytosol (lilley and ploegh, ; ye et al., ) . like their yeast homologue, derlins , , and are tetraspanning er membrane proteins that can homo-and heteroligomerize and could presumably form higher order structures with channel-like properties (lilley and ploegh, ; ye et al., ) . conclusive evidence for a role of derlin- as a channel is still unavailable, and in any case, derlin- is unlikely to be the only channel, as turnover of some erad substrates does not rely on derlin- function lilley and ploegh, ) . derlins and are obvious candidates that could function in place of derlin- . alternatively, derlins may act to deliver a particular substrate to a channel/another adaptor in its cognate dislocation pathway. in fact, derlins form a large, multiprotein complex with p and the hrd p and hrd p mammalian homologues hrd and sel l, respectively (lilley and ploegh, a; ye et al., ) , suggesting a very intimate connection between substrate recognition, export through the membrane, ubiquitination, and extraction into the cytoplasm. the existence of such a complex that would integrate all of these different functions, including formation of a channel or dislocon, would offer obvious advantages in terms of control of both specificity and directionality of the dislocation process. we shall return to this substrate ''guidance'' theme. a cytosolic complex containing the aaa atpase cdc p (yeast) [valosin-containing protein (vcp)/p (in mammals)] and its cofactors nuclear protein localization (npl p) and ubiquitin-fusion degradation (ufd p), was recently shown to participate in er degradation (lord et al., ; romisch, ; ye et al., ) . cdc p/p is an essential protein of the aaa atpase (atpases associated with various cellular activities) family, conserved from archaea to mammals, whose functions include mitotic spindle disassembly, membrane traffic and fusion, nucleic acid repair and replication, and ubproteasome degradation (woodman, ) . cdc p/p is a motor protein that generates energy from atp binding and hydrolysis; it forms a homohexameric barrel structure, with each subunit containing two aaa domains that contain the walker motifs essential for atpase activity, and the subunits arranged in a ring with a pore in the center (zhang et al., ) . cdc p/p interacts with many different adaptor proteins, which regulate its function (dreveny et al., ) . p can recognize denatured proteins nonspecifically (thoms, ) and has an affinity for polyubiquitin chains . when complexed with the polyub-binding ufd p and npl p, cdc p/p activity is directed to er degradation (ye et al., ) . both in yeast and in mammals, the trimeric cdc p(p )/npl /ufd complex is proposed to function in a postubiquitination, preproteasomal step (bays and hampton, ; jarosch et al., ) , in one of two fashions: the atp-hydrolytic activity of the aaa atpase p may provide the driving force to extract substrates through the er membrane, or may be required to liberate already dislocated substrates from the cytosolic face of the er membrane (braun et al., ; flierman et al., ; hirsch et al., ; kostova and wolf, ; meusser et al., ) . the proteasome presumably interacts with the er membrane (hirsch and ploegh, ) , either directly or through a receptor that docks the proteasome to the er membrane, perhaps sec (kalies et al., ) . notwithstanding, the cdc p(p )/npl /ufd complex or other accessory factors might aid substrate feeding to the proteasome (hartmann-petersen and gordon, a; richly et al., ) . ubiquitin-binding factors, such as rad p and dsk p (in yeast), have a ubiquitin-associated (uba) motif that binds polyubiquitin chains and a ubiquitin-like (ubl) motif that binds to the s proteasome, and these are required for efficient degradation of a model erad substrate (elsasser et al., ; schauber et al., ; wilkinson et al., ) . the yeast ub regulatory x domain-containing ubx p/sel p protein, an integral er membrane protein, was recently shown to recruit the cdc p/npl p/ufd p complex to the er membrane, thereby facilitating the transfer of polyubiquitinated substrates from the e ligases hrd p and doa p to cdc /p (neuber et al., ; schuberth and buchberger, ) . these dual function ub-binding factors effectively serve as bridges between the p /npl /ufd complex and the proteasome. as more of these ub-and proteasome-binding proteins are discovered (buschhorn et al., ; decottignies et al., ; medicherla et al., ; mullally et al., ) , a ''guidance'' model, in which erad substrates are escorted from a dislocation channel to the proteasome by a cascade of ub-binding factors, gains strength (hartmann-petersen and gordon, b; hartmann-petersen et al., ; hendil and hartmann-petersen, ; richly et al., ) . these interactions could be responsible for maintaining the substrate in a proteolysis-competent state and protect it from premature deubiquitination, as well as contribute to directionality of dislocation (hendil and hartmann-petersen, ; meusser et al., ; romisch, ) . in fact, it seems cdc p/p may even be capable influencing substrate fate (rumpf and jentsch, ) . cdc p/p can simultaneously bind ufd p, a u-box e that catalyzes polyubiquitin chain extension, and one of two factors that can counteract its action: otu p, a dub, and ufd p protein, a wd repeat protein of unknown function that has been shown to be required for ub-dependent proteolysis (ghislain et al., ; johnson et al., ) . otu p can disassemble the polyub chains, whereas ufd p competes with ufd p for the same docking site on cdc p/p . presumably, cdc p/p can selectively recruit different substrate processing cofactors and thus tip the balance toward substrate degradation or release from the degradation cascade (rumpf and jentsch, ) , suggesting a very tight regulation of proteasomal proteolysis. these aspects are important not only to understand how these pathways contribute to class i mhc-peptide epitope presentation, but also how viruses manipulate these routes to avoid detection. pngase is a cytosolic deglycosylating enzyme that presumably removes n-linked glycan chains from misfolded substrates prior to proteasomal degradation (hirsch et al., ; suzuki et al., ) . both in yeast and mammals, there is generally a tightly knit relationship between pngase and the proteasome: pngase interacts with (at least) the s and s subunits of the mammalian s proteasome and the ub-binding factor rad p (hr b in mammals), which seems to recognize only deglycosylated degradation substrates (katiyar et al., ) . this suggests that misfolded protein substrates may first be deglycosylated by er-associated or free pngase, then identified by the hr b adaptor protein, and subsequently targeted to the nearby proteasome (katiyar et al., ) . mammalian pngase also associates with the er membrane gp e ligase and the cytosolic p and y k, a uba/ubx domain protein (li et al., a) . a gp -y k-p -pngase-hr b complex could therefore be formed that recruits pngase to the cytosolic face of the er membrane that couples the activities of dislocation, ubiquitination, and deglycosylation and escorts misfolded glycoproteins to the proteasome (kim et al., a; li et al., , a . viruses keep evolving and developing sophisticated immune evasion strategies. in particular, they have targeted virtually every step of the class i mhc antigen presentation pathway, inhibiting proteolysis and generation of the antigenic peptide [epstein-barr virus (ebv) nuclear antigen- or ebna- , hcmv e protein pp , and hiv tat], inhibiting peptide loading and assembly in the er (hsv icp , hcmv us , bovine herpes-virus- ul . ), retaining class i mhc molecules in the er (adenovirus e / k and hcmv us ), blocking their exit from the er-to-golgi complex (ergic) (mcmv m ), misdirecting mhc complexes to lysosomal compartments (mcmv m and hhv- u ), internalizing mhc complexes from the cell surface (kshv k and k and hiv nef ), encoding homologues of class i mhc as decoys for nk cells (hcmv ul and ul and mcmv m ), and causing degradation of class i mhc products by the ub-proteasome system (hcmv us and us and mhv- mk ) (fig. ). because these topics have been the subject of numerous reviews (alcami and koszinowski, ; ambagala et al., ; hengel et al., hengel et al., , lybarger et al., ; mocarski, ; yewdell and hill, ) , we shall discuss only a few of these mechanisms in more detail, particularly those exploited by hcmv. the b-herpesvirus hcmv is extremely successful in evolutionary terms: it is a ubiquitous, highly species-adapted pathogen that is able to establish a life-long persistent infection with minimal or no disease symptoms in the immunocompetent host. prolonged latency periods (a dormant state with minimal production of viral proteins and absence of viral progeny) and controlled sporadic reactivation ensure transmission to a new host, and thus survival of both host and virus. perturbation of this delicate balance leads to life-threatening infections in immunocompromised patients, transplant recipients and infected newborns and illustrates how the outcome of this host-virus relationship is dependent on viral manipulation of the host immune response (hengel et al., ; klenerman and hill, ) . the several hcmv-encoded immunoevasins (jones et al., ) are presumably aimed primarily, but not solely, at control of the cd þ t cell and nk cell responses (falk et al., ; mocarski, ; pinto and hill, ; yewdell and hill, ) . here we will discuss the hcmv immunoevasins that interfere with class i mhc antigen presentation, us , us , us , us , us , ul , ul , ul , ul , and ul . in light of our most recent findings, we will elaborate on the mechanism of er dislocation co-opted by the hcmv us and us immunoevasins. more specifically, we discuss the similarities and the differences between the two cellular erad pathways that us and us have allowed us to uncover and the possible implications for er dislocation. we will extend this by comparing the hcmv us -and us -mediated dislocation of human class i mhc hc molecules with dislocation of murine hcs by the mhv- mk immunoevasin. if one goes back to the steps we depicted for antigen presentation (fig. ) and then examines the immunoevasins encoded by hcmv, we will find that this herpesvirus exploits many aspects of the antigen presentation pathway thus defined. the hcmv phosphoprotein pp tegument protein mediates the phosphorylation of the hcmv immediate early antigen- (ie- ) during hcmv infection. phosphorylation of ie- interferes with the presentation of ie- -derived antigens (gilbert et al., ) . the us protein binds to and retains some class i mhc locus products in the er membrane (ahn et al., ; jones et al., ) . us is a type i membrane glycoprotein with an ig-like lumenal domain that is essential for its own retention, albeit transient, in the er (lee et al., ) . us eventually travels to the lysosome where it is degraded (gruhler et al., ) . some evidence suggests that er retention of class i mhc complexes by us depends on the er localization signal on us and perhaps the ability of the us luminal domain to oligomerize (misaghi et al., b) as determinants of retention of both molecules (lee et al., ) . another model suggests that association of us with tapasin, which inhibits tapasin, is sufficient to mediate er retention (park et al., ) . class i mhc alleles that require peptide optimization by tapasin may be retained in the er, whereas tapasin-independent locus mhc products are spared from retention. in fact, there is a perfect correlation between tapasin-dependence and us sensitivity (park et al., ) . both mechanisms, tapasin inhibition and direct binding, may be in place, perhaps allowing us to retain a larger repertoire of class i mhc locus products. us inhibits peptide loading of the class i mhc molecules by blocking the tap transporter (ahn et al., ; lehner et al., ) . us is an er-resident type i membrane glycoprotein with a bulky lumenal domain that binds the core transmembrane domains of the tap subunits, tap and tap , from within the er lumen, inhibiting atp binding (hewitt et al., ; kyritsis et al., ) and thus tap-mediated peptide translocation into the er. the us luminal domain oligomerizes and may form a bridge between the tap and tap subunits to effectively block tap activity (halenius et al., ) . us delays trafficking of class i mhc hcs and stalls them in the er; although the block is not absolute and the mechanism is poorly characterized, us expression results in downregulation of class i mhc from the cell surface (furman et al., a) . us and us catalyze destruction of class i mhc hcs from the er membrane by targeting them to the ub-proteasome system (wiertz et al., a,b) , a process we discuss in detail in a later section. expression of each individual immunoevasin results in reduction of cell surface expression of class i mhc peptide-loaded complexes and evasion of cd þ t cell-mediated lysis (ahn et al., ; jones et al., ) . besides cd þ t cell recognition, hcmv can frustrate nk cell recognition (orange et al., ) . protection of hcmv from nk cell-mediated lysis can be mediated by several immunoevasins, hcmv ul , ul , ul , ul , ul , and pp (lodoen and lanier, ; orange et al., ; rajagopalan and long, ; reyburn et al., ; wills et al., ) . the activating receptor nkg d on the nk cell recognizes divergent families of class i mhc-related ligands, like the mic and ulbp products. hcmv ul retains the ulbp , ulbp , and mic-b nkg d ligands in the ergic compartment of the target cell, preventing nkg d recognition and thus nk cell activation . hcmv ul and pp act at the level of the nk effector cell rather than the apc. the result is, nevertheless, the same: prevention of nk cell activation. ul downregulates the nk cell-activating receptors cd (dnam- ) and cd (tactile) . the pp tegument protein engages the activating receptor nkp and antagonizes its effects, dampening nk cell-mediated cytotoxicity. how a tegument protein gains access to the receptor on the nk cell is unknown, but pp engagement of the nkp receptor causes dissociation of a receptor-associated signaling module (orange et al., ) , disrupting the activating signaling pathway that would lead to nk cell activation (arnon et al., ) . nk cell responses also rely on receptors that recognize class i mhc locus products. the killer cell immunoglobulin-like receptor (kir) genes encode a family of activating and inhibitory receptors that recognize human leukocyte antigen (hla)-a, -b, and -c. the cd /nkg receptors recognize the nonclassical class i mhc molecule hla-e. hla-e presents fragments derived from the signal sequences of classical class i mhc molecules, which delivers an inhibitory signal to cd /nkg receptors on nk cells. ul encodes a peptide whose sequence is exactly homologous to the hla-e binding leader peptide from hla-c locus products. ul therefore loads hla-e and maintains hla-e on infected cells (tomasec et al., ; ulbrecht et al., ) even when other class i mhc products are downregulated. ul encodes a class i mhc-like molecule that engages the inhibitory cd j/lir- /ilt- receptor on the nk cell, thus inhibiting nk cell effector functions (cosman et al., ; reyburn et al., ) . however, ul and ul expression may be insufficient to confer target cell protection (falk et al., ; leong et al., ) . ul and ul may, in fact, be more relevant for control of viral infection by t cells, with hla-e-restricted cd þ t cells playing a role in lysis of cells expressing ul , and non-mhcrestricted cd þ t cells playing a role in lysis of ul -positive cells (pietra et al., ; romagnani et al., ; saverino et al., ) . cd j is an invariant receptor expressed by many t cells and responsible for transduction of inhibitory signals that downregulate antigen-specific t cell functions (merlo et al., ; saverino et al., ) . cd j interaction with ul on cd þ t cells occurs in a tcr-independent manner and leads to activation (not inhibition) of non-mhcrestricted cd þ t cells (saverino et al., ) . this expands the repertoire of t cell activation mechanisms and is probably a viral strategy of ensuring survival of the host, by allowing some level of protection from the initial wave of nk cellmediated antiviral response. in vivo hcmv-infected apcs are faced with multiple immunoevasins displaying allelic preferences and expression patterns that are both spatially and temporally regulated. the many immunoevasins expressed by hcmv are thus likely to have both synergistic and antagonistic interactions (ahn et al., ; farrell et al., ; klenerman and hill, ; mocarski, ; reddehase, ; yewdell and hill, ) , as has been experimentally verified for murine cytomegalovirus (wagner et al., ) . evidence of er-to-cytosol transport or dislocation, a crucial er quality control step now considered of general importance in dispensing with misfolded or misassembled er proteins, was initially provided by studying the mechanism of action of the hcmv us and us immunoevasins (wiertz et al., a,b) . the viral proteins appropriate this cellular quality control process to extract (dislocate) class i mhc hcs from the er membrane. on arrival in the cytoplasm, the dislocated hc molecules are destroyed by the proteasome; destruction of the hc component of the class i mhc complex by us and us abolishes cell-surface expression of class i mhc complexes and, consequently, presentation of viral peptides to cd þ t cells, allowing hcmv to remain undetected (wiertz et al., a,b) . us -and us -mediated hc dislocation from the er membrane is not only an ingenious viral immune evasion strategy, but also a useful case study in er quality control and degradation. one theme that arises from the characterization of this process over the years that have passed since its discovery is that hc dislocation is unique in many respects: hc molecules do not meet the requirement of being either misfolded or misassembled, yet their dislocation takes place by virtue of the presence of us or us ; the speed of hc degradation is unrivaled by that of any other er-associated degradation substrates: hc half-life is reduced from hours to a mere - min in cells infected by hcmv or in ce lls expressin g either us or us ( wiertz et al ., a ,b) ; both us and us have stringent requirements in terms of which hla alleles (barel et al., (barel et al., , machold et al., ) or assembly, folding and ubiquitination status of the class i mhc complex (blom et al., ; furman et al., ; gewurz et al., ) either viral protein is able to target for dislocation and proteasomal destruction. notwithstanding the unique nature of this virus-mediated process, knowledge from the us pathway, and in particular the identification of the derlin proteins, has widened our understanding of the cellular factors involved in er dislocation more generally ploegh, , a) . the us transmembrane domain (tmd) is crucial for us function: more specifically, mutation of a polar amino acid, glutamine (q) , within the us tmd to a hydrophobic leucine (l) residue renders this us q l mutant inactive in dislocating hcs from the er membrane. in a screen for proteins that interact specifically with the active version of us , work from our laboratory showed that the aforementioned derlins, the mammalian der p homologues, are involved in hc dislocation mediated by us , but not by us (lilley and ploegh, ) . the fact that the dislocation mechanism used by us is not dependent on the derlins prompted us to investigate what other erad pathw ay is being coopted by the hcmv us immu noevasin and allow ed us to unco ver an unexpect ed era d pl ayer. us is an er-resident type i membrane glycoprotein of only amino acids, with a noncleavable signal sequence (gewurz et a l., ) , a lumenal domain that dictates an allele-specific association with the lumenal domain of hc (gewurz et al ., ) , a transmembrane segment, and a short cytosolic tail of only amino acids (residues - ), with no obvious sequence homology to known cellular proteins. the us tail is essential for dislocation: us , a cytosolic tail deletion mutant of us , is dislocation incompetent (furman et a l., b) . by using an affinity purification approach similar to that used for us (lilley and ploegh, ) , signal peptide peptidase (spp) was found as a specific interacting partner for dislocation-competent (active) us (loureiro et a l., ) , an interaction that relies solely on the presence of the highly hydrophobic us tail. more importantly, reduction of spp levels by rna interference led to inhibition of class i mhc hc dislocation and to the discovery of spp as a necessary factor for the us -mediated er dislocation pathway. spp is an er -resi dent protein o f ap proximate ly kd a tha t is predic ted to span the er memb rane seven to nine times ( friedmann et al ., ) , an d a memb er of the presenili n (ps)/sp p-like (sppl ) superfam ily of intrame mbran e-cleaving aspartic protea ses ( weihof en et al ., ) . these protea ses are charac terized by the ability to cleave substr ate pol ypeptides with in a transm embran e region and by pos sessing two active site aspartat e (d) resid ues (ita licized) within the con served motifs y d and lglg d in adjacent memb ranespan ning regio ns ( martoglio an d golde , ; wan g et al., a; weihof en et al., ) . there are seven related members of the ps/spp l superfam ily in the human genome : ps - , ps - , spp an d four spp -like pro teins, spp a, spp b, and spp c, an d spp (m artoglio and gold e, ). presen ilins and are the catalytic compone nts of gsec retase, a tetr americ comp lex containin g ps and three other subunits. pss play a role in processing of the b-amyloid precursor protein (app) into ab and ab , peptides that constitute the principal components of the b-amyloid plaques in alzheimer's disease (ad); pss are also required for development due to processing of the notch receptor by g-secretase (selkoe and kopan, ) and might be involved in intracellular traffi cking (si sodia and st george -hyslop , ; wang et al ., c) . the function of the four spp-like proteins is, at this point, unknown, but the role of intramembrane-cleaving proteases is usually to liberate signaling molecules from membrane-bound precursors with consequent activation or repression of signaling cascades (fortini, ; kopan and ilagan, ; martoglio and golde, ; parent et al., ; xia and wolfe, ) . in humans, spp performs an important immunological function as it generates the peptide ligands for the nonclassical class i mhc molecule hla-e. on insertion of secretory or type ii membrane proteins into the er, their signal sequence is cleaved by the er luminal protein signal peptidase, leaving the signal peptide anchored in the er membrane. the er membrane-anchored signal peptide is subsequently cleaved by the intramembrane-cleaving spp within the transmembrane region. the resulting signal peptide fragments are released into the cytosol (n-terminal portion) or into the er lumen (c-terminal portion). the latter peptides may easily bind to class i mhc molecules in the er lumen. the hla-a molecule, for instance, is known to bind signal sequence-derived peptides. the n-terminal signal sequence fragments are tap-transported into the er lumen and bind to hla-e (lemberg et al., ) . by presenting fragments derived from the signal sequences of classical class i mhc molecules, hla-e monitors the presence of classical class i mhc molecules. this is, as we discussed earlier, of crucial importance for nk cell recognition. spp is involved in processing of the hepatitis c virus (hcv) core protein (mclauchlan et al., ) . hcv is a single-stranded rna virus with a single open reading frame encoding a large polyprotein. the n-terminal portion of the hcv polyprotein encodes the structural components of the hcv virion, the core protein (thought to constitute the virion capsid), and the e and e envelope glycoproteins. the mature structural components of the hcv virion are produced through a series of cleavage events catalyzed by cellular proteases. the core protein is the most n-terminal portion of the polyprotein and is followed by the signal sequence of the e envelope glycoprotein. the e signal sequence targets the polyprotein to the er membrane and induces translocation of e into the er lumen. cleavage by signal peptidase liberates the n-terminal end of e , leaving the core protein anchored (by the e signal peptide) in the er membrane. spp-mediated intramembrane proteolysis of the e signal sequence then results in release of the hcv core protein from the er membrane, thus freeing the mature core protein for incorporation into lipid droplets (martoglio and golde, ) . spp-mediated hcv core protein maturation and trafficking to lipid droplets and the outer mitochondrial membrane may be critical for viral assembly and life cycle (ait-goughoulte et al., ) and may also affect cellular lipid metabolism and apoptosis, as hcv core proteintransgenic mice display liver pathologies, mitochondrial injury, and enhanced oxidative stress (chou et al., ; korenaga et al., ; meyer et al., ; okuda et al., ; omura et al., ; schwer et al., ; suzuki et al., ) . spp-mediated cleavage of the hcv core protein may thus modulate these important cellular functions of hcv. spp may regulate the interaction of signal peptide remnants of hiv gp envelope protein and preprolactin (p-prl) with calmodulin. a characteristic feature of a signal sequence is its tripartite structure: a polar n-terminal n-region, a hydrophobic core (h-region) of - residues, and a polar c-terminal c-region that contains the consensus sequence for signal peptide cleava ge (von he ijne, ). the nregion of most signal sequen ces comprise s only a few residues. however, some signal sequences have extended n-regions of up to residues. the function of such long n-regions is not known. both the p-prl and the gp signal sequence have an extended basic n-region that can potentially form a basic amphiphilic alpha-helix, a feature of cam-binding domains (o'neil and degrado, ) , not found in the majority of signal sequences. spp-mediated cleavage releases this cam-binding domain on the n-terminal fragment of the p-prl and gp signal peptides into the cytosol. the functional and physiological significance of an interaction between the p-prl and p-gp signal peptide fragments that are released into the cytosol and calmodulin (cam) could be due to a regulatory function of the signal peptide fragments. cam-dependent processes could be enhanced or inhibited depending on the amounts of cam-binding signal peptide fragments generated and released into the cytosol (martoglio et al., ) . the spp orthologues in drosophila melanogaster, spp, and in caenorhabditis elegans, imp- , play an essential role in development: spp protease activity seems to be essential for larval development both in the fly and in the nematode (casso et al., ; grigorenko et al., ) . although the mechanism by which spp mutations impairs developmental processes in the fly is currently unknown, in c. elegans the molting defect induced by imp- deficiency was mimicked by cholesterol depletion and by deficiency in irp- , a homologue of mammalian lipoprotein receptor-related protein (lrp) receptors suggesting a role in cholesterol and lipid metabolism (grigorenko et al., ) . the possibility of a role for spp in er quality control was first advanced by high and colleagues, who reported an association between spp and a truncated version of a polytopic er protein (opsin) in an in vitro system (crawshaw et al., ) and proposed spp to be implicated in the recognition of misassembled transmembrane domains during membrane protein quality control at the er. us -mediated dislocation of class i mhc hc molecules presents the first functional evidence of such a role for the intramembranecleaving protease spp. the us tail is necessary and sufficient to recruit spp, and structural predictions suggest that the us tail suggests may adopt a helical conformation that could form a protein-protein interaction domain (oresic et al., ) . spp is crucial for dislocation by us , as reduction of its levels by rna interference blocks hc degradation. the question remains as to the detailed mechanism of its involvement. an obvious possibility is involvement of the catalytic activity of spp. this would imply a cleavage event during dislocation, such as within the tmd of us or hc or another factor, unknown at this point; a postcleavage function of one of these protein fragments could play a regulatory role in the process. sppmediated intramembrane proteolysis requires, among other things, a membrane protein substrate to have access to its catalytic core in a type ii orientation martoglio and golde, ) . both us and hc are type i membrane glycoproteins, so the topology of their transmembrane and tail segments is opposite to that of predicted spp substrates. spp-mediated intramembrane cleavage within the hc tmd is inconsistent with the observed recovery of full length hc in the dislocation reaction (blom et al., ; misaghi et al., a; wiertz et al., a,b) . for us , while a suggested protein-protein interaction domain in the us tail (oresic et al., ) may mediate binding to spp, intramembrane cleavage of the us tmd by spp in this inverted orientation would presumably not occur, as seen for other proteases (roques et al., ; tarasova et al., ) . however, the proposed bent-helix conformation on the us tail (oresic et al., ) might allow us to conform to the requirements for spp cleavage. whether spp-mediated us cleavage takes place is unknown at this point. one can speculate that this putative processing of us by spp, as for the hcv core protein (mclauchlan et al., ) , could be necessary for ''maturation'' of us into a dislocation-active form. binding of us to spp could still modulate spp enzymatic activity and thus affect dislocation. the possibility remains that spp mediates cleavage in trans of an unidentified factor whose function is important. experiments with spp inhibitors and catalytic mutants of spp should allow an assessment of the contribution of the proteolytic properties of spp to dislocation. the involvement of spp need not be related to its catalytic activity. substrate recruitment and subsequent cleavage by spp may be separable events, as shown for the related ps (kornilova et al., ; lemberg and martoglio, ) . some intracellular cleavage products of g-secretase are proposed to be intermediates that are destined for degradation (kopan and ilagan, ; parent et al., ) . g-secretase-mediated cleavage of a large number of type i transmembrane proteins releases their c-terminal fragments (ctfs). ps deficiency causes delayed turnover and subsequent accumulation of some g-secretase substrates as full-length proteins (esselens et al., ; wang et al., c; wilson et al., c) , suggesting cleavage of the ctfs as a prelude to degradation. treatment with g-secretase inhibitors, however, does not phenocopy ps deficiency (wang et al ., c) , suggestin g tha t this effec t is in dependen t of gsecre tase activ ity. therefore, spp may be crucial for hc dislocation irrespectively of its catalytic properties. binding of us to spp could presumably modify the spp structure in a way that affects dislocation. alternatively, recruitment of spp by us could perhaps nucleate assembly of a dislocation complex, much like us and the derlins (lilley and ploegh, a) . spp may be a component of an erad pathway for a subset of er degradation substrates that includes misfolded transmembrane proteins, such as truncated opsin (crawshaw et al., ) , and tha t is recru ited by us to dislocat e hc ( fig. , a arrow ) . experi ments to address the identity of spp-associated proteins may prove informative. curiously, another class of intramembrane-cleaving proteases, the rhomboid serine proteases, share a homology domain of unknown function with the derlins (lemberg et al., ) . it is tempting to speculate that our observations extend the connection from regulated intramembrane proteolysis to a direct involvement in er dislocation. an involvement of spp with the upr is also a possibility. cells deficient for the x-box binding protein- transcription factor (xbp- ) show upregulated levels of spp transcripts (shaffer et al., ) , but this aspect of the process remains to be explored. although removal of signal peptide remnants from the er membrane, assigned to spp in animals and plants, is not a function exclusive to higher eukaryotes, a gene that encodes an orthologue of this enzyme is absent from the yeast genome (martoglio, ; weihofen et al., ) . the role of spp in higher eukaryotes might therefore not be limited to signal peptide processing but extend to processes such as protein dislocation from the er. ps and the other spp-like members of the ps/sppl superfamily of intramembrane-cleaving aspartic proteases, so far of unknown function (martoglio and golde, ) , and some of which may not reside in the er (krawitz et al., ) , may likewise be involved in disposal of different degradation substrates. hcmv might just be exploiting a cellular degradation pathway that involves intramembrane-cleaving proteases for disposal of class i mhc hc. the us tmd, although dispensable for interaction with spp, is also required for hc dislocation (loureiro et al., ) . hc dislocation is therefore dependent not just on (us tail-mediated) recruitment of spp, but also on additional (us tmd-mediated) interactions within the plane of the membrane. the us tmd may be involved in further engagement of spp (sppmediated cleava ge or o therwise ) ( fig. , b arrow ) , o r alternativ ely, in the recruitment or engagement of other protein(s) involved in dislocation (fig. , c arrow ). er m embran e e s or their adap tor subunits are like ly cand idates. spp is most certainly not the sole host-derived component of the us dislocation pathway, and a putative multiprotein complex (cascade of adaptor proteins) analogous to that found for us is likely to be found that evokes many (complicated) links between the erad machineries at the level of the er membrane and the cytosol. uncovering the identity of the additional cellular partners of this hcmv us immunoevasin will certainly give us insight into the dislocation mechanism. a theme that arises from analysis of the us -and us -mediated hc dislocation processes is that these are only superficially similar: although hc dislocation by us and us has the same outcome (proteasomal degradation) of hc and shares many if not all of the steps that take place after extraction of the hc from the er membrane (such as deglycosylation and degradation kinetics), the differences between the pathways are rather striking in terms of the steps prior to dislocation. as we mentioned earlier, us and us have different allele and substrate folding, assembly, and ubiquitination requirements. derlin- is crucial for us -but not us -mediated dislocation of hcs (lilley and ploegh, ; ye et al., ) , and, conversely, spp is required by us but not by us (loureiro et al., ) . this suggests that the hcmv immunoevasins us and us are targeting hc molecules to distinct er dislocation pathways, perhaps by serving as adaptor molecules aiding in recruitment and/or assembly of distinct multiprotein complexes at the er membrane ploegh, , b) . the murine g-herpesvirus (mhv- ) mk also targets newly synthesized murine class i mhc hc for dislocation from the er and proteasomal degradation (boname and stevenson, ; lybarger et al., ; wang et al., ; yu et al., ) . mhv- mk belongs to a family of structurally related molecules, the k homologues, which have e ligase activity. k homologues are present in several different g-herpesviruses and poxviruses (coscoy and ganem, ; ishido et al., ; mansouri et al., ; stevenson et al., ) . all k homologues possess a noncanonical ring-finger domain with ubiquitin ligase (e ) activity [also called a plant homeodomain (phd) or leukemia-associated protein (lap) domain], and a conserved integral membrane topology, with the transmembrane domains and cytosolic c-terminal tails mediating interaction with the substrate (coscoy and ganem, ) . the mk phd/lap-family e is a type iii er membrane protein with the phd/lap ring-related domain facing the cytosol (boname and stevenson, ; sanchez et al., ) . mk -mediated degradation of murine hcs is absolutely dependent on components of the plc: association of mk with tap and tapasin presumably imposes the necessary proximity and/or orientation of the mk ring domain that allows mk to specifically ubiquitinate class i mhc hc as they enter the plc wang et al., wang et al., , . mk -mediated dislocation of murine hcs is dependent on the atpase activity of p and physical association with derlin- and vimp (wang et al., d) . thi s is reminiscen t of the pathway that h cmv us is propos ed to co-opt for dislocation of mammalian class i mhc molecules. in a sense, it appears that mk may be a more evolved immunoevasin that can couple the ability to recruit other components of the dislocation machinery, like us and presumably us , with e ligase activity, in one viral polypeptide. there are several mammalian k homologues, the membrane-anchored ring-ch (march) proteins (bartee et al., ; goto et al., ) , which are likely to be the cellular ancestors of mhv- mk . mk -mediated ubiquitination of the murine class i mhc hc cytosolic tail, the portion of the hc molecule more likely to come into contact with the cytosolic ring-ch domain of mk , is not required for dislocation even though ubiquitination and presence of the cytosolic tail are essential for dislocation . in fact, like mk , hcmv us and us also induce ubiquitination-dependent degradation of class i mhc molecules in a cytosolic tail lysine-independent fashion (furman et al., ; shamu et al., ) . how, then, does mk access the luminal domain of hc molecules and trigger its ubiquitination? access of the hc lumenal domain to the cytosol would invoke a ''partial dislocation'' model that has been proposed for hcmv us and us (furman et al., ; shamu et al., ) : the hc luminal domain must begin to emerge in the cytosolic face of the er so ubiquitination can take place. this would mean that the trigger for dislocation would reside upstream from tail ubiquitination. an alternative explanation would be that the class i mhc tail lysine mutant hc molecules are dislocated as a ''bystander effect'' of dislocation of wild type molecules by mk , simply because they are in the proximity of the dislocation machinery that has been recruited by the viral protein for the wild type hc clientele. the hcmv us and us and mhv- mk immunoevasins all target nascent class i mhc hcs for degradation by inducing their dislocation from the er membrane. the mechanisms used by us , us , and mk , however, when analyzed in detail, are strikingly different. for instance, the stage of class i mhc hc biosynthesis that is targeted by each viral protein is distinct: us is the most promiscuous, targeting multiple class i mhc assembly intermediates, whereas us targets only properly folded class i mhc complexes and mk targets predominantly incompletely assembled hc while in association with the peptide-loading complex. the main difference resides in the fact that mk encompasses ubiquitination activity (by means of its ring domain), substrate selection (by means of its association with the peptide-loading complex), and recruitment of er membrane and cytosolic factors necessary for dislocation (like derlin- and p ) all in one polypeptide. hcmv us and us do not possess e ligase activity and possess no obvious sequence similarity with known genes/proteins that would hint at their function. however, at least us , and presumably also us , are still able to induce assembly of a dislocation complex (fig. ) that encompasses all the necessary erad activities. the knowledge that the tmd of us is essential for its function (lilley et al., ) , led to the discovery of derlins (lilley and ploegh, ) and of multiprotein complexes at the er membrane that function in us -mediated dislocation of hcs and us -independent dislocation of a subset of erad substrates (lilley and ploegh, a; oda et al., ; ye et al., ) . us uses its cytosolic tail to recruit spp and its tmd for an additional step (so far unknown) also critical for hc dislocation (loureiro et al., ) , presumably resulting in recruitment of us -specific-components of the er dislocation machinery. the mechanism by which us operates will hopefully be clarified as we continue characterizing the structural and host cofactor requirements for its function in dislocation. the mechanistic details of dislocation catalyzed figure three viral immunoevasins that co-opt distinct erad pathways. the hcmv us immunoevasin delivers class i mhc hc molecules to derlins for dislocation from the er membrane and degradation, whereas hcmv us uses a pathway that is dependent on signal peptide peptidase. the mhv- mk immunoevasin is an e ligase that uses the plc as a platform to target murine class i mhc molecules for ubiquitination and degradation. although the three pathways are superficially similar, the substrate selection and targeting steps at the er membrane are very distinct. ub, ubiquitin; e , ub-activating enzyme; e , ub-conjugating enzyme; e , ub-ligase enzyme; pngase, peptide-n-glycanase; spp, signal peptide peptidase; tap, transporter associated with antigen presentation; crt, calreticulin. by the hcmv us and us and the mhv- mk immunoevasins must be quite diverse and their study will most certainly keep providing new insights into er dislocation. furthermore, it is nothing short of remarkable how sequence and structurally unrelated herpesvirus proteins have converged into (although only superficially) similar mechanisms to dislocate newly synthesized class i mhc molecules from the er membrane. class ii mhc-restricted cd þ t cells are crucial for lymphocyte activation, antibody responses, and coordination of the immune response and rely on activation by the professional apcs. immunoevasins aimed at interfering with class ii mhc antigen presentation are not expected to block presentation of exogenous antigens to cd þ t cells, unless the apc is infected by the virus (yewdell and hill, ) . class ii mhc expression can, however, be modulated by ifn and receptor signaling through the ciita transcription factor (boss and jensen, ) . there are relatively few examples of viruses and bacteria that directly infect apcs and affect ifn-induced class ii mhc expression or directly interfere with class ii mhc-restricted antigen presentation (hmama et al., ; hussain et al., ; miller et al., ; rinaldo, ; schuller et al., ; srisatjaluk et al., ; zhong et al., ) . it is likely that more examples will be found as this important question is being revisited. for viruses that do not infect professional apcs, the route to avoiding helper t cell and antibodymediated responses is to interfere with activation of cd þ t cells by apcs. in this section, we present an overview of some of the mechanisms used mostly by viral pathogens to actively subvert class ii mhc antigen presentation (fig. ) . the degree to which other pathogens, like bacteria and parasites, actively interfere with class ii mhc antigen presentation, by encoding ''immunoevasins'' rather than passively, due to their residence in endocytic compartments, is difficult to discern. epstein-barr virus is an example of a virus that can infect and establish latency in b lymphocytes and is associated with a number of malignancies. the product of the bzlf ebv gene, gp , is a bifunctional protein. first, it functions as the coreceptor for viral entry into b cells by binding to the hla-dr product. second, gp is generated through proteolytic cleavage in the er and matures into a secreted form that binds class ii mhc molecules at the cell surface. gp bound to class ii mhc molecules at the cell surface prevents tcr-(peptide-loaded class ii mhc) interactions and cd þ t cell activation (li et al., ; ressing et al., ressing et al., , spriggs et al., ) . the hiv- nef protein downregulates class ii mhc molecules from the cell surface by restructuring the endocytic pathway such that invariant chain (ii) degradation is impaired and immature class ii mhc complexes (abii) are granted increased access to the cell surface (stumptner-cuvelette et al., ) . nef induces a reduction of surface levels of peptide-loaded class ii mhc as well as a strong accumulation of surface-displayed immature class ii mhc complexes, still containing (and thereby blocked by) intact invariant chain (ii). nef expression results in accumulation of both class ii mhc and invariant chain (ii) in multivesicular bodies (mvbs) (stumptner-cuvelette et al., ) . mvbs are a specialized type of endosome that constitutes a major pathway of delivery of transmembrane proteins for lysosomal degradation . sequestering in mvbs suggests a reduced capacity of immature class ii mhc complexes to reach lysosomes, either due to a defect in class ii mhc sorting to the lysosomes or due to slower internalization of immature complexes. the mechanism is still unclear (stumptner-cuvelette et al., ) . the hcmv us immunoevasin was proposed to downregulate class ii mhc from the cell surface, presumably by targeting hla-dra and hla-dma for degradation by the proteasome, thus inhibiting antigen presentation to cd þ t cells (chevalier et al., ; hegde and johnson, ; tomazin et al., ) . this effect of us , however, was only seen in cell lines in which induction of class ii mhc was induced by stable transfection with the class ii mhc trans-activator (ciita), but not in human dcs or several other cell lines, which express class ii mhc endogenously (rehm et al., ) . presumably the relative expression levels of class ii mhc and/or us could account for the observed differences, and in fact, in ciita-transfected cells, even us was seen to downregulate class ii mhc molecules (hegde et al., ) . hsv- can downregulate surface expression of class ii mhc complexes in b cells and inhibits the ability of b cells to stimulate cd þ t cells. hsv- inhibits synthesis of ii and also encodes an envelope glycoprotein b (gb) that binds both hla-dr and hla-dm (neumann et al., ; sievers et al., ) . by binding to hla-dr, gb affects trafficking of the molecule in the secretory pathway and by binding hla-dm it sequesters this peptide editor and thus prevents peptide loading of class ii mhc molecules that may have escaped (neumann et al., ) . the cd protein serves as the primary cellular receptor for hiv at the surface of cd þ cells. however, its presence inhibits virus budding and interferes with incorporation of the gp protein into the budding virion, not to mention its crucial role in eliciting of a cd þ t cell response (keppler et al., ; lama et al., ; ross et al., ) . not surprisingly, three hiv- proteins, nef, vpu, and gp dramatically reduce the steady state levels of cd on the cell surface piguet et al., a,b) . one of the many mechanisms used by nef involves acceleration of the constitutive endocytosis of cd . in t cells, cd is stabilized at the cell surface by p lck, a src-family tyrosine kinase, which binds a dileucine motif in the cd cytoplasmic tail, preventing cd from being recruited into clathrin-coated pits (aiken et al., ; jin et al., ) . nef can displace lck by directly binding a dileucine sorting motif in the cd tail, overcoming the normal lck phosphorylation-dependent route of cd downregulation. nef itself contains a c-terminal dileucine motif that recruits a subunit of the tetrameric adaptor protein complex- (ap- ), a component of clathrin-coated pits at the cell membrane. thus, nef connects cd to ap- on clathrin-coated pits, triggering rapid cd endocytosis (jin et al., b (jin et al., , mangasarian et al., ; piguet et al., piguet et al., , a . nef can bind not only the ap- components of clathrin-coated pits, but also the regulatory v h subunit of the vacuolar proton (h þ ) atpase. v h (also called nbp or nef binding protein- ) binds ap- in clathrin-coated vesicles. by binding v h, nef strengthens its weak direct interaction with ap- (geyer et al., ; mandic et al., ) . again, by binding both cd (using the aforementioned dileucine motif in the cytoplasmic tail) and v h, nef directs internalization of cd from the cell surface. the endocytosed cd accumulates in early endosomes from where it is sorted to lysosomes for degradation. adaptor protein (ap) complexes mediate transport of proteins to numerous compartments within the cell. whereas ap- initiates early endocytic vesicle formation at the cell membrane, ap- is involved in vesicle formation at the tgn and vesicle targeting to early endosomes and ap- participates in vesicle formation at the tgn and targeting to late endocytic/lysosomal compartments (bonifacino and traub, ) . the ''coat'' on vesicles in the endocytic pathway is composed of ap complexes and another set of coat proteins, clathrin in clathrin-coated vesicles, and cop proteins, in cop-coated vesicles. the cop proteins are involved in vesicle trafficking early in the secretory pathway between the er and the golgi (mcmahon and mills, ) . trafficking in the endocytic pathway can be targeted by hiv nef by direct binding to ap complexes or by interference with the recruitment and release cycles of ap complexes from vesicle membranes. ap complexes cycle from the cytosol to vesicles in a process dependent on the gtpase cycle of adp-ribosylation factor- (arf ). nef can bind to and stabilize the small gtpase arf on the endosomal membrane, preventing ap complexes from being released, affecting trafficking of host molecules (including cd ) along the endocytic pathway. nef can also mediate the formation of a ternary complex composed of nef, arf , and a component of the cop-i coat, bcop. cop-i-coated vesicles are mostly subject to retrograde transport within the golgi and between the golgi and the er, but some are involved in transport from early to late endosomes (mcmahon and mills, ) , and thus association of nef with arf and bcop mediates targeting of cd for lysosomal degradation (faure et al., ) . nef has a preference for ap- and ap- complexes in vitro (janvier et al., b) , suggesting that the nef modus operandi is mostly at the level of endosomal membranes. downregulation from the cell surface by binding ap- does not seem to be a major route for cd downregulation (rose et al., ) . which one of these strategies-downregulation from the cell surface or intracellular retention-or what combinations of these strategies and adaptor protein complexes are used may allow the hiv- nef protein to downregulate cell surface expression of cd perhaps in different cell types (mangasarian and trono, ) . vpu targets newly synthesized cd molecules in the er for proteasomal degradation (kerkau et al., ) by recruiting the cytosolic f-box protein b-trcp, the receptor component of the scf b-trcp e ligase, to the er membrane (margottin et al., ) . a motif in the vpu c-terminus binds a wd repeat on b-trcp, directing scf b-trcp e ligase activity to catalyze the ubiquitination of lysine residues on the cd tail, which serves as the trigger for cd degradation (margottin et al., ; schubert et al., ) . gp retains newly synthesized cd molecules in the er (crise and rose, ; kimura et al., ) . in the absence of cd , hiv gp is posttranslationally cleaved into its gp and gp subunits at the level of the er-to-golgi compartment (ergic). in the presence of cd , gp forms a complex with cd that mediates er retention of both molecules and downregulation of cd from the cell surface. in the context of an hiv-infected cell, coexpression of vpu liberates gp from the complex, ensuring gp translocation to the ergic and ensuing maturation, and simultaneously accelerating cd turnover (crise and rose, ; rose et al., ) . hiv downregulates class i mhc, class ii mhc, cd , and cd d, a class i mhc-like molecule that presents lipid antigens (le gall et al., ; piguet et al., b) , from the cell surface by virtue of its nef, vpu, and gp proteins, reflecting its ability to manipulate various aspects of the immune response (joseph et al., ; piguet et al., b) . furthermore, each of these mechanisms may be more far-reaching than downregulation of each individual receptor. for instance, by directly associating with the proton (h þ ) atpase, which is required for the acidification of lysosomes, nef not only downregulates cd but may also interfere with the ph of class ii mhc-positive compartments, affecting antigen processing by lysosomal proteases and class ii mhc antigen presentation. this strategy is used by other pathogens: the e protein from both human and bovine papillomavirus (andresson et al., ) and the vaca toxin secreted by helicobacter pylori (molinari et al., ) actively manipulate acidification in the endocytic pathway, resulting in impaired cd þ t cell-mediated responses (brodsky et al., ) . by binding to ap complexes or to the small gtpase arf , nef may interfere with trafficking and alter the fate of numerous host molecules in the endocytic pathway (janvier et al., a) , affecting aspects that range from viral replication to modulation of the immune system (lama and ware, ; sol-foulon et al., ; swigut et al., ) . intracellular pathogens exploit the ubiquitin-proteasome system mostly to destroy or avoid destruction of specific cellular proteins. this serves to create a more hospitable environment for themselves in the host cell, or to prevent destruction of their own proteins, and so to ensure replication or avoid detection by the immune system. many events at the initial stage of infection, entry into the cell, are controlled by signaling pathways-for instance, those involved in cytoskeletal rearrangements or in receptor engagement-that rely heavily on the ub-proteasome system. the same is true for signaling cascades involved in cell survival, differentiation, and proliferation. not surprisingly, pathogens have devoted much energy and coding capacity to interfere with cell signaling events through interference with the ub-proteasome system. an illustrative example is that of tumor viruses and degradation of cellular tumor suppressor proteins, like the retinoblastoma protein or p , often resulting in malignant transformation (shackelford and pagano, ) . there have been a number of recent reviews on viral interference with signaling pathways, so we will not describe this aspect in detail here. for comprehensive reviews, the reader is referred to banks et al. ( ) and pagano ( , ) . in this chapter, we will focus on a few examples of viral and bacterial interference with the ub-proteasome system that illustrate how the latter is crucial for aspects of pathogen life cycles that are as distinct as integration into host chromosomes, exit from the host cell, or rna interference, not to mention control of the immune response. as mentioned in the first section of this chapter, proteasome-mediated degradation of cytoplasmic proteins as well as the proteolytic events in the endolysosomal system are important for class i and class ii mhc presentation and cross-presentation. mounting of an immune response involves complicated signaling cascades like nf-kb and jak/stat signaling, which are heavily dependent on the ub-proteasome system, and that can be manipulated from early events of cell-surface receptor-mediated signaling, for example, to later events of ubiquitination and deubiquitination of downstream targets. certainly, because of the central role of the ub-proteasome system in many different aspects of cellular physiology, interference with this pathway will have pleiotropic effects, and which of the observed effects predominates may be difficult to discern. it is not our intention to make the following a comprehensive list. we rather propose to provide an overview of the possibilities of manipulation of this system that have been described for pathogens (fig. ) . we discuss in more detail pathogen-encoded modulators of the ubiquitin-proteasome system, ranging from pathogen-encoded proteolysis-resistant peptides, to ubiquitin ligases and dub, with obvious implications for the viral or bacterial life cycle or with consequences for the control of host immune responses. we elaborate on novel pathogen-encoded dubs and their putative functions. the classic example of a viral protein that interferes with proteasomal processing is the ebna- . the ebna- protein contains an internal repeat exclusively composed of glycines and alanines, the gly-ala repeat, which not only interferes with its own proteasomal proteolysis (levitskaya et al., ) , but also reduces its rate of translation, blocking viral drips formation and inhibiting the presentation figure pathogen interference with the ub-proteasome system. pathogens interfere with the ub-proteasome system not only to manipulate antigen presentation and other aspects of the immune system, but for processes as distinct as chromosomal integration, virus budding, rna interference, and many others. they may rely on hijacking of host e ligase activities or encode their own. pathogens can manipulate host dubs as well as encode dub activities. the function of pathogen-encoded dubs remains a mistery. of ebna- class i mhc-restricted t cell epitopes (yin et al., ) . a single residue change in the mouse leukemia virus (mulv)-derived ctl epitope (from kspwfttl to rspwfttl) can eliminate the proteolytic cleavage site required for its presentation (ossendorp et al., ) . as discussed earlier, phosphorylation of the hcmv ie- by the hcmv pp tegument protein interferes with production of ie- -derived peptides (gilbert et al., ) . the hiv- transcriptional activator (tat) protein manipulates s proteasome function by directly interacting with the lmp and mecl subunits of the proteasome and competing with the s proteasome for binding to the s proteasome (andre et al., ; apcher et al., ) , leading to inhibition of proteolytic activity. tat also acts at the transcriptional level by modifying proteasome composition by upregulating the lmp and mecl subunits and downregulating the lmp subunit, leading to an increased presentation of cryptic and subdominant ctl epitopes (gavioli et al., ) . by preventing display of viral peptides, these viral proteins interfere with cytotoxic t cell recognition. salmonella enterica serovar typhimurium is an important bacterial pathogen, the causative agent of food poisoning and typhoid fever. s. typhimurium temporally regulates the initial phase of bacterial internalization and host cell recovery after invasion through two type iii secretion system (ttss)-delivered substrates, sptp and sope with different proteasomal half lives (kubori and galan, ) . sope is a bacterial guanine nucleotide exchange factor (gef) delivered by a ttss that mimics a host cell gef for the small rho gtpases cdc and rac , involved in actin remodeling and formation of membrane extensions required for bacterial engulfment. sptp, delivered by another ttss, is a rho gtpase activating protein (gap) factor for cdc and rac , which accelerates gtp hydrolysis. spt plays a role once bacterial internalization has taken place, inactivating the rho gtpases, inhibiting actin polimerization, and assuring closure of the plasma membrane and recovery of the normal cellular architecture (pizarro-cerda and cossart, ) . salmonella initially delivers equal amounts of sope and sptb to the host cell. however, - min after infection sope is rapidly degraded, whereas sptb degradation occurs only slowly after h, thereby efficiently timing the actin remodeling events that lead to the initial bacterial engulfment and the later host cell recovery. both processes are proteasome-dependent and catalyzed by the n-terminus of the bacterial proteins, but the mechanism and the host factors involved are currently unknown (kubori and galan, ) . the hiv- vif protein targets the rna editing protein apobec g for degradation by a cellular e ligase to allow production of infectious viral progeny (yu et al., ) . the apolipoprotein b mrna editing enzyme, catalytic polypeptide-like g (apobec g) and related cytidine deaminases are involved in mrna editing and in immunoglobulin gene class switching and hypermutation but are also potent antiretroviral enzymes. the cytosolic apo-bec g is incorporated into budding virions, where on infection of new target cells, its cytidine deaminase activity induces g to a hypermutation on the minus-strand viral dna, resulting in abortive infection (bishop et al., b; zhang et al., ) . vif hijacks the elongin-c-elongin-b-cullin- -e (ecs) complex. the ecs e s recognize substrate receptor proteins containing a bc-box. vif possesses a bc-box motif, through which it binds elongin c . vif hijacks the e ligase complex and by bridging apobec g and elongin c targets apobec g for ubiquitination and degradation (yu et al., ) . this ultimately allows production of infectious virus progeny (bieniasz, ; bishop et al., a; harris and liddament, ) . the endosomal sorting complexes required for transport (escrt) are highly conserved from yeast to mammals and consist of the escrt i, ii, and iii complexes. escrt complexes are composed of vacuolar protein sorting (vps) proteins (in yeast), which are recruited from the cytoplasm to promote sorting of ubiquitinated proteins to mvbs (katzmann et al., ) . mvbs are formed by invagination of the late endosome membrane, which generates internal vesicles into which proteins destined to the lysosomes are sorted; they are critical for receptor downregulation and other normal and pathological cell processes (hierro et al., ; hurley and emr, ; kostelansky et al., ) , as well as for virus budding. the tumor susceptibility gene tsg , the mammalian homologue of yeast vps , is essential for sorting of ubiquitinated proteins to mvbs (babst et al., ) . tsg recruits hepatocyte growth factor-regulated tyrosine kinase substrate (hrs), the mammalian homologue of yeast vps , to the endosomal membrane. hrs nucleates recruitment of the escrt- complex, which in turn recruits escrt and - , leading to formation of the inner membranes of the mvb. tsg is a noncanonical ub e variant (uev) protein-it does not possess ub-conjugating activity, only an n-terminal uev domain that binds ubiquitin. binding of the tsg uev domain to a p(s/t)ap tetrapeptide motif on hrs recruits hrs to the endosomal membrane, triggering assembly of escrt complexes and genesis of the mvb (clague and urbe, ; garrus et al., ; pornillos et al., pornillos et al., , . many viruses such as hiv- and ebola recruit this ub-dependent sorting machinery to the viral release sites at the plasma membrane to promote virus budding from host cells ( li and wild , ; liu, ) , by makin g use of con served p(s/t)ap, ppxy, or fpiv motifs, also called late budding domains ( bieniasz, ) . retroviruse s like hiv use the p(s/t) ap motif on the ldomain contained in their gag protei n to mimic the tsg -rec ruiting activ ity of the hrs protein ( klinger an d schube rt, ; martin -serra no et al., ; sor in and kalpana , ; stuchell et al., ) . oth er viruses can also rec ruit neuro nal precurs or cellexpresse d develop mentally dow nregula t ed (nedd ) and nedd - -like hect e ligases to facilitate viral bud ding ( bieniasz, ; harty et al., ; sakura i et al., ; yasuda et al ., ) . ne dd e s ligase s are a dive rsified group of orthologu es of yeast rsp p and are in volved in a wid e range of proce sses such as recepto r inter nalizati on and degrad ation (presum ably through the endoly sosomal pa thway), mainten ance of ebv latency, and regulation of cytokine sig naling. ned d e s have a catal ytic cterminal hec t domain , two or more centr al nterminal ww dom ains, an d an n-term inal c dom ain. the nterminal c domain seems to be responsi ble for me mbrane associ ation and cellu lar localizatio n of the pro tein. the ww dom ain is a protei n-protei n interactio n modul e o f abou t amino acids with two crucia l tryptop han (w) residues spa ced - amin o acids apart, which appear s to medi ate substr ate selection. it binds mostly pro linerich motifs, such as the ppxy motif present in many cellular proteins , targeting them for degradati on ( ingham et al., ( ingham et al., , . viral pro teins with the ppxy mo tif can therefore bind these ww dom ains on nedd e s, but the na ture of these inter actions and how they facilitate virus budd ing is so far unknown . some viruse s, like ebola, have two different late buddin g motifs, ppxy and p(s/t) ap, and the ebola late domain -con taining vp matrix protein can recruit bot h tsg and nedd for effec tive budding (lic ata et al., ; liu, ; yasuda et al ., ) . the nonen veloped adenov irus pos sesses a ppxy motif on its penton base protein, which is essential for virus internalization that can interact with several nedd hect e ligases (galinier et al., ) . whether or not this interaction or e ligase activity is required for adenovirus entry is currently unknown. agrobacterium tumefaciens exploits a host cell scf e ligase for integration of its t-dna by encoding an fbox pro tein (tzfira et al., b) . agrob acterium is a common phytopathogenic bacterium that induces ''crown gall'' disease in plants by transfer and integration of a segment of its tumor-inducing (ti) plasmid dna into the plant genome. this process relies also on delivery of several virulence (vir) proteins into the host cell, such as the bacterial vire protein, which is thought to package and protect the transported t-dna molecule, and, together with t he host plant vip pro tein, assist its nucl ear import (tzfira et al ., a) . howeve r, di sassembly of this vire /t-dna/ vip complex mus t occur before in tegration and involves intranu clear pro teolysis of vire and vip induced by the virf pro tein, an fboxdomain-co ntain ing protein. t he bacte rial virf f -box hi jacks the scf e plant homolog ue. the virf-contain ing scf virf then leads to degrad ation of vip and vire , and integration of the agrobact erium t-dna (tzfira et al., b) . we conside r it unlikely that this pos sibility has been exploited only by pl ant pathogens. the h ost innate response triggered by type i inter feron (ifn-a and -b) innate response is crucial in early immunity against viruses, bac teria, and some parasites, acting t o lim it pathogen i n fection li mi ting replic ation of t he pathogen a nd constraining cellular permissiveness to infection (smith et al., ) . type i interferon receptor signaling occurs through the jak/stat pathway and leads to transcription of several ifn-stimulated genes (isgs), of which the gene encoding the ub-like modifier isg is one of the most strongly induced (farrell et al., ) . isg and protein modification by isg (isgylation) are induced by viral and bacterial infection or other stresses, suggesting important roles for the isg system in innate immune responses ritchie and zhang, ) . isg is conjugated onto several signaling molecules with immunomodulatory functions, like jak/stat proteins (giannakopoulos et al., ; malakhov et al., ) . the isgylation cascade is initiated by isg activation by an e -like enzyme, ube l, transfer to the isg-conjugating ubch enzyme , and incorporation into ubch -compatible ub e ligases (dastur et al., ; zou and zhang, ) . a deisgylating enzyme, ubiquitin-binding protein (ubp ), also called usp , specifically removes isg from isgylated substrates , and is regulated by ubiquitination by the scf skp e (tokarz et al., ) . usp is unlikely to be the sole enzyme capable of acting on isg conjugates. the role of isg in orchestration of the innate antiviral response sets the ground for pathogen interference. although the mechanism is unclear, viral replication in ubp knockout mice is impaired, a phenotype that was initially attributed to inhibition of deisgylation and deregulation of stat signaling . there are conflicting views on this (dao and zhang, ; kim et al., b; knobeloch et al., ) and ubp may in fact inhibit type i ifn signaling irrespectively of its isg isopeptidase activity, by binding to the ifn receptor and blocking the interaction between jak and the ifn receptor (malakhova et al., ) . ifn-mediated inhibition of hiv replication and budding is dependent on isg (kunzi and pitha, ; pitha, ; poli et al., ) . expression of isg inhibits ubiquitination of the hiv- gag protein and tsg , and disrupts the interaction of the gag late budding domain with tsg (okumura et al., ) . either of these isg effects and/or additional mechanisms of action could lead to failure to recruit escrt complexes and inhibition of hiv budding. isg is strongly induced by infection with influenza b virus. the exact cellular function and targets of isg are not known, but the ns protein of influenza b viruses (ns b) blocks its conjugation to target proteins: the ns b n-terminus binds isg , inhibiting activation of isg by its e enzyme, ube l. influenza a viruses also manipulate cellular isgylation processes, through an even less well characterized mechanism: the influenza a virus ns a protein does not directly bind the isg protein, but little or no isg protein is produced during infection (yuan and krug, ; yuan et al., ) . whether this reflects isgylation, deisgylation, and/or ubp ubiquitination-mediated control of the ifn innate immune response is so far unknown. suppression of nf-kb signaling is a common theme for many viral as well as bacterial pathogens and can be certainly achieved through modulation of ub-dependent events in the nf-kb cascade (bowie et al., ; hiscott et al., ; mason et al., ) . the human enteric flora may influence intestinal epithelium inflammatory tolerance by inhibiting the nf-kb pathway, which can be achieved by blocking any one of the many ub-dependent steps that control it. shigella flexneri, which causes severe diarrhea in humans, injects ttss-effector proteins into host cells to induce their entry into epithelial cells or trigger apoptosis in macrophages. the ospg effector is a serine/ threonine kinase that binds various e s, including ubch , a component of the scf b-trcp e complex. ospg binding to ubch inhibits the scf b-trcp complex and thereby phospho-ikb degradation, blocking nf-kb signaling in response to the bacterial infection. the cullin subunit of the scf b-trcp complex is itself regulated by nedd attachment (pan et al., ) . certain enteric bacteria can lead to rapid deneddylation of cullin- and consequent repression of the nf-kb pathway (collier-hyams et al., ) , but the bacterial activities responsible are still to be determined. in any case, because nf-kb is crucial for both the initial innate inflammatory response and for coordination of the adaptive immune response, dampening of inflammation may endow shigella and other enteric bacteria with the ability to invade and later colonize the gastrointestinal epithelium (kim et al., ) . rna interference (or posttranscriptional gene silencing) in plants and invertebrate animals is important for many regulatory processes and a primitive form of antiviral immunity that is nucleic acid based. consequently, plant viruses have evolved proteins, the so-called silencing suppressors, which directly bind to and inactivate the plant micrornas (ding et al., ; zamore, ) . poleroviruses are small positive-strand rna viruses that cause leafroll phenotypes in many plant species. poleroviruses encode a silencing suppressor p , which is a viral f-box protein (barry and fruh, ) . p has a minimal conserved f-box motif that can bind the plant skp- homologue and form a functional complex with the plant cullin- homologue. the p f-box is required for polerovirus infectivity and its silencing suppressor function (pazhouhandeh et al., ) . p presumably binds the micrornas and targets them for cullin- a-dependent degradation. this constitutes a remarkable finding, as degradation of rna (and not protein) by an e ligase had not been documented before. this primitive form of antiviral immunity, although widely used as a laboratory tool was only recently shown to occur in the context of a natural viral infection in jawed vertebrates (browne et al., ) . hiv- encodes viral small interfering rna (sirna) precursors that provoke rna silencing in human cells, so not surprisingly, the hiv- tat protein also contains a silencing suppressor function (bennasser et al., ) , that prevents dicer from processing the precursor double-stranded rnas into sirnas. suppression of rna silencing by other rna viruses is likely to occur. it will be interesting to see whether the mechanism used by the plant poleroviruses is conserved and whether manipulation of the ub-proteasome system for suppressing rna interference is more widely used by mammalian rna viruses. as mentioned earlier, several g-herpesviruses and poxviruses encode phd/ lap e s, the k homologues, that include the kaposi's-sarcoma-associated herpesvirus (kshv) kk and kk (also called modulator of immune recognition mir- and mir- , respectively), the mhv- mk , and the rabbit myxoma virus m r (coscoy and ganem, ) . we discussed mk which catalyzes ubiquitination and proteasomal degradation of nascent murine class i mhc hcs after dislocation from the er membrane wang et al., ) . kshv kk and kk and myxoma virus m r catalyze ubiquitination of cell surface class i mhc hc, thus providing the trigger for their internalization from the cell membrane, as well as for sorting through mvb formation to lysosomal degradation (duncan et al., ) . in addition to downregulation of class i mhc molecules, some of these k homologues target the lymphocyte costimulatory molecules cd (b . ) and intercellular adhesion molecule icam- , cd d, and cd (coscoy and ganem, ; coscoy et al., ; lehner et al., ; mansouri et al., ) , as well as the fas/cd death receptor (collin et al., ; guerin et al., ) . by targeting not only class i mhc molecules, the viral k homologues are adding an extra layer of protection predicted to be important for viral escape after reactivation from latency (lehner et al., ) . the k homologues are most likely making use of old ideas: the mechanisms used by the viral ub ligases have probably been ''borrowed'' from their mammalian counterparts, the march proteins, and will certainly continue to yield insights into the ub-proteasome system. a bacterial e ligase was recently shown to inactivate another type of immune response in plants (janjusevic et al., ) . antipathogen responses in plants, although not as sophisticated as those afforded by the vertebrate immune system, are nevertheless quite efficient. one mechanism, immunity-induced programmed cell death (pcd), is a response that sacrifices a limited portion of the plant to limit spread of the infection. the pseudomonas syringae bacterium, which causes disease in tomato and arabidopsis, delivers its avrptob protein into plant cells through a type iii secretion system. avrptob can inhibit pcd in susceptible hosts, allowing pseudomonas to cause systemic infection and disease. the avrptob c-terminus encodes a u-box e ligase activity necessary for the pathogenic role of avrptob, since mutation of the putative e -recruitment sites abolishes the anti-pcd and virulence activities of the avrptob protein (janjusevic et al., ) . determining the host targets of the pseudomonas e will be crucial in clarifying its mechanism of action and might even explain plant susceptibility to infection. such experiments may also assist in the identification of similar targets in mammalian species infected with comparable gram-negative microbes. furthermore, it is rather striking that another pathogen uses a mimic of a host e ligase to encode an immunomodulatory function. there are bound to be others. mumps virus and other paramyxoviridae family members hijack the cullin- a-scf b-trcp e ligase to suppress ifn-as well as il- -mediated signaling (ulane et al., ) , important for control of inflammation and apoptosis during inflammation (hodge et al., ) . ifn and il- signaling activates their cognate stat factors and transcription of genes involved in ifn and cytokine signaling. some of these paramyxoviruses use their v protein to bind ddb , the cullin- a-scf b-trcp e substrate adaptor that recruits stat proteins to the e complex, targeting stats for proteasome-mediated degradati on ( li et al., b ; ulane and horva th, ; ulane et al., ) . many other viru ses targe t stat transcri ption factors for protea somal degrad ation (garcin et al., ; lin et al., ; ramaswamy et al., ; zimmermann et al., ) . recruitment of cullin e s is likely to be a more widely used viral strategy of interfering with ifn and cytokine signaling. the interleukin- (il- )-inducible deubiquitinating enzyme dub- is induced by il- stimulation and may regulate il- signaling. the il- -inducible dub- is constitutively expressed in cells transformed by human t cell leukemia virus- (htlv- ). like other cytokines, il- is an important modulator of apoptosis in t cells that acts through the stat pathway. although the mechanism is not at all clear, dub- activity prolongs il- -stimulated phosphorylation and transcriptional activity of stat , inhibiting t cell apoptosis in the aftermath of the immune response (migone et al., ; shackelford and pagano, ) and possibly contributing to cell immortalization. cytokine-inducible dubs could interfere with cytokine signaling, thereby playing an active role in modulation of the immune response. the il- -inducible dub- is specifically induced by interleukin (il- ), gm-csf, and il- , which suggests a role in responses mediated by these cytokines (d'andrea and pellman, ) and might constitute a target for pathogen modulation of the immune response through interference with the ub-proteasome system. although a review of this rapidly expanding field is beyond the scope of this chapter, even this simple example suffices to demonstrate how both ub addition and removal control immune physiology, and consequently are likely targets for interference by pathogens. bacteria of the yersinia genus are the causal agents of plague, septicemia, and gastrointestinal syndromes. enteropathogenic yersinia species are extracellular multiplying gram-negative bacteria that make use of type iii secretion systems to inject virulence factors into host cells. the yersinia yopj virulence factor encodes a protein reported to be a cysteine protease that can cleave ub and sumo. the yopj dub inhibits nf-kb and mitogen-activated protein kinase (mapk) pathways, a function ascribed previously to its somewhat promiscuous deubiquitination of critical cellular proteins, such as traf , traf , and ikb. the mapk/jun pathway is involved in transcriptional control of several cytokine genes. the yopj activity induces macrophage death and blocks their ability to activate these inflammatory pathways (orth, ; orth et al., ; zhou et al., ) . however, recent reports show that the true function of yopj is that of a serine/threonine acetyltransferase (mukherjee et al., ) . the way by which the functions assigned to yopj have changed as the field advances demonstrates the complexity of assigning a function to proteins that lack obvious mammalian counterparts. both salmonella and the plant pathogen xanthomonas secrete proteins with homology to yopj, but their putative dub activity or function in the host have not been assessed yet (gurlebeck et al., ; hardt and galan, ) . a novel viral usp or deubiquitinating enzyme, ul usp , was recently identified in herpes simplex virus- (hsv- ) by labeling with an ub-derived probe . the ul usp is located at the n-terminus of the ul , the large tegument protein of hsv- , an a-herpesvirus. despite the overall low sequence homology-at the exception of almost only the amino acid residues composing the catalytic triad-the ul usp activity is well conserved in all members of the herpesviridae family, as the homologous proteins in murine cytomegalovirus (a b-herpesvirus) and ebv (a g-herpesvirus) also exhibit dub activity in vitro (schlieker et al., ) . one of the two severe acute respiratory syndrome (sars) coronavirus proteases responsible for cleavage of the replicase polyprotein, the sars-cov papain-like protease plpro, is also a dub (barretto et al., ; lindner et al., ) , predicted to be structurally similar to human usp (hu et al., ) . the sars-cov plpro protease activity is involved in the processing of the viral polyprotein, thereby contributing to replication of the viral rna genome. the function of the sars virus deubiquitinating activity, which extends to isg -removal activity (lindner et al., ) , is unknown at this point. the adenovirus proteinase (avp) and the human cytomegalovirus ul protein also encode dub activities, but the viral and/or cellular targets remain unidentified (balakirev et al., ; wang et al., b) . chlamydia trachomatis, an obligate intracellular bacterium that causes a variety of diseases in humans has two genes, chladub and chladub , whose products encode deubiquitinating and deneddylating activitie s (misa ghi et al., ) . unlike c. pneum oniae who se genome is devoid of chladub genes, c. trachomatis is able to block nf-kb signaling and thus the inflammatory response, as well as host cell apoptosis. both processes could be modulated by the c. trachomatis dubs. these unexpected dub activities encoded by pathogens suggest a novel strategy of modulation of host defense by manipulating the cellular ubiquitination machinery. although the experimental evidence is at best tenuous, one can speculate that many of these other pathogen-encoded dubs may be important for modulation of the ub-proteasome system during the pathogen life cycle and/or in the context of immune evasion. the fact that these dub activities are conserved suggests functional importance. they may be delivered to the host cell at the time of infection (for instance, by a bacterial type iii secretion system or by a viral tegument protein) or may be transcribed early during infection. the pathogen-encoded dub could interfere with the levels of ub-or ubl-conjugated proteins, thereby altering cellular processes ranging from signaling pathways, protein degradation, antigen presentation, vesicular trafficking, and many others. we have elaborated on several examples of how the host immune system can be manipulated by a pathogen-encoded e or pathogen hijacking of a host e . similarly-and although the sample pool is rather small at the moment-there is no reason to believe that encoding their own dubs and/or hijacking of host dubs has not ''occurred'' to pathogens. an attractive possibility is that some of these activities are aimed at manipulating the host immune system. possible targets would include pathways involved in ''housekeeping'' processes like life and death of the cells of the immune system, and extend to, for example, interference with antigen presentation. these dub activities could manipulate membrane trafficking in ways that would prevent mhc molecules from reaching the cell surface, or prevent proteasomal degradation of pathogen-derived proteins. this initial window of deubiquitinating activity could help viruses escape detection. bacterial pathogens could benefit from targeting of transcription factors and cytokine signaling networks that are crucial for coordination of macrophage effector functions. other immune responses that could, in principle, be controlled by dubs are ifn and nf-nf-kb signaling, and the 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polyubiquitin serves as a recognition signal, rather than a ratcheting molecule, during retrotranslocation of proteins across the endoplasmic reticulum membrane gamma-secretase-mediated proteolysis in cell-surface-receptor signalling consensus analysis of signal peptide peptidase and homologous human aspartic proteases reveals opposite topology of catalytic domains compared with presenilins the human cytomegalovirus us gene product delays trafficking of major histocompatibility complex class i molecules membrane-specific, host-derived factors are required for us -and us -mediated degradation of major histocompatibility complex class i molecules ubiquitinylation of the cytosolic domain of a type i membrane protein is not required to initiate its dislocation from the endoplasmic reticulum type iii secretion machines: bacterial devices for protein delivery into host cells adenovirus protein involved in virus internalization recruits ubiquitin-protein ligases all four sendai virus c proteins bind stat , but only the larger forms also induce its mono-ubiquitination and degradation tsg and the vacuolar protein sorting pathway are essential for hiv- budding the hrd p ligase complex forms a linchpin between er-lumenal substrate selection and cdc p recruitment hiv- tat protein modulates the generation of cytotoxic t cell epitopes by modifying proteasome composition and enzymatic activity human cytomegalovirus us endoplasmic reticulum-lumenal domain dictates association with major histocompatibility complex class i in a locus-specific manner us , a human cytomegalovirus-encoded type i membrane protein, contains a non-cleavable amino-terminal signal peptide subunit h of the v-atpase binds to the medium chain of adaptor protein complex and connects nef to the endocytic machinery cdc p interacts with ufd p, a wd repeat protein required for ubiquitin-mediated proteolysis in saccharomyces cerevisiae proteomic identification of proteins conjugated to isg in mouse and human cells cytomegalovirus selectively blocks antigen processing and presentation of its immediate-early gene product c-mir, a human e ubiquitin ligase, is a functional homolog of herpesvirus proteins mir and mir and has similar activity the caenorhabditis elegans impas gene, imp- , is essential for development and is functionally distinct from related presenilins structure of s proteasome from yeast at . a resolution a gated channel into the proteasome core particle the many roads to cross-presentation human cytomegalovirus immediate early glycoprotein us retains mhc class i molecules by transient association myxoma virus leukemia-associated protein is responsible for major histocompatibility complex class i and fas-cd down-regulation and defines scrapins, a new group of surface cellular receptor abductor proteins pathways for antigen cross presentation type iii effector proteins from the plant pathogen xanthomonas and their role in the interaction with the host plant ubiquitylation and cell signaling endoplasmic reticulum retention is a common defect associated with tyrosinase-negative albinism physical and functional interactions of the cytomegalovirus us glycoprotein with the transporter associated with antigen processing role of n-linked oligosaccharide recognition, glucose trimming, and calnexin in glycoprotein folding and quality control er-associated degradation in protein quality control and cellular regulation ubiquitin-mediated regulation of -hydroxy- -methylglutaryl-coa reductase role of s proteasome and hrd genes in the degradation of -hydroxy- -methylglutaryl-coa reductase, an integral endoplasmic reticulum membrane protein a secreted salmonella protein with homology to an avirulence determinant of plant pathogenic bacteria retroviral restriction by apobec proteins integral ubl domain proteins: a family of proteasome interacting proteins protein degradation: recognition of ubiquitinylated substrates transferring substrates to the s proteasome rhabdoviruses and the cellular ubiquitin-proteasome system: a budding interaction teb is a c hc ring finger-containing ubiquitin ligase of the endoplasmic reticulum u box proteins as a new family of ubiquitin-protein ligases generation, translocation, and presentation of mhc class irestricted peptides human cytomegalovirus us causes similar effects on both major histocompatibility complex class i and ii proteins in epithelial and glial cells inhibition of hla-dr assembly, transport, and loading by human cytomegalovirus glycoprotein us : a novel mechanism for evading major histocompatibility complex class ii antigen presentation the ultimate nanoscale mincer: assembly, structure and active sites of the s proteasome core roles of n-linked glycans in the endoplasmic reticulum proteasomes: a complex story interference with antigen processing by viruses a viral er-resident glycoprotein inactivates the mhc-encoded peptide transporter immune evasion by cytomegalovirussurvival strategies of a highly adapted opportunist cytomegaloviral control of mhc class i function in the mouse viruses know it all: new insights into ifn networks the ubiquitin system for protein degradation the ubiquitin system the human cytomegalovirus gene product us inhibits atp binding by tap a new ticket for entry into budding vesicles-ubiquitin regulation of membrane protein transport by ubiquitin and ubiquitin-binding proteins structure of the escrt-ii endosomal trafficking complex strategies and mechanisms for host and pathogen survival in acute and persistent viral infections intracellular targeting of the proteasome a role for n-glycanase in the cytosolic turnover of glycoproteins endoplasmic reticulum-associated protein degradation-one model fits all? hostile takeovers: viral appropriation of the nf-kappab pathway attenuation of hla-dr expression by mononuclear phagocytes infected with mycobacterium tuberculosis is related to intracellular sequestration of immature class ii heterodimers sp-ring for sumo: new functions bloom for a ubiquitin-like protein the role of il- and stat in inflammation and cancer lysosomal cysteine proteases regulate antigen presentation multiubiquitylation by e enzymes: 'one size' doesn't fit all role of herp in the endoplasmic reticulum stress response the lysosomal cysteine proteases in mhc class ii antigen presentation crystal structure of a ubp-family deubiquitinating enzyme in isolation and in complex with ubiquitin aldehyde the alpha chain of the t cell antigen receptor is degraded in the cytosol the escrt complexes: structure and mechanism of a membrane-trafficking network mycobacterium avium infection of mouse macrophages inhibits ifn-gamma janus kinase-stat signaling and gene induction by downregulation of the ifn-gamma receptor parkin suppresses unfolded protein stressinduced cell death through its e ubiquitin-protein ligase activity chip is associated with parkin, a gene responsible for familial parkinson's disease, and enhances its ubiquitin ligase activity the nedd family of e ubiquitin ligases: functional diversity within a common modular architecture the epstein-barr virus protein, latent membrane protein a, co-opts tyrosine kinases used by the t cell receptor nod-lrr proteins: role in host-microbial interactions and inflammatory disease downregulation of major histocompatibility complex class i molecules by kaposi's sarcoma-associated herpesvirus k and k proteins htm p, a mannosidase-like protein, is involved in glycoprotein degradation in yeast a bacterial inhibitor of host programmed cell death defenses is an e ubiquitin ligase hiv- nef stabilizes the association of adaptor protein complexes with membranes recognition of dileucine-based sorting signals from hiv- nef and limp-ii by the ap- gamma-sigma and ap- delta-sigma hemicomplexes protein dislocation from the er requires polyubiquitination and the aaa-atpase cdc multiple proteolytic systems, including the proteasome, contribute to cftr processing ring finger specificity in scf-driven protein destruction systematic analysis and nomenclature of mammalian f-box proteins cd phosphorylation partially reverses nef down-regulation of cd hiv nefmediated cd down-regulation is adaptor protein complex dependent ring finger proteins: mediators of ubiquitin ligase activity a proteolytic pathway that recognizes ubiquitin as a degradation signal multiple independent loci within the human cytomegalovirus unique short region downregulate expression of major histocompatibility complex class i heavy chains human cytomegalovirus us impairs transport and maturation of major histocompatibility complex class i heavy chains nef: ''necessary and enforcing factor'' in hiv infection phagocytosis: at the crossroads of innate and adaptive immunity the protein translocation channel binds proteasomes to the endoplasmic reticulum membrane phosphorylation meets ubiquitination: the control of nf-[kappa]b activity a complex between peptide: n-glycanase and two proteasome-linked proteins suggests a mechanism for the degradation of misfolded glycoproteins a deubiquitinating enzyme encoded by hsv- belongs to a family of cysteine proteases that is conserved across the family herpesviridae receptor downregulation and multivesicular-body sorting nedd recruits e -ubiquitin to scf e ligase modulation of specific surface receptors and activation sensitization in primary resting cd þ t lymphocytes by the nef protein of hiv- the human immunodeficiency virus type (hiv- ) vpu protein interferes with an early step in the biosynthesis of major histocompatibility complex (mhc) class i molecules human hrd is an e ubiquitin ligase involved in degradation of proteins from the endoplasmic reticulum the shigella flexneri effector ospg interferes with innate immune responses by targeting ubiquitinconjugating enzymes the png -rad complex regulates glycoprotein turnover ube l and protein isgylation are not essential for alpha/beta interferon signaling monovalent ligation of the b cell receptor induces receptor activation but fails to promote antigen presentation intracellular membrane traffic of human immunodeficiency virus type envelope glycoproteins: vpu liberates golgi-targeted gp from cd -dependent retention in the endoplasmic reticulum t cells and viral persistence: lessons from diverse infections the ubiquitin-proteasome system in hiv replication: potential targets for antiretroviral therapy the proteasome and mhc class i antigen processing proteasome and peptidase function in mhc-class-imediated antigen presentation reexamination of the role of ubiquitin-like modifier isg in the phenotype of ubp -deficient mice der , a novel protein specifically required for endoplasmic reticulum degradation in yeast the substrate translocation channel of the proteasome herp, a new ubiquitin-like membrane protein induced by endoplasmic reticulum stress gamma-secretase: proteasome of the membrane? hepatitis c virus core protein inhibits mitochondrial electron transport and increases reactive oxygen species (ros) production differential effects of inhibitors on the gammasecretase complex. mechanistic implications structural and functional organization of the escrt-i trafficking complex for whom the bell tolls: protein quality control of the endoplasmic reticulum and the ubiquitin-proteasome connection differential localization and identification of a critical aspartate suggest non-redundant proteolytic functions of the presenilin in homologues sppl b and sppl membrane topology of the yeast endoplasmic reticulum-localized ubiquitin ligase doa and comparison with its human ortholog teb (march-vi) the components of the proteasome system and their role in mhc class i antigen processing temporal regulation of salmonella virulence effector function by proteasome-dependent protein degradation a new self: mhc-class-i-independent natural-killer-cell self-tolerance role of interferon-stimulated gene isg- in the interferonomega-mediated inhibition of human immunodeficiency virus replication molecular mechanism and structural aspects of transporter associated with antigen processing inhibition by the cytomegalovirus protein us human immunodeficiency virus type nef mediates sustained membrane expression of tumor necrosis factor and the related cytokine light on activated t cells cell-surface expression of cd reduces hiv- infectivity by blocking env incorporation in a nef-and vpu-inhibitable manner exclusive ubiquitination and sumoylation on overlapping lysine residues mediate nf-kappab activation by the human t-cell leukemia virus tax oncoprotein nk cell recognition nef interacts with the mu subunit of clathrin adaptor complexes and reveals a cryptic sorting signal in mhc i molecules determinant for endoplasmic reticulum retention in the luminal domain of the human cytomegalovirus us glycoprotein the human cytomegalovirus us glycoprotein inhibits transporter associated with antigen processing-dependent peptide translocation downregulation of cell surface receptors by the k family of viral and cellular ubiquitin e ligases requirements for signal peptide peptidase-catalyzed intramembrane proteolysis on the mechanism of spp-catalysed intramembrane proteolysis; conformational control of peptide bond hydrolysis in the plane of the membrane intramembrane proteolysis of signal peptides: an essential step in the generation of hla-e epitopes mechanism of intramembrane proteolysis investigated with purified rhomboid proteases modulation of natural killer cell cytotoxicity in human cytomegalovirus infection: the role of endogenous class i major histocompatibility complex and a viral class i homolog inhibition of antigen processing by the internal repeat region of the epstein-barr virus nuclear antigen- hiv- assembly and budding as targets for drug discovery multiple modes of interaction of the deglycosylation enzyme, mouse peptide n-glycanase, with the proteasome the aaa atpase p links peptide n-glycanase to the endoplasmic reticulum-associated e ligase autocrine motility factor receptor epstein-barr virus uses hla class ii as a cofactor for infection of b lymphocytes the ulp sumo isopeptidase: distinct domains required for viability, nuclear envelope localization, and substrate specificity structure of ddb in complex with a paramyxovirus v protein: viral hijack of a propeller cluster in ubiquitin ligase overexpression of the tumor autocrine motility factor receptor gp , a ubiquitin protein ligase, results in increased ubiquitinylation and decreased secretion of apolipoprotein b in hepg cells overlapping motifs (ptap and ppey) within the ebola virus vp protein function independently as late budding domains: involvement of host proteins tsg and vps- a membrane protein required for dislocation of misfolded proteins from the er multiprotein complexes that link dislocation, ubiquitination, and extraction of misfolded proteins from the endoplasmic reticulum membrane viral modulation of antigen presentation: manipulation of cellular targets in the er and beyond dislocation of a type i membrane protein requires interactions between membrane-spanning segments within the lipid bilayer hepatitis c virus expression suppresses interferon signaling by degrading stat the papain-like protease from the severe acute respiratory syndrome coronavirus is a deubiquitinating enzyme ubiquitin ligases and the immune response immunity by ubiquitylation: a reversible process of modification viral modulation of nk cell immunity er dislocation: cdc p/p gets into the aaact signal peptide peptidase is required for dislocation from the endoplasmic reticulum primate lentiviral virion infectivity factors are substrate receptors that assemble with cullin -e ligase through a hcch motif to suppress apobec g viral immune evasion molecules attack the er peptide-loading complex and exploit er-associated degradation pathways virus subversion of the mhc class i peptide-loading complex the hcmv gene products us and us differ in their ability to attack allelic forms of murine major histocompatibility complex (mhc) class i heavy chains high-throughput immunoblotting. ubiquitiin-like protein isg modifies key regulators of signal transduction ubp (usp ) specifically removes isg from conjugated proteins ubp is a novel regulator of interferon signaling independent of its isg isopeptidase activity negative factor from siv binds to the catalytic subunit of the v-atpase to internalize cd and to increase viral infectivity the multifaceted role of hiv nef nef-induced cd and major histocompatibility complex class i (mhc-i) down-regulation are governed by distinct determinants: n-terminal alpha helix and proline repeat of nef selectively regulate mhc-i trafficking the phd/lap-domain protein m r of myxomavirus is a ubiquitin ligase that induces the rapid internalization and lysosomal destruction of cd a novel human wd protein, h-beta trcp vpu connects cd to the er degradation pathway through an f-box motif virus entry: open sesame role of escrt-i in retroviral budding intramembrane proteolysis and post-targeting functions of signal peptides intramembrane-cleaving aspartic proteases and disease: presenilins, signal peptide peptidase and their homologs signal peptide fragments of preprolactin and hiv- p-gp interact with calmodulin new lessons from old pathogens: what parasitic infections have taught us about the role of nuclear factor-kappab in the regulation of immunity intramembrane proteolysis promotes trafficking of hepatitis c virus core protein to lipid droplets cop and clathrin-coated vesicle budding: different pathways, common approaches the hsc co-chaperone chip targets immature cftr for proteasomal degradation a genomic screen identifies dsk p and rad p as essential components of er-associated degradation cd /lir- / ilt and cd (cytotoxic t lymphocyte antigen ) inhibitory molecules down-regulate the cytolytic activity of human cd þ t-cell clones specific for mycobacterium tuberculosis erad: the long road to destruction inhibition of hepatitis c virus core protein expression in immortalized human hepatocytes induces cytochrome c-independent increase in apaf- and caspase- activation for cell death the deubiquitinating enzyme dub- prolongs cytokine-induced signal transducers and activators of transcription activation and suppresses apoptosis following cytokine withdrawal human cytomegalovirus inhibits major histocompatibility complex class ii expression by disruption of the jak/stat pathway regulatory t cells: friend or foe in immunity to infection? structural and functional analysis of human cytomegalovirus us protein using a small molecule inhibitor of peptide: n-glycanase to probe its role in glycoprotein turnover chlamydia trachomatis-derived deubiquitinating enzymes in mammalian cells during infection immune escape and exploitation strategies of cytomegaloviruses: impact on and imitation of the major histocompatibility system role of edem in the release of misfolded glycoproteins from the calnexin cycle selective inhibition of ii-dependent antigen presentation by helicobacter pylori toxin vaca yersinia yopj acetylates and inhibits kinase activation by blocking phosphorylation doa is a cdc adapter that possesses a novel ubiquitin binding domain dendritic cell maturation by innate lymphocytes: coordinated stimulation of innate and adaptive immunity chip is a chaperonedependent e ligase that ubiquitylates unfolded protein chip: a quality-control e ligase collaborating with molecular chaperones autocrine motility factor and its receptor: role in cell locomotion and metastasis a novel route for f-box protein mediated ubiquitination links chip to glycoprotein quality control ubx links the cdc complex to er-associated protein degradation herpes simplex virus type targets the mhc class ii processing pathway for immune evasion a genomic and functional inventory of deubiquitinating enzymes derlin- and derlin- are regulated by the mammalian unfolded protein response and are required for er-associated degradation mitochondrial injury, oxidative stress, and antioxidant gene expression are induced by hepatitis c virus core protein innate antiviral response targets hiv- release by the induction of ubiquitin-like protein isg core protein of hepatitis c virus induces cardiomyopathy how calmodulin binds its targets: sequence independent recognition of amphiphilic alpha-helices viral evasion of natural killer cells a structural determinant of hcmv us dictates the down-regulation of class i mhc molecules function of the yersinia effector yop disruption of signaling by yersinia effector yopj, a ubiquitin-like protein protease a single residue exchange within a viral ctl epitope alters proteasome-mediated degradation resulting in lack of antigen presentation mechanisms of mhc class i-restricted antigen processing nedd on cullin: building an expressway to protein destruction presenilin attenuates receptor-mediated signaling and synaptic function human cytomegalovirus inhibits tapasin-dependent peptide loading and optimization of the mhc class i peptide cargo for immune evasion f-box-like domain in the polerovirus protein p is required for silencing suppressor function function and regulation of cullin-ring ubiquitin ligases mechanisms underlying ubiquitination back to the future with ubiquitin ubiquitin: structures, functions, mechanisms hla-e-restricted recognition of cytomegalovirus-derived peptides by human cd þ cytolytic t lymphocytes mechanism of nef-induced cd endocytosis: nef connects cd with the mu chain of adaptor complexes nef-induced cd degradation: a diacidic-based motif in nef functions as a lysosomal targeting signal through the binding of beta-cop in endosomes the downregulation of cd and mhc-i by primate lentiviruses: a paradigm for the modulation of cell surface receptors sec p mediates export of a misfolded secretory protein from the endoplasmic reticulum to the cytosol for degradation viral interference with antigen presentation to cd þ t cells: lessons from cytomegalovirus multiple effects of interferon on the replication of human immunodeficiency virus type bacterial adhesion and entry into host cells mutant analysis links the translocon and bip to retrograde protein transport for er degradation endoplasmic reticulum degradation of a mutated atp-binding cassette transporter pdr proceeds in a concerted action of sec and the proteasome viral strategies of immune evasion interferon-alpha but not azt suppresses hiv expression in chronically infected cell lines structure of the tsg uev domain in complex with the ptap motif of the hiv- p protein hiv gag mimics the tsg -recruiting activity of the human hrs protein mutants affecting the structure of the cortical endoplasmic reticulum in saccharomyces cerevisiae translating innate immunity into immunological memory: implications for vaccine development viral evasion of nk-cell activation specific inhibition of type i interferon signal transduction by respiratory syncytial virus survival of the fitters immunology: protein surgery membrane and soluble substrates of the doa ubiquitin ligase are degraded by distinct pathways antigens and immunoevasins: opponents in cytomegalovirus immune surveillance human cytomegalovirus gene products us and us differ in their ability to attack major histocompatibility class i heavy chains in dendritic cells interference with t cell receptor-hla-dr interactions by epstein-barr virus gp results in reduced t helper cell recognition epstein-barr virus gp is posttranslationally modified to produce soluble gp that mediates hla class ii immune evasion the class i mhc homologue of human cytomegalovirus inhibits attack by natural killer cells the ubiquitin binding domain znf ubp recognizes the c-terminal diglycine motif of unanchored ubiquitin a series of ubiquitin binding factors connects cdc /p to substrate multiubiquitylation and proteasomal targeting modulation of major histocompatibility complex antigen expression by viral infection isg : the immunological kin of ubiquitin dysregulation of protein modification by isg results in brain cell injury proteasome function in antigen presentation: immunoproteasome complexes, peptide production, and interactions with viral proteins regulation of proteasome structure and function hla-e-restricted recognition of human cytomegalovirus by a subset of cytolytic t lymphocytes endoplasmic reticulum-associated degradation cdc p is ubx-linked to er ubiquitin ligases complete differentiation between enkephalinase and angiotensin-converting enzyme inhibition by retro-thiorphan cd down-regulation by hiv- and simian immunodeficiency virus (siv) nef proteins involves both internalization and intracellular retention mechanisms inhibition of hiv- progeny virion release by cell-surface cd is relieved by expression of the viral nef protein the specificity of tcr/pmhc interaction functional division of substrate processing cofactors of the ubiquitin-selective cdc chaperone in vivo evidence of chip up-regulation attenuating tau aggregation endoplasmic reticulum stress-inducible protein, herp, enhances presenilin-mediated generation of amyloid beta-protein regulation of human t-cell leukemia virus type (htlv- ) budding by ubiquitin ligase nedd calling in the troops: regulation of inflammatory cell trafficking through innate cytokine/chemokine networks cytokine and chemokine networks: pathways to antiviral defense functional organization of mir , a novel viral regulator of selective endocytosis the cd /lir- /ilt inhibitory receptor is expressed by all human t lymphocytes and down-regulates their functions specific recognition of the viral protein ul by cd j/lir- /ilt on cd þ t cells mediates the non-mhc-restricted lysis of human cytomegalovirus-infected cells rad links dna repair to the ubiquitin/proteasome pathway the hpv- e and e -ap complex functions as a ubiquitin-protein ligase in the ubiquitination of p a deubiquitinating activity is conserved in the large tegument protein of the herpesviridae proteasome-associated proteins: regulation of a proteolytic machine cd glycoprotein degradation induced by human immunodeficiency virus type vpu protein requires the function of proteasomes and the ubiquitin-conjugating pathway membrane-bound ubx recruits cdc to ubiquitin ligases and their substrates to ensure efficient er-associated protein degradation suppression of major histocompatibility complex class i and class ii gene expression in listeria monocytogenes-infected murine macrophages insights into scf ubiquitin ligases from the structure of the skp -skp complex the ubiquitin-domain protein herp forms a complex with components of the endoplasmic reticulum associated degradation pathway a superfamily of protein tags: ubiquitin, sumo and related modifiers targeting of hepatitis c virus core protein to mitochondria through a novel c-terminal localization motif the s proteasome: a dynamic structure notch and presenilin: regulated intramembrane proteolysis links development and degeneration tumor viruses and cell signaling pathways: deubiquitination versus ubiquitination targeting of host-cell ubiquitin pathways by viruses xbp , downstream of blimp- , expands the secretory apparatus and other organelles, and increases protein synthesis in plasma cell differentiation the pathway of us -dependent degradation of mhc class i heavy chains involves a ubiquitin-conjugated intermediate pias proteins and transcriptional regulation-more than just sumo e ligases? all the peptides that fit: the beginning, the middle, and the end of the mhc class i antigen-processing pathway priming of t cells by exogenous antigen cross-presented on mhc class i molecules glycoprotein b from strain of herpes simplex virus type i contains an invariant chain homologous sequence that binds to mhc class ii molecules gamma-secretase, notch, abeta and alzheimer's disease: where do the presenilins fit in? receptor binding and membrane fusion in virus entry: the influenza hemagglutinin type i interferons and the innate immune response-more than just antiviral cytokines regulated sumoylation and ubiquitination of ddmek is required for proper chemotaxis deubiquitinating enzymes: their functions and substrate specificity hiv- nef-induced upregulation of dc-sign in dendritic cells promotes lymphocyte clustering and viral spread gp , a membrane-anchored ubiquitin ligase, associates with insig- and couples sterol-regulated ubiquitination to degradation of hmg coa reductase dynamics of virus-host interplay in hiv- replication the extracellular domain of the epstein-barr virus bzlf protein binds the hla-dr beta chain and inhibits antigen presentation modulation of gamma interferon-induced major histocompatibility complex class ii gene expression by porphyromonas gingivalis membrane vesicles mhc class ii compartment subtypes: structure and function inhibition of mhc class irestricted antigen presentation by gamma -herpesviruses dendritic cells: immunological sentinels with a central role in health and disease interferon-gamma, the functional plasticity of the ubiquitin-proteasome system, and mhc class i antigen processing the human endosomal sorting complex required for transport (escrt-i) and its role in hiv- budding hiv- nef impairs mhc class ii antigen presentation and surface expression human immunodeficiency virus- nef expression induces intracellular accumulation of multivesicular bodies and major histocompatibility complex class ii complexes: potential role of phosphatidylinositol -kinase molecular determinants for subcellular localization of hepatitis c virus core protein png , a yeast gene encoding a highly conserved peptide: n-glycanase a conserved ubiquitin ligase of the nuclear envelope/endoplasmic reticulum that functions in both er-associated and matalpha repressor degradation mechanism for down-regulation of cd by nef transmembrane inhibitors of p-glycoprotein, an abc transporter er-golgi traffic is a prerequisite for efficient er degradation the endoplasmic reticulum degradation pathway for mutant secretory proteins alpha -antitrypsin z and s is distinct from that for an unassembled membrane protein cdc can distinguish between native and non-native proteins in the absence of cofactors the isg isopeptidase ubp is regulated by proteolysis via the scfskp ubiquitin ligase surface expression of hla-e, an inhibitor of natural killer cells, enhanced by human cytomegalovirus gpul downregulation of natural killer cell-activating ligand cd by human cytomegalovirus cytomegalovirus us destroys two components of the mhc class ii pathway, preventing recognition by cd þ t cells viral subversion of the immune system agrobacterium t-dna integration: molecules and models involvement of targeted proteolysis in plant genetic transformation by agrobacterium paramyxoviruses sv and hpiv assemble stat protein ubiquitin ligase complexes from cellular components composition and assembly of stat-targeting ubiquitin ligase complexes: paramyxovirus v protein carboxyl terminus is an oligomerization domain stat ubiquitylation and degradation by mumps virus suppress cytokine and oncogene signaling cutting edge: the human cytomegalovirus ul gene product contains a ligand for hla-e and prevents nk cell-mediated lysis differential processing of class-i-restricted epitopes by the standard proteasome and the immunoproteasome regulated protein degradation misfolded proteins are sorted by a sequential checkpoint mechanism of er quality control distinct retrieval and retention mechanisms are required for the quality control of endoplasmic reticulum protein folding proteases involved in mhc class ii antigen presentation shaping the t cell repertoire signal sequences. the limits of variation major histocompatibility complex class i allele-specific cooperative and competitive interactions between immune evasion proteins of cytomegalovirus c-terminal pal motif of presenilin and presenilin homologues required for normal active site conformation highmolecular-weight protein (pul ) of human cytomegalovirus is a competent deubiquitinating protease: mutant viruses altered in its active-site cysteine or histidine are viable regulation of tyrosinase trafficking and processing by presenilins: partial loss of function by familial alzheimer's disease mutation model for the interaction of gammaherpesvirus ring-ch finger protein mk with major histocompatibility complex class i and the peptide-loading complex requirements for the selective degradation of endoplasmic reticulum-resident major histocompatibility complex class i proteins by the viral immune evasion molecule mk the viral e ubiquitin ligase mk uses the derlin/p endoplasmic reticulum-associated degradation pathway to mediate down-regulation of major histocompatibility complex class i proteins degradation of cftr by the ubiquitinproteasome pathway identification of signal peptide peptidase, a presenilin-type aspartic protease proteasome-dependent endoplasmic reticulum-associated protein degradation: an unconventional route to a familiar fate the human cytomegalovirus us gene product dislocates mhc class i heavy chains from the endoplasmic reticulum to the cytosol sec -mediated transfer of a membrane protein from the endoplasmic reticulum to the proteasome for destruction proteins containing the uba domain are able to bind to multi-ubiquitin chains human cytomegalovirus encodes an mhc class i-like molecule (ul ) that functions to inhibit nk cell lysis degradative organelles containing mislocalized alpha-and beta-synuclein proliferate in presenilin- null neurons a family of mammalian f-box proteins p , a protein coping with multiple identities neddylation and deneddylation regulate cul and cul protein accumulation intramembrane proteolysis by presenilin and presenilin-like proteases parkin phosphorylation and modulation of its e ubiquitin ligase activity novel aspects of degradation of t cell receptor subunits from the endoplasmic reticulum (er) in t cells: importance of oligosaccharide processing, ubiquitination, and proteasome-dependent removal from er membranes nedd regulates egress of ebola viruslike particles from host cells the aaa atpase cdc /p and its partners transport proteins from the er into the cytosol function of the p -ufd -npl complex in retrotranslocation from the er to the cytosol: dual recognition of nonubiquitinated polypeptide segments and polyubiquitin chains a membrane protein complex mediates retro-translocation from the er lumen into the cytosol inaugural article: recruitment of the p atpase and ubiquitin ligases to the site of retrotranslocation at the endoplasmic reticulum membrane viral interference with antigen presentation at the crossroads of cell biology and immunology: drips and other sources of peptide ligands for mhc class i molecules self-inhibition of synthesis and antigen presentation by epstein-barr virus-encoded ebna a novel role for n-glycans in the erad system e ubiquitin ligase that recognizes sugar chains fbs is a new member of the e ubiquitin ligase family that recognizes sugar chains induction of apobec g ubiquitination and degradation by an hiv- vif-cul -scf complex physical association of the k protein of gamma- herpesvirus with major histocompatibility complex class i molecules with impaired peptide and beta( )-microglobulin assembly influenza b virus ns protein inhibits conjugation of the interferon (ifn)-induced ubiquitin-like isg protein structural basis for ubiquitinlike isg protein binding to the ns protein of influenza b virus: a protein-protein interaction function that is not shared by the corresponding n-terminal domain of the ns protein of influenza a virus plant rnai: how a viral silencing suppressor inactivates sirna the cytidine deaminase cem induces hypermutation in newly synthesized hiv- dna structure of the aaa atpase p the ubch ubiquitin e enzyme is also the e enzyme for isg , an ifn-alpha/beta-induced ubiquitin-like protein structure of the cul -rbx -skp -f boxskp scf ubiquitin ligase complex chlamydia inhibits interferon gamma-inducible major histocompatibility complex class ii expression by degradation of upstream stimulatory factor yersinia virulence factor yopj acts as a deubiquitinase to inhibit nf-kappa b activation a cytomegaloviral protein reveals a dual role for stat in ifn-{gamma} signaling and antiviral responses nk cell regulation of t cell-mediated responses the interferon-inducible ubiquitin-protein isopeptide ligase (e ) efp also functions as an isg e ligase we thank howard c. hang for help with the text and the figures and apologize to all colleagues whose work we may have not included. this work was supported by nih grants to h.l.p. key: cord- - bw rm e authors: teraguchi, shunsuke; saputri, dianita s.; anais llamas-covarrubias, mara; davila, ana; diez, diego; aybars nazlica, sedat; rozewicki, john; ismanto, hendra s.; wilamowski, jan; xie, jiaqi; xu, zichang; de jesus loza-lopez, martin; van eerden, floris j.; li, songling; standley, daron m. title: methods for sequence and structural analysis of b and t cell receptor repertoires date: - - journal: comput struct biotechnol j doi: . /j.csbj. . . sha: doc_id: cord_uid: bw rm e b cell receptors (bcrs) and t cell receptors (tcrs) make up an essential network of defense molecules that, collectively, can distinguish self from non-self and facilitate destruction of antigen-bearing cells such as pathogens or tumors. the analysis of bcr and tcr repertoires plays an important role in both basic immunology as well as in biotechnology. because the repertoires are highly diverse, specialized software methods are needed to extract meaningful information from bcr and tcr sequence data. here, we review recent developments in bioinformatics tools for analysis of bcr and tcr repertoires, with an emphasis on those that incorporate structural features. after describing the recent sequencing technologies for immune receptor repertoires, we survey structural modeling methods for bcr and tcrs, along with methods for clustering such models. we review downstream analyses, including bcr and tcr epitope prediction, antibody-antigen docking and tcr-peptide-mhc modeling. we also briefly discuss molecular dynamics in this context. b cell receptors (bcrs) and t cell receptors (tcrs) are key molecules in adaptive immune response that provide protection to perturbations, both from the outside (e.g. pathogens) and from within (e.g. mutated or misfolded proteins). together, bcrs and tcrs constitute a unique class of proteins whose coding sequences are arranged combinatorically in a cell-autonomous manner known as v(d)j recombination. in v(d)j recombination within a given cell, variable (v), diversity (d), and joining (j) segments are selected randomly from among many variants, and joined to make the v (variable) region of a full-length receptor. in addition to v(d)j recombination, bcrs can also undergo subsequent somatic hypermutation (shm) and clonal selection upon antigen encounter, collectively referred to as "affinity maturation". on a cell population level, these processes create a functionally diverse and dynamic set (repertoire) of b and t cells. the number of possible different bcr or tcr sequence combinations is extremely high, with theoretical estimates in the - range [ ] . however, the observed populations of receptor sequences in a given individual follow a power law, where most sequences appear only at very low frequency and a minority of sequences appear at higher frequencies (see for example [ ] for a recent discussion). for both bcrs and tcrs, v regions consist of two polypeptide chains, referred to as "light" (bcrs) or "alpha" (tcrs) and "heavy" (bcrs) or "beta" (tcrs). tcrs are composed of a single set of alpha and beta chains while bcrs contain two sets of light and heavy chains. [ ] for simplicity, in this review, we focus on a single pair of (lightheavy or alpha-beta) chains. both bcrs and tcrs belong to the immunoglobulin-like fold in which the canonical antigen binding site is composed three loops called "complementarity-determining regions" (cdrs), in each receptor chain. the v(d)j recombination junction, in which random nucleotides may be inserted during the recombination, is located in the third cdr (cdr ). as a result, cdr is the most diverse among the three cdrs. [ ] much effort has been spent on cdr modeling, in particular for soluble bcrs (antibodies). bcrs interact directly with antigens, and we refer to interface residues as "paratope" on the bcr side and "epitope" on the antigen side ( figure a) . tcrs, on the other hand, interact with antigen-derived peptide fragments, which are presented by major histocompatibility complex (mhc) proteins ( figure b) . here, generally "epitope" refers to the antigen-derived peptide and not the mhc contacting residues. tcr contacting residues are shown as sticks. b, tcr-peptide-mhc complex for a viral peptide tax and class i hla a- (pdb identifier bd ). the epitope is shown as red spheres and contacting mhc residues are shown as green spheres, while paratope residues are shown as sticks. each human carries up to six class i mhc molecules and up to eight class ii molecules. there are thousands of mhc variants (alleles) in the human population, which can differ in their peptide specificity [ ] . peptide-mhc binding affinity shapes the tcr repertoire, and the particular set of mhc alleles carried by an individual become a source of tcr repertoire diversity, affecting the susceptibility to particular diseases (reviewed in [ ] ). since bcr maturation requires a co-stimulation from activated helper t cells [ ] , the bcr and tcr repertoires are not completely independent. both bcr and tcr sequences can be captured by current sequencing technologies. moreover, molecule and cell barcoding technologies are an area of intense research and development. emerging sequencing and barcoding methods are thus expected to revolutionize our understanding of immune repertoires. as just one example, the number of paired (alpha-beta) tcr sequences for which the peptide-mhc is known has grown by two orders of magnitude in the last two years [ ] , indicating a need for computational tools that can keep pace with this growth. in this review, after briefly reviewing recent technologies for repertoire sequencing, we explore tools for interpreting bcr and tcr sequences in terms of their structures and targeted antigens. in this context, we cover structural modeling, epitope prediction, molecular docking, and molecular dynamics. integration of such tools, along with growth in sequence and associated experimental data, will allow us to more fully describe the immune status of an individual in health and disease. very early approaches to characterize immune repertoires were limited to estimating the length of the cdr loops [ ] . current methods, relying on high-throughput sequencing (hts) technology, can be used for comprehensive quantification of full-length tcr and bcr v region sequences [ , ] . though a comprehensive review on the existing technologies for repertoire sequencing analysis is beyond the scope of this review, hts is the main source of data for subsequent structural analysis. therefore, we briefly describe the basic information contained in bulk and single-cell rna-based repertoire sequencing (fig. ) . in bulk sequencing, the information of receptor pairs will be lost while higher coverage tends to be achieved. in single cell sequencing, the pairing information is preserved while currently sample preparation and sequencing costs tend to be higher than in bulk sequencing. early development of hts repertoire analysis was based on bulk sequencing (i.e. sequencing many cells without preserving their identities). in this approach, the information of light/heavy or alpha/beta pairs is lost. thus, bulk sequence analysis tends to focus on a single (typically the heavy/beta) chain. repertoire sequencing typically uses tcr/bcr enrichment followed by pcr amplification to increase sensitivity and reduce sequencing cost. since a bp fragment is enough to resolve the cdr fragment, short read sequencing is often used. the choice of sequencing technology can have an important impact on quality, since the types and rates of errors can be different. among preferred platforms are illumina miseq (long reads) and hiseq (short reads targeting cdr ). one of the sources of low-quality repertoire data is a biproduct of pcr amplification. without other information, we cannot distinguish between true nucleotide sequence differences and pcr errors. as a result, pcr errors cause the appearance of spurious sequences, in particular from dominant, highly abundant sequences/clonotypes. use of unique molecular identifier (umi) sequences enables correction of pcr amplification biases and quantification of the number of receptors expressed. thus, the use of technologies with umi have a distinct advantage. to date, several pipelines can be used to extract repertoire information from bulk hts data. these tools generally map sequencing reads to tcr/bcr reference sequences. then, contigs, the continuous sequences assembled from the mapped reads, can subsequently be annotated by v(d)j gene usage and cdr ( , ,) amino acid sequences [ , ]. imgt/highv-quest (international immunogenetics information system v-query and standardization) [ , ] uses pairwise alignment and sequence comparison to experimental data to align sequencing reads. igblast [ ] utilizes the blast algorithm [ ] for its search engine. mixcr [ ] is an efficient pipeline equipped with a fast aligner. it can be used for reconstructing tcr/bcr sequences from generic rnaseq data without pcr amplification of tcrs/bcrs [ ] . a detailed assessment on those three tools can be found in [ ] . the immcantation framework [ , ] and trust (tcr repertoire utilities for solid tissue) [ ] can be also used for the same purpose among many other available tools not covered here though single chain information alone is usually not enough to explain the binding of the receptor to the target epitope, there are several methods applicable to bulk sequencing data. for example, diversity analysis of the repertoire sequences can be used for estimating the clonal diversity of an immune repertoire of each individual, as well as repertoire overlap among repertoires of several individuals. this can currently be performed using conventional ecology measures [ ] [ ] [ ] , or repertoire-designed estimators [ ] [ ] [ ] . also, by analyzing repertoire data from many individuals with additional information like human leukocyte antigen (hla) allele profiles or disease status, one can associate each tcr with particular labels with the help of statistical hypothesis testing [ , ] . repertoire information also carries the information of underlying v(d)j recombination. thus, from repertoire sequences, generative models of v(d)j recombination were developed; and, in turn, these models were used to analyze repertoire sequence data [ ] [ ] [ ] [ ] [ ] [ ] . we have collected some of (but not all of) tools used for those sequence analysis as in table . there have also been exciting developments in the application of hts technology for experimental discovery of epitopes. in libra-seq (linking b cell receptor to antigen specificity through sequencing) [ ], the x genomics platform was used to barcode not only bcr sequences but also antigen proteins. by sorting the antigen-bound b cells and then performing single cell sequencing, antigen specific bcrs can be identified from the antigen barcodes. similarly, by using barcoded peptide-mhc complexes, hts allow us to generate a large reference dataset of tcr-epitope pairs [ ] . kula et al. [ ] developed t-scan, a high-throughput method that identifies functional antigen targets of t cells and subsequent next-generation sequencing enabled t-scan to discover cmv antigens as well as the targets of self-reactive tcrs. gee mh et al. [ ] used yeastdisplay libraries of pmhcs and screened for antigens of orphan t cell receptors on tumorinfiltrating lymphocytes. kobayashi et al. [ ] have developed a cloning and expression system called htec (human tcr efficient cloning system within days) that can be used to rapidly determine the antigen specificity of tcrs. they applied their system successfully to peptide specificity and cytotoxic activity of tcrs from ebv infection and cancer. in spite of advances in experiential determination of receptor-antigen interactions, most high-throughput experiments lack residue-level resolution. x-ray crystallography and single-particle electron microscopy (cryo-em), on the other hand, provide such highresolution information, but are not suitable for high-throughput analysis. computational modeling of tcrs and bcrs is now routine and can be performed in a high-throughput manner. building d models of receptors is also the first step in structure-based analysis of receptor antigen interactions. for d structural modeling, tcr or bcr v regions are generally divided into "frameworks" and the three cdrs ( fig. ) . each framework is a double layer of beta sheets that contain the beginning and ending of each cdr loop. there are other loops in v regions, but the cdrs are important because of their high sequence diversity and because they form a continuous surface that constitutes the main antigen binding interface. of the cdrs, cdr is the most diverse in terms of both sequence and structure. cdr modeling has been tackled by a wide range of approaches [ ] . software for cdr modeling ( table ) spans the range from simple sequence alignment methods [ ] , to fragment assembly [ ] , molecular dynamics (md) [ ] and robotics-based loop closure algorithms [ ] . in the most recent antibody modeling assessment (ama-ii) [ ] , the lowest heavy-chain cdr (cdrh ) errors were obtained by our own group using a combination of md, fragment assembly and manual selection [ ] . based on an internal assessment of our ama-ii results, we developed a purely fragment assembly-based tool, kotai antibody builder [ ] . we more recently introduced repertoire builder, which exceeded kotai antibody builder in terms of accuracy, with a factor of improvement in speed [ ]. in the same time frame, several new tools, including abodybuilder [ ], tcrmodel [ ] , and pigspro (prediction of immunoglobulin structure v ) [ ] have been introduced, which show advancement over previously published methods. because of its high accuracy and ability to scale with the number of input sequences, we will briefly outline the repertoire builder approach. in order to improve speed and reduce noise, one aim of repertoire builder was to remove d structure from the key decision-making steps: sampling and scoring. working in three dimensions is computationally expensive and also messy, as protein structure files can contain a plethora of sources of noise. as an alternative, we derived feature vectors from pairwise query-template alignments and trained a machine learning model to recognize the good alignments. feature vectors currently consist of blosum matrix elements or gaps for each aligned residue pair and cover the entire v region. the inclusion of residues outside of the cdr region was intended to take the environment of the cdr into account in the choice of template. we note that scoring at the alignment level is not unique to repertoire builder; all of the methods do this. what is novel here is the alignmentderived feature vectors. another trick used by repertoire builder was to store templates in the form of structure-aware multiple sequence alignments (msas), which can be readily computed using our mafft-dash (multiple alignment using fast fourier transform-database of aligned structural homologs) pipeline and which have been shown to be significantly more accurate than sequence-based msas [ ] . the query sequence can be added to a stored template msa efficiently using mafft's fragmentadding option, which preserves the relationships between the templates in the stored msas [ ] . templates in msas are grouped by their cdr lengths. thus, there is a different template msa stored for each cdr-length combination. the advantage of using mafft-dash in this manner is primarily a combination of speed and msa accuracy. we have not assessed whether use of alternative alignment strategies results in a degradation of model quality. the current repertoire builder can model paired or unpaired sequences in approximately min, which makes it practically useful for highthroughput sequencing discussed above. to our knowledge, repertoire builder is the only server that allows multiple bcr or tcr sequences to be input at one time. as genomic data continues to grow, methods for clustering nucleotide or amino acid sequences will play major role in sequence and structural analysis. since generic sequence clustering methods (e.g. [ , ] ) are beyond the scope of this review, here we focus on methods specific to immune receptors. a common goal when studying immune repertoires is to understand common features of receptors that are shared by a group of donors of interest (fig. ) . the implication here is that receptors target the same antigen and epitope will be more common in the donors of interest than in a control group. this is a very general notion that can be applied to either bcrs or tcrs and approached in a variety of ways. given the broad diversity of immune repertoires, their uneven population distributions, and the relatively low overlap of exact matching sequences among subjects, this task is a significant challenge. to address these issues, several clustering strategies have been developed recently. below, we review some representative examples, including our own efforts. based on the observation that there are specific positions in tcr cdr regions that contact antigen peptides and that the presence of particular sequence motifs can define tcr clusters, glanville et al., developed the gliph (grouping of lymphocyte interactions by paratope hotspots) algorithm [ , ] . this algorithm clusters tcrs based on local sequence motifs, as well as on other parameters such as global cdr similarity, v gene usage, cdr length, mhc profile of donor(s) and clone size. gliph identifies motifs that are enriched in a given dataset relative to a control group, with the goal of producing groups of tcrs targeting the same peptide-mhc (pmhc). by using this approach, the authors were able to design synthetic antigen-specific tcrs to groups, and confirm their specificity experimentally. in a similar study, dash et al. [ ] developed tcrdist; a tool that estimates the similarity of two tcr sequences by computing a weighted hamming distance among the concatenated amino acid sequences of the cdr loops of each tcr. tcrdist assumes a higher weight ( x) for the cdr regions. clusters of highly similar antigen-specific tcrs can be built, and new tcrs of unknown specificity can be assigned to an antigenspecific cluster based on similarity, allowing for the prediction of antigen specificity. additionally, a diversity score (tcrdiv) that robustly calculates the diversity of epitopespecific repertoires by considering both tcr similarity and exact identity in a generalized simpson's diversity index, was developed. tcrdist has recently been used to identify clonal expansion of m. tuberculosis specific tcrs in a south african cohort where it was able to accurately classify active tuberculosis patients [ ] . though they share the same goal, the focus of those two tools are slightly different. the gliph algorithm assumes that the input data is enriched in tcrs targeting a restricted set of epitopes, and tries to cluster these enriched tcrs using common motifs in the dataset. with this approach, they are also able to avoid direct comparison of all pairs of sequences, which is computationally expensive. thus, gliph is suitable for large repertoire analyses of particular disease cohorts. on the other hand, tcrdist is based on direct comparison of each tcrs using a "universal" measure of tcr similarity, and it is thus currently difficult to apply the method to datasets greater than approximately . however, an advantage of tcrdist is that the calculated distance between a pair of tcrs are always the same, regardless of other factors. such "universal" definition of tcr similarity/difference is of use when assumptions about shared antigen/epitope cannot be made. structural studies of antibodies targeting antigens specific to hiv [ ] , influenza [ ] and more recently sars-cov- [ ] have demonstrated that antibodies produced in unrelated donors targeting common antigens and epitopes can share sequence and structural features. we note here that, since b cells can undergo affinity-driven maturation, such receptors need not derive from a similar common clone. recently, the saab+ tool was developed to characterize structural properties of cdrs from differentiated b cells [ ] . it is likely that more tools trained to identify "convergence" of functionally related antibodies will appear in the future as more sequence data from donors with shared bcr epitopes become available. to this end, we recently developed interclone, a method to cluster bcr sequences which are likely to share epitopes [ ] . interclone is based on a comparison of sequence and structural features of pairs of bcrs using a machine learning-based classifier that was trained on known antigen-bcr structures. like tcrdist, interclone assigns a "universal" similarity score to each bcr pair. hierarchical clustering is then used to group sequences of high similarity. as such, interclone can be used without requiring sequences to be enriched in a particular bcr motif. a sensitivity of . % and specificity of . % were obtained when interclone was applied to an independent set of anti-hiv antibody sequences [ ] . a more robust and computationally efficient version of interclone that works for both bcrs and tcrs and can perform high-throughput analysis of up to sequences is currently being developed. in addition to the above clustering methods, networks that describe antibody repertoire architecture can be used to compare repertoires. miho and colleagues [ ] developed a platform that builds similarity networks of hundreds of thousands of antibody sequences from both humans and mice. using this approach, the authors detected global patterns in antibody repertoire architectures that were highly reproducible in different subjects, and tended to converge despite independent vdj recombination. furthermore, these repertoire architectures were robust to clonal deletion of private clones. tcrs recognize short peptides presented on class i or ii mhc complexes. the ability to predict epitope(s) from tcr sequence and mhc allele would be highly valuable in elucidating disease etiology, monitoring the immune system, developing diagnostic assays and designing vaccines. traditionally, identifying epitopes is carried out experimentally [ ], and is both costly and time-consuming. there is necessarily great interest in methods that can accelerate this process computationally. to this end, fischer et al. [ ] developed a deep learning approach on tcr cdr regions to predict the antigen-specificity of single t cells. jokinen et al., [ ] developed tcrgp to predict whether tcrs recognize certain epitopes using a novel gaussian process (gp). their method uses cdr sequences from tcr alpha and beta and learns which cdr recognizes different epitopes. the tool was applied to identify t cells specific to hbv. nettcr by jurtz vi et al. [ ] utilized convolutional networks for sequence-based prediction of tcr-pmhc specificity. nettcr uses the recent explosion of nextgeneration sequencing data to train a sequence based-predictor. ogishi et al. [ ] computationally defined immunogenicity scores through sequence-level simulation of interaction between pmhc complexes and public tcr repertoires. though their focus is more on immunogenicity of peptides presented to mhc molecules, they also observed correlation between individual tcr-pmhc affinities and the features important for immunogenicity score. gielis et al. [ ] applied random forest-based classifiers for epitope specific tcrs to repertoire level analysis. their models successfully detected the increase of epitope specific tcrs upon vaccination in two yellow fever vaccination studies. the works by chain and co-workers [ , ] also addressed related questions. in [ ] , the authors have constructed a classifier to distinguish the tcr beta sequences in expanded repertoires of ovalbumin-stimulated mice from control. their classifier was based on the frequencies of amino acid triplets in cdr and their choice of machine learning algorithm called lpboost (linear programming boosting) allowed them to identify the responsible motifs in cdr . unlike bcrs, which can be expressed as soluble antibodies, tcrs remain attached to the cell surface. this, along with their weaker binding affinities to pmhc complexes, has made experimental structural analysis more difficult than for bcrs. nevertheless, from the known crystal structures of tcr-pmhc complexes, we can see that the range of docking modes is highly restricted, as expected by the similarity of mhcs within a given class (fig. ) . as a result of this restriction, we and others [ ] have approached the problem using structural templates for tcr-pmhc docking. there are currently few methods for modeling tcr-pmhc complexes. to our knowledge, there are two public servers for this purpose: our own immunescape [ ] and the lymphocyte receptor automated modeling or lyra-based [ ] tcrpmhcmodels [ ] . both of these approaches are "template-based" in the sense that existing structures instead of stochastic conformational sampling are used as templates for each of the key modeling steps: tcr, pmhc and tcr-pmhc orientation. they are also both "bottomup" in the sense that models for tcr and pmhc are built and then combined to form the tcr-pmhc complex. one possible conceptual difference is that, in immunescape, cdrs are modeled after the tcr and pmhc templates are combined in order to take the pmhc into account. it will be interesting to compare the two approaches in more detail. tcrpmhcmodels compared favorably to an earlier rigid docking-based approach, tcrflexdock, which suggests that care must be taken in sampling tcr-pmhc orientations beyond that which is observed in typical crystal structures. several computational methods are available to predict bcrs epitopes and paratopes. of the two problems, paratope prediction is much easier, as paratopes tend to correspond to cdr residues, while epitopes can be anywhere on an antigen. this is illustrated in the case of anti-influenza hemagglutinin (ha) antibodies (fig. ) ; a superimposition of all known anti-ha antibodies leaves very little un-targeted surface area. paratope prediction methods include the paratome algorithm [ ] , which is based on structural consensus between bcrs and uses features from sequence or structure; prediction of antibody contacts or proabc [ ] , which applies a random forest learning technique and is based on sequence; parapred [ ] , which uses a deep learning architecture to extract patterns from variable regions in sequence; antibodyinterfaceprediction [ ] , which uses a support vector machine method (svm); ag-fast-parapred [ ], which is based on deep neural networks, and utilizes antigen sequence information to predict paratope. ag-fast-parapred reported improved accuracy over existing methods; however, at the time of this writing, the tool is not available to the public. with regard to epitope prediction there are many tools available. antibody i-patch [ ] algorithm introduces a likelihood score for residue contact as constraints on the local docking to generate the paratope prediction, and thus requires the structure of antigenantibody complex. additionally, epitope predictors have evolved to be specific to cognate antibody. previously, methods were built to predict linear epitopes which are contiguous polypeptide chains, an example of which is lbtope (linear b-cell epitope prediction server) [ ] whose algorithm includes experimentally verified b-cell epitopes to discriminate from background by using svm. however, the majority of epitopes are noncontinuous surface residues characterized by structure as well as sequence. several methods are available to treat such conformational epitopes. sepia [ ] uses a combination of two classifiers (naive bayesian and random forest) from antigen sequence. bepipred- . [ ] uses the random forest algorithm to predict epitopes from primary sequence only. a recent method based on subgraph clustering for the prediction of separated and overlapping epitope, glep [ ] , achieved an f-score of . for singleepitopes. recently, there has been a realization that epitope prediction without reference to a particular antibody is an ill-formed problem, and methods of "antibody-specific epitope prediction" have been introduced [ ] . there are currently few options for antibodyspecific epitope prediction. the pease (predicting epitopes using antibody sequence) [ ] method applies machine learning to predict true contacts of antibody-antigen residue pairs, providing candidates of epitope patches. epipred [ ] identifies the epitope region by rescoring antibody-antigen global docking with its algorithm based on geometric matching of antigen-antibody interfaces and asymmetric potentials. mabtope [ ] predicts epitope residues based on consensus epitopes shared by top-ranked poses; the success of this approach depends on the quality of the docking. although there is a clear awareness of the importance of the antibody information in epitope prediction, the traditional antigen-centric methods cannot easily be extended to include such information. this is partially because of the increase in the number of degrees of freedom when antibody-antigen interactions are considered. the most direct means of tackling antibody-antigen interactions is through protein docking, a technique that requires structure information of antibody and antigen. this introduces additional degrees of freedom for rigid docking and a host of other issues due to the complexity and inherent uncertainty of protein structural information. nevertheless, protein docking is a mature field and steady progress has been made in this area. generally speaking, docking methods can be classified into four categories: fast fourier transform (fft) correlation; monte-carlo (mc) simulated annealing; geometric hashing; and flexible docking [ ] . in table , we give a representative list of molecular docking tools or web servers that can be applied to antibody-antigen docking. of these, cluspro [ ] , patchdock [ ] , frodock [ ] and snugdock [ ] provide [ ] and another three representative tools (cluspro, lightdock [ ] and zdock [ ] ) to systematically analyze antibody-antigen complexes from the well-studied zdock protein-protein interaction benchmark (version . ) [ ] . the results were evaluated using criteria established by the critical assessment of predicted interactions (capri) community where models are classified into the four categories: incorrect, acceptable, medium, or high quality [ ] . it was demonstrated that information-driven docking, even using noisy predictions of epitope and paratope, could significantly improve performance over all four algorithms [ ] . notably, haddock was capable of providing high quality models for all entries based on capri criteria in this test. however, this study did not evaluate the tolerance of the docking methods to typical bcr modeling errors. as with all protein docking from homology models, the success of docking antibody models depends heavily on the quality of the starting structures [ ] . structural uncertainties in the binding regions can occur either from flexibility or modeling errors. moreover, the regions of greatest uncertainty tend to be the cdrs (especially cdrh ), which is highly likely to form part of the paratope [ ] . these issues can be addressed to some extent by use of epitope and paratope predictions. however, few antibody docking methods have been rigorously tested using a large benchmark of realistic models. the bottom line is that structure-based prediction of antibody-antigen interactions from sequence involves a number of interrelated tasks: receptor and antigen model building, initial epitope and paratope prediction, docking, scoring and refinement. the combination of so many critical steps results in complexity, both in terms of software integration and in parameter optimization. fortunately, the emergence of larger and better bcr sequence datasets will be a motivation to develop well-integrated structure prediction pipelines. in this review, we have focused primarily on high-throughput structure-based methods that can be applied to bcr or tcr repertoires. as is clear from the previous section, combining software methods that work well in isolation introduces complexity. such complexity arises from conceptual considerations (e.g. parameter optimization) and technical issues (code interoperability). in this regard, md is conceptually simple: it applies newtonian mechanics to molecular systems. the force fields describing the interatomic interactions can be taken as given and generally do not have to be optimized. therefore, even though md is not a high-throughput method, it can be used to independently confirm bcr-or tcr specific calculations. as with all proteins, the dynamics of bcrs and tcrs is intimately tied to their functions. most studies focusing on the t cell receptor only study the dynamics of t cell receptors when bound to a pmhc. in contrast, dominguez and knapp compared the dynamics of t cell receptors bound to pmhc and free t cell receptors. in their study they found, apart from expected results as an increased flexibility and increased solvent accessible surface of the cdrs in the free t cell receptor, also differences in the hydrogen bond network of the cdr α chain in the free tcr versus the pmhc bound tcr [ ] . a study combining steered molecular dynamics and single-molecule biophysical experiments [ ] studied the formation of catch bonds between the pmhc and the tcr. catch bonds are a special type of bond in which the lifetime increases when more force is applied. this study suggests that catch bond formation is influenced by conformational changes in the pmhc. a downside of molecular dynamics simulations are the high computational requirements. fodor et al. were able to distill conformational data from pmhc class i x-ray structures using ensemble refinement, which is a refinement technique to obtain dynamic data without the need of more computationally intensive molecular dynamics simulations [ ] . another way to reduce the computational requirements is by using coarse grained simulations, in which atoms are grouped together into beads. coarse graining allows for the study of much larger systems on longer time scales. friess et al. modeled the transmembrane domains of the immunoglobulin m (igm) b cell receptor, which have been unresolved so far, and subsequently used coarse grained simulations to study their aggregation behavior and association with lipid rafts [ ] . recent advances in sequencing technology enable the study of immune responses in unprecedented breadth and depth. as discussed above, the emerging data has spawned the development of a wide range of modeling methods that are applicable to b cells, t cells or both. current challenges include the integration of data and methodologies. for example, sequence and structural information can, in principle, be combined to yield more accurate descriptions of receptors sharing antigen and epitope specificity. structural modeling is still not in the mainstream of repertoire analysis; nevertheless, d modeling methods present a straightforward direction to encompass "shared features" of functionally related receptors in different donors. in the context of repertoire analysis, we are often interested in the target antigens and epitopes; however, the scale of publicly available data on targeted antigens and epitopes is currently smaller than that of bcr/tcr sequences, and vastly smaller the actual bcrantigen or tcr-peptide-mhc interactome. as barcoding methods evolve to include antigens themselves [ ] , there may soon be new and valuable data available to train methods for functional classification of bcrs and tcrs. at the point where we are asking not only what is targeted but also why or why not, the use of structural modeling is likely to play a critical role in our understanding of bcr and tcr molecular recognition. as a case in point, at the time of this writing, we are in the midst of the covid- pandemic. this is an example where the target antigens, along with their structures, are largely known, and understanding host immune responses to these antigens is of vital importance in the development of diagnostics, biomarkers, vaccines and therapeutics [ ] . structural similarity among neutralizing antibodies targeting sars-cov- [ ] or between sars-cov- and sars-cov- [ ] have been noted. with such high stakes driving research and development, integration of emerging technologies in the repertoire analysis domain, including structural analysis, is expected. as the saying goes, "necessity is the mother of invention," and the need for understanding human immune repertoires has never been greater. we would like to thank all members of the systems immunology lab for helpful janeway's immunobiology how many different clonotypes do immune repertoires contain? current opinion in systems biology structural determinants of t-cell receptor bias in immunity cytokine-secreting follicular t cells shape the antibody repertoire vdjdb in : database extension, new analysis infrastructure and a t-cell receptor motif compendium statistical analysis of cdr length distributions for the assessment of t and b cell repertoire biases characterizing immune repertoires by high imgt/highv quest paradigm for t cell receptor imgt clonotype diversity and next generation repertoire immunoprofiling igblast: an immunoglobulin variable domain sequence analysis tool gapped blast and psi-blast: a new generation of protein database search programs mixcr: software for comprehensive adaptive immunity profiling antigen receptor repertoire profiling from rna-seq data benchmarking immunoinformatic tools for the analysis of antibody 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features define epitope-specific t cell receptor repertoires a diverse lipid antigen-specific tcr repertoire is clonally expanded during active tuberculosis sequence and structural convergence of broad and potent hiv antibodies that mimic cd binding vaccine-induced antibodies that neutralize group and group influenza a viruses convergent antibody responses to sars-cov- in convalescent individuals structural diversity of b-cell receptor repertoires along the b-cell differentiation axis in humans and mice functional clustering of b cell receptors using sequence and structural features large-scale network analysis reveals the sequence space architecture of antibody repertoires t cell antigen discovery predicting antigen-specificity of single t-cells based on tcr cdr regions. biorxiv tcrgp: determining epitope specificity of t cell receptors. biorxiv quantitative prediction of the landscape of t cell epitope immunogenicity in sequence space specificity, privacy, and degeneracy in the cd t 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protein-dna/rna docking based on a hybrid strategy hexserver: an fft-based protein docking server powered by graphics processors a web interface for easy flexible protein-protein docking with attract gramm-x public web server for proteinprotein docking sedat aybars nazlica, jiaqi xie and martin de jesus loza lopez contributed to the experimental epitope determination sections. john rozewicki, ana davila and jan wilamowski wrote most of the in-house software sections. floris j. van eerden wrote the md section. zichang xu wrote the bcr docking section. daron m. standley wrote the overall manuscript and coordinated the efforts of the other members standley and songling li are shareholders in kotai biotechnologies, inc. and are co-applicants on us patent app. / , . all authors declare no other conflicts of interest key: cord- -f jtufja authors: benedictus, lindert; otten, henny g; van schaik, gerdien; van ginkel, walter gj; heuven, henri cm; nielen, mirjam; rutten, victor pmg; koets, ad p title: bovine neonatal pancytopenia is a heritable trait of the dam rather than the calf and correlates with the magnitude of vaccine induced maternal alloantibodies not the mhc haplotype date: - - journal: vet res doi: . /s - - - sha: doc_id: cord_uid: f jtufja bovine neonatal pancytopenia (bnp), a bleeding syndrome of neonatal calves, is caused by alloantibodies absorbed from the colostrum of particular cows. a commercial bvd vaccine is the likely source of alloantigens eliciting bnp associated alloantibodies. we hypothesized that the rare occurrence of bnp in calves born to vaccinated dams could be associated with genetic differences within dams and calves. we found that the development of bnp within calves was a heritable trait for dams, not for calves and had a high heritability of %. to elucidate which genes play a role in the development of bnp we sequenced candidate genes and characterized bnp alloantibodies. alloantigens present in the vaccine have to be presented to the dam’s immune system via mhc class ii, however sequencing of drb showed no differences in mhc class ii haplotype between bnp and non-bnp dams. mhc class i, a highly polymorphic alloantigen, is an important target of bnp alloantibodies. using a novel sequence based mhc class i typing method, we found no association of bnp with mhc class i haplotype distribution in dams or calves. alloantibodies were detected in both vaccinated bnp and non-bnp dams and we found no differences in alloantibody characteristics between these groups, but alloantibody levels were significantly higher in bnp dams. we concluded that the development of bnp in calves is a heritable trait of the dam rather than the calf and genetic differences between bnp and non-bnp dams are likely due to genes controlling the quantitative alloantibody response following vaccination. electronic supplementary material: the online version of this article (doi: . /s - - - ) contains supplementary material, which is available to authorized users. since an increase in newborn calves with the bleeding syndrome bovine neonatal pancytopenia (bnp) was observed all over europe [ ] [ ] [ ] . epidemiological studies showed a strong association between the occurrence of bnp in calves and vaccination of their dams with the pregsure© bvd vaccine (pfizer animal health) [ ] . symptoms of bnp are severe internal and external bleeding, first seen around - days of age. hematological signs are severe leukopenia and thrombocytopenia. in addition, trilineage hypoplasia of the bone marrow can be observed upon post-mortem examination [ ] [ ] [ ] . colostrum of dams that had previously given birth to a calf which developed bnp contained alloantibodies recognizing bovine leukocytes [ ] [ ] [ ] [ ] . feeding this colostrum to healthy neonatal calves induced the symptoms of bnp [ , , ] . proteins from the bovine kidney cell line mdbk [ ] , used to grow the bvd type virus present in pregsure© bvd, are the likely source of alloantigens that induce alloantibody production in vaccinated dams. the alloantibodies bind mdbk cells and it was shown that an important target of these antibodies were mhc class i proteins [ , , ] . moreover, mdbk derived mhc class i proteins were detected in the pregsure© bvd vaccine [ , ] and immunization of calves with pregsure© bvd induced alloantibodies recognizing mdbk cells [ , ] . since the incidence of bnp calves born to pregsure© bvd vaccinated dams was estimated to be lower than . % [ , , ] , it was hypothesized that factors other than vaccination per sé play a role in the etiology of bnp. the prevailing hypothesis is that the pathogenesis of bnp resembles a histocompatibility (mis)match between dam and calf and is based on immunization of the dam with mdbk derived mhc class i [ , ] . first, in the dam mdbk cell derived proteins, present in the preg-sure© bvd vaccine, are presented in the context of mhc class ii. the resulting t cell help to b cells recognizing allogeneic differences between mdbk cells and the dam will result in the generation of alloantibodies which are also present in the colostrum. due to tolerance to self-antigens, dams do not exhibit adverse effects after vaccination, i.e. the vaccine induced alloantibodies do not recognize alloantigens expressed in the dam. the maternal alloantibodies transferred to the calf via the colostrum will recognize alloantigens in case of a partial alloantigen match between mdbk cells and the calf. we hypothesized that the rare occurrence of bnp after pregsure© bvd vaccination may depend both on the capability of the dam's immune system to present the mdbk alloantigens via mhc class ii, as well as the degree of alloantigen (mis)match between the dam and the mdbk cell line (and the calf and the mdbk cell line, respectively) and the ensuing immune response of the dam. since alloantigens (including mhc i and mhc class i associated b m) and mhc class ii are genetically determined and therefore heritable, we studied whether differences in these genes between dams and/or calves may explain why bnp only occurs in part of the calves born to pregsure© bvd vaccinated dams. first we studied the heritability of the development of bnp in the calf as a potential dam or calf trait. next, to elucidate if these genes genes play a role in the development of bnp we sequenced and compared the mhc and b m candidate genes and characterized bnp associated alloantibodies. the data used for the heritability study were a subset of data from a large multi country epidemiological study on bnp [ ] and concerned dutch farms that participated in this study. data on herd matched bnp and non-bnp calves were collected by on farm questionnaires. we looked at the heritability of the development of bnp within the calf as a trait of pregsure© bvd vaccinated dams as well as of calves born to these dams. the definitions for bnp and non-bnp calves used, were according to jones et al. [ ] . a bnp calf was defined as a calf that showed one or more bnp clinical signs on or before days of age; bone marrow depletion as assessed by histopathology and/or thrombocytopenia (< × /litre) and leucopenia (< × /litre). a non-bnp calf was defined as a calf on the same farm as a case, aged - days at the time of case reporting, no clinical signs of bnp up to days of age, and normal blood parameters (thrombocytes ≥ × /litre, leucocytes ≥ × /litre). to ensure that the correct phenotype, bnp or non-bnp, was assigned to the dam, only calves that were fed colostrum from their own dam were included. furthermore, dam-calf combinations without pedigree information were excluded. pedigrees of calves and dams were provided by the dutch cattle improvement organization (crv, arnhem, the netherlands). the pedigree of dam-calf combinations meeting the inclusion criteria were traced back up to generations and the final pedigree included records. the first generation of the pedigree was a % complete for calves and % complete for dams. the data were analyzed using the software package asreml [ ] , a statistical package that fits generalized linear mixed models using residual maximum likelihood. the heritability of the development of bnp within the calf as a dam and calf trait was estimated from the dam and sire variance components of a sire-dam model. only alloantigens inherited from the sire can be recognized by maternal alloantibodies and therefore the heritability of the development of bnp as a calf trait was estimated by calculating bnp as a sire trait. variables included in the data set were: bnp was fitted as a binomial variable using the logistic link function to relate binomial outcome of bnp to the linear predictor used for the generalized linear mixed model. the following general model was used: where bnp is the outcome of bnp, μ is the general mean, (x i ) n is one or more of the aforementioned variables, sire j is the random effect of the jth sire; dam k is the random effect of the kth dam and e (i)n jk is the vector of residuals. heritability was calculated using the variance components of the model, as follows: bnp as dam trait h = σ dam /σ p ; bnp as a sire trait h = σ sire /σ p ; σ p = σ dam + σ sire + (π )/ , where σ p is the phenotypic variance, σ dam is the dam variance, σ sire is the sire variance and the residual variance was fixed at (π )/ . first we looked at the effect of each individual variable on bnp in a sire-dam model. next all variables with a p-value < . were included in the final sire-dam model. immune responses normally decline with time and to test if the incidence of bnp after the last preg-sure© bvd vaccination also declines with time, the variable time since last pregsure© bvd vaccination was forced into the final model despite having a p-value higher than . in the univariate model. because the estimates for time since last pregsure© bvd vaccination appear to have a linear effect on bnp, the variable was added as a linear covariable in the final model. there were only eight dams with one pregsure© bvd vaccination and because the vaccination scheme consists of an initial prime and subsequent boost vaccination which may have been interpreted as one vaccination by the farmer, in the final model animals with one or with two pregsure© bvd vaccinations were grouped. blood of calves was drawn as part of the multi country epidemiological study on bnp [ ] . farms with more than one living bnp dam were revisited in to collect blood-and colostrum-samples from dams. throughout our study we used the following definitions for dams and calves: non-bnp dam -dam that had been vaccinated with pregsure© bvd and had not given birth to a calf that developed bnp following colostrum feeding. -bnp dam -dam that had been vaccinated with pregsure© bvd and had given birth to a calf which developed bnp following colostrum feeding. -non-bnp calf -calf born to a pregsure© bvd vaccinated dam, that upon receiving colostrum from its dam did not show signs of bnp, confirmed via hematology and/or pathology. -bnp calf -calf born to a pregsure© bvd vaccinated dam, that upon receiving colostrum from its dam showed clear signs of bnp, confirmed via hematology and/or pathology. this study was approved by the animal ethical committee of utrecht university and conducted according to their regulations. sequence based typing of mhc class i was done using gene specific primers aligning with intron and intron of mhc class i genes , , and [ ] (additional file ). these primers amplify exon and , which encode the most polymorphic regions of the mhc class i gene. for genes , and pcr was carried out in μl containing . u expand high fidelity taq (roche diagnostics, indianapolis, usa), . mm mgcl , . mm each dntp and . μm, or . um in the case of primers with ambiguous nucleotide, of each primer. the thermal cycling profile was °c for min, cycles of °c for s, °c for s, °c for s followed by °c for min. for gene pcr conditions were similar, except . u of amplitaq® (applied biosystems, life technologies) was added, the mgcl concentration was mm and the annealing temperature was °c. sequencing of pcr's resulting in a product were performed on the dna analyzer (applied biosystems) using the same primers used for the pcr and the bigdye® terminator v . cycle sequencing kit (applied biosystems). sequence products were analyzed using seqscape© (v . , applied biosystems). forward and reverse sequences were aligned to a reference sequence to produce a consensus sequence. using the ipd mhc database [ ] a library of the exon and sequences of known mhc class i alleles was constructed using seqscape©. seqscape© is able to cope with ambiguous nucleotides and, in the case of heterozygous pcr products, matches the consensus read to the best combinations of alleles from the library. consensus read basecalling of the amplified genomic dna and library matches to known full length mhc class i cdna sequences were checked. using the assigned mhc class i alleles, mhc class i haplotypes were determined using haplotypes defined in codner et al. [ ] and this study (additional file ). mhc class i haplotypes define a set of mhc class i alleles that are inherited together and haplotype differences between animals do not give information on differences in mhc class i as an alloantigen. to better estimate allogeneic differences between mdbk cells and dams/calves we looked at mhc class i protein differences between mdbk cells and dams/calves. alloantibodies recognize the extracellular part of expressed proteins and we therefore looked at protein differences within the extracellular part of mhc class i (exon - ). dna sequences of exon - were translated into protein sequences and the difference in protein sequence between two mhc class i alleles was calculated, expressed as percentage of the protein sequence that was different. dams can recognize mdbk alleles (listed in additional file ) as non-self if there are differences between the dam and mdbk mhc class i and for dams we calculated the difference between the mdbk allele that was most different to the dam mhc class i alleles. for alloantibodies to recognize mhc class i in the calf, there has to be a (partial) match between the mdbk and paternally inherited calf mhc class i and for calves we calculated the difference between the most similar mdbk and paternally inherited calf mhc class i allele. beta- -microglobulin (b m) primers (additional file ) flanking exon were designed using the bovine whole genome assembly umd . . pcr was carried out in μl containing . u pfuturbo cx hotstart dna polymeras (agilent, santa clara, usa), mm mgcl , . mm each dntp and . μm each primer. the thermal cycling profile was °c for min, cycles of °c for s, °c for s, °c for s followed by °c for min. sequencing was performed as described for mhc class i. forward and reverse sequences were aligned to the umd . reference sequence using seqscape©. drb sequence based typing was based on the method described by miltiadou et al. [ ] . primers aligning with intron and of the drb locus (additional file ) amplify exon , the most polymorphic region of the drb gene. pcr was carried out in μl containing . u amplitaq gold (applied biosystems), . mm mgcl , . mm each dntp and . μm each primer. the thermal cycling profile was °c for min, cycles of °c for s, °c for s, °c for s followed by °c for min. sequencing was performed as described for mhc class i. sequence reads were analysed using seqscape© as described for mhc class i. total alloantibody levels were assessed as serum antibody levels specific for mdbk cells. the latter were suspended in serum diluted : in pbs supplemented with % fcs and . % sodium azide. bovine igg binding was detected using polyclonal biotinylated sheep anti-bovine igg antibodies (abd serotec, bio-rad laboratories inc, hercules, usa) and streptavidin-phycoerythrin (bd biosciences, franklin lakes, usa). isotype specific alloantibodies were measured in a similar way. mdbk cells were suspended in serum or colostrum diluted : and alloantibody binding was detected by bovine isotype specific mouse monoclonal antibodies [ ] and fitc conjugated polyclonal goat anti-mouse antibodies (bd biosciences). total leukocytes were isolated from blood collected from ten healthy randomly selected dams at the slaughterhouse by hypotonic lysis of erythrocytes. whole blood was suspended in parts of distilled water, after lysis of erythrocytes isotonicity was restored using volume of x pbs. total leukocytes, used to detect alloantibody binding to peripheral blood mononuclear cells (pbmc), were suspended in serum or colostrum diluted : . alloantibody binding was detected by anti-bovine igg mouse monoclonal antibodies and fitc conjugated polyclonal goat anti-mouse antibodies. in all alloantibody binding experiments serum from non pregsure© bvd vaccinated dams were used as (isotype) controls. flow cytometry (bd facscanto™, bd biosciences) was used to measure alloantibody binding and data was analyzed using flowjo software (tree star inc., ashland, usa). pbmc were selected based on forward and sideward scatter. data are depicted as geometric mean fluorescent intensity (gmfi). in the case of alloantibody binding to pbmc depicted gmfi values are gmfi values subtracted by the gmfi of the isotype controls. in order to be able to compare alloantibody binding of pbmc irrespective of total alloantibody levels in serum or colostrum, relative alloantibody binding was calculated by dividing the gmfi of each sample by the gmfi of alloantibody staining of mdbk cells, representing total alloantibody binding. a positive pbmc sample was defined as a sample that had a higher geometric mean fluorescent intensity (gmfi) than the average of all measured samples or in the case of alloantibody level compensated values defined as having a higher relative signal than the average of the relative signal of all samples. the wald test was used to test whether a variable improved the fit of the sire-dam model. haplotype/allele frequencies were analyzed using fisher's exact test. alloantibody binding levels were compared by two tailed simple t-tests for unequal variance. to adjust for multiple comparisons the false discovery rate (fdr) was controlled using the method by benjamini and hochberg [ ] . this method controls the chance of falsely declaring the result of a statistical test as significant. the largest p-value lower than its fdr-derived significance threshold and all p-values smaller were considered to be significant. the number of significant p-values that are false positive was controlled at %. correlation was tested with pearsons correlation. normality was tested with d' agostino and pearsons omnibus normality test. effects were considered significant at p < . . when applicable, values were given as mean ± the standard error of the mean, with the latter between brackets. heritability of the development of bnp within the calf as a trait for pregsure© bvd vaccinated dams and for calves based on the inclusion criteria dam-calf combinations were selected for the heritability analysis. the calves were born from dams, fathered by sires and comprised bnp cases. the effect of each individual variable on bnp is summarized in additional file . the parameter estimates and odds ratios for the final model are shown in table . for pregsure© bvd vaccinated dams the heritability estimate for the development of bnp within the calf was . ( . ) and for sires it was . ( . ). the odds of bnp increased with an increased number of pregsure© bvd vaccinations. the odds of bnp increased up to the third lactation and was lower for the fourth and fifth lactation. the effect of time since last pregsure© bvd vaccination on bnp was not significant. sequence based typing was used to determine mhc class i haplotypes in vaccinated non-bnp and bnp dams ( table ). the largest frequency differences between dams were seen for variants of the a mhc class i haplotype, but with a p-value of . , which was much higher than the fdr-threshold of . , this was not significant. assuming an incidence of bnp of . % for pregsure© bvd vaccinated dams [ ] , the positive predictive value of the a haplotypes was . . implying that bnp only occurred in . % of calves born to preg-sure© bvd vaccinated dams with the a mhc class i haplotype. the difference in protein sequence between the extracellular parts of the mdbk and dam mhc class i alleles was . % ( . %) for vaccinated non-bnp dams and . % ( . %) for bnp dams, with a p-value of . this was not significantly different between both groups (additional file ). the paternal mhc class i haplotype frequencies of non-bnp and bnp calves are shown in table . based on the fisher's exact test the frequency of the a haplotype was significantly higher in bnp calves, however with a p-value of . this value was higher than the fdr threshold of . . assuming an incidence of bnp of . % for pregsure© bvd vaccinated dams [ ] , the positive predictive value of the a haplotype was . . which implies that only . % of calves with a paternally inherited a mhc class i haplotype born to pregsure© bvd vaccinated dams get bnp. in five non-bnp and three bnp calves fathered by the same sire, the mhc class i haplotypes were also typed (additional file ). since all eight calves had the a mhc class i haplotype, it is likely that the sire was a homozygous. in that case both non-bnp and bnp calves inherited the a haplotype from their father and for these calves there was no association between the paternally inherited a haplotype and the development of bnp. the protein difference between the extracellular part of the mdbk mhc class i alleles and paternally inherited to adjust for multiple comparisons the false discovery rate (fdr) was controlled at % using the principle from benjamini and hochberg [ ] . the largest p-value lower than its fdr-derived significance threshold and all p-values smaller are significant. calf mhc class i alleles was . % ( . %) for non-bnp calves and . % ( . %) for bnp calves, with a p-value of . this was not significantly different between both groups (additional file ). exon of the beta- -microglobulin (b m) gene, encoding % of the mature protein, was sequenced in mdbk cells and in five vaccinated non-bnp dams and five bnp dams that were farm matched. the b m sequences of all vaccinated non-bnp dams, bnp dams and mdbk cells were identical. results of the drb typing of vaccinated non-bnp dams and bnp dams are shown in table . the largest frequency differences were seen for the drb alleles and both with a higher frequency in vaccinated non-bnp dams. however, the p-values were well above the fdr threshold and not significant. total alloantibody levels in dams not vaccinated with pregsure© bvd and in pregsure© bvd vaccinated non-bnp and bnp dams were assessed as serum antibody levels specific for mdbk cells using flow cytometry ( figure ). alloantibody levels in bnp dams were significantly higher than in both non-bnp dams and dams not vaccinated with pregsure© bvd, levels in vaccinated non-bnp dams are significantly higher than in dams not vaccinated with pregsure© bvd. isotype specific alloantibody binding of mdbk cells is shown in figure . igg alloantibodies were most abundant and the levels were significantly higher in serum of non-bnp dams and in serum and colostrum of bnp dams compared to dams not vaccinated with pregsure© bvd. igg alloantibody levels were significantly higher in serum and colostrum of bnp dams compared to dams not vaccinated with pregsure© bvd. igg alloantibody levels tended to be higher in non-bnp dams as well, but due to higher variation among dams, did not differ significantly from that in dams not vaccinated with pregsure© bvd. for igm and iga there were no significant differences between groups. antibodies present in serum and colostrum from non-bnp and bnp dams bind pbmc ( figure a ). alloantibody binding of pbmc was significantly higher for both serum and colostrum of bnp dams compared to non-bnp dams. the number of pbmc samples that were positive were also higher for both serum and colostrum of bnp dams. however, average alloantibody binding of pbmc and alloantibody binding of mdbk cells had a high correlation ( figure b ) and when alloantibody binding of pbmc was compensated for mdbk specific alloantibody levels to enable comparison of the binding of pbmc irrespective of total alloantibody levels, the relative signal was to adjust for multiple comparisons the false discovery rate (fdr) was controlled at % using the principle from benjamini and hochberg [ ] . the largest p-value lower than its fdr-derived significance threshold and all p-values smaller are significant. c denotes a local name and is not included in the ipd bovine mhc class ii database. the same for bnp dams and non-bnp dams for serum as well as colostrum ( figure c) . also, the number of pbmc samples that were positive were similar in both groups for serum as well as colostrum. results of individual serum and colostrum samples are shown in additional file . we hypothesized that the rare occurrence of bnp after pregsure© bvd vaccination depends both on the capability of the dam's immune system to present the mdbk alloantigens via mhc class ii, as well as the degree of alloantigen (mis)match between the dam and the mdbk cell line (and the calf and the mdbk cell line, respectively) and the ensuing immune response of the dam. as a corollary we hypothesized that genetic differences in mhc class ii in dams and alloantigens in dams and calves (e.g. mhc i and mhc class i associated b m) would then explain why bnp only occurs in part of the calves born to pregsure© bvd vaccinated dams. the present study demonstrates that the development of bnp in calves is a heritable trait for pregsure© bvd vaccinated dams with the high heritability estimate of %, which shows that genetic differences between dams explain in part why only the colostrum of some pregsure© bvd vaccinated dams cause bnp in the calf. genetic variation in the paternal haplotype of the calves is not related to the development of bnp in the calf, since the heritability of the development of bnp in calves born to pregsure© bvd vaccinated dams is %. demasius et al. [ ] found that in an experimental german holstein x charolois crossbred herd with a limited number of sire lines, all bnp cases were restricted to a single maternal grandsire, also indicating the importance of the genetic background of the dam. in addition from a limited number of bnp dams was shown to induce bnp in randomly selected healthy calves [ ] [ ] [ ] which supports the notion that the genetic background of the calf is not critical. the phenotype of the calf was based on very strict objective criteria, whereas the phenotype of the dam was based on the phenotype of the calf. bnp is caused by alloantibodies present in the colostrum and the phenotype of the calf therefore depends on the quality, quantity and source (own dam or other dam) of the ingested colostrum. this means that the phenotype of the calf, may not always be the proper phenotype of the dam. much of this information was farmer reported and although we have tried to control for these aspects, the possibility exists that non-differential misclassification of the phenotype of the dam occurred in this study and implies that the heritability for the development of bnp within calves of % for dams is potentially underestimated. results were compared by two tailed simple t-tests for unequal variance. to adjust for multiple comparisons, the false discovery rate (fdr) was controlled at % using the principle from benjamini and hochberg [ ] . the largest p-value lower than its fdr-derived significance threshold and all p-values smaller are significant and are depicted by an asterisk (*). in our more in depth analyses of the genetic differences between pregsure© bvd vaccinated non-bnp and bnp dams we sequenced a number of specific candidate genes. an important target of bnp alloantibodies is mhc class i [ , ] , a highly polymorphic alloantigen [ ] . hence mhc class i was genotyped to see if differences in mhc class i alloantigen repertoire of dams and/or calves were associated with the development of bnp in the calf. we did not find an association between the mhc class i of the pregsure© bvd vaccinated dams and the occurrence of bnp. although the number of bnp calves in the mhc class i haplotyping analysis was limited, it showed that bnp calves do not have a single paternal mhc class i haplotype and that most of the paternal haplotypes are shared between bnp and non-bnp calves ( table , additional file ), together indicating that the paternally inherited mhc class i of calves is not associated with the occurrence of bnp. this result supports our finding that the heritability of the development of bnp in calves is zero and shows that bnp and non-bnp calves do not have a different allogeneic background. ballingall et al. [ ] found no differences in drb allele frequencies between bnp and non-bnp calves. since drb and mhc class i are in linkage disequilibrium, this corroborates our mhc class i typing result in calves. the binding of certain monoclonal antibodies to the b m-mhc class i heavy chain heterodimer can depend on the associated b m allele [ ] or mhc class i allele [ ] . although polymorphisms within the bovine b m gene are known, none lead to changes in the amino acid sequence [ ] . nevertheless we wanted to exclude the possibility that an unknown rare allelic variant of b m influences the recognition and immune response to mdbk mhc class i proteins present in the vaccine. since sequences of b m were identical in the mdbk cell line and all typed pregsure© bvd vaccinated non-bnp and bnp dams, it is highly unlikely that allelic variations of b m play a role in the etiology of bnp. another aspect of immune recognition of mdbk alloantigens present in the pregsure© bvd vaccine is their presentation to the dam's immune system via mhc class ii. mhc class ii haplotypes have been associated with disease resistance and susceptibility [ , ] and influence antibody responses after vaccination [ , ] . we found no association between mhc class ii haplotypes, as assessed by sequencing the highly polymorphic drb locus, and the occurrence of bnp in pregsure© bvd vaccinated dams. pregsure© bvd vaccinated bnp dams had significantly higher serum alloantibody levels compared to pregsure© bvd vaccinated non-bnp dams. nonetheless figure isotype characterization of alloantibodies from pregsure© bvd vaccinated dams. flow cytrometry was used to measure the isotype of alloantibodies binding to mdbk cells in serum (ser) or colostrum (col) from i) dams not vaccinated with pregsure© bvd (bnp-vacc-) ii) pregsure© bvd vaccinated non-bnp dams (bnp-vacc+) and iii) pregsure© bvd vaccinated bnp dams (bnp + vacc+). all results were compared by two tailed simple t-tests for unequal variance. within each isotype, all groups are compared to the non pregsure© bvd vaccinated dams (ser bnp-vacc-). to adjust for multiple comparison, the false discovery rate (fdr) was controlled at % using the principle from benjamini and hochberg [ ] . the largest p-value lower than its fdr-derived significance threshold and all p-values smaller are significant and are depicted by an asterisk (*). gmfi = geometric mean fluorescent intensity. alloantibodies were produced both in pregsure© bvd vaccinated non-bnp and bnp dams, confirming results from a previous study [ ] . alloantibody production by all pregsure© bvd vaccinated dams indicated there were allogeneic differences between the bovine mdbk proteins and both pregsure© bvd vaccinated non-bnp and bnp dams. this corroborated the sequencing results, where we did not find a difference between mhc class i or b m between pregsure© bvd vaccinated non-bnp and bnp dams. it also indicated that all dams were able to present alloantigens from the pregsure© bvd vaccine in the context of mhc class ii and fitted with the lack of an association between drb and the occurrence of bnp within pregsure© vaccinated dams. the antibody isotype produced by b-cells depends on the cytokines that are produced during an (vaccine induced) immune response [ , ] . the type of vaccine induced immune response may therefore influence the quality of the ensuing antibody response. as different antibody isotypes induce different biological effector functions, such as complement activation and neutralization, we studied the quality of the antibody response in pregsure© bvd vaccinated non-bnp and bnp dams to determine if bnp dams only differ in alloantibody levels or also in the isotype and specificity of alloantibodies produced. bnp is caused by alloantibodies from colostrum and for bnp dams serum and colostrum alloantibodies were compared to see if results for serum alloantibodies can be extrapolated to colostrum derived alloantibodies. alloantibody isotypes were similar in serum of pregsure© bvd vaccinated non-bnp dams and serum and colostrum of bnp dams, indicating a similar response to vaccination in both groups. likewise, studying cattle responding with high or low antibody levels after vaccination with hen-egg white lysozyme or candida albicans extract heriazon et al. [ ] also did not find any differences in igg and igg levels between animals, whereas antibody levels following vaccination varied significantly. when stained with serum or colostrum from pregsure© bvd vaccinated bnp dams higher numbers of (random) pbmc samples were positive for alloantibody binding and on average staining intensity was higher than when stained with serum or colostrum from pregsure© bvd vaccinated non-bnp dams. however, when compensated for alloantibody binding of mdbk cells to enable comparison of binding to pbmc irrespective of total alloantibody levels, numbers of positive pbmc samples and the relative staining intensity with alloantibodies were comparable between pregsure© bvd vaccinated bnp and non-bnp dams, indicating that the specificity for allogeneic cells was also comparable. based on the similar antibody isotypes and relative staining of pbmc we argue that alloantibodies from pregsure© bvd vaccinated non-bnp and bnp dams are qualitatively similar and that only the level of alloantibodies is higher in pregsure© vaccinated bnp dams. the high correlation between binding of alloantibodies to mdbk cells and pbmc corroborates the notion that the most important alloantigens in the pregsure© bvd vaccine are derived from the producer cell line. bastian et al. [ ] found that bnp dams also had higher bvd neutralizing antibody levels than pregsure© bvd vaccinated non-bnp dams, showing that bnp dams generally respond with higher antibody levels to components in the pregsure© bvd vaccine. in combination with the high heritability estimate for the development of bnp in calves for preg-sure© bvd vaccinated dams, it is likely that genetic differences between vaccinated non-bnp and bnp dams are due to genes that determine the level of antibody production after pregsure© bvd vaccination. in cattle high heritability estimates have been found for antibody production after vaccination, these ranged from % to % [ , ] . high antibody production after brsv vaccination was associated with single nucleotide variants of tlr and tlr [ ] . likewise, differences in responsiveness of the innate immune system of bnp dams to the adjuvant of the pregsure© bvd vaccine may have led to higher antibody production to antigens in the vaccine. the occurrence of bnp shows that in an outbred population some individuals may respond very differently to vaccination than the general population. this emphasizes the importance of monitoring adverse effects of both existing and new vaccines, but may on the other (see figure on previous page.) figure binding of peripheral blood mononuclear cells by alloantibodies from pregsure© bvd vaccinated dams. a: peripheral blood mononuclear cells (pbmc) from ten random dams were stained with serum (n = ) and colostrum (n = ) of different pregsure© bvd vaccinated non-bnp dams (bnp-vacc+, n = ) and with serum (n = ) and colostrum (n = ) of pregsure© bvd vaccinated bnp dams (bnp + vacc+, n = ). igg alloantibody binding was measured by flow cytometry. gmfi subtracted by isotype control is plotted on the y-axis. the horizontal dotted line depicts the overall average geometric mean fluorescent intensity (gmfi) and the number above the plots describes the number of samples with a signal above the horizontal line. b: correlation between the average igg alloantibody binding of pbmc's from ten dams to igg alloantibody binding of mdbk cells by serum or colostrum samples as in figure a . c: the data from figure a were divided by the gmfi signal of the alloantibody staining of mdbk cells by the respective serum or colostrum. the horizontal dotted line depicts the overall average relative signal and the number above the plots describes the number of samples with a signal above the horizontal line. mean ± standard error of the mean is depicted in all graphs. two tailed simple t-tests for unequal variance was used to compare serum or colostrum alloantibody binding of pbmc's between pregsure© bvd vaccinated non-bnp and bnp dams. correlation was tested with pearsons correlation. normality was tested with d'agostino and pearsons omnibus normality test. hand also provide opportunities for selective breeding for an increased humoral immune response. bovine mhc class i has an unusual organization, with six putative genes of which a variable number of genes are functionally present per haplotype [ ] , making mhc class i typing in cattle difficult. several techniques with different (dis)advantages have been used to type mhc class i in cattle, including serology [ ] , cloning and sequencing of full length cdna [ ] and next generation sequencing of polymorphic regions [ ] . in this study we use gene specific primers for four of the six mhc class i genes to amplify exon and [ ] , encoding the most polymorphic region of the mhc class i. alleles are distinguished based on exon and sequence and full length sequences are imputed from the ipd bovine mhc class i database [ ] , a method commonly used for hla typing (e.g. [ ] ). advantages of this typing method are that the amplified gene specific sequence normally only contains two alleles, allowing the use of traditional sanger sequencing, and that a relatively large number of samples can be typed, as was necessary for the present study. however, there are also some limitations to this method. the gene specific primers are only validated for holsteins, which was not a problem in this study as all dams were of holstein origin, and alleles from mhc class i gene and are not directly typed. however, genes and are the least polymorphic of the bovine mhc class i genes with only seven documented alleles of which only two have been reported in holsteins [ ] . one of these alleles ( * ) can be imputed based on haplotype and the other ( * ) is amplified by gene specific primers. mhc class i haplotypes define a set of mhc class i alleles that are inherited together and because different haplotypes can define very similar mhc class i alleles, haplotype differences between animals do not accurately represent allogeneic or immunological differences between animals. it has been hypothesized that the occurrence of bnp depends on allogeneic (mis)matches between the dam, the mdbk cell line and the calf [ , ] . the likelihood of an alloimmune response is directly related to the number of epitope mismatches between the foreign alloantigen and the host [ ] and in order to better estimate allogeneic (mis)matches between animals and mdbk cells, we analyzed protein differences in the extracellular domain of the mhc class i protein between mdbk cells and dams/calves. although this method is potentially a better estimate of allogeneic differences between animals than only relying on mhc class i haplotypes, accurate prediction of antibody epitopes is much more complicated and depends on many other factors, such as conformation, non-linear epitopes and flanking residues [ ] . since genes and are not directly typed in the mhc class i method used in this paper, in some animals the presence of certain alleles was imputed from the defined haplotype for that animal and for newly defined haplotypes the presence of additional alleles cannot be excluded, giving an extra level of uncertainty to the analysis of the mhc class i protein differences. however, previously published haplotypes comprise the majority of the haplotypes typed in this study and the results from the mhc class i halpotyping corroborates the results from other experiments in this study. together, the results indicate that the occurrence of bnp is not associate with a specific allogeneic background of bnp dams or calves. the only difference we found between pregsure© bvd vaccinated non-bnp and bnp dams were in the alloantibody levels and this would imply that the development of bnp in the calf primarily depends on the alloantibody dose the calf absorbs. the finding by jones et al. [ ] that the odds of bnp increases with increased colostrum intake, and thus alloantibody intake, strengthens this hypothesis. the risk of bnp increases with increased number of pregsure© bvd vaccinations and this can be explained by boosting of antibody production, increasing the alloantibody levels in the colostrum and thus increasing the alloantibody dose of the calf after colostrum ingestion. furthermore, our findings that the heritability for the development of bnp in the calf was % for calves, whereas as a dam trait the heritability was % and the observation that bnp can be induced in unrelated healthy calves by alloantibodies/ colostrum from bnp dams [ ] [ ] [ ] show that the dam and not the calf plays a pivotal role in determining whether a calf gets bnp or not. we conclude that the development of bnp in calves is a heritable trait of the dam rather than the calf and that genetic differences between bnp and non-bnp dams are likely due to genes controlling the quantitative alloantibody response following vaccination. additional file : primers used for the amplification of mhc class i genes, bèta- -microglobulin and drb . the table lists the sequences and the location of the forward and reverse primers used to amplify the mhc class i genes, bèta- -microglobulin and drb genes. additional file : list of mhc class i haplotypes and mhc class i typing results of the mdbk cell line. list of mhc class i haplotype definitions used in this article and the mhc class i typing results of the mdbk cell line. the newly defined haplotypes, containing an uu prefix or suffix, are provisional haplotypes. these haplotypes have not been confirmed using different mhc class i typing methods and because the gene specific primers used in this study do not amplify gene and and have not been validated for all known mhc class i alleles, the presence of additional alleles cannot be excluded. in some cases previously defined haplotypes [ ] have been renamed to accommodate for additional haplotype variants within a group. allele nomenclature refers to the ipd bovine mhc class i database [ ] . additional file : summarizing results of the univariable analysis of the effect of independent variables on bnp, including sire and dam heritability estimates (n = ). the table provides results from the univariable analysis of the effect of independent variables on bnp. the number of records per category, the estimate (β), the odds ratio and the p-value from the wald test are given. the heritability estimates for bnp as a sire trait and bnp as a dam trait are also depicted. additional file : comparison of the difference in protein sequence of the extracellular part of the mhc class i protein (exon - ) between the the mdbk mhc class i allele that is most different to the mhc class i alleles of pregsure© bvd vaccinated non-bnp and bnp dams. dna sequences of the extracellular part of mhc class i, exon - , were translated into protein sequences and the percentage of sequence difference between the mdbk mhc class i allele that was most different to the dam mhc class i alleles was calculated. results for pregsure© bvd vaccinated non-bnp and bnp dams were compared using an unpaired t-test for unequal variance. additional file : mhc class i haplotypes of calves (n = ) fathered by the same sire. the table lists sequence based mhc class i haplotyping results for three bnp and five non-bnp calves fathered by the same sire. additional file : comparison of the difference in protein sequence of the extracellular part of the mhc class i protein (exon - ) between the most similar mdbk and paternally inherited mhc class i allele from non bnp and bnp calves. dna sequences of the extracellular part of mhc class i, exon - , were translated into protein sequences and the percentage of sequence difference between the most similar mdbk and paternally inherited calf mhc class i allele was calculated. results for non-bnp and bnp calves were compared using an unpaired t-test for unequal variance. additional file : binding of peripheral blood mononuclear cells by alloantibodies from pregsure© bvd vaccinated dams. a: peripheral blood mononuclear cells (pbmc) from ten random dams were stained with serum (ser, n = ) and colostrum (col, n = ) of different pregsure© bvd vaccinated non-bnp dams (bnp-vacc+, n = ) and with serum (n = ) and colostrum (n = ) of pregsure© bvd vaccinated bnp dams (bnp + vacc+, n = ). igg alloantibody binding was measured by flow cytometry. gmfi subtracted by isotype control is plotted on the y-axis. the horizontal dotted line depicts the overall average geometric mean fluorescent intensity (gmfi) and the number above the plots describes the number of samples with a signal above the horizontal line. b: the data from additional file a were divided by the gmfi signal of the alloantibody staining of mdbk cells by the respective serum or colostrum. the horizontal dotted line depicts the overall average relative signal and the number above the plots describes the number of samples with a signal above the horizontal line. mean ± standard error of the mean is depicted in all graphs. two tailed simple t-tests for unequal variance was used to compare alloantibody binding of pbmc's between pregsure© bvd vaccinated non-bnp and bnp dams. this study was funded by zoetis. authors' contributions lb participated in the design of the study, carried out the experiments, the data collection and analysis and prepared the manuscript. hgo and wgjvg participated in the design, the experiments, the data collection and analysis of the sequencing experiments and revised the manuscript. hcmh, mn and gvs participated in the design, the experiments, the data collection and analysis of the heritability study and revised the manuscript. vpmgr and apk participated in the design of the study, the experiments, the data collection and analysis and prepared the manuscript. all authors read and approved the final manuscript. gehäuftes auftreten von hämorrhagischer diathese infolge knochenmarkschädigung bei jungen kälbern calf-level factors associated with bovine neonatal pancytopenia -a multi-country case-control study haemorrhagic diathesis in neonatal calves: an emerging syndrome in europe reproduction of bovine neonatal pancytopenia (bnp) by feeding pooled colostrum reveals variable alloantibody damage to different haematopoietic lineages bone marrow depletion with haemorrhagic diathesis in calves in germany: characterization of the disease and preliminary investigations on its aetiology immunophenotyping and characterization of bnp colostra revealed pathogenic alloantibodies of igg subclass with specifity to platelets, granulocytes and monocytes of all maturation stages bovine neonatal pancytopenia: is this alloimmune syndrome caused by vaccine-induced alloreactive antibodies? vaccine detection of colostrum-derived alloantibodies in calves with bovine neonatal pancytopenia alloantibodies against mhc class i: a novel mechanism of neonatal pancytopenia linked to vaccination ingestion of colostrum from specific cows induces bovine neonatal pancytopenia (bnp) in some calves established kidney cell lines of normal adult bovine and ovine origin vaccine-induced antibodies linked to bovine neonatal pancytopenia (bnp) recognize cattle major histocompatibility complex class i (mhc i) effect of the vaccination scheme on pregsure (r) bvd induced alloreactivity and the incidence of bovine neonatal pancytopenia asreml user guide. release . . hemel hempstead, uk: vsn international ltd generation and maintenance of diversity in the cattle mhc class i region constraints on haplotype structure and variable gene frequencies suggest a functional hierarchy within cattle mhc class i establishment of a sequence-based typing system for bola-drb exon monoclonal-antibodies against bovine immunoglobulins and their use in isotype-specific elisas for rotavirus antibody controlling the false discovery rate -a practical and powerful approach to multiple testing bovine neonatal pancytopenia (bnp): novel insights into the incidence, vaccination-associated epidemiological factors and a potential genetic predisposition for clinical and subclinical cases lack of evidence for an association between mhc diversity and the development of bovine neonatal pancytopenia in holstein dairy cattle acquisition of hla class i w / defined antigenic determinant by heavy-chains from different species following association with bovine beta- -microglobulin biochemical characterization of activation-associated bovine class i major histocompatibility complex antigens beta- -microglobulin haplotypes in us beef cattle and association with failure of passive transfer in newborn calves association of blv infection profiles with alleles of the bola-drb . gene characterization of lymphocyte subpopulations and major histocompatibility complex haplotypes of mastitis-resistant and susceptible cows genes controlling vaccine responses and disease resistance to respiratory viral pathogens in cattle a single amino acid deletion in the antigen binding site of bola-drb is predicted to affect peptide binding type and type responses in regulation of ig isotype expression in cattle isotype-specific antibody responses of cattle to salmonella dublin lipopolysaccharide and porin following salmonella dublin vaccination and acute and chronic infection immunoglobulin isotypes of lactating holstein cows classified as high, average, and low type- or- immune responders quantitative evaluation of genetic and environmental parameters determining antibody response induced by vaccination against bovine respiratory syncytial virus a quantitative approach to classifying holstein cows based on antibody responsiveness and its relationship to peripartum mastitis occurrence polymorphism of bovine mhc class i genes cdna sequence of cattle mhc class i genes transcribed in serologically defined haplotypes a and a two-way calf to dam major histocompatibility class i compatibility increases risk for retained placenta in cattle hla class i sequence-based typing using dna recovered from frozen plasma the number of amino acid triplet differences between patient and donor is predictive for the antibody reactivity against mismatched human leukocyte antigens hlamatchmaker: a molecularly based algorithm for histocompatibility determination bovine neonatal pancytopenia is a heritable trait of the dam rather than the calf and correlates with the magnitude of vaccine induced maternal alloantibodies not the mhc haplotype we thank the dutch dairy farmers and veterinary practitioners who enabled the sample collection. we also thank matthijs schouten for taking the on farm questionnaires, crv (arnhem, the netherlands) for supplying the pedigrees and gd animal health service (deventer, the netherlands), especially ingrid den uijl and iwan kristens, for help with processing the serum samples and data of the calves. submit your next manuscript to biomed central and take full advantage of: key: cord- - cdqhrcw authors: seliger, barbara; ruiz‐cabello, francisco; garrido, federico title: chapter ifn inducibility of major histocompatibility antigens in tumors date: - - journal: adv cancer res doi: . /s - x( ) - sha: doc_id: cord_uid: cdqhrcw interferons represent a protein family with pleiotropic functions including immunomodulatory, cytostatic, and cytotoxic activities. based on these effects, interferons are involved in innate as well as adaptive immunity, thereby shaping the tumor host immune responses. these cytokines, alone or in combination, have been successfully implemented for the treatment of some malignancies. however, it has been recently demonstrated that tumor cells could be resistant to interferon treatment, which may be associated with an escape of tumor cells from immune surveillance. therefore, the aim of this chapter is to summarize the frequency of impaired interferon signal transduction, their underlying molecular mechanisms, and their clinical relevance. ag, antigen; apc, antigen presenting cells; apm, antigen-processing machinery; bh, bleomycin hydrolase; bp, base pairs; ciita, class ii transactivator protein; clip, class ii invariant chain peptide; ctl, cytotoxic t lymphocyte; dc, dendritic cell; er, endoplasmic reticulum; gas, gammainterferon-activated site; ifn, interferon; ifn-r , interferon-receptor- ; il, interleukin; irf, interferon regulatory factor; isg, interferon-stimulated genes; isgf , ifn-stimulated gene factor ; isre, interferon-stimulated response element; jak, janus kinase; lps, lipopolysaccharide; mapk, mitogenactivated protein kinase; mca, methylcholanthrene; mhc, major histocompatibility complex; nf, nuclear factor; nk, natural killer; pkc, protein kinase c; rcc, renal cell carcinoma; sclc, small-cell lung carcinoma; socs, suppressor of cytokine signaling; stat, signal transducer and activator of transcription; ta, tumor antigen; tap, transporter associated with antigen processing; tcr, t cell receptor; tfbs, transcription factor-binding sites; tnf, tumor necrosis factor; tpn, tapasin; tppii, tripeptidyl peptidase ii; tsa, trichostatin a; tyk, tyrosine kinase; uirr, upstream interferon response region; usf , upstream stimulatory factor ; wt, wild type. interferons (ifns) represent proteins that are secreted from cells in response to various stimuli and provide the basis for the understanding of the evolution, structure, and function as well as the pathways of other cytokines and their receptors (pestka, ; pestka et al., ) . they exert pleiotropic effects and are involved in host responses to bacterial and viral infection, in tumor surveillance mechanisms as well as in innate and adaptive immune responses (decker et al., ; pestka et al., ; stetson and medzhitov, ; takaoka and yanai, ) . in addition, ifns were the first cytokines used for the treatment of tumor patients. however, it has been suggested that tumor cells might develop either a transient or a permanent ifn insensitivity. this phenotype is linked to cytotoxicity resistance and might lead to escape of tumor cells from immune surveillance. we here summarize the current knowledge about (i) pleiotropic functions of ifns that mediate various biological responses, (ii) mechanisms of action and transduction pathways, (iii) the effect of type i and type ii ifns on the expression levels of molecules involved in proper major histocompatibility complex (mhc) class i and class ii antigen processing and presentation of tumor cells, (iv) the frequencies and the underlying molecular mechanisms of ifn resistance in tumors in association with alterations of the mhc class i and ii antigen-processing machinery, and (v) the clinical relevance of aberrant ifn signaling. the elucidation of the mechanisms leading to dysregulation of ifn signal transduction cascades triggering immune dysfunction and to tumor immune escape will benefit the design of strategies reversing these deficiencies, which could be of clinical relevance. interferons (ifns) are a family of multifunctional cytokines, which were originally described as antiviral cytokines, thereby protecting cells from viral infection (isaacs and lindenmann, ) . however, based on the current knowledge they exhibit a broad spectrum of activities including antiproliferative, immunomodulatory, anti-inflammatory, apoptosis-inducing, stress-mediated effects as well as regulation of cell differentiation steps and angiogenesis (amadori, ; baccala et al., ; theofilopoulos et al., ) . the ifn family is divided into type i, type ii, and type iii ifns. type i ifns consist of ifn-members and single members of ifn-, ifn-, ifn-, and ifn-e, respectively, which are all clustered on chromosome . in contrast, type ii ifn is represented only by a single gene, ifn-, encoded by chromosome (decker et al., ) . recently, type iii ifns have been discovered as a novel class of antiviral cytokines which are classified into ifn- , - , and - (oesterlund et al., ; sheppard et al., ; uze and monneron, ) . ifns bind to two distinct cell surface receptors. type i and ii ifn signal through a common -chain, thereby activating discrete, but related pathways leading to the transcriptional activation of the so-called interferonstimulated genes (isgs) ( table i; fig. ). isgs represent a functionally diverse group of genes involved in many cellular activities such as transcription, translation, regulation of cell cycle and apoptosis, intracellular communication as well as the processing and presentation of antigens. the transcriptional activity of isgs is necessary to mediate the effect of ifns. because of their diverse activities, ifns have been used for the treatment of various diseases such as chronic viral infections, like hepatitis c, multiple sclerosis, hematopoietic malignancies as well as solid tumors including renal cell carcinoma (rcc) and melanoma. the ifn therapy has been shown to reduce the rates of relapses and mortality by between and % in tumor patients (kirkwood et al., ) . however, during the last decade no further progress concerning the adjuvant therapy of tumor patients has been achieved. therefore, a better knowledge of the underlying molecular mechanisms of ifn action may lead to improved and more effective applications and the design of innovative, intelligent treatment strategies using ifns alone or in combination with other therapeutics. a wealth of information is available on the molecular processes underlying some of the ifn-induced signaling cascades. binding of ifns to their specific receptors lacking intrinsic kinase activity induces oligomerization of receptor subunits triggering diverse signaling pathways ( fig. ) , thereby leading to the transcriptional regulation of a plethora of target genes (kaur et al., ; li et al., ; schindler et al., ) . the physiologic relevance of ifn-dependent signal transduction cascades including the stat/jak pathway was established by generating and characterizing mice with targeted disruption of genes encoding stat /stat or jak , respectively (platanias, ; ramana et al., ) . both type i and type ii ifn receptors (ifn-r) initiate the activation of the jak/stat cascade, which consists of four janus kinases (jak , jak , jak , and jak ) and seven signal transducers and activators of transcription (stat , stat , stat , stat , stat a, stat b, stat c; fig. ifn signal transduction cascade and defects in this pathway. the type i and type ii receptors are transmembrane glycoproteins whose extracellular domains serve as ifn-binding sites, whereas their cytoplasmic domains associate with members of the jak kinase family and initiate signal transmission (dunn et al., ) . upon binding to their specific receptors both type i and type ii ifns induce a number of signal transduction cascades, which involve the phosphorylation of various components such as tyk , jaks, and stats. after recruitment to the receptor, stats become phosphorylated, form homo-or heterodimers, and migrate to the nucleus to bind to specific sequences in the promoter of target genes. type i ifn-induced signaling then induces homodimerization of stat and heterodimerization of stat and stat . stat and stat associate with the cytosolic transcription factor ifn-regulatory factor (irf ), forming a trimeric complex known as ifn-stimulated gene factor (isgf ) and activates transcription by binding to the isres. type ii ifn associates kinases, jak and jak phosphorylate stat , which then forms homodimers, translocates to the nucleus, and activates transcription by binding to the gas sequences. ifn-mediated signaling is controlled by several mechanisms including dephosphorylation of ifn-r , jak , and stat (mediated by sh -domain-containing protein tyrosine phosphatase , shp ), inhibition of the jaks (mediated by suppressor of cytokine signaling , socs ), proteasomal degradation of the jaks, and inhibition of stat (mediated by protein inhibitor of activated stat , pias ). shin-ya et al., ; yu and jove, ) . stats, sh -containing transcription factors, represent cytosolic proteins of - amino acids and are composed of (i) an extracellular domain that plays an important role in the association of stat with receptor molecules, (ii) a ligand-binding domain, and (iii) an intracellular domain that is responsible for the stat dimer formation. stat induces the expression of ifn-responsive genes through the activation of ifn-stimulated response element (isre)-containing promoters (yu and jove, ) . however, it has now become apparent that the activation of jak-stat pathways alone is not sufficient for the generation of all biological activities of ifns. there exists accumulating evidence that several other ifn-regulated signaling elements and cascades are required for the generation of many ifn responses. some of them operate independently of the jak-stat pathway, whereas others cooperate with stats to optimize the transcriptional regulation of target genes. these include in particular pathways linked to cellular stress and cell death like the mitogenactivated protein kinase (mapk), the stress-induced kinase p , and protein kinase c (pkc) signaling cascades. pkcs are known to be involved in both ifn-andsignaling pathways (kwon et al., ) . in this context, it is noteworthy that the ifn-, ifn-, and ifn-cascades exhibit overlapping activities, but also clearly different features ( fig. ; levy et al., ) . after the engagement with the type i ifn receptors (ifn-r), ifnbinding stimulates the cross-linking between the ifn-r chain (ifn-r ) and (ifn-r ), thereby bringing the receptor-associated kinases tyk and jak into close proximity. this triggers the activation of jak and tyk leading to the phosphorylation of tyr- of the ifn-r , which serves as a docking site for stat . the activated kinase subsequently phoshorylates stat and stat on tyr- and tyr- , respectively. both phosphorylated stats form a heterodimer and associate with the interferon regulatory factor (irf) , which does not undergo tyrosine phosphorylation to form the ifn-stimulated gene factor (isgf ), which, in turn, translocates to the nucleus and binds specific elements known as isres that are present in the promoters of certain isgs initiating the transcription of a broad variety of genes. in addition, phosphorylated stat , other stat complexes, and combinations of different stat-containing complexes can be formed which translocate to the nucleus and bind to the ifn--activated site (gas) leading to the transcription of further genes (caraglia et al., ) . it is noteworthy that ifn-can also activate stat and stat , but the role of stat in the ifn--mediated activity has still to be elucidated (uddin et al., ) . in contrast, ifn-mainly activates stat b. however, one can speculate that a fine balance between different stat complexes might account for specific responses and represent a key mechanism for ifn--induced activities. ifn-acts through a heterodimer consisting of the ifn-receptor- (ifn-r ) and ifn-r expressed on most cells, thereby upregulating specific genes. binding of ifn-initially leads to the formation of an ifn-r homodimer, which consecutively attracts the ifn-r chains. the ifn-r and -r homodimer is constitutively associated with jak and jak , which phosphorylate the tyrosine at the intracellular domain of the ifn-r serving as a docking site for the latent cytosolic transcription factor stat . stat is subsequently phosphorylated on tyrosine and serine leading to the homodimerization of phospho-stat molecules. these form a complex named the -activating factor (gaf) that translocates into the nucleus and upregulates the transcription of ifn--regulated genes including in particular the interferon-regulated factors (irf) and irf which represent transcriptional activators, whereas the constitutively expressed irf generally acts as a transcriptional repressor (harada et al., ) . irf subsequently activates the transcription of caspase genes involved in apoptosis next to genes encoded in the major histocompatibility complex (mhc) in particular components of the mhc class i and class ii antigen-processing machinery (apm) as well as -microglobulin ( -m) located on chromosome . the molecules of the antigen-processing pathway are required for the initiation and triggering of proper cd þ or cd þ t-cell responses, respectively. in addition, stat and irf cooperate with the ubiquitously expressed transactivating factor upstream stimulatory factor (usf) to activate the transcription of the class ii transactivator protein promoter iv (ciita-piv) that controls the expression of mhc class ii molecules (chen et al., ) . the expression of mhc class i and class ii molecules is critical for the presentation of antigens and essential for the generation of an adaptive immune response (cresswell et al., ; jensen, ) . in the last decades, cd þ cytotoxic t lymphocytes (ctl) have been implicated as main effector cells in antitumor responses. they recognize and attack tumor cells presenting intracellular antigens derived from different nonself peptides on their surface through the interaction of the t-cell receptor (tcr) with mhc class i peptide complexes. the generation and presentation of these antigens (ag) requires a coordinated expression of several genes ( fig. a) . briefly, endogenously synthesized proteins are cleaved by the multicatalytic proteasome complex, in particular the ifn--regulated proteasome subunits, such as the low molecular weight proteins (lmp) , lmp , and lmp . these peptides are further trimmed by cytosolic enzymes such as, for example, the tripeptidyl peptidase (tpp)ii and the bleomycin hydrolase (bh) generating the correct n-terminus (kloetzel, ; rock et al., ) . then the peptides are transported from the cytosol into the endoplasmic reticulum (er) via the transporter associated with antigen processing (tap), a heterodimer consisting of the tap and tap subunits. in the lumen of the er the mhc class i assembly occurs, which is assisted by various chaperones such as calnexin, calreticulin, the oxido thiol reductase erp , and tapasin (tpn). tpn facilitates the peptide loading onto mhc class i molecules. after successful peptide loading, mhc molecules are released from the peptide loading complex and the trimer consisting of mhc class i heavy chain (hc)/ -m/peptide is then transported through the trans-golgi apparatus to the cell surface and presented to cd þ ctl. thus, proper expression of the major components of the complex mhc class i apm components is obligatory for effective t-cell recognition of tumors (groettrup et al., ; jensen, ; seliger et al., ) . recently, it has been demonstrated that cd þ t cells are also important for proper antitumor immune responses (drozina et al., ; jensen, ) . these t cells recognize via their tcr antigens presented on mhc class ii molecules. in contrast to mhc class i antigens which are expressed on all nucleated adult cells, the expression of the heterodimeric mhc class ii molecules also representing transmembrane glycoproteins is highly restricted and preferentially found on the cell surface of professional antigen presenting cells (apcs). however, mhc class ii antigen expression can be induced in other cell types by various cytokines, in particular ifn-. mhc class ii expression is mainly controlled by the class ii transactivator protein (ciita), which acts as a master regulator for its coordinated constitutive and ifn--induced expression which also involves pkc delta (kwon et al., ; giroux et al., ) . ciita interacts with the transcription factors rfx, nfy, and creb (van den in the cytosol, endogenous peptides are generated by the proteasome, which were further trimmed by other peptidases and then transported into the er via the heterodimeric tap. erap is involved in the final aminoterminal trimming of peptides. the loading of mhc class i molecules with peptides is further assisted by the chaperone tapasin which is also a component of the plc. upon peptide loading, the plc dissociates and then transported via the trans golgi to the cell surface and there exposed to cd þ cytotoxic t lymphocytes. (b) mhc class ii pathway. mhc class ii molecules assemble in the er with the invariant chain (li), which contains an endosomal targeting signal. this complex is then transported to the endosomal compartment and there the ii is cleaved by a number of proteases leaving only the clip fragment, which occupies the peptide-binding groove. hla-dm and -do catalyze the release of clip, which is exchanged by antigenic peptides. hla-dm edit the repertoire of the mhc class ii-peptide complexes, which are then transported to the cell surface for recognition by cd þ t lymphocytes. exogenous proteins are internalized into the endosomal pathway by different mechanisms then unfolded and cleaved which is catalyzed by different proteases. in addition, the yielded peptides are further trimmed after binding to mhc class ii molecules. elsen et al., ) , thereby forming an enhanceosome governing the mhc class ii transcription. in addition, a coordinated expression of various mhc class ii apm components exists. mainly exogenous antigens are phagocytosed by apcs, directed then to lysosomes where they are cleaved into small peptide fragments (fig. b) . mhc class ii antigens are assembled in the er. the peptide-binding groove of these molecules is initially occupied by the invariant chain which is degraded into the class ii invariant chain peptide (clip) fragment by a series of key cleavage events, thereby protecting the mhc class ii-binding groove. the loading of mhc class ii molecules with exogenously derived peptides is assisted by the chaperone-like components hla-dm and -do, which results in an exchange of the clip fragment by these antigens. hla-dm is editing the peptides presented to cd þ t cells by catalyzing multiple rounds of peptide exchanges possibly favoring the most stable complexes. the peptide-loaded mhc class ii molecules are then transported to the cell surface and presented to cd þ t lymphocytes. in professional apc, exogenous antigens can gain access to the mhc class i pathway through distinct cross-presentation mechanisms. furthermore, the endosomal mhc class ii loading pathway could also receive peptides derived from endogenous antigens through autophagy and other mechanisms (dengjel et al., ; schmid et al., ) . the promoters of the mhc class i and class ii apm components have been intensely characterized and exert some similarities, but also unique properties. concerning the promoter of mhc class i apm components, some of them contain tata and caat boxes, whereas others completely lack these regulatory domains in the promoters). in addition, it is noteworthy that both tap and lmp are transcribed from a shared bidirectional promoter of only base pairs (bp) separating their atg translation initiation codon (wright et al., ) . the promoter of the major mhc class i apm components contain a combination of distinct transcription factor-binding sites (tfbs), like sp , creb, the nuclear factor (nf)-b, e f, and p , but all exhibit ifn-response elements, which hint toward their regulation by irfs chatterjee-kishore et al., , fig. ) . in terms of the mhc class ii pathway, the promoters of the invariant chain, hla-dm/-do and the mhc class ii hc, respectively, contain similar, but also distinct transcription factorbinding sites, whereas all of them contain an ifn-response element in their promoter. an exception is represented by ciita, which is regulated by multiple promoters differing in their tfbs composition. there exist three tissue-specific promoters for ciita, pi, pii, and piii. one promoter controls the constitutive ciita expression in dendritic cells (dc), whereas another is specific for the constitutive expression in b cells. the ciita-piv regulates the induction of ciita expression in different cell types. it contains several cis elements including a putative nf-b site overlapping with an ap site, the ifn-activating sequence (gas), the e box, and an irf element (dong et al., ; muhlethaler-mottet et al., ) . thus, the activity of the different mhc class i and ii apm component promoters can be induced, but to a different extent, by type i and type ii ifns, respectively. ifn-is a stronger inducer when compared to type i ifns, whereas a combination of both substances exerts additive or even synergistic effects on mhc class i and ii apm components. activation of adaptive immune responses by ifn, in particular ifn-, is partially due to transcriptional activation of genes encoding the mhc class i and class ii antigens and respective apm components such as the invariant chain, hla-dm/-do, ciita, tap, tpn, the lmps, and erap / . fig. promoter structure of major apm components. the structure of representative promoters of the major apm components is schematically illustrated, demonstrating a number of transcription factor-binding sites such as nf-b, ap , sp , and creb as well as interferon regulatory response elements (isre), which are involved in the inducibility by this cytokines. decrease in or absence of mhc class i molecules has been observed in a diversity of human tumor types (garrido and algarra, ; garrido et al., garrido et al., , ). an increasing proportion of tumors were found with total or selective hla allelic losses supporting the theory that altered hla expression phenotypes represent a major mechanism of tumor escape from t-cell recognition due to downmodulation of presentation of immunodominant tumor antigens. distinct hla class i abnormalities, including total loss or downregulation of hla class i antigens (paschen et al., ) , hla haplotype loss (ramal et al., ) , hla locus or allele loss (jimenez et al., ) has been described in tumors originating from different tissues and multiple molecular mechanisms have been identified as responsible for these changes (garrido and algarra ) . the mechanisms that underlie total or partial loss of hla class i antigens (table ii) include mutations of the -microglobulin ( -m) gene (perez et al., ) and loss of heterozygosity (loh) of mhc genes (maleno et al., ) . other causes of total hla class i downregulation comprise defects in the regulation of different components of the mhc class i antigen processing. structural defects of apm components cannot be corrected by cytokine treatment; therefore, it does not restore hla class i surface antigen expression. t-cell-based therapy may not be effective due to the irreversible loss of hla class i molecules. this is important when selecting the appropriate immunotherapy for a given cancer patient. abnormalities in the expression of various mhc class i apm components occur at a high frequency in human tumors of distinct origin like small-cell lung carcinoma (sclc), melanoma, colon carcinoma, breast carcinoma, renal cell carcinoma, and hematological malignancies and are frequently associated with malignant transformation (table ii) . this phenotype allows the tumor cells to evade recognition by mhc class i-restricted, tumor antigen (ta)-specific ctl. mutations in different apm components appear to be a rare event postulating that dysregulation rather than structural alterations is the major cause for aberrant apm component expression (fernandez et al., ; ramal et al., ; seliger et al., ; table ii) . this hypothesis is supported by experiments (i) identifying only few mutations in these molecules, (ii) characterizing the apm promoter activity in tumors, (iii) determining posttranscriptional regulatory mechanisms, and (iv) treating tumor cells with ifns to analyze whether deficiencies of apm component expression could be overcome by cytokines. indeed, impaired apm component expression of tumor cells could be often restored by ifn-/ and/or ifn-treatment. the ifn-mediated upregulation of apm components often results in enhanced mhc class i surface expression, which is required for the generation of an effective antitumor-specific immune response. indeed, the ifn-induced upregulation of apm components improves antitumor-specific ctl responses (seliger et al., ; tajima et al., ) and therefore represent a valuable strategy for the treatment of patients with apm component deficiencies. however, in some cases, tumors remain insensitive to ifn treatment despite the lack of structural alterations in apm components, rather suggesting an impaired ifn signal transduction. the unresponsiveness to ifn treatment was analyzed in a number of different tumor types and according to kaplan et al. ( ) can be frequently found in human cancers. approximately % of melanoma and non-adenocarcinoma lung tumor cell lines analyzed exhibit a quantitative reduction in ifn-sensitivity, while out of lung adenocarcinoma cell lines were totally unresponsive to ifn-. these data were extended in a recent study in which melanoma cell lines were analyzed for the ability to upregulate mhc class i surface antigens in response to stimulation with ifn-. a total unresponsiveness to ifn-was found in out of melanoma cell lines (rodriguez et al., b) . however, the number of tumor types and tumor samples analyzed for ifn resistance is still limited and requires further studies in order to determine the frequency, relevance, and molecular mechanisms of these deficiencies. it is noteworthy that an impaired ifnresponse despite a functional ifn-induction may exist. on the other hand, a lack of ifn-responsiveness can also be found in the presence of ifnsensitivity, suggesting that the ifn signal transduction cascades are not coordinately regulated in tumor cells. the importance and involvement of ifn signal transduction pathways in the transcriptional regulation of apm promoters have been established, but there exists only limited information about the underlying molecular mechanisms of defective ifn-inducible apm component expression. the impairment could occur at different steps along the ifn signal transduction pathways and might involve sequence abnormalities and/or different regulatory processes such as transcriptional, posttranscriptional, and epigenetic control ( fig. ; table iii ). the physiological relevance of the stat/jak and pi k pathway has been established in mice with a targeted disruption of these genes. the lack of jak activity was associated with a loss of ifn-to induce growth arrest and apoptosis as well as an increased tumorgenicity (sexl et al., ) . however, the observed ifn-response with respect to growth inhibition might also be attributable to the ifn-inducibility of lmp (hayashi et al., ) . so far, there exists only limited information regarding the molecular mechanisms of ifn resistance in tumors (huang et al., ; lesinski et al., ; wellbrock et al., ; wong et al., ) . based on the current knowledge that stat and irf are involved in the transcriptional regulation of the dual tap and lmp promoter, the loss of tap and lmp expression may be attributable to deficiencies of these regulatory factors. regarding the ifn-resistance of rcc cell lines, it is associated with a defective induction of stat that could be restored by the addition of a supernatant from pma-stimulated peripheral mononuclear cells (brinckmann et al., ) . this effect appears to be mediated by ifn-although other cytokines might also be involved in this process. in addition, the loss of the ifn--mediated upregulation of mhc class i apm components in some rcc cell lines appears to be due to the lack of irf -and stat -binding activities upon ifn-stimulation. the stat , jak , and jak proteins were expressed but not phosphorylated in the presence of ifn-. the ifn--mediated inducibility was not restored by gene transfer of jak and/or jak into rcc cells, whereas jak overexpression increased both tap and lmp expression independent of ifn-. therefore, the loss of tap and lmp induction was associated with a defect of an early step in the ifn-signal transduction pathway (dovhey et al., ) . furthermore, an association of impaired stat phosphorylation with the loss of ifn-mediated hla class i induction was also found in melanoma cell lines (rodriguez et al., b) . the absence of stat phosphorylation was at least partially due to the constitutive expression of the suppressor of cytokine signaling (socs)- protein, which could be mediated by the jak kinase inhibition via the socs phosphatase. socs- modulates the ifn-mediated signaling by binding to the autophosphorylation site of jak and by targeting bound jak to the proteasome for degradation (waiboci et al., ) . in addition, socs- expression correlates with melanoma progression and confers growth advantage (komyod et al., ; li et al., ) . in another study, the ifn-resistance was associated with socs expression. the resistant cell lines differed from the sensitive cells by a constitutive expression of socs , by the absence or a low degree of socs - activation following ifn-treatment, and by a short duration of the cytokine activatory signal (fojtova et al., ) . the expression of ifn--responsive genes is also reduced in the choriocarcinoma cells jeg and jar in comparison to the epithelial cell line hela (choi et al., ) . this is mediated by a compromised tyrosine phosphorylation of jak and stat at tyrosine and the reduced expression of irf . in addition, inhibition of the tyrosine phosphatases results in increased jak and stat phosphorylation and ifn--induced gene expression in these cells (choi et al., ) . the impaired expression of irf and deficient phosphorylation of stat were also observed in primary trophoblast cell lines suggesting that these defects are of clinical relevance. besides the posttranslational regulation of components of the ifn signal cascades, the absence of the ifn--mediated mhc class i expression can be controlled by epigenetic alterations in this pathway. indeed, methylation affects the binding of irf leading to an abrogation of the irf transactivation (rodriguez et al., b) . treatment with the demethylating agent deoxyazacytidine (dac) restored the irf expression and consecutively led to the reconstitution of the ifn--mediated mhc class i inducibility. other studies have identified that the ifn unresponsiveness is attributed to low expression of stat rather than to an absence of its phosphorylation (abril et al., ; xi et al., ) . the absence of stat expression has been correlated with the methylation of its promoter (xi et al., ) . finally, there exists evidence that genetic instability in tumor cells may lead to modulation of the expression of the ifn-r, which in some cases has been reported to be associated with cancer prognosis. for instance, the loss of ifn-r independently predicts poor prognosis in ovarian cancer and may be responsible for the limited success in the outcome of treatment of ovarian cancer with ifn- (duncan et al., ) . the multiple activities of ifns on tumor cells might coordinate the antitumor immune responses so that the early recognition and/or elimination of cancer cells by the innate immune system transitions to immune attack by the adaptive immune system (dunn et al., ) . the ifn-on the tumor cell immunogenicity mediate the immune response directed against tumor cells through distinct mechanisms. ifn-can downregulate the expression of the nkg d ligands and at the same time increase the expression of mhc class i molecules (bui et al., ) . in vitro treatment with ifn-decreased the death by nk cells independently from the expression of hla class i molecules, whereas an increased mhc class i expression increased the sensibility ctl-mediated lysis. besides these in vitro results, there also exist information that abnormalities in the ifn signaling occurs in vivo. lmp -/-mice exhibit an impaired proteasome function and % of female lmp -/-mice develop uterine leiomyosarcomas by months of age. thus, the development of spontaneous human uterine leiomyosarcomas might be probably due to defects in early steps of the ifn signal cascade. indeed, the defective tap and lmp expression in these tumors is associated with a g e mutation in the atp-binding region of the jak kinase domain, thereby affecting jak kinase activity, but neither jak expression and production nor its degradation (hayashi et al., ) . this allows the tumor cells to evade antitumor-specific immunity. in different tumor types, immunosuppression associated with stat activation and stat -mediated inhibition of dc function has been reported (yu and jove, ) . the biological function of stat and stat differs in terms of cell growth and induction of an antitumor immune response. whereas stat abrogates growth and mediates antitumor effects, stat promotes cell proliferation and tumorigenicity as it has been shown in melanoma and head neck squamous carcinoma. in both tumor entities, stat expression is associated with tumor progression and mediates immune suppression. in addition, unphosphorylated or phosphorylated stat and stat are coordinately upregulated by both ifn-and ifn-and may represent a marker for the dynamic mechanism of melanoma progression and host response. using methylcholanthrene (mca) and untreated ifn-r -/-, a significant tumor development was observed in the ifn-r control mice. the crossing of ifn-r and stat -/mice with p -/mice resulted in a spontaneous and more rapid tumor development in particular teratomas, hemangiomas, and chondrocytomas, whereas lymphoid tumors generally develop in ifn--sensitive p -/mice. interestingly, the ifn-sensitive tumor cells transfected with the dominant negative ifn-r mutant grew faster than untransfected tumors and were not rejected upon their treatment with lipopolysaccharide (lps) effectively eliminating control tumors . furthermore, downregulation of the ifn-r in association with loss of fas function is linked to tumor progression (yang et al., ) . thus, the ifn-responsiveness is an important mechanism in the control of tumor growth. an increased responsiveness to metastasespromoting agents might be induced by many mediators in the microenvironment of melanoma including type i and type ii ifns. both cytokines cooperate with tnf-, which involves a positive interplay between jak and pkc signal transduction (bianchini et al., ) . these data suggest that multiple signals were generated by the host inflammatory cells, which are accompanied by cooperate with the invasive properties of tumor cells. therefore, strategies targeting this cross-talk among tumor and host cells in the microenvironment are needed to prevent tumor growth. the chimeric ret/ptc (rearranged in transformation/papillary thyroid carcinoma) oncoproteins were constitutively expressed in papillary thyroid cancer and are able to phosphorylate the y of stat , which is accompanied by irf expression (hwang et al., ) . this is associated with an enhanced transcription of ciita and consequently with mhc class ii expression of papillary thyroid carcinoma cells and explain the immune cell infiltration of ret/ptc-positive cancers. furthermore, a synergistic activity of tnf-and ifn-on ciita was found in thyroid carcinoma (rahat et al., ) the ciita-independent mhc class i expression could be upregulated by histone deacetylases like trichostatin a (tsa) (chou et al., ; gialitakis et al., ) . ciita was refractory to ifn induction in many tumors. in colorectal and gastric carcinoma cells, ciita is silenced by epigenetic mechanisms resulting in the lack of ifn--induced mhc class ii expression (satoh et al., ) . in order to correlate the ifn unresponsiveness with the expression profile of isgs, cdna microarray analyses were employed using a customized microarray consisting of isg (holko and williams, ) . expression of genes associated with transcription precedes the expression of genes involved in signal transduction, whereas no differences in the stat induction were observed. however, subtle alterations in the expression profile might be responsible for the insensitivity to this cytokine. the maintenance of transcriptional activation following ifn treatment appeared to enhance ifn sensitivity. ifns have been used in various clinical settings, since they are potent negative regulators of cell growth either by modulating the cell cycle or by inducing pro-apoptotic genes. ifn-has been extensively studied in the treatment of various malignancies during the last two decades demonstrating improved clinical outcome of hematological malignancies (chronic myeloid leukemia, cutaneous t-cell lymphoma, hairy-cell leukemia, multiple myeloma), solid tumors including malignant melanoma, renal-cell carcinoma (rcc), aids-related kaposi's sarcoma, and viral syndromes (hepatitis c, hepatitis b, severe acute respiratory syndrome). ifn-has shown positive results in the treatment of chronic granulomatous disease, multiple sclerosis, and severe malignant osteopetrosis (parmar and platanias, , for review). however, the resistance to ifns has been described, which limits their anticancer activity. the impaired expression of ifn-responsive genes might have important implications not only in immunotherapy but also in transplantation, pregnancy, and the development of tumors such as choriocarcinoma. despite proven clinical efficacy in malignancies, viral infections, and multiple sclerosis, a substantial number of patients fail to develop positive clinical response to ifn therapy. although ifn- b is a clinically active therapeutic agent for malignant melanoma and rcc, only - % patients with metastatic melanoma respond to ifn therapy (marincola et al., ) . other reviews report even lower response rates of only % of treated melanoma patients (quesada et al., ; umeda and niijima, ) . in rcc, the best results of ifn treatment as determined by the response rate and the duration of the effect were obtained in patients with a previous nephrectomy without chemotherapy, in a good functional state, and with preferentially lung metastasis. in these patients the survival rate increased from to weeks upon ifn-administration (logothetis, ) . despite these positive results, there exist many aspects of these response factors which are not well understood. actually, none of these factors has been proved to be associated in an unambiguous way with the cytokine response and the patients' survival. the key aspect may be the right selection of patients, since currently all of them independent of previous nephrectomy and the presence of metastasis are enrolled into the treatment with poor clinical outcome. unlike type i ifns, ifn-has not been approved for cancer treatment by the fda. ifnproduces numerous antitumor effects and plays a central role in promoting natural immune responses directed against developing tumors. however, its practical application in immunotherapeutic protocols has been very limited. in clinical trials, an improved survival was observed in patients with ovarian cancer of stage ic-iiic treated with ifn- (windbichler et al., ) , when ifn was intravesically administered to patients with transitional-cell bladder carcinoma (giannopoulos et al., ) or when ifn was used in isolatedlimb perfusion of individuals with non-melanoma cancers of the extremities (lienard et al., ) . however, no effect was detected upon ifn-treatment of patients with metastatic rcc (gleave et al., ) , advanced colon cancer (wiesenfeld et al., ) , or small-cell lung cancer (jett et al., ) . the limited success of the therapeutic use of ifn-might reflect the inability to target ifns in the right place with an efficient concentration (dunn et al., ) . despite the proven pivotal role of endogenously produced antitumor immunity of ifn-in animal models, the limited success of this cytokine in cancer immunotherapy trials in humans might be explained by the resistance of tumor cells to ifn- (kaplan et al., ; rodriguez et al., a; wong et al., ) . in this context, it is important to note that unlike type i ifns, ifn-has a direct effect on tumor cells during the antitumor immune response supporting the relevance of ifn-in the cancer immunoediting process (dunn et al., ) . the targets of the immunologic unresponsiveness represent genes encoding components of the mhc apm components or the constituents of the ifn-r signaling pathway. in this context, in two recent studies from our laboratory, the physiological relevance of hla class i surface expression during the tumor rejection process in patients receiving different protocols of immunotherapy was assessed (cabrera et al., ; carretero et al., submitted) . in the first study, a significant difference in the immunotherapeutic response of patients exhibiting metastases with low levels of mhc class i surface antigens and those with high levels of mhc class i expression was detected. in a second trial, the impact of cytokine unresponsiveness was demonstrated by determination of hla class i antigen expression levels on metastatic melanoma lesions during the course of the disease in one patient undergoing ifn- b and autologous vaccination plus bcg (m-vax). bcg triggers the il- /ifn-axis and induces upregulation of genes associated with antigen presentation (feinberg et al., ; saban et al., ) . the level of the mhc class i antigen expression was dependent on the ifn response since neither of the progressor metastases increased the expression of hla class i antigens after vaccination. however, a significant increase in the hla class i surface expression was detected in the regressor metastases. therefore, the hla class i surface antigen on tumor cells significantly contributed to the therapeutic effect of bcg. in connection with these findings, downregulation of hla class i surface antigens in cancer cells has been considered a significant risk factor for recurrence in patients with intravesical bcg immunotherapy for bladder cancer (kitamura et al., ) . based on these results, a better understanding of the molecular mechanisms by which tumors modulate the cytokine signaling may be essential for the development of immunotherapeutic strategies with the aim to enhance mhc class i surface antigen expression in tumor cells. the balance of stat phosphorylation versus socs expression might be crucial in the activation of immunologic response through apm and mhc class i transactivation (wang et al., ) . for instance, the effects of high-dose ifn are associated with immunologic processes such as an upregulation of tap , tap , tpn, and lmp . the stat and stat pathways in melanoma cells are sensitized to ifn-by pretreatment of the cells with ifn-. thus, the biological response to ifn-might be mediated by a direct effect on melanoma cells and suggests also a potential role for ifn-in the treatment of this disease (carson, ) . in addition, it has recently been demonstrated that ifntreatment of patients with cutaneous melanoma significantly modulates the balance of stat /stat in tumor cells and host lymphocytes. this results in an upregulation of tap and an increased immune response (wang et al., ) . an increased knowledge of the factors responsible for the resistance to ifns might lead to an improved use of these cytokines in malignant diseases. the application of the molecular analysis of tumor tissues has now advanced to the point where better classification schemes and prognostic variables are used leading to an optimization of specific treatment programs and patients' selection. the identification of tumor lesions with the capacity to upregulate mhc gene expression will determine the ability to present new antigenic peptides to t lymphocytes favoring regression of primary or metastatic tumor lesions. in contrast, the identification of tumors with mhc irreversible genetic lesions will maintain an unaltered mhc expression, thereby not exposing new antigenic peptides to t cells, which subsequently favors tumor and/or metastases progression. we propose that suppression of ifn signaling in tumors contributes to tolerance by inhibiting expression of genes encoding subunits of hla class i/ii antigens and/or components of the mhc class i/ii apm that could be detrimental to successful antitumor responses. unresponsiveness to interferon associated with stat protein deficiency in a gastric adenocarcinoma cell line the role of ifn-alpha as homeostatic agent in the inflammatory response: a balance between danger and response? interferons as pathogenic effectors in autoimmunity expression of a metastatic phenotype in ifns-primed/tnfalpha-activated b murine melanoma cells: role of jak /pkcdelta signal transduction factors interferon-alpha resistance in renal carcinoma cells is associated with defective induction of signal transducer and activator of transcription which can be restored by a supernatant of phorbol -myristate -acetate stimulated peripheral blood mononuclear cells regulation of murine tap and lmp genes in macrophages by interferon gamma is mediated by stat and irf- ifn-dependent down-regulation of the nkg d ligand h on tumors hla class i expression in metastatic melanoma correlates with tumor development during autologous vaccination alpha-interferon and its effects on signal transduction pathways interferon-alpha-induced activation of signal transducer and activator of transcription proteins in malignant melanoma different requirements for signal transducer and activator of transcription alpha and interferon regulatory factor in the regulation of low molecular mass polypeptide and transporter associated with antigen processing gene expression how stat mediates constitutive gene expression: a complex of unphosphorylated stat and irf supports transcription of the lmp gene positive regulatory domain i-binding factor mediates repression of the mhc class ii transactivator (ciita) type iv promoter dampening of ifngamma-inducible gene expression in human choriocarcinoma cells is due to phosphatasemediated inhibition of the jak/stat- pathway histone acetylation regulates the cell type specific ciita promoters, mhc class ii expression and antigen presentation in tumor cells mechanisms of mhc class i-restricted antigen processing and cross-presentation the yin and yang of type i interferon activity in bacterial infection autophagy promotes mhc class ii presentation of peptides from intracellular source proteins ifn-gamma regulation of the type iv class ii transactivator promoter in astrocytes loss of interferon-gamma inducibility of tap and lmp in a renal cell carcinoma cell line expression of mhc ii genes loss of ifn gamma receptor is an independent prognostic factor in ovarian cancer the immunobiology of cancer immunosurveillance and immunoediting interferons, immunity and cancer immunoediting bacillus calmette guerin triggers the il- /ifn-gamma axis by an irak- -and nemodependent, non-cognate interaction between monocytes, nk, and t lymphocytes beta -microglobulin gene mutation is not a common mechanism of hla class i total loss in human tumors development of ifn-gamma resistance is associated with attenuation of socs genes induction and constitutive expression of socs in melanoma cells mhc antigens and tumor escape from immune surveillance natural history of hla expression during tumour development implications for immunosurveillance of altered hla class i phenotypes in human tumours coordinated changes of histone modifications and hdac mobilization regulate the induction of mhc class ii genes by trichostatin a the immunomodulating effect of interferon-gamma intravesical instillations in preventing bladder cancer recurrence ifn-gamma-induced mhc class ii expression: transactivation of class ii transactivator promoter iv by ifn regulatory factor- is regulated by protein kinase c-alpha interferon gamma- b compared with placebo in metastatic renal-cell carcinoma peptide antigen production by the proteasome: complexity provides efficiency structurally similar but functionally distinct factors, irf- and irf- , bind to the same regulatory elements of ifn and ifn-inducible genes the mutation in the atp-binding region of jak , identified in human uterine leiomyosarcomas, results in defective interferongamma inducibility of tap and lmp functional annotation of ifn-alpha-stimulated gene expression profiles from sensitive and resistant renal cell carcinoma cell lines stat negatively regulates angiogenesis, tumorigenicity and metastasis of tumor cells regulation of signal transducer and activator of transcription (stat ) and stat -dependent genes by ret/ ptc (rearranged in transformation/papillary thyroid carcinoma) oncogenic tyrosine kinases virus interference. i. the interferon. by a. isaacs and j. lindenmann, recent advances in antigen processing and presentation phase iii trial of recombinant interferon gamma in complete responders with small-cell lung cancer a nucleotide insertion in exon is responsible for the absence of expression of an hla-a* allele in a prostate carcinoma cell line demonstration of an interferon gamma-dependent tumor surveillance system in immunocompetent mice the pi kinase pathway in interferon signaling a pooled analysis of eastern cooperative oncology group and intergroup trials of adjuvant highdose interferon for melanoma effect of human leukocyte antigen class i expression of tumor cells on outcome of intravesical instillation of bacillus calmette-guerin immunotherapy for bladder cancer generation of major histocompatibility complex class i antigens: functional interplay between proteasomes and tppii constitutive suppressor of cytokine signaling expression confers a growth advantage to a human melanoma cell line role of pkcdelta in ifn-gamma-inducible ciita gene expression melanoma cells exhibit variable signal transducer and activator of transcription phosphorylation and a reduced response to ifn-alpha compared with immune effector cells synergistic interaction between interferon-alpha and interferon-gamma through induced synthesis of one subunit of the transcription factor isgf expression of socs- , suppressor of cytokine signalling- , in human melanoma isolated limb perfusion in primary and recurrent melanoma: indications and results treatment of chemotherapy-refractory metastatic urothelial tumors combination therapy with interferon alfa- a and interleukin- for the treatment of metastatic cancer distribution of hla class i altered phenotypes in colorectal carcinomas: high frequency of hla haplotype loss associated with loss of heterozygosity in chromosome region p stat regulates lipopolysaccharide-and tnf-alpha-dependent expression of transporter associated with antigen processing and low molecular mass polypeptide genes in macrophages by distinct mechanisms activation of the mhc class ii transactivator ciita by interferon-gamma requires cooperative interaction between stat and usf- ifn regulatory factor family members differentially regulate the expression of type iii ifn (ifn-) genes complete loss of hla class i antigen expression on melanoma cells: a result of successive mutational events a new beta microglobulin mutation found in a melanoma tumor cell line the human interferon alpha species and receptors interferons, interferon-like cytokines, and their receptors mechanisms of type-i-and type-ii-interferon-mediated signalling antitumor activity of recombinant-derived interferon alpha in metastatic renal cell carcinoma increased binding of ifn regulating factor mediates the synergistic induction of ciita by ifn-gamma and tumor necrosis factor-alpha in human thyroid carcinoma cells molecular strategies to define hla haplotype loss in microdissected tumor cells stat -dependent andindependent pathways in ifn-gamma-dependent signaling post-proteasomal antigen processing for major histocompatibility complex class i presentation patterns of constitutive and ifngamma inducible expression of hla class ii molecules in human melanoma cell lines distinct mechanisms of loss of ifn-gamma mediated hla class i inducibility in two melanoma cell lines repeated bcg treatment of mouse bladder selectively stimulates small gtpases and hla antigens and inhibits single-spanning uroplakins epigenetic inactivation of class ii transactivator (ciita) is associated with the absence of interferon-gamma-induced hla-dr expression in colorectal and gastric cancer cells antigen-loading compartments for major histocompatibility complex class ii molecules continuously receive input from autophagosomes jak-stat signaling: from interferons to cytokines ifn-gamma-mediated coordinated transcriptional regulation of the human tap- and lmp- genes in human renal cell carcinoma molecular mechanisms of hla class i antigen abnormalities following viral infection and transformation jak deficiency leads to enhanced abelson-induced b-cell tumor formation il- and their class ii cytokine receptor il- r intracellular interferon triggers jak/stat signaling cascade and induces p -dependent antiviral protection type i interferons in host defense interferon-gamma differentially regulates susceptibility of lung cancer cells to telomerasespecific cytotoxic t lymphocytes interferon signalling network in innate defence type i interferons (alpha/beta) in immunity and autoimmunity role of stat in type i interferon-signaling and transcriptional regulation phase ii study of alpha interferon on renal cell carcinoma. summary of three collaborative trials il- and il- : newcomers to the interferon family transcriptional regulation of antigen presentation both the suppressor of cytokine signaling (socs- ) kinase inhibitory region and socs- mimetic bind to jak autophosphorylation site: implications for the development of a socs- antagonist modulation of signal transducers and activators of transcription and signaling in melanoma by high-dose ifnalpha b stat contributes to interferon resistance of melanoma cells controlled clinical trial of interferon-gamma as postoperative surgical adjuvant therapy for colon cancer interferon-gamma in the first-line therapy of ovarian cancer: a randomized phase iii trial interferon-resistant human melanoma cells are deficient in isgf components, stat , stat , and p -isgf gamma coordinate regulation of the human tap and lmp genes from a shared bidirectional promoter decreased stat expression by promoter methylation in squamous cell carcinogenesis downregulation of ifn-gammar in association with loss of fas function is linked to tumor progression the stats of cancer -new molecular targets come of age this work was supported by grants from the fondo de investigaciones sanitarias (fis), red genomica del cancer (retic rd / ), plan andaluz de investigacion (group cts ), consejeria andaluz de salud (sas), proyecto de excelencia de consejeria de innovacion (cts ), proyecto de investigacion iþd (saf - ) in spain; and from the integrated european cancer immunotherapy project (oj /c , ) and by grants from the deutsche forschungsgemeinschaft dfg se - / and - (b.s). in addition, we thank tarish abbas for providing figure and anne wasilewski for excellent secretarial help. key: cord- -y ev authors: magor, katharine e.; miranzo navarro, domingo; barber, megan r.w.; petkau, kristina; fleming-canepa, ximena; blyth, graham a.d.; blaine, alysson h. title: defense genes missing from the flight division date: - - journal: dev comp immunol doi: . /j.dci. . . sha: doc_id: cord_uid: y ev birds have a smaller repertoire of immune genes than mammals. in our efforts to study antiviral responses to influenza in avian hosts, we have noted key genes that appear to be missing. as a result, we speculate that birds have impaired detection of viruses and intracellular pathogens. birds are missing tlr , a detector for single-stranded rna. chickens also lack rig-i, the intracellular detector for single-stranded viral rna. riplet, an activator for rig-i, is also missing in chickens. irf , the nuclear activator of interferon-beta in the rig-i pathway is missing in birds. downstream of interferon (ifn) signaling, some of the antiviral effectors are missing, including isg , and isg and isg (ifits). birds have only three antibody isotypes and igd is missing. ducks, but not chickens, make an unusual truncated igy antibody that is missing the fc fragment. chickens have an expanded family of lilr leukocyte receptor genes, called chir genes, with hundreds of members, including several that encode igy fc receptors. intriguingly, lilr homologues appear to be missing in ducks, including these igy fc receptors. the truncated igy in ducks, and the duplicated igy receptor genes in chickens may both have resulted from selective pressure by a pathogen on igy fcr interactions. birds have a minimal mhc, and the tap transport and presentation of peptides on mhc class i is constrained, limiting function. perhaps removing some constraint, ducks appear to lack tapasin, a chaperone involved in loading peptides on mhc class i. finally, the absence of lymphotoxin-alpha and beta may account for the observed lack of lymph nodes in birds. as illustrated by these examples, the picture that emerges is some impairment of immune response to viruses in birds, either a cause or consequence of the host-pathogen arms race and long evolutionary relationship of birds and rna viruses. lymph node duck chicken major histocompatibility complex a b s t r a c t birds have a smaller repertoire of immune genes than mammals. in our efforts to study antiviral responses to influenza in avian hosts, we have noted key genes that appear to be missing. as a result, we speculate that birds have impaired detection of viruses and intracellular pathogens. birds are missing tlr , a detector for single-stranded rna. chickens also lack rig-i, the intracellular detector for singlestranded viral rna. riplet, an activator for rig-i, is also missing in chickens. irf , the nuclear activator of interferon-beta in the rig-i pathway is missing in birds. downstream of interferon (ifn) signaling, some of the antiviral effectors are missing, including isg , and isg and isg (ifits). birds have only three antibody isotypes and igd is missing. ducks, but not chickens, make an unusual truncated igy antibody that is missing the fc fragment. chickens have an expanded family of lilr leukocyte receptor genes, called chir genes, with hundreds of members, including several that encode igy fc receptors. intriguingly, lilr homologues appear to be missing in ducks, including these igy fc receptors. the truncated igy in ducks, and the duplicated igy receptor genes in chickens may both have resulted from selective pressure by a pathogen on igy fcr interactions. birds have a minimal mhc, and the tap transport and presentation of peptides on mhc class i is constrained, limiting function. perhaps removing some constraint, ducks appear to lack tapasin, a chaperone involved in loading peptides on mhc class i. finally, the absence of lymphotoxin-alpha and beta may account for the observed lack of lymph nodes in birds. as illustrated by these examples, the picture that emerges is some impairment of immune response to viruses in birds, either a cause or consequence of the host-pathogen arms race and long evolutionary relationship of birds and rna viruses. Ó elsevier ltd. all rights reserved. a survey of genomic resources demonstrates that the avian immune gene complement is reduced compared to mammals. an initial investigation of the immune genes in the chicken genome, a red jungle fowl, suggested that birds have a reduced immune gene repertoire (consortium, ) . as this sequence assembly and annotation has been improved, some of these missing genes have been identified, however others are clearly not present. as genomes are sequenced for other birds, including turkey (dalloul et al., ) , zebrafinch and duck (http:// pre.ensembl.org/anas_platyrhynchos/info/index), synteny along the chromosome allowed identification of genes. thus, immune genes could be identified even if significantly diverged. a comparison of immune genes between three species of birds, confirmed that immune genes show greater divergence between species than other genes, with higher dn/ds ratio than other parts of the genome and evidence of positive selection on specific codons within genes . the sequencing of cdna libraries as expressed sequence tags (ests) (carre et al., ) , and blast homology searches helped to identify the genes. nonetheless, some genes are still unaccounted for. this appears true for all birds, although species differences exist. for some of these genes missing from the avian defense arsenal, the evidence is overwhelming, while others are less certain. in all cases, the completion and quality of the genome sequence and annotation determines whether a gene can be identified or not. gaps exist in the genome sequences, and immune genes are often present in gene families, which are particularly prone to problems with assembly. est libraries are incomplete, and immune gene expression may be restricted to certain tissues or cell types, and most importantly, only following immune activation. thus, until genomes are complete and error-free it may be premature to say that a gene is not there. nonetheless, claiming that a gene is missing certainly inspires research aimed at confirming or disproving this, or demonstrating that another gene plays an analogous or compensatory role. thus, it is worth highlighting the genes that appear to be missing. the contracted immune gene repertoire of birds was discussed in recent review of the progress in avian immunology since the availability of the chicken genome (kaiser, (kaiser, , . in comparison with mammals, birds have partial repertoires of pattern recognition receptors including tlr receptors (boyd et al., ; brownlie and allan, ; cormican et al., ) and rig-like receptors (barber et al., ; karpala et al., ) . others have extensively examined the repertoire of avian cytokines (kaiser et al., ) and chemokines (hughes et al., ; kaiser et al., ) interferons (schultz et al., ) (schultz and magor, ) and defensins (lynn et al., ) noting the genes missing from these repertoires. the immunoglobulin locus been characterized in ducks (lundqvist et al., ) , and encodes just three antibody isotypes (magor, ) . finally, the chicken major histocompatibility complex (kaufman, ) is a minimal mhc, where only the most essential genes have been retained. these reviews of each system, although excellent, do not dwell on the genes not found. over the course of our analysis of immune systems of ducks, we have often invested significant effort to identify homologues of the chicken or mammalian immune system. despite our best efforts, some genes have eluded our search. here we will focus on components of three parts of the immune system that we are investigating in ducks (pattern recognition, antibodies and mhc) and identify the genes that are not there in the duck or the chicken or both. we will assess the strength of the data suggesting the absence of the gene, and consider the effect of the gene loss on the immune system of the animal. finally, we will speculate on the selective forces that may have led to the loss of the gene. innate immunity provides the first line of defense against pathogens. recognition of the pathogen through the molecular patterns of conserved pathogen components, or pattern recognition activates a signaling cascade to turn on genes for the effectors of the immune response. toll like receptors (tlrs) detect foreign invaders by sensing pathogen-associated molecular patterns. binding of agonists to tlrs on the cell surface, or within the endosomal compartment, activate signal transduction pathways to turn on antimicrobial peptides, cytokines, interferons and cellular killing mechanisms. birds possess genes for ten tlrs. these include two tlr genes, two tlr genes, tlr , tlr , tlr , tlr , tlr and tlr . several excellent reviews have been written recently on avian tlr genes (brownlie and allan, ; cormican et al., ) . two genes are missing in comparison to fish and mammals, tlr and tlr . tlr , which detects cpg, has been functionally compensated by tlr (brownlie et al., ; keestra et al., ) . tlr and tlr are phylogenetically related as the product of an ancient gene duplication and both can recognize single-stranded rna, oligoribonucleotides, and nucleic acid analogues in the endosomal compartment (reviewed by cervantes et al., ) . tlr , which is present in fish and mammals, is absent in birds. sequencing downstream of chicken tlr showed only fragments of tlr (philbin et al., ) . further, pcr evidence suggested that tlr was disrupted in all the galliform birds, but not anseriform birds, and there was speculation that this could account for the increased susceptibility of chickens to influenza relative to ducks (philbin et al., ) . to follow up on this observation, we cloned duck tlr cdna, and isolated a genomic clone for duck tlr , sequenced it, and examined the region downstream. as seen for chickens, we could identify only small fragments of tlr , and a cr element disrupted the gene in both ducks and chickens (macdonald et al., ) . tlr is also absent from the zebra finch (cormican et al., ) and turkey genome (ramasamy et al., ) . given the evolutionary distance of galliform birds and zebra finch, tlr is likely missing from the entire avian lineage. for several years, mouse tlr had been presumed non-functional, based on the lack of response to tlr / agonists in the tlr À/À mouse (hemmi et al., ) . however, when peripheral blood monocytes from mice are treated with selective tlr agonists, imidazoquinoline m and poly t oligonucleotides, mouse tlr activation is demonstrated while tlr is suppressed (gorden et al., ) . tlr is expressed in monocytes/macrophages and myeloid dcs, while tlr is expressed in pdcs and b cells (hornung et al., ) . tlr also plays a role in detecting bacterial rna, including rna from borrelia burgdorferi, the agent of lyme disease, inducing production of ifn-beta through irf (cervantes et al., ) . tlr is upregulated by the phagocytosis of mycobacterium, including the attenuated bcg vaccine strain, mycobacterium bovis, and mycobacterium tuberculosis (davila et al., ) . human tlr allelic variants are associated with increased susceptibility to pulmonary tuberculosis (davila et al., ) . the protective allele is associated with decreased translation of tlr , presumably resulting in a decrease in sensing and activation, and less inflammation (davila et al., ) . effectively, loss of tlr expression is protective against tuberculosis. it is not clear why the loss of tlr was selected for in birds. the simplest explanation is the similarity of function between tlr and tlr rendered the second gene non-functional. in this scenario, however, there is no selection for the deletion of the gene. alternatively, tlr became detrimental, perhaps by recognizing self-antigens and initiating autoimmunity. negative selection would then likely lead to the loss of this receptor. tlr has been implicated in induction of autoimmunity (mills, ) . early experiments used chickens to demonstrate thyroid autoimmunity (sundick et al., ; wick et al., ) but it is not known to what extent avian species suffer autoimmunity in nature. ironically, knockout of tlr in mice leads to autoimmunity through overexpression and disregulation of tlr (demaria et al., ) . intriguingly, nucleic acid sensing tlrs are implicated in preventing reactivation of host retroviral elements and consequent tumor production (yu et al., ) . tlr has been directly implicated in this immunosurveillance, as lack of antibodies against endogenous retroviral elements correlates with absence of tlr in knockout mice strains. this crucial role of tlr in immunosurveillance of endogenous retroviruses would provide the selective pressure to retain tlr in the genome, regardless of how tlr was lost. since tlr appears to have been inactivated by a cr repetitive element, it is tempting to speculate that tlr was lost in a hypothetical reactivation of endogenous retroviral elements that disrupted the genome in a distant avian ancestor. jim kaufman has alluded to such a catastrophic 'avian big bang' in describing the loss of several avian mhc genes (kaufman and wallny, ) . another theoretical possibility is that tlr became the target of a pathogen that subverted it for its own benefit (discussed in barber, ) . viral subversion of tlr is such that its absence increases host survival from many pathogens. tlr -induced host proinflammatory cytokines allow west nile virus to cross the blood brain barrier (wang et al., ) . tlr activity has also been implicated in influenza-induced pneumonia (le goffic et al., ) and morbidity from vaccinia infection (hutchens et al., ) . along these lines, we can envision a pathogen that subverted avian tlr for increased susceptibility. this could include viral targeting of tlr receptor for increased inflammation and pathology, or subversion of an endosomal tlr for entry of a mycobacterial pathogen into the cell. mycobacteria are initially engulfed by macrophages, but survive and multiply intracellularly. thus, bacterial or viral subversion of a prr may drive selection to disable the gene. whether cause or effect, the lack of tlr in avian monocytes/ macrophages likely does contribute to the susceptibility of birds to rna viruses (west nile virus, newcastle disease virus, influenza virus and others) and intracellular bacterial infections, including mycobacteria. mycobacterium avium is a significant pathogen of birds, particularly those raised in small flocks, while modern flock hygiene has reduced the incidence in commercial poultry. susceptibility to mycobacteriosis in birds varies, with chickens, pheasants, partridges being most susceptible, ducks and geese moderately resistant, and pigeons being very resistant (reviewed in tell et al., ) . rig-i is a cytoplasmic pattern recognition receptor for singlestranded -triphosphate rna with short double-stranded conformation, such as panhandle structures of viral genomes (hornung et al., ; pichlmair et al., ; schlee et al., ; yoneyama et al., ) . both rig-i, and the related pattern recognition receptor for intracellular rna, mda , share the same pathway signaling through mavs on the mitochondrion (fig. ). after detection of viral rna by rig-i, a conformational change releases the card domains (kolakofsky et al., ; kowalinski et al., ; luo et al., ; takahasi et al., ) . trim , an e ubiquitin ligase, interacts with the card domains of rig-i to activate it through attached (gack et al., ) or unanchored k -polyubiquitin chains (jiang et al., ; zeng et al., ) . the relative importance of these two mechanisms in the activation of rig-i is still controversial, but activation leads to oligomerization and rig-i translocation to the mitochondria. translocation of rig-i and trim to the mitochondrial membrane involves the mitochondrial chaperone - - e , allowing interaction with mavs at the mitochondrion. this interaction induces prion-like aggregates of mavs (hou et al., ) that initiate signaling leading to irf / translocation and the production of type i interferons and proinflammatory cytokines. the gene encoding rig-i, ddx , is not annotated in the chicken genome sequence, and is missing in some fish species, but mda homologues are present in all vertebrate families (zou et ). we demonstrated that ducks have a functional rig-i (barber et al., ) . in contrast, the ddx gene appears absent in chickens by analysis of the syntenic region of the z chromosome, although we can identify the flanking gene. we also cannot find rig-i in a search of the expressed sequence tag database for chickens. thus rig-i is missing in the genome of the ancestral chicken represented by the red jungle fowl, and the sequences from modern commercial chicken breeds. our southern blots show a duck rig-i probe cross-hybridizes with pigeon dna, but not with chicken dna (barber et al., ) . we also cannot detect the gene in dna of turkey or partridge, suggesting that the gene is missing in galliformes (barber, ) . furthermore, we showed that chicken df- cells cannot detect rig-i ligand, but if we transfect the cells with duck rig-i we can reconstitute the pathway (barber et al., ) . the loss of rig-i likely contributes to the susceptibility of chickens to infection compared to ducks to a variety of singlestrand rna viruses, including influenza a virus and newcastle disease virus, both of which cause more harm in chickens than ducks. the related rna detector, mda , can partially compensate and detect avian influenza in chicken cells to generate an interferon response (karpala et al., ; liniger et al., ) . it is difficult to speculate on the selective forces resulting in loss of rig-i in some birds. some have suggested the possible existence of a compensatory yet-to-be identified alternate receptor (karpala et al., ) , which could certainly facilitate the loss from the genome. rig-i was initially identified in a leukemia cell line upregulated by retinoic acid (liu et al., ) , and indeed it is upregulated by a variety of stress inducers. rig-i is implicated in a number of other biological events including cell proliferation, apoptosis, senescence, and acute and chronic inflammatory diseases (liu and gu, ) . it is possible that selection to eliminate rig-i from aberrant activation in one of these alternate roles had resulted in the loss of rig-i in galliform birds. finally, there remains the intriguing possibility that the rig-i receptor was the prey of one of the many single-strand rna viruses that infect birds, including influenza virus, newcastle disease virus, west nile virus, and coronaviruses, and was usurped for the virus' advantage. the tlr and rlr signaling pathways are targets for viral subversion (reviewed by es-saad et al., ; ramos and gale, ) . influenza virus interferes in the mammalian rig-i pathway in several places, through the action of ns protein (gack et al., ) . in a similar manner, paramyxoviruses make the v protein that interferes with signaling by the chicken mda receptor (childs et al., ) involving direct protein-protein interaction and preventing interaction with the rna ligand (motz et al., ) . while this interference renders the receptor non-functional during an infection, it would not necessarily lead to selection to eliminate the receptor. however, we can envision interactions with the rig-i receptor where regulation is aberrant, or excessive activation leads to death. in this scenario, loss of rig-i could provide a selective advantage to survive a lethal infection with an unknown pathogen. suggesting that rig-i is also involved in development in some capacity, rig-i knockout mice are embryonic lethal due to liver damage (kato et al., ) . aberrant or loss of expression of rig-i during development due to pathogen subversion, and associated embryonic lethality, could result in selective loss of this receptor. while this raises the question of how the birds without rig-i survived, we note that rig-i is not absolutely essential for development, since rig-i knockout mice have since been made on a different genetic background which are fertile and viable (wang et al., ) . given that rig-i expression is impaired in lethal infections (kobasa et al., ) , it is possible to envision a scenario by which aberrant expression of rig-i leads to death, and the gene is selectively lost in a common ancestor of chickens and turkeys. human rig-i is regulated through polyubiquitinylation, isgylation, sumolyation, and phosphorylation and alternate splicing (eisenacher and krug, ; loo and gale, ; maelfait and beyaert, ; oshiumi et al., ; wang et al., ) . a major on-off switch for human rig-i upon viral infection is polyubiquitination by host e ubiquitin ligase, tripartite motif protein (trim ) (gack et al., ) . sequences at the t residue implicated in interaction with trim and the site of attachment of polyubiquitin chains, k , are not conserved in duck or zebra finch rig-i (barber et al., ) or goose rig-i . thus activation of avian rig-i involves ubiquitination at alternate residues, or interaction with unanchored polyubiquitin chains, not attached to any protein, can activate rig-i (zeng et al., ) . given the lack of rig-i in chickens, the recent observation that knockdown of chicken trim impairs the interferon response of chicken cells is intriguing (rajsbaum et al., ) . perhaps trim is involved in the activation of chmda . the binding of unanchored k polyubiquitin chains can activate human mda in vitro (jiang et al., ) . a role for trim in generating or attaching ubiquitin chains to mda could explain the importance of chtrim in the interferon response of chicken cells. riplet/rnf is a cytoplasmic e -ligase identified by yeast two-hybrid as one of the proteins binding rig-i, and is essential for rig-i activation in human cell lines upon infection with an rna virus (oshiumi et al., ; oshiumi et al., ) . riplet shares . % identity with trim in humans (oshiumi et al., ) , and also has an n-terminal ring domain and c-terminal pry/spry domain. the ring domain confers ubiquitin e ligase activity (nisole et al., ) and also contributes to other protein-protein interactions (borden, ) . riplet also mediates k -polyubiquitination of rig-i (gao et al., ; oshiumi et al., ) . however, there is debate as to whether riplet interacts with the card domains of rig-i (gao et al., ) the c-terminal repressor domain of rig-i or both (oshiumi et al., ) . riplet is crucial for rig-i activation in cells regardless of expression of trim (oshiumi et al., ) . knockout of riplet (oshiumi et al., ) or trim (gack et al., ) impaired the rig-i dependent innate immune response, suggesting that both are required. knockout of riplet resulted in animals that were deficient in the production of interferon in response to rna, but not dna viruses (oshiumi et al., ) . riplet is present in zebra finch (taeniopygia guttata), but we were unable to find the ortholog in the chicken (gallus gallus) genome. in the duck, we have located a putative riplet coding region, but it lacks exon . in repeated race experiments, all clones recovered contain sequences that correspond to an intact open reading frame, but lack the expected ring domain. in mice, deletion of the ring domain prevents rig-i activation (oshiumi et al., ) therefore we hypothesize that deletion of the ring domain in ducks may render it functionally inactive. nonetheless, we saw upregulation of riplet and trim in duck lung at dpi with highly pathogenic avian influenza virus (fleming-canepa x. et al., unpublished data) . because riplet may not be functional in ducks, but still highly upregulated during influenza infection, we speculate that riplet is acting as a decoy for the viral ns . influenza a ns protein interacts with trim (gack et al., ) and riplet (rajsbaum et al., ) causing inhibition of innate immune signaling. alternatively, riplet may dimerize with other e ligases to function, as recently shown for trim (bell et al., ) . comparison of embryonic fibroblast cells from rig-i knockout and wild-type mice upon influenza infection, reveal genes that are downstream of rig-i signaling. these genes have been referred to as the rig-i bioset, the genes induced by influenza infection in a rig-i dependent manner. in mouse fibroblast cells, the genes include ifnb, irf , irf , stat , stat , pkr, oas, mx , ifit (isg ), ifit (isg ) and rsad (viperin) (loo et al., ) . while the overlap between rig-i and mda inducible genes downstream of mavs signaling in chicken cells is unknown, we used a microarray approach to examine the genes turned on by rig-i in chicken cells. using chicken df- cells, transfected with duck rig-i, the expression of the rig-i gene bioset in avian species is augmented (barber et al., ) . we noted that some essential genes of the mouse rig-i bioset are missing in avian species, including irf , isg , and ifit (isg ) and ifit (isg ). interferon regulatory factor- is a critical player in the induction of type i ifns following virus infection (au et al., ) . irf and irf have different and crucial roles in the induction of infa/b . irf is constitutively expressed, and is activated by c-terminal phosphorylation that allows dimerization and nuclear localization (lin et al., ) . this led to the suggestion that irf was responsible for the initial upregulation of the ifnb gene, followed by interferon dependent induction of irf . however, irf À/À knockout mice are severely impaired in interferon production upon infection with ssrna viruses (honda et al., ) , suggesting the contribution of irf is minor. although a gene has been named irf in chickens (grant et al., ) , it is interferon inducible and more similar to irf . others have noted the absence of irf in chickens (huang et al., ) and in avian species (cormican et al., ). it is not known which irf is translocating to the nucleus to activate interferon in the rig-i/mda pathway in avian species. we speculate that irf fulfills the nuclear translocation and activation of type i ifns in both tlr and rlr signaling, but this has not been experimentally examined. interferon stimulated gene (isg ) is highly up regulated by interferon treatment and was the first ubiquitin-like modifier identified. the amino acid sequence of isg is similar to a linear ubiquitin dimer (reviewed in zhang and zhang, ) . isg is conjugated to proteins like ubiquitin, through a process called isgylation. among the identified isgylated substrates are interferon-induced proteins like pkr, rig-i, mxa (zhao et al., ) . irf isgylation by herc (the main isg e ligase in human) increases stability of irf , exerting a positive regulation in the rig-i pathway . negative feedback on rig-i expression and signaling is mediated by isg conjugation to rig-i (kim et al., ) . isg is also involved in a direct antiviral mechanism where isgylation of influenza a virus ns protein impairs viral replication . no genes homologous to human isg have been annotated in any of the available avian genomes. in the chicken, genes located adjacent to human isg were predicted; including hes (homologous to human hes ) and agrn. within this syntenic region of the chicken genome no ubiquitin-like gene was present. similarly, homologs of hes and argn genes were found in the duck scaffolds (scaffold and , respectively) but synteny analysis cannot be performed because the scaffolds do not overlap. enzymes involved in the isgylation system (including ube l, ubch , herc and usp ) are present in the chicken and duck genomes, but there is not yet any functional evidence of isgylation in these species. usp , which is responsible for cleavage of isg from isgylated substrates, correlates with survival of influenza-infected chickens, indirectly suggesting some functionality of the isgylation system (uchida et al., ) . isg conjugation plays many roles in mammalian antiviral immunity, including isgylation of mx, pkr, rig-i, and irf , and influenza ns protein (reviewed in skaug and chen, ) . however, given the absence of several isg targets, including rig-i in chickens, irf in birds, and evidence that mx is non-functional in chickens (schusser et al., ) and ducks (bazzigher et al., ) , the absence of this ubiquitin modifier in birds would be less significant. it is not known whether ns is modified by isg in avian hosts. we cannot rule out the possibility that we have failed to identify the avian isg homolog because of low sequence conservation with human isg . it is also possible that another unknown ubiquitin-like modifier in birds plays the role of isg within the isgylation system. intriguingly, the most similar sequence to isg in the duck and chicken genome lies within the c-terminal end of , -oligoadenylate synthetase-like (oasl) gene. oasl has two tandem ubiquitin-like domains that share % amino acid identity to human isg . the antiviral activity of the human p protein, encoded by human oasl, is dependent on the c-terminal ubiquitin-like domain (marques et al., ) . however, the biological function of the oasl ubiquitin-like domains is not yet clear and its role as ubiquitin-like modifier has not been described. the interferon-induced proteins with tetratricopeptide repeats (ifit) genes are highly upregulated by type i ifns or by viral infection (bluyssen et al., ; levy et al., ; wathelet et al., ) . the human ifit gene family consists of ifit (isg ), ifit (isg ), ifit (isg ), and ifit (isg ), while the mouse ifit family lacks ifit and contains ifit , ifit , and ifit (bluyssen et al., ) . the ifit family appears to be limited to a single gene in marsupials, birds, frogs and fish (reviewed by zhou et al., ) . while these proteins have served as markers of viral infection, only recently have their functions in the innate antiviral response been elucidated (daffis et al., ; fensterl et al., ; mcdermott et al., ; pichlmair et al., ; schmeisser et al., ) . ifit proteins reside within the cytoplasm of cells, and all contain multiple tetratricopeptide repeats (tprs) (lamb et al., ) . the tprs within these proteins consist of a helix-turn-helix motif and facilitate protein-protein interactions (blatch and lassle, ) . ifit and ifit mediate their antiviral activity by a disruption of translation via an interaction with eukaryotic initiation factor (eif ) . ifit and ifit also inhibit the translation of viral mrnas lacking a -o methylation cap structure (daffis et al., ) . ifit and ifit have the ability to bind to, and sequester viral -triphosphate rna (pichlmair et al., ) . the crystal structure of ifit has revealed an rna binding domain that also may function in an antiviral context (yang et al., ) . the multi-functional ifit protein can also restrict the replication of human papilloma virus (hpv) by binding the viral helicase e , and restricting its function in viral replication (saikia et al., ) . interestingly, ifit has also been associated with negative feedback regulation of genes upregulated during viral infection, further demonstrating the diverse function of these genes . the ifit gene family represents a significant contributor to the broad-ranged antiviral activity of interferons, and plays an important role in the cellular, innate antiviral response. in avian species, the only identifiable ifit gene encodes a protein that aligns with other ifit proteins in a phylogenetic tree (fig. ) . the upregulation of ifit following viral infection of chicken cells expressing duck rig-i ( barber et al., ) or infection of ducks (vanderven et al., ) suggests ifit is an important antiviral effector in avian species. the apparent absence of an expanded ifit gene family in avian species suggests that several of the functions attributed to ifit proteins will be missing. indeed, the specific role of avian ifit during a viral infection is unknown. birds have only three antibody isotypes, igm, iga and igy. igy, the avian serum ig most similar to mammalian igg, is a precursor to igg and ige that has composite function of both isotypes (warr et al., ) . ducks make a truncated version of igy (magor et al., ) . in addition, birds use a single light chain gene of the k type (magor et al., a; reynaud et al., ) . ducks have three immunoglobulin heavy chain genes arranged in the gene order ighm, igha and ighy encoding the mu, alpha and upsilon chains for igm, iga and igy, respectively (lundqvist et al., ; magor et al., ) . the igha gene, encoding alpha is inverted in the locus, and ighd (delta) is absent. despite availability of chicken, zebrafinch and turkey genomes, no other avian immunoglobulin heavy chain locus has yet been assembled. from the limited analysis that has been published for chicken igh , it shares the same organization. the transposition of igha from the most position in the locus, to an inverted position downstream of ighm, may have also resulted in the loss of ighd. lack of ighd is evident from genomic sequencing for ducks. early studies reported a d chain in chickens (chen et al., ) , but it is generally accepted that there is no avian homologue of igd. since igd has been identified in teleosts (bengten et al., ; wilson et al., ) frogs (zhao et al., ) and reptiles (cheng et al., ; wei et al., ) the ighd gene was lost in birds. igd is an enigmatic antibody that exists in a wide variety of forms in different species, except birds. the function of igd is beginning to emerge from observations first made for fish, and subsequently for human igd. igd functions at the interface of innate and adaptive immune responses. in fish, igd specific b cells have been identified, and secreted igd lacks the variable region suggesting it functions more like a pattern recognition receptor (edholm et al., ) . igd is found on the surface of granulocytes in fish, which do not make the igd transcript, and involves a specific receptor (edholm et al., ) . in humans, circulating igd binds to basophils and activates antimicrobial and inflammatory factors . igd from igd+ igm-b cells binds to basophils, and can also bind to certain bacteria in the respiratory tract. the basophil binds igd through a specific receptor, and cross-link-ing of igd leads to the production of b cell activating factors (baff) and pro-inflammatory cytokines. serum igd is elevated in patients with chronic infections, and specific igd antibodies could be demonstrated in a number of these infections (reviewed by chen and cerutti, ) . this ancient surveillance system serves to instruct the b cells of the type of pathogens in the respiratory tract. as the specific functions of igd are elucidated, the consequences of the lack of igd antibody in birds will become evident. birds have basophils, but it is unclear whether a different ig isotype can bind to the basophil igd receptor to compensate, or whether the receptor exists in birds. indeed, it remains to be demonstrated that a homologous receptor is involved in the igd binding by basophils of humans and fish. duck igy is made in two secreted forms, a full-length form and a truncated form. the truncated form, called igydfc, lacks the fc region entirely. it arises from alternate splicing that adds an exon encoding just two amino acids after the ch and ch domains, and uses an alternate polyadenylation site (magor et al., ) (magor et al., b) . what controls the alternate splicing is unknown, but the truncated form predominates later in the immune response. the igydfc antibodies would be expected to be defective in several processes such as antigen internalization, which is required for appropriate presentation of antigens needed to generate t cell help. the truncated igy also does not participate in complement fixation, opsonization, precipitation reactions, and reportedly also cannot participate in hemagglutination inhibition (hi) (higgins et al., ) . of benefit to ducks, perhaps the truncated igy helps prevent viral internalization through receptor-mediated endocytosis and subsequent infection of macrophages and other leukocytes (magor, ) . the chicken ig-like receptor (chir) genes (dennis et al., ) are counterparts of the leukocyte immunoglobulin-like receptor family (lilr). the chir genes constitute a large and diverse family of genes in the chicken, with more than members located in a region syntenic to the mammalian leukocyte receptor complex fig. . a phylogenetic tree showing similarity of avian and mammalian ifit sequences. sequences were aligned and phylogenetic tree generated using a maximum likelihood estimation using a program called phyml using www.phylogeny.fr. (dereeper et al., ) . accession numbers for the ifit sequences were: chicken ifit (xm_ . ), turkey ifit (xm_ . ), zebra finch ifit (xm_ . ), human ifit (nm_ . ), mouse ifit (nm_ . ), human ifit (nm_ . ), mouse ifit (nm_ . ), human ifit (nm_ . ), mouse ifit (nm_ . ), human ifit (nm_ . ). note the duck ifit sequence is a partial sequence. (lrc) (laun et al., ; nikolaidis et al., ; viertlboeck and gobel, ; viertlboeck et al., ) and vast diversity within an individual (viertlboeck et al., ) . chir are expressed in a variety of myeloid and lymphoid cells, with individual receptors expressed in a cell-type restricted manner (viertlboeck et al., ) . receptor diversity includes variation within a hypervariable region, the putative binding region, alternate transcript splicing, and presence or absence of functional activation or inhibitory motif. the extensive expansion and diversification of this family in chickens, and leukocyte expression, suggests their evolution is in response to the pressure of pathogens, as suggested for human lilr and mouse pir genes (barclay and hatherley, ) . human lilr receptors are involved in self/non-self recognition and some engage mhc class i targets, as well as pathogen mimics of mhc proteins (anderson and allen, ; brown et al., ) . staphylococcus aureus targets the mouse inhibitory receptor pir-b for increased virulence (nakayama et al., ) . in turn, activating pir-a receptors may have evolved in response to the selective pressure from pathogens, as indicated by the relict itims in the pir-a gene suggesting it is derived from a pir-b ancestor (nakayama et al., ) . thus, counterbalance through inhibitory and activating chir proteins may have evolved in response to pathogen manipulation of immune signaling through these receptors. we have searched unsuccessfully for immunoglobulin superfamily members homologous to the chicken chir receptors in ducks. in high and low stringency southern blots, genomic dna from chickens shows an extensive pattern of hybridization, while dna from ducks shows no significant hybridization (macdonald et al., ) . all efforts to amplify these genes by polymerase chain reaction using several sets of degenerate primers are completely unsuccessful. our searches of the draft assembly of the duck genome, and , expressed tag sequences generated by sequencing, also find no evidence of chir homologues. we cannot rule out the possibility that we have simply missed the chir genes due to weak homology, if they have evolved to be quite different in ducks. this in itself is quite intriguing. the rapid species-specific divergence of primate lilr genes, with only some genes showing clear orthologous relationships between species (canavez et al., ) , while others have evolved to be unique in each species is thought to reflect their species-specific interactions with pathogens. alternatively, these genes are truly absent from ducks, despite their presence in chickens. there are several examples where different vertebrates have employed different families of leukocyte receptors (parham and moffett, ) . for example, cattle use kir as nk cell receptors, which have undergone expansion (mcqueen et al., ) while horses use ly (takahashi et al., ) . although there are hundreds of chir genes, the only chir with a known ligand is chir-ab , which functions as the chicken igy fc receptor (viertlboeck et al., ) . chickens have a large number of chir-ab genes that have varying specificities for igy . remarkably, we cannot find identifiable homologues of the chir-ab in ducks. duck full-length igy does not bind the chicken fc receptor (viertlboeck et al., ) . as noted above ducks also make a truncated igydfc that would be expected to not to bind fc receptor. göbel speculated that the loss of the igy fc fragment, and the duplication and divergence of the chicken chir-ab (fc receptor) family were both strategies to evade a pathogen interfering with the igy-fc receptor interaction in birds (purzel et al., ) . ducks evade this pathogen by production of an anti-body lacking the fc region, retaining the specificity for the antigen, as this truncated form predominates in the later immune response. in chickens, selection favored the duplication of the chir receptor family to make a large number of potential 'decoy receptors' for igy. while no known pathogen targets the fc-igy interaction in birds, this is a very interesting hypothesis. chickens may elude this pathogen through the binding of decoy receptors, while ducks may avoid the internalization of an intracellular pathogen through the production of the igydfc. the vertebrate mhc is the most dynamic part of the genome, showing repeated cycles of 'birth-and-death' evolution (kelley et al., ) . polygeny and polymorphism are hallmarks of the region, with varying numbers of genes between species (and sometimes between individuals). usually it is not possible to identify orthologous genes between species. the mhc class i and class ii genes are the most polymorphic genes in the vertebrate genome. the mhc of the chicken has been referred to as the 'minimal mhc' (kaufman et al., ) , fulfilling all the requirements of an mhc region, with a limited set of genes. the b locus, or genomic mhc region, contains just genes within kb (kaufman et al., ) . mhc class i genes flank either side of the transporters for antigen processing (tap) genes. they are referred to as the major (bf ) and minor (bf ) mhc class i loci. similarly, the mhc class ii genes (blb and blb ) are located on either side of tapasin (tap-bp), and in close proximity to the chaperones involved in mhc class ii loading (dma and dmb). several genes are notably absent, including the proteasome genes lmp and lmp , as well as genes encoding tnf alpha, and lymphotoxin alpha and beta. kaufman argues the mhc organization critically affects function because proximity of the genes involved in antigen transport and presentation, allows their encoded proteins to evolve to work together. indeed, tap and tap genes are also polymorphic, and using a peptide translocation assay, kaufman recently showed that tap determines specificity for the linked dominant mhc class i gene (walker et al., ) . the limitation to one mhc class i gene in chickens, impairs defense against viral pathogens, as ability to defend against a particular pathogen is completely dependent on whether or not it can load peptides from that pathogen. the best illustration of the consequences of limited mhc class i presentation is the ability of chickens of one genotype to defend against rous sarcoma virus, while other strains cannot (wallny et al., ) . the duck has a functionally similar mhc class i region, with mhc class i genes encoded adjacent to the tap genes (moon et al., ) . ducks predominantly express one gene, which is adjacent to the tap gene, which is also polymorphic (mesa et al., ) . in ducks, as in chickens, this is expected to have functional consequences for the defense against viruses. viruses can easily change the one or two epitopes that can be presented by alleles encoded by one mhc class i gene, and thus escape the cytotoxic t cells focused on these epitopes. we have been unable to identify the tapasin (tapbp) gene in the duck mhc. tapasin bridges the gap between the tap transporter and empty mhc class i molecules, bringing them into close proximity to the translocation core where peptides are loaded (sadasivan et al., ) . in the absence of tapasin, empty mhc class i molecules weakly associate with tap leading to binding and cell surface expression of less than optimal peptides (grandea et al., ). tapasin has been identified adjacent to mhc class ii in other birds, including chicken (frangoulis et al., ) , quail (shiina et al., ) , turkey (chaves et al., ) , pheasant and zebra finch and black grouse . also, the tapasin gene is polymorphic in chicken, turkey and pheasant (sironi et al., ) . through analysis of the unannotated duck genome (preensemble) we can identify the location of the duck mhc class ii genes, but a search for tapasin within proximity is unsuccessful. using primers based on the chicken sequence, or conserved regions identified in aligned avian tapasin sequences, our attempts to amplify tapasin from mallards or a domestic duck by rt-pcr or from genomic dna fail to yield a tapasin product. it is possible that failure to amplify tapasin from ducks is due to sequence divergence of tapasin between avian species. the galliform tapasin proteins are about % identical , but the human and chicken tapasin share only % amino acid identity (frangoulis et al., ) . in low stringency southern blot analysis, distinctive bands could be detected in chicken, however the probe does not hybridize to duck genomic dna (petkau, ) . although tapasin seems to play an important role in antigen presentation, certain human mhc class i alleles can function in a tapasin-independent manner (park et al., ; lewis et al., ) . a single amino acid substitution in hla-b from asp to tyr allows the latter to function in a completely tapasin independent manner (sieker et al., ) . it also appears that tapasin influences the peptide repertoire presented to mhc class i, favoring certain peptides over others. in the absence of tapasin antigen presentation is altered, rather than deficient, and is still sufficient to induce in an immune response (boulanger et al., ) . similarly, the absence of immunoproteasomes in mice, results in a change in % of the loaded peptide repertoire (kincaid et al., ) . we could speculate that loss of tapasin was advantageous in ducks. we presume that ducks, like chickens, already had constraints on presentation of antigens by mhc class i due to potential co-evolution of the tap transporters and adjacent mhc class i genes. evidence for this is that both tap and tap are polymorphic in ducks, and they express one dominant mhc class i gene (mesa et al., ) . perhaps the loss of tapasin permits closer interaction of the tap transporter and the specific mhc class i molecule intended for loading. proteins encoded by genes co-evolving along a haplotype reach a best fit. tapasin as the bridge between tap and mhc class i molecules could serve to bring the incorrect mhc class i into proximity of the tap transporters from the other haplotype, unless it was also evolving to keep step. the genes encoding tnf-alpha (tnf-a), and lymphotoxin-alpha and beta (lta and ltb) are missing from the avian mhc. extensive efforts by pcr, est mining, and hybridization to identify tnf-a in chickens and ducks have failed. similarly, the two genes encoding lta and ltb are missing in chickens (kaiser, ) , and a scan of the genome sequence shows they are also absent in ducks. lymphotoxin-alpha knockout mice lack lymph nodes (de togni et al., ) , and similarly chickens have no lymph nodes. primitive lymph nodes were previously described in ducks (berens von rautenfeld and burdras, ) and immunoglobulin transcripts were analyzed in lymphatic tissues isolated from ducks (bando and higgins, ; magor et al., a) . however, we question whether these tissues contain recognizable lymph nodes containing secondary lymphoid tissue, as we are unable to identify anything in the lymphatic tissue that resembles a lymph node, even tracking with injected india ink. we examined isolated lymphatic tissues in ducks for mrna expression of ccl and ccl , the two chemokines which are responsible for recruiting naïve t cells and dendritic cells to lymph nodes. we showed expression of these chemokines was negligible in lymphatic tissues, and abundant in spleen and influenza-infected lung tissues (fleming-canepa et al., ) . clearly, the lymphatic tissues of ducks are not sites of recruitment of lymphocytes and dendritic cells as expected for secondary lymphoid tissues. thus, ducks and chickens, like all other non-mammalian vertebrates (hofmann et al., ) , lack true lymph nodes. through comparison of the immune arsenal of ducks and chickens we highlight several immune genes that are 'mia or missing in action' from the flight divisions. we refer to these genes as mia, because we cannot say with certainty that these genes are not present, at least until the sequencing and annotation of avian genomes is more complete. if these genes are truly absent, what emerges is a picture in which chickens, missing tlr and rig-i, have less ability to detect rna viruses and intracellular bacteria than ducks, which lack only tlr . in addition, birds are missing components in the rig-i pathway, and interferon-responsive antiviral effectors. chickens have a large expanded family of leukocyte receptors, which are apparently missing in ducks, which include the igy fc receptors. notably, ducks make a truncated igy that is lacking the fc region as their most abundant serum antibody. by similarity to their human homologues, the lilr receptors, other members of the chir family are presumed to be involved in self/non-self recognition, and their expansion may somehow compensate for the deficit due to the minimal avian mhc. in contrast, ducks may have lost tapasin, and with it lost some of the constraint on antigen presentation due to co-evolving linked mhc class i and tap transporter genes. whether the host-pathogen arms race is cause or consequence of these gene losses, 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the early phase of viral infection ubiquitin-mediated modulation of the cytoplasmic viral rna sensor rig-i the ubiquitin ligase riplet is essential for rig-i-dependent innate immune responses to rna virus infection variable nk cell receptors and their mhc class i ligands in immunity, reproduction and human evolution a single polymorphic residue within the peptide-binding cleft of mhc class i molecules determines spectrum of tapasin dependence allelic diversity of tap in wild mallards identification and characterization of a functional, alternatively spliced toll-like receptor (tlr ) and genomic disruption of tlr in chickens reis e sousa, c, . rig-i-mediated antiviral responses to single-stranded rna bearing -phosphates chicken igy binds its receptor at the ch /ch interface similarly as the human iga: fc alpha ri interaction species-specific inhibition of rig-i ubiquitination and ifn induction by the influenza a virus ns protein expression analysis of turkey (meleagris gallopavo) toll-like receptors and molecular characterization of avian specific tlr rig-i like receptors and their signaling crosstalk in the regulation of antiviral immunity complete sequence of a chicken lambda light chain immunoglobulin derived from the nucleotide sequence of its mrna roles for calreticulin and a novel glycoprotein, tapasin, in the interaction of mhc class i molecules with tap the inhibitory action of p on select functions of e mediates interferon's effect on human papillomavirus dna replication recognition of triphosphate by rig-i helicase requires short blunt double-stranded rna as contained in panhandle of negative-strand virus identification of alpha interferon-induced genes associated with antiviral activity in daudi cells and characterization of ifit as a novel antiviral gene the interferon system of non-mammalian vertebrates comparative immunology of agricultural birds mx is dispensable for interferon-mediated resistance of chicken cells against influenza a virus positive regulation of interferon regulatory factor activation by herc via isg modification gene organization of the quail major histocompatibility complex (mhccoja) class i gene region comparative molecular dynamics analysis of tapasin-dependent and -independent mhc class i alleles single nucleotide polymorphism discovery in the avian tapasin gene emerging role of isg in antiviral immunity goose rig-i functions in innate immunity against newcastle disease virus infections the role of iodine in thyroid autoimmunity: from chickens to humans: a review natural killer cell receptors in the horse: evidence for the existence of multiple transcribed ly genes nonself rna-sensing mechanism of rig-i helicase and activation of antiviral immune responses mycobacteriosis in birds identification of host genes linked with the survivability of chickens infected with recombinant viruses possessing h n surface antigens from a highly pathogenic avian influenza virus avian influenza rapidly induces antiviral genes in duck lung and intestine complexity of expressed chir genes the chicken leukocyte receptor cluster the chicken leukocyte receptor complex: a highly diverse multigene family encoding at least six structurally distinct receptor types the chicken leukocyte receptor complex encodes a primordial, activating, high-affinity igy fc receptor the chicken leukocyte receptor complex encodes a family of different affinity fcy receptors the dominantly expressed class i molecule of the chicken mhc is explained by coevolution with the polymorphic peptide transporter (tap) genes peptide motifs of the single dominantly expressed class i molecule explain the striking mhc-determined response to rous sarcoma virus in chickens sequencing of the core mhc region of black grouse (tetrao tetrix) and comparative genomics of the galliform mhc mitochondrion: an emerging platform for host antiviral signalling tolllike receptor mediates west nile virus entry into the brain causing lethal encephalitis rig-i À/À mice develop colitis associated with downregulation of g alpha i igy: clues to the origins of modern antibodies the genome of a songbird molecular cloning, full-length sequence and preliminary characterization of a -kda protein induced by human interferons expression of igm, igd, and igy in a reptile, anolis carolinensis a review: the obese strain (os) of chickens: an animal model with spontaneous autoimmune thyroiditis a novel chimeric ig heavy chain from a teleost fish shares similarities to igd crystal structure of isg reveals a novel rna binding structure and potential functional mechanisms isolation of a -kb minimal essential mhc b locus from a new reverse- d bac library of the golden pheasant the rna helicase rig-i has an essential function in double-stranded rna-induced innate antiviral responses nucleic acid-sensing toll-like receptors are essential for the control of endogenous retrovirus viremia and erv-induced tumors reconstitution of the rig-i pathway reveals a signaling role of unanchored polyubiquitin chains in innate immunity interferon-stimulated gene and the protein isgylation system human isg conjugation targets both ifn-induced and constitutively expressed proteins functioning in diverse cellular pathways isg conjugation system targets the viral ns protein in influenza a virus-infected cells identification of igf, a hinge-region-containing ig class, and igd in xenopus tropicalis mapping of the chicken immunoglobulin heavy-chain constant region gene locus reveals an inverted alpha gene upstream of a condensed upsilon gene interferon induced ifit family genes in host antiviral defense origin and evolution of the rig-i like rna helicase gene family the idea for this review came from a conversation with martin flajnik in the beer tent at the comparative immunology workshop, held in waterloo, ontario in . key: cord- - bf eiix authors: nan title: coronavirus induction of class i major histocompatibility complex expression in murine astrocytes is virus strain specific date: - - journal: j exp med doi: nan sha: doc_id: cord_uid: bf eiix neurotropic strains of mouse hepatitis viruses (mhv) such as mhv-a (a ) and mhv- (jhmv) cause acute and chronic encephalomyelitis and demyelination in susceptible strains of mice and rats. they are widely used as models of human demyelinating diseases such as multiple sclerosis (ms), in which immune mechanisms are thought to participate in the development of lesions in the central nervous system (cns). the effects of mhv infection on target cell functions in the cns are not well understood, but a has been shown to induce the expression of mhc class i molecules in glial cells after in vivo and in vitro infection. changes in class i expression in infected cells may contribute to the immunopathogenesis of mhv infection in the cns. in this communication, a large panel of mhv strains was tested for their ability to stimulate class i expression in primary astrocytes in vitro. the data show that the more hepatotropic strains, such as mhv-a , mhv- , mhv- , mhv- , mhv-d, mhv-k, and mhv-nuu, were potent inducers of class i expression in astrocytes during acute infection, measured by radioimmunoassay. the kb molecule was preferentially expressed over db. by contrast, jhmv and several viral strains derived from it did not stimulate the expression of class i molecules. assays of virus infectivity indicated that the class i-inducing activity did not correlate with the ability of the individual viral strain to replicate in astrocytes. however, exposure of the viruses or the supernatants from infected astrocytes to ultraviolet light abolished the class i-inducing activity, indicating that infectious virus is required for class i expression. these data also suggest that class i expression was induced directly by virus infection, and not by the secretion of a soluble substance into the medium by infected astrocytes. finally, analyses of a /jhmv recombinant viral strains suggest that class i-inducing activity resides in one of the a structural genes. m ouse hepatitis viruses (mhv) are members of the coronaviridae, which cause gastrointestinal, neurological, and respiratory diseases in a wide variety of mammalian species ( ) ( ) ( ) ( ) ( ) ( ) ( ) . although some mhv strains, such as a and mhv- , can cause both gastrointestinal and neurological disease in mice and rats, many tend to induce pathology that is restricted to either system, prompting their classification as hepatotropic or neurotropic. there has been considerable interest in the study of the more neurotropic strains of mhv, such as jhmv and a , because of their ability to produce demyelinating lesions that resemble the demyelinating plaques observed in the human neurological disease, multiple sclerosis (ms). in addition, mhv and human coronavirus iso- lates are capable of inducing demyelination in primates ( ) , and coronavirus rna and antigen have been detected in demyelinating lesions in the brains of ms patients ( , ) . as coronaviridae, all mhv strains are enveloped viruses that contain a single-stranded, positive-sense ilna genome of -kb that is expressed as or mrnas encoding both structural and nonstructural proteins ( ) . the structural proteins have been well characterized, and include the nucleocapsid (n) protein, which interacts with the viral rna, and two envelope glycoproteins, the spike (s) and membrane (m) proteins. the s protein forms the virion surface spikes and is responsible for binding the cellular mhv receptor, inducing cell-to-cell fusion and providing a target for neutralizing antibodies. it is the least conserved of the structural proteins among the mhv strains ( ) ( ) ( ) ( ) ( ) . m interacts with the n protein-r.na comply, and may be involved in virus assembly. it is the most conserved structural protein among the mhv. a third envelope glycoprotein, hemagglutinin-esterase (he), has been identified in mhv-s and several jhmv isolates, and may participate in the pathogenesis of jhmv infection in the central nervous system (cns) ( , ) . nonstructural proteins include an rna-dependent rna polymerase, and three additional proteins that do not appear to be required for viral replication, but whose functions and biological activities have not been identified. after intracerebral (i.c.) inoculation in mice and rats, the parent strains of both jhmv and a cause an acute encephalomyelitis and demyelination from which a varying number of animals survive to exhibit chronic demyelination ( ) ( ) ( ) ( ) ( ) ( ) ( ) ) . the severity of the disease varies with the individual virus strain and with the age, genetic background, and immune status of the infected host. the neuropathological features of acute infection can be somewhat distinguished from those of chronic disease by a prevalence of gray matter involvement in which virus replicates in neurons, oligodendrocytes, and astrocytes to cause extensive cns damage ( ) ( ) ( ) ( ) . in chronic disease, lesions are more prevalent in the white matter and consist of primary demyelination with axonal sparing ( , ) . infectious virus is rarely isolated during chronic jhmv disease, though viral rna can be detected by reverse transcriptase (rt)-pcr in the brains of infected mice as late as yr post infection (p.i., fleming, j., personal communication). viral antigen and rna tend to be restricted to astrocytes in chronic infection ( , ( ) ( ) ( ) ( ) , though there is evidence that neurons are also affected ( ) . this pattern of white matter involvement is also characteristic of infection with attenuated or mutant strains of a ( , ) or jhmv ( , ( ) ( ) ( ) , which cause demyelination in the relative absence of encephalitis. in vitro, jhmv and a cause a lytic infection in primary oligodendrocyte cultures that is rapidly selflimiting, but both virus strains can establish a productive and relatively nonlytic, acute, and long-term chronic infection in mixed glial cell cultures and in cultures enriched for astrocytes ( ) ( ) ( ) . these data support early reports that acute demyelination is the result of virus-mediated oligodendrocyte death ( , ) , but more importantly, they also suggest that the astrocyte plays an important role in acute encephalitis and chronic demyelination. currently, there is very little known about the ability of mhv strains to influence specific activities of the host cells that they infect. early reports indicate that a infection enhances the expression of mhc class i molecules in primary cultures of murine glial cells ( ) and that the enhancement also occurs in the brain after i.c. infection in c b / mice ( ). since class i molecules play a key role in the interaction between infected target cells and cd + cytotoxic t cells (ctl; , ) , any change in their expression after virus infection has potential implications for the outcome of the infection. cells in the cns generally express little, if any, constitutive class i antigen in vivo, but astrocytes have been consistently reported to express both class i and class ii antigens in vitro after the addition of ifn- ' and/or tnf-ot to the culture medium ( ) ( ) ( ) ( ) ( ) ( ) . several reports indicate that astrocytes are capable of acting as antigen-presenting cells for in vitro antigen-or a ospecific cd § and cd § t cell responses ( , ) , which suggests that they also have potential to participate in immune responses occurring within the confines of the cns. since clearance of infectious jhmv from the cns requires class i-restricted cd + t cells ( , ( ) ( ) ( ) , it is of particular interest to characterize the effect of mhv infection on class i expression in one of its principal cellular targets. in this communication, we have examined the ability of a panel of mhv strains to induce class i expression in cultures enriched at least % for astrocytes. the data indicate that class i expression occurred in response to all the mhv strains tested except jhmv and virus strains derived from jhmv. additional studies show that class i expression requires the presence of infectious virus. finally, the testing of a /jhmv recombinant viral strains suggests that the class i-inducing activity resides in the ' end of the a genome, possibly in one of the genes encoding the structural proteins. primary astrocyte cultures. astrocytes were isolated from mixed glial cell cultures prepared from the brains of newborn c b / mice (bantin and kingman, fremont, ca) at postnatal day - according to mccarthy and devellis ( ) . briefly, single cell suspensions were prepared from cerebri dissected free of brain stems and cerebelli, plated at - brains per t- flask and allowed to grow to confluence at - d in vitro. culture medium consisted of dmem/ham's f ( : ; jrh biosciences, lenexa, ks) supplemented with % fcs (gemini bioproducts, inc., calabasas, ca), mm hepes, . mm t-glutamine, and penicillin/streptomycin ( u/ml- ~g/ml). at confluence the cultures were mechanically shaken to dislodge microglia and oligodendroglia, resulting in preparations enriched % or greater for cells staining for glial fibrillary acidic protein (gfap). immunoperoxidase or immunofluorescent staining (described below) revealed that the cell preparations contained ,- - % of cells of microglia/macrophage lineage, expressing f / , t- , the mouse equivalent of human common leukocyte antigen (cd ), and/or mac- surface markers. coronavirus strains and infection. table presents a summary of the mhv strains used and the principal types of diseases they cause in mice. the derivation, propagation, and sources of mhv-a- , jhm-dl, jhm-ds, mhv- , mhv- , mhv- , mhv-d, mhv-k, and mhv-nuu have been described ( ) . the neutralization-resistant mhv- strains . -v- and . / . -v- ( , ) were the kind gift of dr. john fleming (university of wisconsin, madison, wi). the development and properties of jhm-x and the jhm/a recombinant mhv strains have been reviewed ( ) . all virus strains were propagated using the murine astrocytoma dbt as previously described ( ) . virus titers were determined for each virus preparation using dbt as indicator cells. infectious center assays. the number of infected astrocytes was determined on day p.i. to provide a measure of the relative efficiency of infection by several mhv strains. briefly, astrocytes were trypsinized into single cell suspensions and, after washing, were plated on dbt cell monolayers at . , , , , and , cells/ mm petri dish. cells were allowed to attach for h at ~ before the addition of agarose at . % in ikpmi supplemented with % heat-inactivated fcs, mm hepes, and penicillin/streptomycin. plaques were counted after h incubation at ~ virus inactivation by exposure to ultraviolet light. inactivation of virus was accomplished by exposure to uv light under a uvp transilluminator (uvp inc.,; san gabriel, ca) at mw/cm z for the possible presence of oligodendrocytes was identified using polyclonal rabbit antigalactocerebroside (gal c, a gift from m. smith, stanford university, stanford, ca). finally, viral antigen was identified using j. . , a mab specific for the n protein of jhmv that crossreacts with all of the mhv strains used in this study ( ) . rta. the expression of class i molecules was measured in astrocytes cultured in flat-bottomed -well plates at a density of cells/well and infected with various virus strains at a multiplicity of infection (m.o.i.) of - . mock infected cells served as controls. on day or p.i., cells were washed twice using a wash buffer of . % bsa in . m pbs before the addition of /a of the appropriate mabs in triplicate. after a rain incubation at room temperature, cells were washed three times, followed by the addition of , cpm/well of si-labeled protein a ( /~ci//zg; icn biomedicals, costa mesa, ca). at the end of a second min incubation, cells were extensively washed in pbs to remove unbound radiolabeled protein a and detached with . % trypsin- . % edta (jrh biosciences). data are presented as cpm bound radioactivity + sd, or percent increase in class i expression in infected cells compared with that in uninfected controls, corrected for background binding in the absence of antibody. a result was considered positive when the percent increase in expression was % or greater. nonspecific staining was identified by the inclusion of kd-specific mab sf - . . . in some experiments, supernatants from infected cells were substituted for virus and ria performed to detect class i d later. immunoperoxidase and immunofluorescent staining. cell phenotypes and the number of infected ceus were determined by immunoperoxidase staining in astrocytes cultured in tissue culture chamber slides. avidin-biotin immunoperoxidase staining kits (vectastain; vector laboratories inc., burlingame, ca) were used according to the manufacturer's instructions as previously described ( ) . cells were infected with virus d after plating and fixed on day or p.i. in acetone/methanol ( : ). the number of positively stained cells in each well was determined in three fields/well at a magnification of x and reported as the percentage of the number of total cells in the same fields. background staining was determined in cells stained in the absence of primary antibody. class i expression was also evaluated by facs | using an indirect immunoftuorescent staining procedure. briefly, single cell suspensions of astrocytes were adjusted to to cells/tube before the addition of class -specific primary antibodies. fitc-labeled goat anti-mouse igg f(ab) was used as secondary antibody (jackson immunoresearch laboratories, west grove, pa). fluorescence data were collected on x to viable cells, indicated by forward light scatter intensity, using a facstar cell sorter (becton dickinson & co.). again, background fluorescence was determined in cells stained in the absence of primary antibody. detection oflnterferons. the possible presence oflfn in the supernatants of infected astrocytes was determined by bioassay in a vesicular stomatitis virus (vsv) neutralization assay using vital dye uptake as a spectrophotometrically measured endpoint in l cells ( , ) . supernatants were collected from infected and control uninfected astrocytes on day and p.i., uv irradiated, and added in triplicate to l cells plated at s cells/well in -well plates. after a -h incubation at ~ and % co , supernatants were aspirated and cells infected with vsv (indiana strain) at approximately tissue culture infectious doses (tcids )/well. control wells included uninfected l cells (cell control) and vsv-infected l cells cultured in the absence of interferon (virus control). at - h p.i., when cpe was maximum in the control wells, cells were stained for h at ~ with . % neutral red. they were then lysed to release retained dye using . m hc in % ethanol, and ods measured at nm in an automated microelisa reader (dynatech laboratories, inc., chantilly, va). ifn concentrations were deduced by comparison of sample ods with those of standard concentrations of recombinant mouse ifn- ' (genzyme corp., cambridge, ma) and murine ifn-c~// (lee biomolecular, san diego, ca). immunoneutralization was also used to identify the possible presence of ifn in uv-irradiated supernatants of a qnfected astrocytes. for this purpose, rat monoclonal anti-mouse ifn- ' antibody (hybridoma r - a ; atcc hb ) or rabbit polyclonal anti-mouse ifn-a/ (lee biomolecular) was added to the astrocytes before the addition of a , and class i expression measured by ria on day p.i. anti-ifn- ' ( - /zg/ml) completely inhibited the protective activity of u/ml of recombinant mouse ifn- ', whereas - u/ml anti-ifn-a/r effective in neutralizing u/ml purified mouse ifn-c~//~. detection of tne tnf activity was measured in a cytotoxicity assay using actinomycin d-treated l fibroblasts as targets, again with vital dye uptake as a spectrophotometric endpoint. serial -fold dilutions of uv-irradiated supernatants, or -fold dilutions of murine recombinant tnf-ot (genzyme corp.), as a standard were added to l monolayers in -well plates in the presence of actinomycin d ( #g/ml; sigma chemical co., st. louis, mo). after an -h incubation at ~ % co , neutral red was added as described for the ifn assay, and od read at nm. to investigate the effects of coronavirus infection on mhc class i expression in primary astrocytes~ cultures were routinely used at - d in vitro, or - d after mechanical shaking to remove oligodendrocytes and microglia. fig. shows that a induced abundant class i expression in astrocytes, measured by ria using the kb-specific mab af . . . . class i expression was routinely measured on days and p.i. after infection at an m.o.i, of , though it was detectable within - h p.i. k b was preferentially expressed over d b after a infection. this was not due to a lack of inducibility of the d b molecule, since both k b and d b were expressed spontaneously with time in culture, and also in response to u/ml of ifn- /(data not shown). under these conditions, k b was expressed earlier than d b, and usually at significantly higher levels. in infected astrocytes, the class i-inducing activity of a was consistently - % more potent than the single dose of u/ml of ifn- ' (fig. ) . finally, facs | analyses revealed that k b was expressed on '~ % of the astrocytes in the infected cultures. by contrast, the large plaque morphology variant of jhmv designated jhm-dl did not stimulate class i at all, or at best, stimulated minimal expression at - % over that observed in uninfected ceus ( fig. and ) . these data indicate that the ability to induce class i expression is not a general property of mhv strains. to determine whether the lack of ability to stimulate class i expression in astrocytes is specific for jhmv, a panel of virus strains derived from wild-type jhmv or from jhm-dl were tested. the results indicate that none of the jhmv strains isolated in our laboratories stimulated class i ( table ). in addition, jhm-x, which was derived from jhmv passage in japan and has been shown to have extensive deletions in the s gene ( ), did not affect dass i expression. by contrast, all of the viral strains in a panel of more hepatotropic isolates of mhv, including mhv-nuu, mhv-k, mhv-d, mhv- , mhv- , and mhv- , were effective stimulators of class i expression. the magnitude of stimulation, expressed as percent change relative to uninfected cells, varied with the individual viral strain - fold over that in uninfected cells. a and mhv- were the most potent inducers of the class i k b molecule, and both inconsistently induced a low-level expression of d b. it should be mentioned that the kd-specific mab sf - . . occasionally yielded a low level of nonspecific binding; however, it was significantly lower than the specific binding observed with the kb-specific antibody. determine whether a specific a gene or genes are responsible for the class i-inducing activity, a panel of recombinant viruses between a and jhmv that retain various portions of a sequences were tested. the results, represented in fig. and summarized in fig. , indicate that all strains that retain a sequences at the ' end of the recombinant genome were potent class i inducers. thus, ca and ca , which retain • - % jhmv sequences at the ' end, were completely devoid of class i-inducing activity. this end of the mhv genome contains the genes encoding the structural coronavirus induction of class i mhc expression is virus strain specific proteins s, m, and n in addition to two nonstructural proteins, which suggests that the class i-inducing activity resides in one of these proteins. often, though not exclusively, the most potent inducers of class i were strains that retain the highest percentage of a sequences at the ' end, while those retaining the least a character, or exhibiting more than one crossover site for recombination, were significantly less potent. in addition, it was observed in three out of four experiments that rl was • % less potent than el , as illustrated in fig. b. el retains slightly more a sequences in the s gene than rl . this observation suggests that class i-inducing activity may involve s. however, it was not possible to attribute the class i-stimulating activity to an individual gene or gene product, since none of the recombinants had crossover sites that sufficiently isolated individual a from jhmv genes. however, it was clear that ifjhmv sequences were retained at the ' end, class -inducing activity was abolished, and that ' retention of a sequences did not salvage it. the ability_ of mhv to induce class i is not dependent on replication efficiency. in our laboratory, a grows to higher titer in vitro than the jhmv strains in dbt cells, often ex-ceeding jhmv growth - -fold. in addition, recombinant viruses containing a leader predominate over those containing jhmv leader, suggesting that a leader provides a growth advantage ( ) . thus, it was important to determine whether the lack of class i inducing activity by jhmv was due to poor replication efficiency in primary astrocytes. for this purpose, the number of astrocytes staining for viral antigen was determined by immunoperoxidase staining, and the yield of infectious viral particles/cell was measured in infectious center assays. the data, presented in table , indicate that on day p.i., % of the jhm-dl-infected astrocytes were positive for viral antigen, compared with % of cells infected with a . in addition, cells infected with the recombinant virus ca , which retains ' jhmv and ' a sequences and does not stimulate class i expression, showed % antigen positive staining. cells infected with ca produced the highest level of infectious virus at . plaques/cell, in spite of its inability to stimulate class i expression (table ) . by contrast, el , which was an effective class i inducer, replicated at relatively low efficiency at . plaques/cell. thus, class -inducing activity was not a function of the ability of the virus to infect or replicate in pri- infected glial cell cultures is not a direct consequence of infection, but is instead due to the release of a soluble factor into the medium that requires continual virus production ( , , ) . the presence of a non-ifn-like soluble factor was demonstrated in supernatants from a -infected glial cells that had been exposed to uv light to inactivate the virus. similarly, our data show that uv-inactivated a and a like recombinant coronavirus strains are not able to induce class i activity in purified astrocytes (table ). virus inactivation in the uv-treated virus preparations was confirmed by plaque assay using dbt cells (data not shown). however, class i-inducing activity was not detected in the supernatants collected on days and p.i. from a -infected astrocytes and exposed to uv light to inactivate the virus (fig. ) , while supernatants that were not exposed to uv light and thus, contained infectious virus, were able to induce class i. induction was not inhibited by the addition of antibodies specific for ifn-'y or ifn-ot/~ to the astrocytes before a infection (table ). finally, there was no evidence of the presence of tnf-ot in uv-treated supernatants of a -infected astrocytes (data not shown). these data suggest that class i induction is a direct consequence of a infection itself, and does not occur indirectly as a result of the release of a soluble class i inducer into the astrocyte medium. data are also included for uninfected astrocytes exposed to ifn-y ( u/ml) for h before assay on day p.i. a and b are two experiments that are representative of four experiments yielding similar results. in this communication, we report that the ability of a to stimulate mhc class i expression during acute infection in primary murine astrocytes, previously observed by suzumura et al. ( , , ) , is not an inherent property of mhv strains. it is interesting to note that the strains that did not stimulate class i are derivatives of the highly neurotropic mhv- , or jhmv strain, whereas most of the mhv strains that did are the more hepatotropic strains. however, class i-inducing in jhmv was also not due to poor replication efficiency in astrocytes, since jhmv infected the same percentage of cells and produced similar levels of infectious virus as the class i-inducing mhv strains (table ) . thus, some other mhv characteristic must be responsible for class i-inducing activity, or the lack thereof. since the expression of class i genes is regulated by interferons ( ) , it seemed possible that class i induction might correlate with the ability of mhv strains to stimulate ifn production. this possibility gains credibility from reports that jhmv is a notoriously poor inducer of interferon after in vitro ( ) and in vivo ( ) infection. however, suzumura et al. ( ) did not detect ifns in uv-treated supernatants from a -infected mixed glial cells in spite of the presence of class i-inducing activity, and our findings indicate that a -induced class i expression was not inhibited by antibodies to ifn-a/ or - ' (table ). unlike suzumura et al. ( ), we did not find that class i-inducing activity was retained after uv-inactivation of a or the a - ike recombinant mhv strains (table ) , and it was not detected in supernarants from a -infected astrocytes that were uv-treated to inactivate infectious virus (table and fig. ). in addition, we have recently found that class i expression is no longer observed when a replication is controlled by the culture of persistently infected astrocytes in the presence of mhvspecific polyclonal antiserum, in spite of the persistence of viral protein and rna ( ) . overall, our data suggest that class i induction by mhv in astrocytes requires infectious virus and does not involve the secretion of a soluble factor. in this respect, it is interesting to note that the class i-inducing activity originally reported by suzumura et al. ( , ) was also dependent on continuous virus production in glial cell cultures prepared from the brains of infected mice ( ) . however, it is possible that our lack of detection of class i-inducing activity in the supernatants could reflect culture, or other technical conditions. although it was not possible to attribute class i-inducing activity to a single gene or gene product using the a /jhmv recombinant virus strains, the data clearly indicate that class i-inducing activity maps to the ' end of the a genome, which contains the genes encoding the primary structural proteins s, m, and n, and two poorly understood nonstructural proteins. however, there are some indications that s is the most likely candidate. for example, recombinant strains which retain more a sequences in the s gene tended to be more potent than those retaining less a , or morejhmv character. in addition, the s gene shows the greatest antigenic divergence of the structural proteins between a and jhmv ( ) ( ) ( ) ( ) ( ) and so might be more likely to exhibit differences in activity. this possibility is supported by data indicating that a andjhmv differ considerably in their ability to cause receptor-independent fusion and syncytium formation ( ) . in addition, preliminary data suggest that a and jhmv may show selective binding to the cellular receptors for mhv ( ) , which are members of the carcinoembryonic antigen (cea) family of proteins ( ) . the mechanism by which a , mhv- , and the a like recombinant mhv strains induce class i expression in astrocytes is currently unknown. massa et al. ( , ) have shown that mhc class i promoter activity is upregulated in astrocytes treated with ifn-y and that the upregulation is associated with an increase in the binding activities of the mhc class i regulatory element (mhc-cre) and the ifn consensus sequence (ics). whether or not these regulatory elements are engaged by a infection remains to be determined. perhaps one of the more interesting findings in these studies is the prevalence of k b expression over that of d b after mhv infection, and to a lesser extent, ifn- " treatment. differential modulation of h- k and h- d molecules has been reported to follow in vitro jhmv infection in mouse brain endothelial cells, and the data indicate that the nature of the regulation was dependent on the mouse strain and did not necessarily correlate with susceptibility to jhmv-induced cns disease ( ) . thus, k was decreased and d increased in endothelial cells from susceptible balb/c mice, but both were upregulated in susceptible b .s and (balb/c x sjl)f and resistant sjl endothelial cells. differential expression of k and d molecules has not always been observed after virus infection of neural cells; both k and d molecules appear to be equally enhanced by west nile virus infection in astrocytes from cba/h mice ( ) , and the murine neuroblastoma c does not show differential k and d expression when persistently infected with measles virus ( ) . however, the possibility that differential modulation of class i molecules may be linked to cns disease, or may be a marker of susceptibility to virus-induced cns disease, is suggested by studies using theiler's murine encephalomyelitis virus (tmev; , ). after i.c. tmev inoculation, resistant b mice showed minimal class i expression in the cns that did not differ between k and d, whereas susceptible b .q and b .rbq mice showed a greater increase in d expression compared with that of k ( ) . the inability of jhmv to stimulate class i expression in c bl/ astrocytes in the current studies was not reported for astrocytes from balb/c, cxj- , sjl, and b .s mice by joseph et al. ( ) . in these experiments, jhmv infection at an m.o.i, of . was followed by a two-to threefold increase in the expression of h- k molecules, measured by facs | analyses, but astrocytes from c bl/ mice were not tested. the disparity in our findings may again reflect differences in the regulation of class i expression that are mouse strain specific. however, it is also possible that the disparity may be due to the cellular composition of the astrocyte cultures used for infection. in their report, joseph et al. ( ) , did not indicate whether or not they purified the astrocytes from mixed glial cell cultures. in our hands, mixed glial cells that have not been subjected to mechanical shaking for purification of astrocytes show up to % contamination by microglia, or cells staining for macrophage/monocyte surface antigens (data not shown). microglial cells readily express class i molecules both in vivo and in vitro ( ) ( ) ( ) , and so might be expected to express class i after jhmv infection. the cultures used in our experiments routinely show % or greater purity, containing - % microglial cells, and "~ % express class i after a infection. thus, class i expression in these cultures occurs primarily in astrocytes, with little, if any contribution by microglia. finally, it should also be mentioned that class i expression in astrocytes, like that in endothelial cells, does not appear to be linked to susceptibility to jhmv-induced cns disease, since both balb/c and c bl/ develop encephalitis and demyelination in spite of their differences in class i inducibility after jhmv infection. in addition, astrocytes from sjl mice express class i after jhmv infection in spite of being resistant to cns disease. the different effect of a and jhmv on class i expression in astrocytes suggests that class i may play different roles in their pathogenesis. as discussed by maudsley et al. ( ) , the classical picture of mhc expression after virus infection is that of an increase that is most likely mediated by the release of ifns by infiltrating immune cells, and is thought to facilitate the ability of t cells to recognize the infected cells and control or eliminate the infection. unfortunately, clearance of virus from infected tissue by ctls is often accompanied by significant cellular destruction, which is not well tolerated in tissues with limited regenerative capacity, especially the cns. thus, the ability of a virus to stimulate class i expression in host cells may facilitate a rapid ctl response that in turn accelerates tissue destruction originally begun by virus-mediated cell lysis ( ) . this may be the case for a infection, since the a strains used in these experiments cause a severe encephalitis after i.c. inoculation that begins on day p.i., and results in death of the majority of infected mice by day - p.i. (our unpublished data). however, it is not known if the infiltration of t cells into the cns after i.c. a infection is associated with the onset of encephalitis. by contrast, jhmv causes a similar clinical dis-ease, but the onset of encephalitis is delayed in comparison with that of a , beginning on day - p.i. ( ) . death occurs on day - at a similar incidence. the onset of encephalitis coincides with the appearance of immune cells in the cns, which, after jhm-ds infection, peaks on days - p.i. ( ) . it is possible that class i expression is upregulated at this time by the secretion of interferon by the infiltrating immune cells, facilitating jhmv-specific, ctl-mediated tissue destruction. thus, the difference in the ability of jhmv and a to stimulate class i expression in astrocytes may contribute to the speed of disease progression in the cns. it is interesting to note that fazakerley et al. ( ) reported that the jhmv variant, v a . , is neuroattenuated relative to parental jhmv by its slower rate of spread in the cns of balb/c mice. variant v a . differs from parental jhmv by the deletion of amino acids in one of the subunits of the s protein ( , ) , suggesting that s plays an important role in the rate of virus spread in the cns. in this respect, it would be of interest to test the ability of neuroattenuated a strains to stimulate class i expression in astrocytes. in conclusion, we present data indicating that significant differences exist among mhv strains in their ability to stimulate class i expression in murine astrocytes, and that these differences may contribute to their ability to cause cns disease. since class i expression has been reported on astrocytes in the brains of ms patients ( ) , the data have implications for understanding the role of class i in human cns disease. we wish to thank michael lai for the recombinant virus strains and steve stohlman, michael lai, and minnie mcmiuan for helpful critiques of the data and manuscript. we are also grateful for help with the manuscript provided by sonia garcia. a murine virus (jhm) causing disseminated encephalomyelitis with extensive destruction of myelin pathogenesis of demyelination induced by a mouse hepatitis virus ohm virus) pathogenic murine coronaviruses. iii. biological and biochemical characterization of temperature-sensitive mutants of jhmv hydrocephalus in suckling rats infected intracerebrally with mouse hepatitis virus mhv-a in vivo and in vitro models of demyelinating diseases chronic central nervous system demyelination in mice after jhm virus infection relapsing subacute demyelinating encephalomyelitis in rats during the course of eoronavirus jhm infection coronavirus infects and causes demyelination in primate central nervous system coronavirus isolates sk and sd from multiple sclerosis patients are serologically related to routine coronaviruses a and jhm gilmore et al. and 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glial cells is dependent on persistent mouse hepatitis virus infection interferon-activated genes interferon production and activity in mouse neuroblastoma cells mouse hepatitis virus type infection of primary glial cultures from genetically susceptible and resistant mice the effect of persistent mouse hepatitis virus infection on mhc class i expression in murine astrocytes cell receptor-independent infection by a neurotropic murine coronavirus mouse hepatitis virus utilizes two carcinoembryonic antigens as alternative receptors receptor for mouse hepatitis virus is a member of the carcinoembryonic antigen family of glycoproteins expression of major histocompatibility complex (mhc) class i genes in astrocytes correlates with the presence of nuclear factors that bind to constitutive and inducible enhancers cell typespecific regulation of major histocompatibility complex (mhc) class i gene expression in astrocytes, oligodendrocytes, and neurons differential modulation of mhc class i antigen expression on mouse brain endothelial cells by mhv- infection flavivirus infection up-regulates the expression of class i and class ii major histocompatibility antigens on and enhances t cell recognition of astrocytes in vitro persistent measles virus infection enhances histocompatibility complex class i expression and immunogenicity of murine neuroblastoma cells differential expression of h- k and h- d in the central nervous system of mice infected with theiler's virus coexpression of class i major histocompatibility antigen and viral rna in central nervous system of mice infected with theiler's virus: a model for multiple sclerosis regulation of mhc class i and ii antigens on cerebral endothelial cells and astrocytes following mhv- infection perivascular microglia cells of the cns are bone-marrow derived and present antigen in vivo mhc antigen expression on bulk-isolated macrophage-microglia from newborn mouse brain: induction of ia antigen expression by y-interferon isolation and direct characterization of resident microglial cells from the normal and inflamed central nervous system modulation of mhc antigen expression by viruses and oncogenes viral persistence characterization of brain-infiltrating mononuclear cells during infection with mouse hepatitis virus strain jhm neutralization-resistant variants of a neurotropic coronavirus are generated by deletions within the amino-terminal half of the spike glycoprotein multiple sclerosis: relevance of class i and class ii mhc-expressing cells to lesion development key: cord- -yt pmxi authors: de sousa, eric; ligeiro, dário; lérias, joana r.; zhang, chao; agrati, chiara; osman, mohamed; el-kafrawy, sherif a; azhar, esam i; ippolito, giuseppe; wang, fu-sheng; zumla, alimudin; maeurer, markus title: mortality in covid- disease patients: correlating association of major histocompatibility complex (mhc) with severe acute respiratory syndrome (sars-cov- ) variants date: - - journal: int j infect dis doi: . /j.ijid. . . sha: doc_id: cord_uid: yt pmxi abstract as the (covid- ) pandemic caused by the novel coronavirus, sars-cov- spreads globally, differences in adverse clinical management outcomes have been associated with associated with age > years, male gender, and co-morbidities such as smoking, diabetes, hypertension, cardiovascular comorbidity and immunosuppression. ethnicity has been the focus of attention after data from the united kingdom showed a disproportionate number of deaths among healthcare workers from black, asian and other ethnic minority backgrounds ( ). in addition to ethnicity, socio-economic factors, prior vaccinations and exposure to other coronaviruses, other factors need to be considered to explain geographical and regional variations in susceptibility, severity of clinical expression of covid- disease and outcomes. in the united states there have been disproportionate covid- death rates among african americans at around . times higher than that of other groups. although these data could be due to multiple cultural and socioeconomic factors an underlying genetic susceptibility to sars-cov- infection may be a factor. as the (covid- ) pandemic caused by the novel coronavirus, sars-cov- spreads globally, differences in adverse clinical management outcomes have been associated with associated with age > years, male gender, and co-morbidities such as smoking, diabetes, hypertension, cardiovascular comorbidity and immunosuppression. ethnicity has been the focus of attention after data from the united kingdom showed a disproportionate number of deaths among healthcare workers from black, asian and other ethnic minority backgrounds ( ). in addition to ethnicity, socio-economic factors, prior vaccinations and exposure to other coronaviruses, other factors need to be considered to explain geographical and regional variations in susceptibility, severity of clinical expression of covid- disease and outcomes. in the united states there have been disproportionate covid- death rates among african americans at around . times higher than that of other groups. although these data could be due to multiple cultural and socioeconomic factors an underlying genetic susceptibility to sars-cov- infection may be a factor. genetic factors were thought to play a causative role in the pathogenesis of the sars outbreak in in a group of taiwanese patients, where the hla-b* haplotype was associated with severity of sars infection ( ) . in hong kong chinese patients a strong association was shown j o u r n a l p r e -p r o o f between hlab* and hla-drb * alleles and an increased susceptibility to sars infection ( ) . in contrast, l-sign homozygote individuals seem to have a significantly lower risk of sars infection ( ) . generally, peripheral blood lymphocytes counts of black americans show lower neutrophil counts and proportionally higher frequency of lymphocytes compared to the rest of the population ( ). hla-association studies of sars-cov- with hla-ligands for sars-cov- have been compiled ( ) . the biological and clinical relevance of immune responses to sars-cov- requires further discussion: . autoimmune associations with covid- . some individuals with covid- experienced neurological symptoms, e.g. guillain barre syndrome ( ), suggesting an autoimmune background, which has been associated with mhc alleles ( ) . the role of mhc variants in increased susceptibility to infections or, vice versa, immune protection is well known for a number of viral diseases, e.g. the role of mhc alleles in hiv-control, or increased risk for chronic hepatitis b ( ). dampens autoimmune responses and confers protection from type i diabetes ( ) ( ) associated with strong ifn-production. hla-dqb * : has been selected for increased resistance to yersinia pestis in immigrants from africa to europe, engagement of cd + t-cells to hla-dqb * : leads to increased, pro-inflammatory il- production, independent of the mhc class ii presented peptides ( ) and confers increased risk to the development of anti-myelin directed autoimmune responses ( ) . the haplotypes hla-dr -dq , dr -dq , and dr -dq accommodate peptides from infectious pathogens to cd + t-cells from europeans who survived the bottleneck of different, life-threatening infections prevalent in europe ( ). these alleles have also shown to be associated with increased risk for autoimmune diseases, for instance to dietary antigens (celiac disease) ( ) in part due to their intrinsic capacity to stimulate stronger il- production, that facilitates central nervous system (cns)associated disease manifestations ( ) . ( ) . we used for this prediction a residue peptide with the mutation site in the middle (vnkitygacpkyvkqntlklatgmrnvpekqtr) and thebest fitting peptide with residues that was predicted to binds to hla-drb * : , is exactly the peptide reported earlier ( ) recognized by a flu epitope specific t-cell clone. in contrast, the variant (t k) peptide does not bind ( table ) . ( ), type diabetes ( , ) , and lyme disease induced arthritis ( ) . drb * : is associated with early childhood myasthenia gravis ( ) . drb * : is linked to grave's disease ( ) , serum igg antibodies to chlamydia pneumoniae with essential hypertension ( ) and acute necrotizing encephalopathy ( ) in conclusion, there appears to be no selective pressure from mhc class i alleles for sars-cov- variants tested. most likely there is selective pressure from mhc class ii alleles in regard to binding of the orf (l s) variants assuming that this mutation may be biologically relevant ( , ) . this data underlines the need to examine sars-cov- variants and mhc-associations along with clinical outcomes, a detailed longtime observation with a particular focus on cns-associated symptoms, particularly in individuals with increased risk to develop autoimmune responses. is ethnicity linked to incidence or outcomes of covid- ? association of hla class i with severe acute respiratory syndrome coronavirus infection association of human-leukocyteantigen class i (b* ) and class ii (drb * ) genotypes with susceptibility and resistance to the development of severe acute respiratory syndrome homozygous l-sign (clec m) plays a protective role in sars coronavirus infection black/white differences in leukocyte subpopulations in men hla studies in the context of coronavirus outbreaks guillain-barre syndrome associated with sars-cov- infection: causality or coincidence? association between human leukocyte antigen-dr and demylinating guillain-barre syndrome the mhc locus and genetic susceptibility to autoimmune and infectious diseases genetics of the hla region in the prediction of type diabetes crystal structure of hla-dq that protects against type diabetes and confers strong susceptibility to narcolepsy hla class ii molecules influence susceptibility versus protection in inflammatory diseases by determining the cytokine profile dqb * : -associated pathogenic anti-myelin autoimmunity in multiple sclerosis-like disease: potential function of dqb * : as a disease-predisposing allele the prevalence of hla dq and dq in patients with celiac disease, in family and in general population mechanisms regulating regional localization of inflammation during cns autoimmunity the autoimmune basis of narcolepsy hpv variants and hla polymorphisms: the role of variability on the risk of cervical cancer association between human papillomavirus e variants and human leukocyte antigen class i polymorphism in cervical cancer of swedish women structural analysis of a peptide--hla class ii complex: identification of critical interactions for its formation and recognition by t cell receptor cytotoxic tcell activity antagonized by naturally occurring hiv- gag variants natural variants of cytotoxic epitopes are t-cell receptor antagonists for antiviral cytotoxic t cells separation of il- production from th cell proliferation by an altered t cell receptor ligand longitudinal analysis of mycobacterium tuberculosis -kda antigen-specific t cells in patients with pulmonary tuberculosis: association with disease activity and cross-reactivity to a peptide from hivenv gp human papillomavirus type e peptide-directed cd + t cells from patients with cervical cancer are cross-reactive with the coronavirus ns protein phylogenetic network analysis of sars-cov- genomes on the origin and continuing evolution of sars-cov- . national science review tepitool: a pipeline for computational prediction of t cell epitope candidates peptide binding predictions for hla dr, dp and dq molecules netmhciipan- . , a common pan-specific mhc class ii prediction method including all three human mhc class ii isotypes quantitative predictions of peptide binding to any hla-dr molecule of known sequence: netmhciipan a review of hla allele and snp associations with highly prevalent infectious diseases in human populations chemistry of peptides associated with mhc class i and class ii molecules structure of a complex of the human alpha/beta t cell receptor (tcr) ha . , influenza hemagglutinin peptide, and major histocompatibility complex class ii molecule, hla-dr (dra* and drb * ): insight into tcr cross-restriction and alloreactivity balancing selection and heterogeneity across the classical human leukocyte antigen loci: a meta-analytic review of population studies association of hla-drb /dqb polymorphism with high-risk hpv infection and cervical intraepithelial neoplasia women from shanghai impact of host molecular genetic variations and hiv/hpv co-infection on cervical cancer progression: a systematic review drb * /drb * , and drb * are susceptibility genes for graves' disease in north american caucasians, whereas drb * is protective e and e gene polymorphisms in human papillomavirus types- and identified in southwest china multiple sclerosis and autoimmune diseases: epidemiology and hla-dr association in north-east italy analysis of hla dp, dq, and dr allesles in adult italian rheumatoid arthritis patients genes for insulin-dependent diabetes mellitus (iddm) in the major histocompatibility complex (mhc) of african-americans hla-encoded genetic predisposition in iddm: dr subtypes may be associated with different degrees of protection identification of lfa- as a candidate autoantigen in treatment-resistant lyme arthritis a variant of childhood-onset myasthenia gravis: hla typing and clinical characteristics in japan association of hla-drb * and serum igg antibodies to chlamydia pneumoniae with essential hypertension in a highly homogeneous population from majorca molecular analysis of hla class iiassociated susceptibility to neuroinflammatory diseases in korean children hla-dpb and hla class i confer risk of and protection from narcolepsy dna methylation as a mediator of hla-drb * : and a protective variant in multiple sclerosis professor ippolito, sir zumla and prof mohamed osman are co-investigators investigators of the pan-african network on emerging and re-emerging infections (pandora-id-net) funded by the european and developing countries clinical trials partnership the eu horizon framework programme for research and innovation. sir zumla is in receipt of as uk-nihr senior investigatorship. professor maeurer is a member of the innate immunity advisory group of the gates foundation and his work is funded by the champalimaud foundation. all authors are members of the global cancer and infectious diseases consortium for host-directed therapies: weblink: https://fchampalimaud.org/covid /aci key: cord- -qbcb qi authors: agarwal, ajay title: in-silica analysis of sars-cov- viral strain using reverse vaccinology approach: a case study for usa date: - - journal: biorxiv doi: . / . . . sha: doc_id: cord_uid: qbcb qi the recent pandemic of covid that has struck the world is yet to be battled by a potential cure. countless lives have been claimed due to the existing pandemic and the societal normalcy has been damaged permanently. as a result, it becomes crucial for academic researchers in the field of bioinformatics to combat the existing pandemic. the study involved collecting the virulent strain sequence of sars-ncov for the country usa against human host through publically available bioinformatics databases. using in-silica analysis and reverse vaccinology, two leader proteins were identified to be potential vaccine candidates for development of a multi-epitope drug. the results of this study can provide further researchers better aspects and direction on developing vaccine and immune responses against covid . this work also aims at promoting the use of existing bioinformatics tools to faster streamline the pipeline of vaccine development. the situation of covid a new infection respiratory disease was first observed in the month of december , in wuhan, situated in the hubei province, china. studies have indicated that the reason of this disease was the emergence of a genetically-novel coronavirus closely related to sars-cov. this coronavirus, now named as ncov- , is the reason behind the spread of this fatal respiratory disease, now named as covid- . the initial group of infections is supposedly linked with the huanan seafood market, most likely due to animal contact. eventually, human-to-human interaction occurred and resulted in the transmission of the virus to humans. [ ]. since then, ncov- has been rapidly spreading within china and other parts of world. at the time of writing this article (mid-march ), covid- has spread across countries. a count of , cases have been confirmed of being diagnosed with covid- , and a total of deaths have occurred. the cumulative cases have been depicting a rising trend and the numbers are just increasing. who has declared covid- to be a “global health emergency”. [ ]. current scenario and objectives currently, research is being conducted on a massive level to understand the immunology and genetic characteristics of the disease. however, no cure or vaccine of ncov- has been developed at the time of writing this article. though, ncov- and sars-cov are almost genetically similar, the respiratory syndrome caused by both of them, covid- and sars respectively, are completely different. studies have indicated that – “sars was more deadly but much less infectious than covid- ”. -world health organization currently, research is being conducted on a massive level to understand the immunology and genetic characteristics of the disease. however, no cure or vaccine of ncov- has been developed at the time of writing this article. though, ncov- and sars-cov are almost genetically similar, the respiratory syndrome caused by both of them, covid- and sars respectively, are completely different. studies have indicated that -"sars was more deadly but much less infectious than covid- ." the spread of sars epidemic has provided with any useful insights as during that time the virus epidemic was contained only through general prevention means and treatment of the individual symptoms. as a result, there exists only a limited number of tools available to test the coronavirus for their ability to infect humans. this has acted as a major limitation for predicting the next zoonotic viral outbreak. [ ] [ ] [ ] . in response to the current medical crisis, the world health organization has activated the r&d blueprint for the acceleration of the development of diagnostics, therapeutics and vaccines for treatment of this novel coronavirus. the objective of this study lies in compliance to the guidelines for research activated by who. this study aims to utilize a reverse-vaccinology approach in order to identify potential vaccine candidates for covid for the country usa. we shall be deploying open-access bioinformatics tools for our analysis of the same. the virulent strain of sars-cov- was selected for in-silico analysis. the genome of the viral strain is made available by ncbi -national center for biotechnology information (https://www.ncbi.nlm.nih.gov.in). the reference identification for sars-cov- is given by refseq nc_ . viral protein sequences of the sars-cov- were obtained from the vipr -virus pathogen database and analysis. [ ] . these protein sequences were identified and downloaded in a tabular format for the host -humans and the country -usa. the fasta-file for all the protein sequences was downloaded and loaded in r using "seqinr" and "biostrings" packages. the different physicochemical properties were analysed using the "peptides" package in the r. [ ] [ ] [ ] . vaxijen . is used to predict the antigenicity of the protein based on the fasta-files that contain their respective amino acid sequences. the online software predicts the antigenic score for the same. [ ] [ ] . the t-cell and b-cell epitopes of the selected two leader protein sequences were predicted using the iedb (the immune epitope database). iedb is a freely available resource funded by niaid. it is a catalog that contains the experimental data on antibody and t cell epitopes studied in humans, non-human primates, and other animal species in the context of the infectious disease, allergy, autoimmunity, and transplantation. it also assists in hosting tools for predicting and analysing the epitopes. [ ] . for mhc class-i t-cell epitope prediction for orf ab leader proteins, netmhcpan el . method was used. this method was applied for the hla-a* - allele. for mhc class-ii t-cell epitope prediction for orf ab leader proteins, sturniolo prediction method was used. this method was applied for the hla drb * - allele. for b-cell lymphocyte epitope prediction, bepipered prediction method was used. [ ] . ten epitopes were selected on random for analysis of their antigenicity, allergenicity, topology and conservancy. allertop v (https://www.ddg-pharmfac.net/allertop/) was used for determining the allergenicity of selected epitopes. toxinpred server (https://webs.iiitd.edu.in/raghava/toxinpred/protein.php) was used to determining the toxicity of the selected epitopes. the prediction of transmembrane helices in proteins was determined using the tmhmm server v . (http://www.cbs.dtu.dk/services/tmhmm/). the conservancy of the selected epitopes was analysed using the conservancy analysis tool of the iedb server. for this analysis, the parameter for the sequence identity threshold was adjusted to '>= '. [ ] . in order to generate the d structure of the epitopes choses, the pep-fold tool was used. pep-fold (http://bioserv.rpbs.univ-paris-diderot.r/services/pep-fold /) uses a de novo approach to predict the peptide sequences from the amino acid sequences. [ ] [ ] [ ] . it utilizes the structural alphabet sa letters to describe the conformations of four consecutive residues. the pre-docking of the selected epitopes was done using the ucsf chimera. it was also used to perform pre-docking of the selected alleles hla-a* - (for mhc class-i) and hla drb * - . later, the docking of the peptide-protein was done using hpepdock. hpepdock is a web server of performing blind peptide-protein utilizing hierarchical algorithm. [ ] . instead of performing length stimulations to refine peptide conformations, hpepdock studies the peptide flexibility through an ensemble of peptide conformations produced by the modpep program. the sars-cov- strain was identified. all the protein sequences were analysed on the basis of the physicochemical properties to select the top five candidates for further analysis. for the physicochemical analysis, the number of amino acids, instability index, aliphatic index, and the grand average of hydrophobicity (gravy) scores of the all the , proteins were taken using the peptides package in r. this packages allows the identification, selection and analysis of multiple amino acid sequences in the same fasta-file. hence, it was utilized. the physicochemical study unveiled five potential candidates having the lowest score of gravy and instability index less than (hence, displaying stability). these five candidates were individually analysed for their molar extinction coefficients and antigenicity. the vaxijen . tool was used to analyse the antigenicity of the potential five candidates and the molar extinction coefficient was analysed using the expasy's online tool -protparam. out of the five potential candidates, only two were selected for further analysis. this was based on the criteria of having the highest score of predicted antigenicity and highest values of the extinction coefficients. both the leader proteins were then used for further analysis. the t-cell epitopes of the mhc class-i for both the leader protein were determined using the netmhcpan el . prediction method of the iedb server keeping the sequence length at . this tool allows the generation of the epitopes and sorts them on the basis of their percentile scores. randomly, ten potential epitopes were selected randomly for the antigenicity, allergenicity, toxicity, and conservancy tests. for mhc class-ii, t-cell epitopes (hla drb * - allele) of the proteins were also determined using the iedb analysis tools. the sturniolo prediction methods were used for the same. again, ten potential candidates were chosen based on the same criteria as that of mhc class-i. the b-cell epitopes of the proteins were selected using the bepipered linear epitope prediction method of the iedb server. the topology of the chosen epitopes was determined using the tmhmm v . server (http://www.cbs.dtu.dk/services/tmhmm/). the potential t-cell epitopes, whose topology, antigenicity, allergenicity, toxicity and conservancy was analysed, for orf ab leader proteins mt and mt are depicted in the following tables. table generation. on the analysis of the antigenicity, allergenicity, toxicity, and conservancy analysis of the tcell epitopes, it was found that most of them were antigenic, simultaneously being nonallergenic, non-toxic and higher values of conservancy. from the ten selected mhc class-i and mhc class-ii t-cell epitopes, one from each category were selected from both the leader proteins. the criteria for being selected was having the higher antigenic scores, nonallergenicity, non-toxicity, and conservancy value above %. the selected epitopes were: rsdartaph, vqlnnnnnn, lslpvlqvr, and vqlslpvlq. the b-cell epitopes of the orf ab leader proteins are displayed in the following two figures. the following figures depict the pep-fold generated d structures of the selected t-cell epitopes of mhc class-i and class-ii: rsdartaph, vqlnnnnnn, lslpvlqvr, and vqlslpvlq. leader protein mt . vipr: an open bioinformatics database and analysis resource for virology research seqinr . - : a contributed package to the r project for statistical computing devoted to biological sequences retrieval and analysis biostrings: efficient manipulation of biological strings peptides: a package for data mining of antimicrobial peptides vaxijen: a server for prediction of protective antigens, tumour antigens and subunit vaccines identification of novel vaccine candidates against campylobacter through reverse vaccinology the immune epitope database (iedb) . . nucleic acids research exploiting the reverse vaccinology approach to design novel subunit vaccine against ebola virus pep-fold: an updated de novo structure prediction server for both linear and disulfide bonded cyclic peptides improved pep-fold approach for peptide and miniprotein structure prediction pep-fold : faster de novo structure prediction for linear peptides in solution and in complex hpepdock: a web server for blind peptide-protein docking based on a hierarchical algorithm this research didn't receive any specific grant from funding agencies. the author declares that there is no conflict of interest. the study doesn't contain any work/experiment performed with human participants or animals done by the author. hpepdock server was used to perform docking of the peptide and protein. the purpose of the same was to analyse which of the two selected t-cell mhc class-i epitope: rsdartaph and lslpvlqvr had the lowest global energy. the epitope having the lowest global energy acts as a better vaccine candidate. the docking was performed against the hla-a* - allele whose pdb format was obtained through pre-docking using ucsf chimera.the global energy for the selected class-i epitopes were: - . and - . respectively for rsdartaph and lslpvlqvr. out of the two mhc class-i epitopes selected for leader proteins orf ab mt and mt , the global energy was lowest for lslpvlqvr. for the mhc class-ii t-cell epitope, the docking was performed against the hla drb * - allele. same procedure was followed as mentioned before. the two selected epitopes after the previous analysis were: vqlnnnnnn and vqlslpvlq. the global energy for the selected epitopes were - . and - . respectively.out of the two t-cell mhc class-ii epitopes selected for the orf ab leader proteins mt and mt , the lowest global energy was of vqlslpvlq. the scope of this study involved performing an in-silica analysis of the sars-cov- viral strain for the country of usa against human host. the vipr database was used for the same to obtain all the protein sequences. a total of viral protein sequences were obtained whose extensive physicochemical analysis was done to select a group of two. this extensive analysis was performed using peptides package in the r software.it was revealed that the two leader proteins orf ab mt and mt had the highest extinction coefficient and the lowest score on the gravy. along with the given parameters, these leader proteins were highly stable and were also antigenic in nature.the fasta-formatted files of these selected proteins were taken and analysed to obtain the potential t-cell and b-cell epitopes. the t-cell epitopes of mhc class- and mhc class-ii were analysed on the basis of their scores. ten randomly selected t-cell epitopes from both the classes were taken for further analysis of allergenicity, toxicity, conservancy scores and antigenicity. only one epitope from both the classes was selected which possessed a higher conservancy score (more than %), was non-toxic, non-allergic and antigenic in nature.the two selected epitopes were then docked against their respective alleles to obtain the global energy scores. the epitopes which displayed the lowest global energy score on docking with the alleles were selected and proposed as successful and potential vaccine candidates funding: key: cord- - goj ej authors: karl, julie a.; bohn, patrick s.; wiseman, roger w.; nimityongskul, francesca a.; lank, simon m.; starrett, gabriel j.; o’connor, david h. title: major histocompatibility complex class i haplotype diversity in chinese rhesus macaques date: - - journal: g (bethesda) doi: . /g . . sha: doc_id: cord_uid: goj ej the use of chinese-origin rhesus macaques (macaca mulatta) for infectious disease immunity research is increasing despite the relative lack of major histocompatibility complex (mhc) class i immunogenetics information available for this population. we determined transcript-based mhc class i haplotypes for chinese rhesus macaques from five different experimental cohorts, providing a concise representation of the full complement of mhc class i major alleles expressed by each animal. in total, mamu-a and mamu-b haplotypes were defined in the full chinese rhesus macaque cohort. we then performed an analysis of haplotype frequencies across the experimental cohorts of chinese rhesus macaques, as well as a comparison against a group of indian rhesus macaques. notably, of the mamu-a and mamu-b haplotypes observed in indian rhesus macaques were also detected in the chinese population, with % of the chinese-origin rhesus macaques expressing at least one of these class i haplotypes. this unexpected conservation of indian rhesus macaque mhc class i haplotypes in the chinese rhesus macaque population suggests that immunologic insights originally gleaned from studies using indian rhesus macaques may be more applicable to chinese rhesus macaques than previously appreciated and may provide an opportunity for studies of cd (+) t-cell responses between populations. it may also be possible to extend these studies across multiple species of macaques, as we found evidence of shared ancestral haplotypes between chinese rhesus and mauritian cynomolgus macaques. macaque monkeys are used extensively to model and understand human diseases. historically, rhesus macaques (macaca mulatta; mamu) of indian origin have been preferred for such research. however, exportation of rhesus macaques from india was halted in (southwick and siddiqi ) . as it became more difficult to obtain indian rhesus macaques, researchers turned to other sources, including chineseorigin rhesus macaques. chinese rhesus macaques have been used to study infectious diseases including simian immunodeficiency virus/ human immunodeficiency virus, avian influenza, malaria, plague, and severe acute respiratory syndrome, as well as studies of xenotransplantation, stem cell therapies, reproduction, and neurobiology (shen et al. ; chen et al. chen et al. , chen et al. , choi et al. ; liu et al. ; tian et al. ; wei et al. ; hutnick et al. ; li et al. b; ling et al. ; mumbauer et al. ). yet comparatively little is known about the immunogenetics of these animals. the genome of a single chinese rhesus macaque was sequenced in , but whole-genome sequencing technology struggles to accurately characterize highly polymorphic, duplicated regions such as the major histocompatibility complex (mhc) loci and killer immunoglobulin receptor loci (fang et al. ) . the mhc is an exceptionally polymorphic region of the genome responsible for presentation of peptides to t cells (bontrop ) . because of its pivotal role in immune response, understanding mhc genetics is essential for infectious disease research. our group and others have shown that the repertoire of mhc class i alleles expressed by chinese rhesus macaques is largely distinct from that observed in indian rhesus macaques (otting et al. (otting et al. , karl et al. ; copyright © karl et al. doi: . /g . . manuscript received march , accepted for publication may , this is an open-access article distributed under the terms of the creative commons attribution unported license (http://creativecommons.org/licenses/ by/ . /), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. supporting information is available online at http://www.g journal.org/lookup/ suppl/doi: . /g . . /-/dc . sequence data from this article have been deposited with the embl/genbank data libraries under accession nos. kc -kc for novel mamu mhc alleles. otting et al. ; wu et al. ; ma et al. ; wiseman et al. ; naruse et al. ; doxiadis et al. ) . consequently, we inferred mhc class i restricted cd + t-cell responses in these two populations would be unlikely to target many of the same epitopes. here we reconsider the relationship between mhc class i genetics of chinese-and indian-origin rhesus macaques by focusing on mhc class i transcript defined haplotypes inherited together, rather than simply analyzing individual allelic variants. most studies that currently control for the complex rhesus macaque mhc class i region only account for the presence of one or a few widely studied alleles (mamu-a à , etc.), with no regard for the majority of alleles expressed by the animal. analysis on a haplotype level as presented here provides a more global view of the mhc class i alleles transcribed by each animal, creating the potential for a greater degree of allele matching between animals within a study. to define haplotypes with high resolution, we deep sequenced complementary dna (cdna) amplicons spanning most of exons of mamu-a and mamu-b genes, the most polymorphic area of mhc class i corresponding to the peptide binding region. a total of chinese-origin rhesus macaques from five different experimental cohorts were examined, as well as indian-origin rhesus macaques from two distinct sources. unexpectedly, we discovered that of of the mamu-a and mamu-b haplotypes observed in indian rhesus macaques also were expressed by a significant majority of chinese rhesus macaques. conservation of these haplotypes suggests that functional cd + t-cell immunity to pathogens may be more similar between chinese-and indian-origin rhesus macaques than previously appreciated. whole blood and peripheral blood mononuclear cell samples from chinese rhesus macaques were obtained over the span of yr from five different sources, including battelle biomedical research center, etubics corporation, the national institute of allergy and infectious diseases/national institutes of health, and harlan laboratories, inc. cohorts are detailed in table . the core set of chinese rhesus macaque samples included samples from cohort chrh # and six samples from cohort chrh # . the expanded set of chinese rhesus macaque samples included an additional animals from these two cohorts, as well as animals from the three other sources. it is unknown from which provinces in china the animals or their ancestors were initially obtained, or any degree of relation between the animals sampled. whole blood samples from indian rhesus macaques were obtained from the wisconsin national primate research center (wnprc) and new england primate research center (neprc) breeding colonies. the wnprc cohort was selected to be unrelated based on pedigree records whereas the neprc cohort was taken from active adult breeding stock. macaques at the wnprc were cared for according to protocols approved by the university of wisconsin-madison graduate school animal care and use committee, whereas macaques housed at the neprc were maintained in accordance with standards of the association for assessment and accreditation of laboratory animal care and the harvard medical school animal care and use committee. rna isolation, cdna synthesis, and polymerase chain reaction (pcr) amplification (normalization and pooling) rna was isolated from whole blood or peripheral blood mononuclear cell samples using the roche magna pure instrument and rna high performance kit (roche, indianapolis, in), following manufacturer's protocols (supporting information, file s ). synthesis of cdna and pcr amplification were performed as previously described, generating either a -bp or -bp genotyping amplicon (the shorter amplicon dates back to studies performed before roche/ sequencing technology advanced to~ -bp read lengths) (wiseman et al. ; fernandez et al. ). the amplicons for each sample were tagged during primary pcr with a multiplex identifier sequence, a unique -bp tag for each sample (file s ). amplification was confirmed by running pcr products on a flash gel (lonza group ltd, basel, switzerland), and bead-based purification was performed using ampure xp spri beads (agencourt bioscience corporation, beverly, ma) to remove primer dimers. quantification of purified products was performed using the quant-it dsdna hs assay kit and a qubit fluorometer (invitrogen, carlsbad, ca). products were then normalized to . ng/ml and pooled in equal volumes. sequencing for all amplicons was performed using roche/ secondgeneration methods following manufacturer's protocols for emulsion pcr and pyrosequencing (roche, indianapolis, in). the core set of chinese rhesus macaques and indian rhesus macaques were sequenced in a total of five runs on a gs junior pyrosequencing instrument. the expanded set of chinese rhesus macaques were sequenced on a combination of gs junior and gs flx platforms; both platforms utilize the same basic chemistry, differing only in scale of the run (a single gs junior run is~ / the scale of a full gs flx run). for the core set of chinese-origin and indian-origin rhesus macaques, sequences were analyzed by trimming all sequences from the end to fixed -bp lengths, binning sequences into separate animal groups by parsing for the unique multiplex identifier tags, then creating uni-directional contigs of sequence reads assembled at % n stringency using the program geneious pro (biolegend, san diego, ca). fasta format sequences of all contigs for each sample were then exported, and bidirectional reads were assembled at % stringency using codoncode aligner (codoncode, dedham, ma). bidirectional contigs were interrogated against a curated database of known rhesus macaque mhc class i alleles using the basic local alignment search tool (i.e., blast), and novel alleles were submitted to genbank (kc -kc ). putative haplotypes were inferred by looking for combinations of shared alleles between animals (file s ). analyses of the mamu-a and mamu-b regions were performed independently, since these gene clusters are separated by~ . million base pairs and recombination is relatively common at the population level (daza-vamenta et al. ) . once putative haplotypes were defined, a simpler workflow was used to assign haplotype designations to the additional chinese rhesus macaque samples from the expanded set. all individual sequence reads obtained for each sample were directly compared against our rhesus macaque mhc class i allele database using blast-like analysis tool (i.e., blat) in a local instance of galaxy (penn state, state college, pa; emory, atlanta, ga), following the data analysis workflow detailed in figure s . haplotypes were assigned by identifying groups of shared major alleles from the previously defined putative haplotypes. the unparalleled complexity of the nonhuman primate mhc class i region makes it difficult to describe simply the full diversity of class i alleles expressed by an animal. macaques may encode as many as distinct mhc class i transcripts expressed at widely differing steady state levels in peripheral blood. however, combinations of distinct alleles are inherited together on a chromosome as haplotypes, making it possible to assign a simplified name (i.e., haplotype a ) to each specific combination of alleles. here, unique haplotypes were defined on the basis of transcriptionally abundant "major" mamu-a and mamu-b alleles; an example of this simplification for the a haplotype is illustrated in figure . our operational criterion for defining a "major" allele is that it must exceed a % transcript abundance relative to all class i transcripts identified per animal. we previously showed that cd + t-cell responses are principally restricted by highly transcribed major mhc class i alleles expressed in macaques, so we believe functionally defining haplotypes based on configurations of major alleles is appropriate for assessing conservation of mhc alleles involved in antigen presentation to cd + t cells (budde et al. ) . to further streamline haplotype definitions, only lineage level allele names were considered (commonly referred to as two-digit resolution in human hla genotyping, e.g., all mamu-a à allelic variants were grouped with the designation mamu-a à ; figure ). alleles are named by the nonhuman primate immuno polymorphism database-mhc group based on similarity to other known macaque mhc class i alleles, such that alleles sharing a common lineage level designation are identical or nearly so within the peptide binding regions. a total of unique haplotypic configurations were defined in the full cohort of chinese rhesus macaques, consisting of distinct mamu-a haplotypes and mamu-b haplotypes (table s ). names were assigned to each haplotype based on the presence of what we call a "diagnostic major" allele. mamu-a region haplotypes typically contained a single major allele, usually from the mamu-a locus, so that allele was considered the "diagnostic major." a few of the mamu-a haplotypes and most of the mamu-b haplotypes expressed two or more major alleles; for these haplotypes, the most transcriptionally abundant allele expressed on the haplotype was typically considered the "diagnostic major" allele. occasionally the "diagnostic major" allele was selected based on known biological significance. for instance, even though the mamu-b à allele was not typically the most abundant transcript, it was considered the "diagnostic major" due to its known association with spontaneous control of siv (yant et al. ) . different versions of a base haplotype, denoted with a lowercase letter following the haplotype name (for instance, b a and b b), were designated to indicate differences in the complement of major transcripts accompanying a shared diagnostic allele. by this method of haplotype definition, four simple haplotype names represent the full complement of mhc class i major alleles expressed by each macaque (table s ). because the geographical source of macaques within china has been indicated as a factor in frequency of specific mhc class i alleles, we wanted to explore the differences in chromosomal haplotype frequencies between our five experimental cohorts (li et al. a; liu et al. ) . after identifying the most common chinese rhesus macaque mamu-a and mamu-b haplotypes across the data set as a whole, we examined the differences in the frequencies for those haplotypes between the five cohorts ( figure ). frequencies were calculated based on the total number of chromosomes examined ( chinese rhesus macaque chromosomes), to account for each animal expressing pairs of mamu-a and mamu-b haplotypes. some haplotype frequencies, such as a , a a, and b a, were relatively consistent across the cohorts; however, some frequencies varied greatly from cohort to cohort. one example is the a haplotype, which was observed in~ % of the total chromosomes examined for cohort chrh # , but only~ % for cohort chrh # . overall, frequency differences between cohorts were more pronounced for the mamu-b haplotypes, and some cohorts did not contain one or more of the most common mamu-a and mamu-b haplotypes. this finding is not unexpected for cohort chrh # , because it only contained macaques. the remaining cohorts each contained at least macaques, minimizing the effect of sample size on observed frequencies in these cohorts. these results emphasize the point that not all research figure simplification of mhc class i allele calls from full allele down to haplotype designation. schematic representation of reducing the a haplotype observed in chinese-origin rhesus macaques, from full allele name to major alleles to lineage designation to final haplotype call. cohorts of chinese rhesus macaques are equal from the standpoint of mhc class i diversity, as the complement of major mhc class i alleles expressed by chinese rhesus macaques can vary greatly between different sample groups. however, despite frequency differences, common haplotypes can still be identified across all five cohorts. we next performed mhc class i haplotype analysis on a group of indian rhesus macaques, allowing for haplotype comparison between the two geographically isolated populations. in total, haplotypes were identified in the indian rhesus macaque cohort, including mamu-a haplotypes and mamu-b haplotypes. interestingly, only haplotypes (four mamu-a and mamu-b haplotypes) were unique to indian rhesus macaques in this comparison (figure ) . these findings indicate that although the allelic variation between chinese-and indian-origin rhesus macaques is largely distinct (table s ) , the two populations frequently inherit shared combinations of closely related allelic variants. overall, of the mhc class i haplotypes present within indian rhesus macaques have closely related homologs in the chinese population. the frequencies of these shared haplotypes in each population are shown in figure ; frequencies were again based on total number of chromosomes examined. although several of these shared haplotypes were not among the most abundant in this study, there were four haplotypes (a , a , b a, and b b) observed at a chromosomal frequency of at least % in each population. remarkably, % of the chinese rhesus macaques in this study express at least one of the shared mamu-a or mamu-b haplotypes ( % carry at least one of the shared mamu-a haplotypes, and % carry one or more of the shared mamu-b haplotypes). likewise, % of the indian rhesus macaques in this cohort express at least one haplotype shared with chinese-origin animals ( figure ). therefore, there is a high likelihood that these shared haplotypes were present in prior studies using indian-origin rhesus macaques that researchers might reproduce or expand upon today using chinese-origin rhesus macaques. mhc class i genotyping would need to be performed to confirm presence of shared haplotypes between populations for any specific studies. we also compared our chinese-and indian-origin rhesus macaque haplotypes against the well-characterized panel of mauritian cynomolgus macaque (macaca fascicularis; mafa) haplotypes to explore possible ancestral haplotype sharing between the two species (wiseman et al. ; budde et al. ) . mauritian cynomolgus macaques exhibit extremely limited mhc class i diversity, as the population arose from a small group of founder cynomolgus macaques deposited on the island in the s (lawler et al. ) . the mhc class i diversity of mauritian cynomolgus macaques is effectively described by five mafa-a and seven mafa-b haplotypes (one mafa-a haplotype is commonly associated with three distinct mafa-b haplotypes) (wiseman et al. ; budde et al. ) . three of our mamu-a haplotypes (a , a , and a ) and two of our mamu-b haplotypes (b a and b ) were homologous to mauritian cynomolgus macaque haplotypes (table s ). four of these haplotypes were observed exclusively in chinese rhesus macaques; the fifth haplotype (b a) was detected in both chinese-origin and indian-origin rhesus macaques. it has previously been shown that the specific mhc class i alleles expressed by chinese-and indian-origin rhesus macaques are largely distinct, and those distinctions were thought to equate to an unfathomable mhc class i diversity between the two geographically divided populations (otting et al. (otting et al. , karl et al. ; wu et al. ; ma et al. ; wiseman et al. ; naruse et al. ; doxiadis et al. ) . however, our model of defining mhc class i haplotypes of coinherited highly expressed major alleles provides a novel approach for assessing putatively functional similarities between macaque populations. most notably, more than two thirds of the haplotypes observed in indian rhesus macaques were shared with chinese rhesus macaques. this indicates that ancestral haplotypes were retained in both populations after the split from their most recent common ancestor~ , years ago, despite subsequent shrinking and expansion of the indian and chinese rhesus macaque populations, respectively (hernandez et al. ) . although chinese rhesus macaques overall display a greater repertoire of mhc class i haplotypes private to the population than indian rhesus macaques, % of the chinese rhesus macaques in this study express at least one of the shared ancestral mamu-a or mamu-b haplotypes described here ( figure ). these homologous haplotypes may provide a valuable resource for cross-population studies of functional cd + t-cell immune response to pathogens. does conservation of mhc class i haplotypes correspond to conservation of cd + t-cell immune responses? though this remains to be tested at the haplotype level, a previous study demonstrated that it was possible to create mamu-b à : (a chinese rhesus macaquespecific allelic variant of the mamu-b à lineage) monomers and tetramers with the siv nef - peptide iw (ouyang et al. ). this amino acid sequence was characterized as a ctl epitope restricted by mamu-b à : in indian rhesus macaques, an allele that has been associated with control of sivmac viral replication (mothe et al. ; yant et al. ) . mamu-b à : differs from mamu-b à : by four amino acids, three of which occur in the peptide binding domain encoded by exons - . these amino acid substitutions did not appear to alter the ability of mamu-b à : to bind siv nef - peptide iw (ouyang et al. ). if the highly similar allelic variants expressed on homologous haplotypes are also able to bind the same peptides, it is very likely those haplotypes would mount similar cd + t-cell responses in both chinese-and indianorigin rhesus macaques. to extend these observations, class i alleles of specific interest sharing a lineage designation would need to be analyzed for predicted binding preferences as well as the functional ability to present specific peptides. there is also early evidence that sharing of homologous haplotypes may be a pan-macaque phenomenon. comparison of the rhesus macaque haplotypes identified in this study to an established panel of mauritian cynomolgus macaque haplotypes revealed a total of five putative orthologous haplotypes, including three of the five mafa-a haplotypes. interestingly, the cynomolgus macaque haplotypes with putative rhesus macaque orthologs account for more than % and % of the mafa-a and mafa-b diversity, respectively, in mauritian cynomolgus macaques (budde et al. ) . it may therefore be possible to also explore whether rhesus and cynomolgus macaques with conserved ancestral mhc class i haplotypes mount similar cd + tcell immune responses. additional haplotype similarities are also likely to exist between rhesus macaques and cynomolgus macaques of other geographic populations, or between rhesus macaques and other macaque species, but a current lack of defined haplotypes for these other populations make comparisons difficult. overall, this study provides the first large-scale description of mhc class i transcript-based haplotypes in rhesus macaques. it begins to explore some of the variability of haplotypes observed between independent experimental research cohorts of chinese rhesus macaques, and provides a valuable chromosomal haplotype frequency survey for groups looking to study high-frequency chinese rhesus macaque alleles. importantly, it highlights the unexpected sharing of indian rhesus macaque 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histocompatibility complex class i allele mamu-bà is associated with control of simian immunodeficiency virus sivmac replication we thank battelle biomedical research center, etubics corporation, the national institute of allergy and infectious diseases/national institutes of health, and harlan laboratories, inc., for providing us with chinese rhesus macaque samples, and the new england primate research center for providing an independent source of indian rhesus macaque samples. we also thank the staff of the w.m. keck center for comparative and functional genomics at the university of illinois at urbana-champaign for performing roche/ gs flx sequencing. this work was supported by the national institute of allergy and infectious diseases (hhsn c, hhsn c), the national center for research resources (p rr ), and the office of research infrastructure programs (p od ) of the national institutes of health. this research was conducted at a facility constructed with support from the research facilities improvement program (rr - , rr - ). key: cord- -u ys xcm authors: zhang, qian‐jin; seipp, robyn p.; chen, susan s.; vitalis, timothy z.; li, xiao‐lin; choi, kyung‐bok; jeffries, andrew; jefferies, wilfred a. title: tap expression reduces il‐ expressing tumor infiltrating lymphocytes and restores immunosurveillance against melanoma date: - - journal: int j cancer doi: . /ijc. sha: doc_id: cord_uid: u ys xcm many immune therapeutic strategies are under development for melanoma to treat metastatic disease and prevent disease reoccurrence. however, human melanoma cells are often deficient in antigen processing and this appears to play a role in their expansion and escape from immunosurveillance. for example, expression of the transporters associated with antigen processing (tap and tap ) is down‐regulated in the mouse melanoma cell line b f . this results in a lack of tumor‐associated antigen processing, low surface expression of mhc class i molecules and low immunogenicity. we observe that restoration of tap expression by transfection resurrects the processing and presentation of viral antigens, and the melanoma‐associated antigen, trp‐ . immunization with irradiated b f /rtap transfected cells generates ctls that are capable of killing b f /rtap transfected targets and b f targets deficient in tap . furthermore, b f /rtap transfectants grow at a significantly slower rate in mice than b f cells. in an experimental model that closely recapitulates the clinical situation, treatment of b f tumors in mice with a vaccinia virus vector expressing tap also significantly decreases tumor growth in vivo. furthermore, tumors treated with vaccinia tap had significantly reduced numbers of immunosuppressive, cd (+)/il‐ positive, tumor infiltrating lymphocytes. therefore, tap expression restores both antigen presentation and immunogenicity in b f melanoma cells and concomitantly reduces immunosuppressive il‐ production at the local tumor site, thereby increasing immunosurveillance mechanisms against tumors. © wiley‐liss, inc. melanomas (maas) are neoplasms that arise from pigmented melanocytes differentiated from the neural crest. cutaneous maa represents only % of all skin cancers but accounts for almost all of the deaths associated with the disease. the incidence of maa over the last years has more than doubled in the united states with , new cases and , deaths in . intermittent acute sun exposure and lifetime sun exposure, age, skin pigment type, immune suppression, atypical nevi and family history are risk factors for maa. the early stage disease is curable but reoccurrence is common for later stage disease and the prognosis is poor if the disease has spread to the lymph nodes and other tissues. standard treatment is biopsy and -cm wide excision after histological determination that the lesion is malignant. in some cases, regional and sentinel lymph nodes are also removed and, if positive, adjuvant therapy consisting of interferon-a or il- may be administered but response rates low and long term benefit are not significant. [ ] [ ] [ ] however, cases of spontaneous remission and reoccurrence of the disease many years after remission or resection, and correlation of remission with immune cell infiltrates all suggest that there is an immune surveillance process that could be leveraged to develop treatments for late stage disease. [ ] [ ] [ ] [ ] [ ] the discovery of tumor antigens associated with maa has led to research and development of therapeutic vaccines designed to gener-ate antitumor responses with the aim of controlling metastasis. a wide variety of vaccination approaches and protocols are currently being tested. the results show that even though specific cellular immune responses are generated against the antigens, the response of the disease to the treatment is low. the reasons for the low response rates are thought to be low immunogenicity of the antigens, which are usually self-antigens, and the natural selection of tumor variants that have immune-suppressive phenotypes arising through a process called immuno-editing. the nature of the immune suppression may include secretion of immunosuppressive cytokines, the expression of ligands (fas-l) that initiate apoptosis in cytotoxic t-cells and tumor variants that are deficient in antigen processing and presentation. variants that are deficient in the mhc class i antigen processing pathway do not express mhc class i antigens on the cell surface. as a consequence, specific cytotoxic t-cells generated by the vaccine protocol are unable to recognize and kill these tumor variants due to defective presentation of tumor associated antigen-derived peptides recognized by the ctls. the mhc class i restricted antigen presentation pathway consists of a number of genes encoded in the mhc class i locus of human chromosome . the pathway generates peptides from endogenous proteins by the degradative action of proteolytic enzymes lmp and lmp , located in a proteolytic complex called the proteasome. the peptides are transported from the cytoplasm into the endoplasmic reticulum (er) by the abc transporter, transporters associated with antigen processing (tap), a heterodimer that is composed of subunits, tap and tap . within the er, the peptide may be trimmed further by resident er proteases and subsequently loaded onto an mhc class i molecule, which consists of mhc heavy chain and beta- -microglobulin (b m). the assembly of this complex is aided by a number of chaperone proteins; these include calreticulin and calnexin, which are responsible for the folding of mhc class i heavy chains and stabilization of their association with b m, and tapasin, which is responsible for anchoring the mhc molecules to tap and loading the peptides onto the mhc class i molecules. the properly assembled complex is then transported to the cell surface by the secretory pathway. on the cell surface, the functional mhc class i molecules offer a ligand to the tcr of cd t-cells. if the tcr binds with high enough affinity, activation of the t-cell occurs and the target cell can be destroyed. any alterations or deficiencies in the antigen presentation pathway can lead to variants, which give rise to nonimmunogenic tumors. these deficiencies can be due to chromosomal lesions leading to loss of heterozygosity or mutations in the genes of the pathway (such as b m) and can be referred to as hard lesions. in most cases however, deficiencies in mhc class i antigen expression on the cell surface are due to soft lesions characterized by the down-regulation of components of the antigen presentation pathway. , down-regulation of tap is a critical factor in mhc class i antigen deficiencies and has been associated with disease progression and death in cutaneous and orbital maa. [ ] [ ] [ ] conversely tap expression has been associated with tumor infiltrating lymphocytes (tils), a characteristic of good clinical outcome , , and spontaneous regression of maas. in our study, we examine the effect of the restoration of tap expression on mhc class i antigen surface expression in the murine maa cell line, b f . b f cells are a subclone of the mouse b maa cell line that are weakly immunogenic and have been widely used as a tumor model for tumor-host immune interactions. this tumor, like most metastatic carcinomas, has deficiency in components of mhc class i antigen-processing pathway, including tap, mhc class i antigen surface expression, proteasome subunits lmp , lmp and lmp , pa a and b, and the chaperone tapasin. , this down-regulation of the antigen presentation pathway can be reversed by ifn-g treatment. we test the hypothesis that restoration of tap expression in b f cells increases mhc class i antigen surface expression and immunogenicity, making these cells visible to immune surveillance mechanisms. the mouse strain c bl/ (h- b ) was obtained from the jackson laboratory (bar harbor, me), and maintained at the biotechnology breeding facility (university of british columbia, vancouver, bc, canada). the mice were maintained according to the guidelines of the canadian council on animal care. mice were kept on a standard diet with water ad libitum. the colony was routinely screened for mycoplasma pulmonis and mycoplasma arthritidis, rodent coronaviruses (including hepatitis) and sendai virus, using the murine immunocomb test (charles river laboratories, wilmington, ma). the mice used in the experiments were between and weeks of age. vesicular stomatitis virus, indiana strain (vsv), a gift from frank tufaro (university of british columbia), was cultured on vero cells [american type tissue culture (atcc), rockville, md]. recombinant vaccinia virus (vv) either carrying rat-tap genes (vv-rtap ) or the empty plasmid pjs- (vv-pjs- , vector negative control) is described previously. all vv strains were grown in cv- cells (atcc). vsv and vv titres were determined by tissue culture infective dose (tcid ) assay or standard plaque assay (pfu), using vero cells and cv- cells, respectively. rma, rma-s, vero and b f cells (murine maa) were cultured in rpmi- medium supplemented with % heat inactivated fetal bovine serum (hyclone, logan, ut), l-glutamine ( mm), penicillin( iu/ml), streptomycin ( lg/ml) and hepes ( mm). cv- , cmt. (murine lung carcinoma) and murine tapasin / fibroblasts (a kind gift from dr. luc van kaer, vanderbilt university school of medicine, nashville, tn) cell lines were cultured in dmem with the same supplements. the clones of rat tap (rtap ) and vector-only transfectants of b f cells were created by transfecting the cells with the rtap cdna in mammalian expression vector ph (apr- neo) and maintained in geneticin (invitrogen, burlington, on, canada) selecting rpmi- medium. two rtap -transfected clones were designated as b / rtap - and b /rtap - and a clone to control for the transfection vector was designated as b /phb - . rtap and mouse tap expression in b /rtap - cells and b /rtap - cells was examined by immunoblotting. total extracts from cells were separated on % polyacrylamide-sds gels and blotted onto nitrocellulose filters. the blots were probed for mouse or rtap and tap with relevant specific rabbit antiserum at a : , dilution. the blots were then incubated with horseradish peroxidase-labelled anti-rabbit igg antibodies at a : , dilution. the immune complexes were visualized by enhanced chemiluminescence (ecl) according to the instructions of the manufacturer (ge healthcare, chalfont st. giles, uk). the rabbit antiserum against mouse and rtap protein was created by immunizing rabbits with a common tap peptide sequence, rggcyramvealaapad-c with a cysteine at the c-terminal, linked to keyhole limpet hemocyanin (pierce biotechnology, rockford, il). the specificity of the antiserum was confirmed by the detection of a band of kda in size in lysates from tap-expressing cells (rma) that was absent in lysates of fibroblasts derived from tap / mice. the rabbit serum against mouse and rat tap ( / ) was kindly provided by dr. geoff butcher (university of cambridge, cambridge, uk). for mouse tapasin expression, . cells were lysed in % np- lysis buffer min on ice, spun at , g for min at °c and precleared with protein g-sepharose ( ll) (ge healthcare). tapasin was immunoprecipitated with a rabbit antitapasin antiserum (number , courtesy of dr. ted hansen, washington university school of medicine, st. louis, mo) and protein g sepharose, followed by separation by % sds-page, transferred to pdvf membranes (ge healthcare) and probed with the same antiserum followed by ecl as described earlier. b f or b /rtap clone - cells were infected with vv-pjs- or vv-rtap [multiplicity of infection (moi) of ] and incubated for days ( °c, % co ), followed by fixation and preparation for facs analysis. indirect immunofluorescence staining with conformational specific monoclonal antibodies (abs) for h- k b (y- , atcc) and h- d b ( . . .s, atcc) detected mhc class i surface expression. aliquots of cells were incubated ( min at °c) with the primary ab ( ll) for h- k b or h- d b antigens. after washing twice with pbs, the cells were resuspended and incubated ( min at °c) in fitc-conjugated rabbit anti-mouse igg secondary ab ( ll) (dakopatts, glostrup, denmark). for i-a b antigen, pe-conjugated anti-mouse i-a b af - . (bd biosciences, mississauga, on, canada) was used at a : dilution for staining cells min at °c, then washed times with pbs. a facscan analyzer (becton dickinson, mountain view, ca) measured the mean logarithmic fluorescence intensity associated with the staining of surface antigens. all splenocytes were cultured in rpmi- complete medium containing % heat-inactivated hyclone fbs, l-glutamine, iu/ml penicillin, lg/ml streptomycin, mm hepes, . mm nonessential amino acids, mm na-pyruvate and lm -me at °c, % co . h- k b antigen-restricted, vsv-specific ctls were generated by infection of mice (i.p.) with vsv ( tcid ). splenocytes were harvested days after infection and cultured for an additional days in media ( cells/ml) containing vsv-np - peptide (rgyvyqgl) ( lg/ml) at °c, % co . to generate h- k b antigen-restricted tyrosinase related protein- (trp- )-specific ctls, trp- peptide (vydffvwl) ( lg) was mixed with -ll titermax adjuvant (cedarlane laboratories, hornby, on, canada) and -ll pbs and injected subcutaneously into mice. this procedure was repeated after days. fourteen days after the initial injection, mice received an additional injection (i.p.) with g-irradiated rma-s cells ( cells in ll). the irradiated rma-s cells were prepared by incubating cells with trp- peptide ( lg/ml peptide in ml media) overnight at room temperature followed by g-irradiation ( , rads). cells were washed and resuspended in pbs ( ll). seventeen days after the initial injection, the immunized spleen was removed, and the splenocytes ( cells) were cultured for days with g-irradiated naÿve splenocytes ( cells) pulsed with vydffvwl peptide ( lg/ml). to generate b f tumor-specific ctls, c bl/ mice were injected (i.p.) with g-irradiated ( , rads) b f cells, b / phb - or b /rtap - cells ( cells/mouse). about days after immunization, splenocytes were removed and cultured with stimulators at a : (stimulator/splenocyte) ratio for another days at °c, % co . the stimulators were prepared by incubating b f cells, b /rtap - , or b /phb - cells ( hr at °c) with mitomycin c ( lg/ml) followed by g-irradiated ( , rads) and washed times before addition to the splenocyte culture. cytotoxicity assay for vsv, trp- specific and b f tumor-specific effector ctls the cytotoxic activities were measured in standard hr cr release assays. overnight vsv (moi of ) infection of b f , b /rtap - or b /phb - cells provided targets for vsv-specific ctl assays. for trp- -specific and tumorspecific killing, b f cells, b /rtap - or b /phb - target cells were untreated. all targets were labeled with na cro ( lci/ cells) (ge healthcare) for hr at °c and washed extensively. mice (n ) were injected subcutaneously into the hindquarter with . to detect il- -producing, cd -positive cells, the tumor masses were homogenized, passed through a nylon cell strainer ( lm), and the tils were isolated by centrifugation on ficoll-paque. the tils were then stimulated with phorbol -myristate -acetate ( ng/ml) (sigma-aldrich canada, oakville, on, canada), calcium ionophore a ( lg/ml) (sigma-aldrich) and golgiplug (becton dickinson) at °c for hr. afterwards, the fc-receptors of the stimulated tils were blocked by antimouse cd /cd (bd biosciences) for min at °c. the cells were then fixed, permeabilized and double stained with pe-conjugated anti-mouse cd and fitc-conjugated anti-mouse il- (bd biosciences) according to the protocol provided in the cytofix/cytoperm plus kit (bd biosciences). finally, the mean logarithmic fluorescence intensity was measured, using a facscan analyzer (becton dickinson). the effect of vv-rtap infection on surface mhc class i antigen expression was analyzed, using the probability binning chi (t) test (flowjo, ashland, or). results were considered statistically different if the t(x) value was greater than , implying that the distributions are different with a p < . ( % confidence). the effect of rtap transfection and treatment with vaccinia vectors on the growth of b f tumors was analyzed by -way anova. the tukey hsd test was used for multiple comparisons to determine the differential effects of the treatments on tumor growth. the effect of vaccinia vectors on the percentage of cd tils producing il- was analyzed by anova. the p values less than . , after corrections for multiple comparisons, were considered significant. h- d b antigen surface expression, but not mhc class ii (i-a b ) antigen expression rtap cdna was used for transfection to allow for distinction, by polymerase chain reaction, between endogenous mouse tap and transfected rtap during the determination of transfection efficiency and clone stability (data not shown). rtap , mouse tap and mouse tapasin (tpn) protein expression was examined in b f cells transfected with rtap by immunoblotting. rma cells were used as a positive control for tap , tap and tapasin expression, and cmt. cells were used as a negative control for tap and tap expression. , fibroblasts derived from tapasin / mice were used as a negative control for mouse tapa- sin. b /rtap - , b /rtap - and b /phb - cells were tested for rtap and mouse tap and tapasin expression (fig. a) . normal b f cells and b /phb - cells were negative for both tap and tap , but expressed some mouse tapasin. both b /rtap - and b /rtap - cells were positive for rtap expression. the expression of rtap also induced and/ or stabilized the expression of endogenous mouse tap in b / rtap - and b /rtap - cells. rtap expression also greatly increased endogenous levels of mouse tapasin. mhc class i and class ii antigen surface expression on b f cells was compared to surface expression on b /rtap - , b /rtap - and b /phb - cells, using facs analysis. the antibodies used for the facs analysis are conformationspecific and only bind to h- k b , h- d b and i-a b antigens properly folded and loaded with antigen peptide. both b /rtap - and b /rtap - cells exhibited significant expression of conformation specific, mature h- k b and h- d b antigens on the cell surface (fig. b) . this was in contrast to b f cells and b /phb - cells, which had undetectable levels of h- k b antigen on the cell surface and only a small amount of h- d b antigen. both rtap and vector-alone transfected b f cells exhibited a small population of cells positive for surface i-a b antigen expression, between and % of the total population. however, this population was neither increased nor decreased in cells expressing tap compared to untransfected or vector-alone transfected cells. we tested whether tap expression and the subsequent increase in h- antigen surface expression restores immunogenicity and t-cell recognition of b f cells by measuring the ability of b /rtap - , b /phb - and b f cells to process and present the h- k b antigen-specific, immuno-dominant vsv antigen: vsv-np - . vsv-np - -specific cytotoxic splenocytes were able to kill vsv-infected b /rtap - cells but not vsv-infected b /phb - or b f cells in a cr release assay (fig. a) . b /rtap - cells were able to correctly process and present viral antigens but b /phb - or b f cells were not. we also examined if rtap expression was able to restore the surface presentation of the h- k b antigen-restricted tumor-associated antigen, trp- . the cr release assay showed that both b /rtap - and b /rtap - cells were sensitive to killing by trp- -specific splenocytes compared to b f cells or b /phb - cells, which were resistant to killing (fig. b) . both b /rtap - and b /rtap - cells were therefore able to present tumor-associated antigens, in the context of h- k b antigen, making the cells sensitive to killing by trp- specific splenocytes. a ctl assay measured the immunogenicity of b f cells expressing tap . splenocytes from mice immunized with b / rtap - cells were able to kill all target cell lines in contrast with splenocytes from mice immunized with b /phb - or b f cells, which possessed diminished cytotoxic activity (fig. c) . these experiments demonstrate that rtap expression restores antigen processing sufficiently to make b f cells sensitive to killing by antigen-specific cytotoxic cells. in addition, rtap expression by b f cells stimulates immune responses that can generate cytotoxic cells capable of killing not only cells that express surface h- antigens but also those that have very low levels of h- antigen expression. we infected b f or b /rtap - cells with either vv-pjs- or vv-rtap and examined the effect after days on mhc class i antigen surface expression. facs analysis demonstrated that h- k b antigen is upregulated by . -fold [p < . ; t(x) table i) . the effect of tap expression on tumor growth rate was examined in a syngeneic mouse model. b /r tap - and b f tumors were grown in the hindquarters of mice and treated with either a vaccinia rtap expression vector (vv-rtap ), vaccinia vector control (vv-pjs- ) or pbs. tumor growth rates were analyzed by -way anova, which revealed significant reductions in tumor mass due to the effects of both rtap transfection (p < . ) and the vaccinia treatments (p < . ). there was a significant interaction (p < . ), however, between the main effects. the nature of the interaction was further characterized by comparisons between treatment groups. multiple comparisons showed that the growth rates of b f tumors were significantly slowed by treatment with vv-rtap (p < . ) but not by treatment with vv-pjs- . on the other hand b /r tap - tumor growth rates were significantly retarded by both vv-rtap and vv-pjs- (p < . ) but there was no difference between the vaccinia vectors (p > . ) (fig. ) . the expression of il- , a th cytokine capable of inhibiting cytotoxic t-cell responses, was measured in t lymphocytes infiltrating b f cells tumors treated with vv-rtap , vv-pjs- or pbs. the percentage of tils expressing il- in tumors was determined by facs analysis, and the treatments compared using -way anova (fig. ) . tumors treated with vv-rtap had significantly reduced cd lymphocytes expressing il- compared to vv-pjs- (p < . ) and pbs (p < . ) treatments. the treatment of tumors with vv-pjs- also significantly reduced the number of il- expressing cd lymphocytes compared to tumors treated with pbs (p < . ). data represent the fold increase in mean fluorescence intensity of vv-rtap -infected cells with the fold increase in vv-pjs- -infected cells subtracted, as detected by flow cytometry, using antibodies to h- k b and h- d b antigens. the restoration of antigen processing, mhc class i antigen expression, and immunogenicity by transfection or infection of tap alone into b f maa cells occurs despite numerous other deficiencies in the antigen presentation pathway. this has also been demonstrated in other cell lines with similar antigen presentationdeficient phenotypes, such as the murine nsclc cell line cmt. , human maa, human small cell lung carcinoma, human squamous cell carcinoma of the head and neck and human renal cell carcinomas. , , [ ] [ ] [ ] [ ] [ ] in b f cells, tap expression stabilized the expression of tap and increased the expression of endogenous tapasin. this indicates that the re-expression of tap may lead to a general reconstitution of several other components of the mhc class i antigen-processing pathway, and may therefore increase the amount of antigenic peptide available for assembly onto mhc class i molecules in the er. restoration of tap expression in tap-deficient cancer cells should make a wide variety of peptides derived from tumor-specific and -associated antigens available and perhaps this may compensate for any unpredictable deficiencies in the cell's mhc class i antigen allele repertoire. in the case of b f cells, rtap gene transfer was able to resurrect the presentation of the appropriate trp- peptide on h- k b antigens to allow for trp- -specific ctl killing. b /rtap cells present h- d b antigen-specific peptides derived from gp , making b /rtap cells susceptible to specific ctl lysis both in vitro and in vivo . vaccination by irradiated cells expressing tap enhances greatly the ctl activity not only towards b / rtap target cells but also to untransfected b f target cells. this indicates that it is likely not necessary for every cell to reexpress tap for immune tolerance to the tumor to be broken, allowing cd cytotoxic t-cell responses to occur. this is further supported by our previous in vivo study with cmt. lung carcinoma, in which mice initially immunized with cmt. expressing tap were better able to reject a challenge with untransfected cmt. , unlike mice initially immunized with untransfected cmt. . perhaps encouraging for applications to metastatic disease, the lysis of b f cells by splenocytes generated by vaccination with irradiated b /rtap cells demonstrates that there is sufficient h- antigen on the surface of b f cells to facilitate cytolytic activity. tap activity or the products of tap activity in b /rtap cells must be transferred in some way to the dendritic cells involved in the cross-presentation of mhc class i tumor antigens, a crucial step in generating specific cd cytotoxic tcell responses. tap expression in b /rtap cells results in a source of antigen that may be bound to mhc class i antigens on the surface of b /rtap cells and these mhc class i antigenrestricted antigens are transferred to the dendritic cell mhc class i antigens. alternatively, dendritic cells may access processed tumor antigens from the er compartment of b /rtap cells during internalization and antigen cross-presentation. the enhanced immunogenicity of b /rtap cells has a significant effect on tumor growth in mice. vaccination of mice with maa antigens specific for h- d b antigen are protected from b / rtap tumor challenge but not by challenge with b f cells by cd t-cells. in our study, b /rtap tumor growth was retarded without prior vaccination with tumor differentiation antigens, though the protection was not as complete as with prior vaccination. the effect also appears when tap is transferred in vivo by vaccinia expression vectors. in this case, not only does tap expression have an effect on tumor growth, but also the vector provides an adjuvant effect. in addition, we found that in vitro infection of b f cells transfected with tap (b / r tap - cells) with vv-rtap did not greatly enhance h- k b and h- d b antigen surface expression relative to vv-pjs- infected cells. this may indicate that tap activity is already at a maximum as a result of transfection, and that additional expression of tap from the vv construct does not further upregulate mhc class i antigen expression to a great extent. we propose that in addition to tumor antigens, upon infection the tumor cells also present viral antigens in the presence of tap that provide further epitopes for ctl recognition. the presence of the vv during the processing tumor antigens by dcs may also assist in breaking the tolerance of the immune system towards the tumor cells by acting as a ''danger signal'' that contributes to the priming of tumor antigenspecific immune responses. , furthermore, b f tumors are known to harbor regulatory t-cells that secrete cytokines, such as il- , which energize cd t-cells in tumors. tap gene transfer by vaccinia vectors reduced the number of cd /il- secreting lymphocytes in the tumors, one of the modulating factors that have been implicated in the resistance of maas to antitumor immune responses. tap expression, in conjunction with viral gene transfer vector, promotes a th type response that can function to retard tumor growth. thus, immunotherapeutic approaches that utilize tap have the potential to restore the priming and expansion of specific t-cells and the subsequent ability of the tumors to process and present tumor antigens. prospective randomized trial of interferon a- b and interleukin- as adjuvant treatment for resected intermediate-and highrisk primary melanoma without clinically detectable node metastasis adjuvant interferon in high-risk melanoma: the aim high study-united kingdom coordinating committee on cancer research randomized study of adjuvant low-dose extended-duration interferon a- a in high-risk resected malignant melanoma eggermont am; eortc melanoma group in cooperation with the german cancer society (dkg). final results of the eortc /dkg - randomised phase iii trial. rifn-a b versus rifn-g versus iscador m versus observation after surgery in melanoma patients with either high-risk primary (thickness > mm) or regional lymph node metastasis metastatic malignant melanoma of unknown primary origin: a study of cases regression in skin tumours: a common phenomenon complete regression of primary cutaneous malignant melanoma immune response against human primary malignant melanoma: a distinct cytokine mrna profile associated with spontaneous regression association of tap downregulation in human primary melanoma lesions with lack of spontaneous regression a gene encoding an antigen recognized by cytolytic t lymphocytes on a human melanoma immunotherapy of melanoma identification of multiple antigens recognized by tumor-infiltrating lymphocytes from a single patient: tumor escape by antigen loss and loss of mhc expression tumour escape from immune surveillance through dendritic cell inactivation tumor-induced death of immune cells: its mechanisms and consequences impaired surface antigen presentation in tumors: implications for t cell-based immunotherapy hla class i antigen abnormalities and immune escape by malignant cells down-regulation of hla class i antigen-processing molecules in malignant melanoma: association with disease progression tap down-regulation in primary melanoma lesions: an independent marker of poor prognosis reduced expression of tap- and tap- in posterior uveal melanoma is associated with progression to metastatic disease intratumoral t cells, recurrence and survival in epithelial ovarian cancer immune escape of melanoma: first evidence of structural alterations in two distinct components of the mhc class i antigen processing pathway characterization of the major histocompatibility complex class i deficiencies in b melanoma cells tap expression provides a general method for improving the recognition of malignant cells in vivo association of erp with mouse mhc class i molecules is tapasin dependent and mimics that of calreticulin and not calnexin heterogeneity in a spontaneous mouse lung carcinoma: selection and characterisation of stable metastatic variants induction of immunogenicity of a human renal-cell carcinoma cell line by tap -gene transfer transfection of tap gene restores hla class i expression in human small-cell lung carcinoma comparison of cell lines deficient in antigen presentation reveals a functional role for tap- alone in antigen processing role of antigen-processing machinery in the in vitro resistance of squamous cell carcinoma of the head and neck cells to recognition by ctl constitutive transduction of peptide transporter and hla genes restores antigen processing function and cytotoxic t cell-mediated immune recognition of human melanoma cells ctl-dependent and -independent antitumor immunity is determined by the tumor not the vaccine when two strands are better than one: the mediators and modulators of the cellular responses to double-stranded rna recognition of double-stranded rna and activation of nf-jb by toll-like receptor interleukin- expressed at early tumour sites induces subsequent generation of cd ( ) t-regulatory cells and systemic collapse of antitumour immunity the authors acknowledge the assistance of ms. eunice yao, ms. kyla omilusik and dr. anna reinicke in article preparation. the authors also thank dr. geoff butcher for the tap antiserum, dr. luc van kaer for the murine tapasin / fibroblasts and dr. ted hansen for the rabbit antiserum to mouse tapasin. key: cord- -z xbn authors: namvar, ali; bolhassani, azam; javadi, gholamreza; noormohammadi, zahra title: in silico/in vivo analysis of high-risk papillomavirus l and l conserved sequences for development of cross-subtype prophylactic vaccine date: - - journal: sci rep doi: . /s - - - sha: doc_id: cord_uid: z xbn human papillomavirus (hpv) is the most common sexually transmitted infection in the world and the main cause of cervical cancer. nowadays, the virus-like particles (vlps) based on l proteins have been considered as the best candidate for vaccine development against hpv infections. two commercial hpv (gardasil and cervarix) are available. these hpv vlp vaccines induce genotype-limited protection. the major impediments such as economic barriers especially gaps in financing obstructed the optimal delivery of vaccines in developing countries. thus, many efforts are underway to develop the next generation of vaccines against other types of high-risk hpv. in this study, we developed dna constructs (based on l and l genes) that were potentially immunogenic and highly conserved among the high-risk hpv types. the framework of analysis include ( ) b-cell epitope mapping, ( ) t-cell epitope mapping (i.e., cd (+) and cd (+) t cells), ( ) allergenicity assessment, ( ) tap transport and proteasomal cleavage, ( ) population coverage, ( ) global and template-based docking, and ( ) data collection, analysis, and design of the l and l dna constructs. our data indicated the -epitope candidates for helper t-cell and ctl in l and l sequences. for the l and l constructs, combination of these peptides in a single universal vaccine could involve all world population by the rate of . % and . %, respectively. in vitro studies showed high expression rates of multiepitope l (~ . %) and l (~ . %) dna constructs in hek- t cells. moreover, in vivo studies indicated that the combination of l and l dna constructs without any adjuvant or delivery system induced effective immune responses, and protected mice against c tumor cells (the percentage of tumor-free mice: ~ . %). thus, the designed l and l dna constructs would represent promising applications for hpv vaccine development. www.nature.com/scientificreports www.nature.com/scientificreports/ charge and secondary structure. at first step, the conserved region sequences were analyzed by bepipred- server to predict potential b-cell epitopes (table ). in l protein, l [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] (eatvylppvpvskvv-type ), l - (pppggtledtyrfv-type ) and l - (nfgvppppttslvd-type ) epitopes had the best b cell epitope identification scores. for l protein, l [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] (kqsgtcppdvvpkv-type ), l - (psdpsivslveets-type ), l - (epvgptdpsivtli-type ) and l [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] (glgigtgsgtggrt-type ) epitopes showed the highest epitope identification score between their own protein sequences. since a linear form of t-cell epitopes are bound to mhcs, the interface between t-cells and ligands can be accurately modeled. in this study, we used three different algorithms (published motifs, ann and quantitative matrix) for mhc-i and two algorithms for mhc-ii (ann and quantitative matrix). prediction of mhc-i. at first step, the l and l conserved regions were analyzed to find the most immunodominant peptides using netmhcpan . , syfpeithi and propred i. in each protein, peptides with the highest binding affinity scores were determined as high-potential ctl epitope candidates (tables and ). the analysis showed that l [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] (ylppvpvskv-type and ylpppsvarv-type ), l - (dqfplgrkfll-type ) , l - (dqyplgrkflv-type ), l [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] (krasatqlyk-type and krasvtdlyk-type ), l - (dpdfldivalhr-type ) and l - (dsdfmdiirlhr-type ) epitopes had the highest binding affinity among their own protein sequences. in general, the results of three different algorithms confirmed each other. conservancy and allergenicity analyses were done on the selected epitopes. the sequence of all the epitopes were well conserved among high-risk hpv types and none of them were allergens (tables and ). in addition, there was no cross-reactivity between peptide and human proteome. in this study, we used netmhciipan and propred servers for mhc-ii epitope identification analysis (table ). since a suitable t-cell epitope should be predicted to bind to different hla alleles, epitopes with the maximum number of binding hla-dr alleles were selected as high-potential helper t-cell epitope candidates. among predicted epitopes, l [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] (eatvylppvpvskvv-type ) , l - (tqrlvwacvgvevgrgq-type and tqrlvwacagveigrgq-type ), l - (dtyrfvtsqaiacqk-type ) , l - (dtyrfvqsvaitcqk-type ), l - (dpsivtliedssvvtsgap-type ) , l - (pdfldivalhrpaltsr-type ) and l - (sdfmdiirlhrpaltsr-type ) had the highest scores of binding affinity. also, the sequence of all the epitopes were well conserved among high-risk hpv types and none of them were allergen (tables and ) . also, there was no cross-reactivity between peptide and human proteome. population coverage analysis. hla distribution varies among the diverse geographic regions around the world. thus, while designing an effective vaccine, population coverage must be taken into consideration to cover the maximum possible populations. in this study, population coverage was estimated separately for each putative epitope in specified geographic regions of the world (tables and ) . for ctl epitopes, the highest population coverage of world's population was calculated for l a clear band of ~ bp and ~ bp on agarose gel for l and l , respectively (data not shown). the recombinant endotoxin-free plasmids (i.e., pcdna-l and pcdna-l ) had a concentration range between . and . mg/ml. into the eukaryotic cell line (hek- t) was performed by turbofect as a transfection reagent. the levels of dna expression were evaluated using fluorescence microscopy and flow cytometry at h post-transfection. the data indicated that pegfp-l and pegfp-l can effectively penetrate into hek- t cells in vitro. the cellular uptake of the l and l genes into the hek- t cells was ~ . % and ~ . %, respectively. the delivery of pegfp-n as a positive control was detected in approximately ~ . % of hek- t cells (fig. ) . moreover, the spreading green regions were observed for l and l dna delivery using turbofect carrier by fluorescent microscopy in hek- t cells. on the other hand, western blot analysis indicated the successful expression of l and l proteins fused to gfp (i.e., l -gfp and l -gfp) using anti-gfp antibody. the data indicated the clear bands of ~ , ~ and ~ kda for l -gfp, l -gfp and gfp, respectively using dab substrate (fig. ). to evaluate the prophylactic effects of the designed l and l dna constructs, tumor growth and survival percentage were assessed in all groups for days after challenging with c tumor cells. as shown in fig. a , all test groups immunized with dna constructs (g , g & g ) demonstrated significantly lower tumor growth than that in control groups (pbs and empty vector, g & g , p < . ). our data showed progressive tumor growth in control groups on approximately - days (survival rate or tumor-free mice percentage: %). it was interesting that groups vaccinated with l dna, l dna and l + l dna constructs similarly reduced the tumor growth (p > . ). as shown in fig. b , group vaccinated with the mixture of l + l dna constructs showed a higher survival rate (g , ~ . %) than l and l dna constructs, alone (g & g , ~ . %). antibody assay. the levels of total immunoglobulin g (igg), igg a and igg b in mice immunized with the mixture of l + l dna constructs (g ) were significantly higher than other groups (p < . , fig. a ,c,d). moreover, our data showed that the levels of igg were similar in all groups vaccinated with dna constructs (g , g & g , p > . , fig. b ). there are no significant differences in the secretion of igg a and igg b isotypes between groups receiving the l and l dna constructs, alone (g & g , p > . , fig. c ,d). no significant anti-(l + l ) antibody responses could be detected in the sera of control groups, thus, the seroreactivities were completely l + l antigen-specific responses in mice. cytokine assay. the results of cytokine assay in each group showed that the levels of (l + l )-specific ifn-γ, il- and il- secretions in groups immunized with l (g ), l (g ) and l + l (g ) dna constructs were significantly higher than control groups (p < . , fig. www.nature.com/scientificreports www.nature.com/scientificreports/ fig. ) . furthermore, our data indicated that the ratios of ifn-γ/il- and ifn-γ/il- were higher in all test groups as compared to control groups; therefore, they could trigger th immune response. granzyme b secretion. the secretion of granzyme b in all test groups was significantly higher than the control groups (p < . , fig. ). the group immunized with the l + l dna construct (g ) produced significantly higher concentrations of granzyme b than other groups (g & g , p < . ). the level of granzyme b in group receiving l dna construct was similar to that in group receiving l dna construct (p > . ). in recent years, development of bioinformatics tools applied in vaccine researches could potentially save time and resources. indeed, the immunoinformatics tools help to identify antigenic domains for designing a multi-epitope vaccine. with sequence-based technology advancement, now we have enough information about the genomics and proteomics of different viruses . thus, using various bioinformatics tools, we can design peptide vaccines based on a neutralizing epitope. for example, in silico design of an epitope-based vaccine against human immunodeficiency virus , , coronavirus , dengue virus , and saint louis encephalitis virus has already been reported. while around high-risk hpvs were recognized, current vaccines just protect humans from few types. an important limitation of the current vaccines is their narrow coverage. the accessibility of fully sequenced proteome from high-risk hpv strains provides a prospect for in silico screening of reliable peptide-based therapeutic vaccine candidates among billions of possible immunogenic peptides. in silico approaches are intended to reflect the possibilities for overcoming the above-mentioned difficulties in hpv multi-type vaccine. gupta and coworkers designed prophylactic multiepitopic dna vaccine using all the consensus epitopic sequences of hpvs l capsid protein. they also evaluated how engineering cpg motifs by bioinformatics tools could increase immunogenicity of dna vaccines . hosseini et al. applied in silico analysis of l and l protein of hpv , , , and types to identify universal peptide vaccine in order to protect against mentioned types . in , singh et al. analyzed e , e , e and e proteins of high-risk hpv types to identify cd + t-cell epitopes. they suggested a pool of peptides ( to amino acids) to provide the protection against high-risk hpv types . www.nature.com/scientificreports www.nature.com/scientificreports/ panahi and colleagues used a two-step method (consist of molecular docking and sequence-based approach) to determine immunogenic epitopes for induction of immune system against the oncoproteins of hpv , , and types . in this research, we designed a framework for the comprehensive analysis of l and l conserved regions of high-risk hpv types containing both mhc-i and mhc-ii epitopes. the framework begins with conservancy analysis of all high-risk hpv strains following with ( ) b-cell epitope mapping, ( ) t-cell epitope mapping (cd + and cd + ), ( ) allergenicity assessment, ( ) tap transport and proteasomal cleavage, ( ) population coverage, ( ) global and template-based docking and ( ) data collection, analysis, and design of the l and l dna constructs. for experimental analysis, the final l or l dna constructs were cloned into mammalian expression vector with green fluorescent tag (pegfp vector) and their expression was evaluated in the eukaryotic cells using flow cytometry, fluorescent microscopy and western blotting. moreover, the l /l -specific antibody and t-cell immune responses induced by l and l dna constructs were assessed in mouse tumor model. at first, l and l sequences obtained from high-risk hpv types were aligned using muscle algorithms. conservancy analysis showed that five regions of hpv , l protein ( - , - , - , - and - ) and four regions of hpv , l protein ( - , - , - and - ) were more conserved among other subtypes and could be analyzed as an immunoinformatics input. in b-cell epitope prediction, l [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] , l - , l - , l - , l - , l - and l - had the highest epitope prediction scores. unfortunately, a reliable method for prediction of b-cell epitope has not been revealed up to now and the sensitivity and specificity of existing methods were very low (the specificity and sensitivity of this method were . and . , respectively). in the case of t-cell epitope prediction, in silico analysis has been significantly improved, thus, the results are more reliable. in this study, for mhc-i epitopes, l - (ylppvpvskv-type and ylpppsvarv-type ), l - (dqfplgrkfll-type ), l - (dqyplgrkflv-type ), l - (krasatqlyk-type and krasvtdlyk-type ), l - (dpdfldivalhr-type ) and l - (dsdfmdiirlhr-type ) epitopes had the highest binding affinity scores. in addition, above-mentioned epitopes had the highest t-cell epitope prediction scores which were obtained from proteasomal cleavage and tap transport analysis. high degree of conservancy was observed between subtypes for these epitopes ( [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] (ffgglgigtgsgtggr-type ) epitopes had the highest binding affinity scores. among them, l - had the greatest degree of conservancy (high similarity with all of the high-risk hpv types). one of the remarkable points is that l [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] and l - epitopes are the same (or overlapping with little difference (among b-cell and mhc-ii selected epitopes. due to a limitation of mhc-peptide binding prediction such as the gap between the peptides that are predicted to bind to mhc and those that experimentally bind www.nature.com/scientificreports www.nature.com/scientificreports/ been employed to address this problem and raise the accuracy of mhc-peptide prediction. in the current study, template-based docking and also global docking were performed on the selected peptides to determine which peptide would get into the groove of mhc with the highest modeling scores. for mhc-i epitope, l - , l - and l - sequences had the highest interaction similarity and cluster density scores. for mhc-ii epitopes, l - , l - , l - and l - sequences had the highest docking scores. in this study, mhc-i-peptide docking scores confirmed mhc-i-peptide binding affinity scores because the same epitopes had the highest scores in both methods but in mhc-ii molecular docking, the results were slightly different. one of the reasons is the significant conformational changes during the process due to the longer epitope length. as a general rule: the longer the length of the query peptide, the more torsions and conformational flexibilities . herein, due to longer peptide sequences, docking results in mhc-ii were less accurate than mhc-i. for example, average similarity score in mhc-i was variable ( . - . ), but in mhc-ii was . - . after the completion of the analysis and according to all of the above-mentioned parameters, two separate constructs were designed. in addition, accumulative population coverage of helper t-cell and ctl epitopes for the designed constructs www.nature.com/scientificreports www.nature.com/scientificreports/ were estimated. for the l and l constructs, the combination of epitope candidates for helper t-cell and ctl in a single universal vaccine could involve all world population by the rate of . % and . %, respectively (fig. ) . in previous studies, ylppvpvskv (hpv l ) and krasvtdlyk (hpv l ) have been reported as potentially immunogenic epitopes. the ability of in vitro expression of the designed l and l dna constructs was determined in hek- t cells using flow cytometry and western blot analysis. the transfection efficiency of the l and l dna constructs was ~ . % and ~ . %, respectively indicating their high potency for delivery into the eukaryotic cells. as known, the use of a polytope dna vaccine containing multiple t-cell and b-cell epitopes is an attractive strategy for developing a therapeutic and prophylactic vaccine against hpv infections. after in vitro assay, immunological experiments were performed in mice to determine the efficiency of the designed l and l dna constructs without the use of adjuvant or delivery system for vaccine development. similarly, some studies used the pcdna vector harboring the gene of interest for immunization without any adjuvant , . our data indicated that the groups immunized with l , l and l + l dna constructs increased antibody and t-cell responses as compared to control groups. furthermore, the (l + l )-specific immunity in mice receiving the mixture of l + l dna constructs (g ) resulted in higher secretion of total igg, igg a, igg b, ifn-γ, il- and il- cytokines as well as granzyme b than other groups. the higher levels of igg a and igg b as well as ifn-gamma (as a th cytokine) in this group drive t-cell responses toward th -type immunity. the studies showed that immunoglobulin g (igg ) is related to a th -type response, while a th response is associated with the induction of igg a and igg b in mice . regarding to our observations in protective studies, this regimen (l + l dna construct: g ) could confer further protection against c tumor-challenged mice (survival rate: ~ . %) depending on stimulation of cd + t cell-dominated th responses as well as granzyme b secretion (indicating ctl activity) as compared to the l or l dna constructs, alone (survival rate: ~ . %). these data showed high potency of the combined l + l dna constructs versus each dna construct alone as a prophylactic hpv vaccine. taken together, immunoinformatics approaches have been emerged as a critical field for accelerating immunological researches. yet, the immunoinformatics techniques applied to t-cells have more advancement than those dealing with b-cells . moreover, recently, due to the limited options for choosing an adjuvant in clinical trials, bioinformatics analyses have been developed to predict the best adjuvant. in this way, in silico studies help researchers saving time and resources, and also can guide the experimental work with higher probabilities of finding the desired solutions and with fewer trial and error repeats of assays. the accessibility of hpv genomic sequences and functional characterization of the genes involved in the virulence has significantly improved our understanding of the molecular foundation for the pathogenesis of hpv and offered a wealth of data that can be used to design new plans for vaccine design. nowadays, powerful immune system simulators have been developed using bioinformatics tools which predict artificial immunity provided by the vaccine. these approaches could predict the best adjuvant for using in human vaccine studies. there is a multi-scale computational infrastructure approach which can stimulate the dynamics of the immune response induced by several vaccination formulations and predict optimal combination in terms of adjuvant type, dosage and timing. netlogo is an agent-based modeling of the immune system running different simulations with different parameter settings. it also can interact with different modeling strategies including the investigation of pathogen growth, life cycle modeling environment for simulation complex phenomena [ ] [ ] [ ] . therefore, using these methods can increase efficiency and reduce costs in vaccine studies. in this study, for the first time, comprehensively integrated methods (using sequence-based tools in combination with flexible peptide-protein docking) were used to design highly immunogenic and protective vaccine candidates which were able to boost both humoral and cellular table . mhc-ii -peptide docking scores of selected helper t-cell epitopes. *higher rate shows better quality of peptide-mhc interactions. www.nature.com/scientificreports www.nature.com/scientificreports/ immune responses against all high-risk hpv types. in addition, in vivo analysis demonstrated high potency of the designed l and l constructs as combined in dna-based vaccines without the use of adjuvant or delivery system. however, we will improve the efficiency of these dna-based vaccines using a delivery system and also will compare their efficacy with the designed peptide-based vaccines along with adjuvants in near future. table . physicochemical properties of l and l dna vaccine constructs. *higher rate shows high degree of peptide antigenicity. **higher rate shows high degree of peptide solubility. protein alignments and conservancy analysis. to determine conserved epitopes between different subtypes, l and l sequence datasets were first aligned using snapgene software . . (from gsl biotech; available at snapgene.com). after protein alignments analysis using muscle algorithms, the conserved epitopes of each protein were selected for immune-bioinformatics analysis such as b-and t-cell epitope prediction. also, to calculate the degree of variability and conservancy of each epitope, iedb epitope conservancy tools (http://tools.immuneepitope.org/tools/conservancy/) were used. linear b-cell epitope prediction. a successful vaccine must elicit a strong t-cell and b-cell immune response, but above all, provide protection against the disease being targeted. therefore, it is essential to show that constructed immunogens are able to induce protective cellular and humoral immunity. since the antibodies are induced against linear b-cell epitopes, it would be very difficult to synthesize long peptides with the native protein conformation resembling for the induction of protective antibodies. however, optimal peptide-based vaccines should be presented in a desired secondary structure of peptides in order to induce a specific humoral response , . for the b-cell epitope prediction of conserved regions in l and l proteins, bepipred- . server (http://www.cbs. www.nature.com/scientificreports www.nature.com/scientificreports/ dtu.dk/services/bepipred- . /) was employed. in this study, epitope threshold value was set as . (the specificity and sensitivity of this method are . and . , respectively) . t-cell epitope prediction. mhc-i epitope prediction: the initial step on applying bioinformatics to vaccine researches is to assess potentially immunoprotective epitopes. t-cell epitopes presented by mhc molecules are typically in a linear form containing to amino acids. this fact facilitates accurate modeling for the interaction of ligands and t-cells . thus, the most selective step in the presentation of antigenic peptide to t-cell receptor (tcr) is the binding of the mhc molecule . in this study, we tried to use three different algorithms including artificial neural networks (netmhcpan . server (http://www.cbs.dtu.dk/services/netmhcpan/), quantitative matrix (propred i (http://crdd.osdd.net/raghava/propred /) and published motifs (syfpeithi server (http://www.syfpeithi.de) to predict high-potential t-cell epitopes. for netmhcpan, percentile rank was set at . % for strong binders and % for weak binders and for propred i threshold was set at %. mhc-ii epitope prediction: for mhc class ii, netmhciipan . server (http://www.cbs.dtu.dk/services/ netmhciipan/) and propred (http://crdd.osdd.net/raghava/propred/) were employed to predict potential interaction of helper t-cell epitope peptides and mhc-ii. in this case, the threshold for strong and weak binders was set at % and %, respectively. prediction of mhc-i peptide presentation pathway. investigating the tap transport and proteasomal cleavage as well as affinity prediction of binding is essential in mhc-i presentation pathway. in this study, we used netctl . server combined with tap transport/proteasomal cleavage tools (http://www.cbs.dtu.dk/services/netctl/) to access the prediction of antigen processing through the mhc class i antigen presentation pathway. in this method, parameters of weight on the c-terminal cleavage, tap transport efficiency, and epitope identification were set to default ( . , . and . , respectively) . population coverage. since the response to t-cell epitopes is restricted by mhcs, the selection of epitopes with multiple hla-binding increases population coverage in defined geographical regions where the peptide-based vaccine might be employed. the coverage rate of population for each epitope was computationally validated using the iedb population coverage tool (/population/iedb_input). in this study, individual epitope and its binding to hla alleles were analyzed, and different geographic areas were also selected. allergenicity and cross-reactivity assessment. since proteins are very important in inducing allergenic reactions, the prediction of potential allergenicity is an important item in the safety assessment especially in the field of genetically modified foods, therapeutics, bio-pharmaceuticals etc. . the food and agriculture organization (fao) and world health organization (who) protocol includes three terms to evaluate the allergenicity of proteins which are defined as following: the term sensitivity refers to correctly predicted allergens (%), whereas www.nature.com/scientificreports www.nature.com/scientificreports/ specificity refers to correctly predicted non-allergens (%), and also accuracy refers to the proportion of correctly predicted proteins . the allergenicity of the epitopes was analyzed by the pa p (http://lpa.saogabriel.unipampa. edu.br: /pa p/pa p/pa p.jsp) using allergen online ( aa and wordmatch) and afds-motif algorithms based on amino acid composition. the specificity of these methods is . % ( aa), . % ( aa) and . % (adfs) . to assess cross-reactivity between peptide and human proteome, top-ranked epitope were analyzed by peptide matching program (https://research.bioinformatics.udel.edu/peptidematch/index.jsp) . peptide-protein flexible docking. computational docking methods have been known as an important tool for drug design . with the rapid development of peptide therapeutics in rational drug design, the use of new techniques such as protein-peptide docking is inevitable. in this study, two different algorithms (template-based docking and global docking) were performed by galexypepdock server (http://galaxy.seoklab.org/cgi-bin/ submit.cgi?type=pepdock) and cabs dock server (http://biocomp.chem.uw.edu.pl/cabsdock). to estimate the formation of mhc-peptide complex, the galaxypepdock server effectively models the structural d peptide-protein complexes from input peptide and protein sequences using the structure database and energy-based optimization (template-based docking). cabs-dock server performs global docking procedure which at first explicit fully flexible docking simulation and then clustering-based scoring. receptor flexibility was limited by default to small backbone fluctuation but could be increased to include selected receptor fragments , . this study presented an example of mhc-peptide docking performed by each individual epitope and available pdb file (table ) of hla alleles, separately. physicochemical properties of the designed l and l constructs. based on l and l top-ranked epitopes, two different constructs were designed. the physicochemical properties of top-ranked epitopes such as solubility, molecular weight, estimated half-time, instability index and antigenicity were determined by protparam (https:// web.expasy.org/protparam/) tools , vaxijen (http://www.ddg-pharmfac.net/vaxijen/vaxijen/vaxijen.html) and protein-sol (https://protein-sol.manchester.ac.uk/) server . www.nature.com/scientificreports www.nature.com/scientificreports/ experimental studies construction of the recombinant plasmids. after bioinformatics analysis, the selected peptides were assembled in two separated constructs (fig. ) . the puc -l and puc -l constructs were synthesized by biomatik company. for in vitro experiments, the puc -l and puc -l vectors were digested by xhoi/hindiii, and the l and l genes were subcloned into xhoi/hindiii sites of pegfp-n vector, individually (i.e., pegfp-l and pegfp-l ). all the recombinant vectors were transformed into escherichia coli (e. www.nature.com/scientificreports www.nature.com/scientificreports/ coli) dh α strain. after extraction of plasmids from single colonies using mini-kit (qiagen), the presence of inserted l and l fragments was confirmed by digestion with restriction enzymes and sequencing. for in vivo immunological assessment, the puc -l and puc -l vectors were digested by bamhi/hindiii and the l and l genes were subcloned into bamhi/hindiii sites of pcdna . (-) vector containing cytomegalovirus early promoter and enhancer sequence, individually (i.e., pcdna-l and pcdna-l ). indeed, we used the pcdna vector harboring cpg motif for in vivo studies. as a final point, the recombinant dna vectors harboring l and l genes were purified by an endotoxin-free plasmid extra ef kit (macherey nagel, germany). the concentration and purity of the recombinant l and l dna constructs were determined by nanodrop spectrophotometry . in vitro expression of l and l dna constructs in hek- t cells. human embryonic kidney cells (hek- t) were cultured in rpmi supplemented with % fetal bovine serum (fbs) at °c and % co atmosphere. after some passages, the cells were seeded in a -well plate. the optimal cell confluency for effective transfection was considered - %. for the generation of turbofect-plasmid dna complex, μl of turbofect (thermo scientific) and μg of each plasmid (pegfp-l , pegfp-l and pegfp-n as a positive control) were mixed and incubated for min at room temperature. then, the complex was added to each well in serum-free media. in addition, the non-transfected hek- t cells were used as negative control. after six hours, the media was replaced with the completed rpmi medium. finally, the cells were harvested, washed and resuspended in pbs buffer, to analyze the expression of l and l dna constructs using flow cytometry, fluorescent microscopy and western blotting at hr after transfection . western blot analysis. hek- t cells were scraped from their plates and washed with pbs x. after washing steps, the cells were lysed in whole-cell lysis buffer ( % glycerol, nm dtt, mm natrium fluoride, . % triton x- , . edta in pbs ph = . ). the extracted protein samples (l -gfp, l -gfp and gfp) were separated by sds-page in . % (w/v) polyacrylamide gel and transferred to nitrocellulose membrane (millipore). the membrane was equilibrated with tbst (tris-buffered saline tween- ) solution containing . % bsa (bovine albumin serum) overnight. the anti-gfp polyclonal antibody ( : v/v; acris antibodies gmbh) was used to recognize the expressed proteins under standard procedures. the immunoreactive protein bands were visualized by detection of peroxidase activity using a substrate named as , ′-diaminobenzidine (dab, sigma) . peptide constructs synthesis. for immunological assay (i.e., secretion of antibody, cytokine and granzyme b), two peptide constructs (l and l peptides, fig. ) were synthesized by biomatik co. with more than % purity. mice immunization. five groups of six female c bl/ mice (obtained from the breeding stocks maintained at pasteur institute of iran; mhc haplotype b/h- kb/h- db) were immunized on days , , and (i.e., three times with a -week interval) with µg of each plasmid dna (pcdna-l or pcdna-l : g or g ) or their combination (pcdna-l + pcdna-l : g ) at the right footpad as shown in table . the control groups (g and g ) received pcdna . and pbs, respectively. all mice were maintained under specific pathogen-free conditions . moreover, all of the animal experimental procedures were approved by animal care and use committee of pasteur institute of iran and carried out according to the animal experimentation regulations of pasteur institute of iran (national guideline) for scientific purposes (code: ). for in vivo protection assay, vaccinated mice were subcutaneously challenged in the right flank with c tumor cells ( × cells), two weeks after the last injection. the c tumor cells contain whole hpv genome, and the presence of l and l genes was confirmed in the previous studies . tumor growth and the percentage of tumor-free mice were monitored twice a week by palpation for days post-challenge. at each time, tumor volume was calculated by this formula: v = (a b)/ (a = the smallest diameter and b = the biggest diameter) . antibody assay secreted from b-cells. two weeks after the last injection, serum samples were collected from each group. the levels of goat anti-mouse immunoglobulin g (igg ), igg a, igg b and total igg antibodies (diluted : , in % bsa/pbs-tween, sigma) secreted from b-cells were measured in the pooled sera of each group by indirect elisa. the coated antigens were the mixture of l and l synthetic peptides ( μg/ml). moreover, mice sera were diluted : in % bsa/pbs-tween . cytokine assay secreted from t-cells. three mice from each group were sacrificed and the spleens were removed. the red blood cell-depleted pooled splenocytes ( × cells/ml) were cultured in -well plates for h in the presence of μg/ml of l + l peptides, rpmi % as negative control and μg/ml of concanavalin a (cona) as positive control in complete rpmi culture medium. the supernatants were harvested to assess the secretion of ifn-γ, il- and il- from t-cells using the sandwich-based elisa method (r&d systems) according to the manufacturer's instructions. all data were represented as mean ± sd for each sample . granzyme b assay (in vitro ctl activity). to measure granzyme b (grb) by elisa, the p target cells (t) were seeded into -well plates ( × cells/well) incubated with the mixture of l and l peptides (~ μg/ml) for h. then, the prepared splenocytes (effector cells: e, before section) were counted and added to the target cells at e: t ratio of : in complete rpmi culture medium for h incubation. finally, the supernatants were harvested to measure the concentration of grb by elisa (ebioscience kit) according to the manufacturer's instruction . table . immunization program for in vivo analysis. worldwide burden of cancer attributable to hpv by site, country and hpv type estimate of the global burden of cervical adenocarcinoma and potential impact of prophylactic human papillomavirus vaccination hpv vaccination: the promise & problems pros, cons, and ethics of hpv vaccine in teens-why such controversy? virus-like particles for the prevention of human papillomavirus-associated malignancies therapeutic human papillomavirus vaccines: current clinical trials and future 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requests for materials should be addressed to a.b.reprints and permissions information is available at www.nature.com/reprints.publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.open access this article is licensed under a creative commons attribution . international license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons license, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons license, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this license, visit http://creativecommons.org/licenses/by/ . /. key: cord- - czobqy authors: byun, hyewon; gou, yongqiang; zook, adam; lozano, mary m.; dudley, jaquelin p. title: erad and how viruses exploit it date: - - journal: front microbiol doi: . /fmicb. . sha: doc_id: cord_uid: czobqy endoplasmic reticulum (er)-associated degradation (erad) is a universally important process among eukaryotic cells. erad is necessary to preserve cell integrity since the accumulation of defective proteins results in diseases associated with neurological dysfunction, cancer, and infections. this process involves recognition of misfolded or misassembled proteins that have been translated in association with er membranes. recognition of erad substrates leads to their extraction through the er membrane (retrotranslocation or dislocation), ubiquitination, and destruction by cytosolic proteasomes. this review focuses on erad and its components as well as how viruses use this process to promote their replication and to avoid the immune response. although endoplasmic reticulum (er)-associated degradation (erad) has been most thoroughly defined in yeast, recent studies in higher organisms have revealed the conservation of this process and its components. multiple diseases, including parkinson's, alzheimer's, cancer, and infectious processes, result from failure of erad, confirming its significance for correct cell function. predictably, viruses have exploited various aspects of this key cellular machinery to further their propagation. nonetheless, the complexity of erad and the number of players involved necessitates a review of its features prior to a description of how viruses have manipulated erad to their advantage. in understanding how viruses exploit erad, we learn more about the cellular process, but also how we might alter the outcome of viral diseases. a majority of newly synthesized proteins in mammalian cells are either misfolded or misassembled (hoseki et al., ) . approximately % of new proteins are synthesized in association with the er (brodsky and wojcikiewicz, ) . the er quality control system both senses and disposes of terminally misfolded proteins by erad, a process that is conserved in eukaryotes (vembar and brodsky, ; merulla et al., ) . this process detects misfolded proteins in the er lumen, and then extracts them through membrane channels in an energy-dependent manner for delivery to cytosolic proteasomes (olzmann et al., ) . protein extraction through er membrane channels is known as dislocation or retrotranslocation (hampton and sommer, ) . because protein folding depends on multiple cellular components (merulla et al., ) , protein overexpression or the presence of mutant proteins may sequester limiting components, leading to accumulation of misfolded proteins in the er lumen. a more general failure of the erad process may occur if proteins are unable to fold within a reasonable time, resulting in inefficient retrotranslocation and proteasomal degradation. levels of erad-associated factors also may be affected by the intraluminal concentration of misfolded proteins. inability of the erad system to destroy misfolded proteins is associated with more than diseases, including neurological illnesses (alzheimer's and parkinson's), cystic fibrosis, infectious diseases, diabetes, and cancer (guerriero and brodsky, ) . particularly relevant to the subject of this review, viruses can produce large quantities of glycoproteins in a short period of time, which may overwhelm erad, leading to the accumulation of misfolded proteins, cell death, and associated pathology (franz et al., ) . although erad is vital to the maintenance of healthy cells, many parts of this process are not well characterized. multiple aspects of erad have been described in yeast (thibault and ng, ) , including the nature of the er channel and the components needed to identify misfolded proteins during and after translation. protein translocation across the er membrane is the prerequisite for erad. translation of many transmembrane proteins involves recognition of a hydrophobic signal peptide (sp) emerging from the ribosome by signal recognition particle (srp), which is associated with the trimeric sec complex. many of the sps are cleaved by signal peptidase, which is associated with the luminal side of the translocon (auclair et al., ) . the sec complex provides the aqueous channel for co-translational transfer of proteins across the er membrane (loibl et al., ) . recent evidence indicates that translocation across the er membrane can occur through an srp-independent process (denic, ; johnson et al., ) . based on recent experiments in yeast, more than % of signal-containing proteins fail to use srp, including tail-anchored (ta) proteins and short secretory proteins (johnson et al., ; ast et al., ) . instead, these proteins are targeted by the get pathway to the sec translocon that is associated with the sec / complex rather than through docking to the srp receptor (rapoport, ; ast et al., ) . one large class of srp-independent proteins includes the glycosylphosphatidylinositol (gpi)-anchored proteins, which contain both an n-terminal signal sequence and a c-terminal gpi anchor (ast et al., ) . this n-terminal signal is less hydrophobic than typical srp targets. furthermore, the sec translocon has been implicated as the channel for retrotranslocation (kiser et al., ) , and it has been proposed that protein transfer can be either forward or reverse with respect to the er lumen (johnson and haigh, ) . therefore, sec appears to complex with a number of different proteins, leading to a highly flexible and dynamic structure, where association with different proteins/protein complexes leads to transit in or out of the er (figure ). reports in yeast indicate that proteins can be o-mannosylated prior to n-glycosylation (ecker et al., ) , and both types of glycosylation are believed to occur co-translationally (loibl et al., ) . these glycosylases also have been shown to be associated with the translocon (chavan and lennarz, ) , and experiments indicate competition for different glycosylation sites (loibl et al., ) . the protein o-mannosyl transferases (pmts) and the oligosaccharyltransferases (osts) are transmembrane proteins, but the latter catalyzes addition of oligosaccharides to nascent polypeptides on asparagine residues (breitling and aebi, ) . the osts prefer nxt/s sequences in an unfolded or flexible protein domain, and the unfolded state may be facilitated by the ost complex associated with the translocon (breitling and aebi, ) . glycosylation near the c-terminal end of the protein is less efficient, perhaps due to competition between osts and protein folding (ben-dor et al., ; breitling and aebi, ) . pmts also are essential for erad in yeast. a pmt mutant showed increased degradation of a typical erad substrate (arroyo et al., ) . moreover, addition of oligosaccharides can be prevented by nearby cysteines and disulfide bond formation (allen et al., ) . thus, glycosylation is one determinant of the correct folding of a protein in the er lumen (breitling and aebi, ; figure a ). the oligosaccharides on er luminal proteins are critical for their correct folding or selection for erad. the nascent n-glycosylated protein has a three-branch structure with glucose mannose -n-acetylglucosamine -asparagine (aebi et al., ; merulla et al., ) . trimming of the first two glucose residues on one branch then allows interactions with two er-resident chaperone/lectin proteins, calnexin and calreticulin, which may lead to protein folding (brodsky, ) . removal of the third glucose causes release from these lectins and exit from the er (smith et al., ; olzmann et al., ) , but re-addition of this glucose by udp-glucose:glycoprotein glucosyltransferase allows reassociation (shenkman et al., ) . proteins retry folding until removal of three or four mannose residues triggers erad (lederkremer and glickman, ; shenkman et al., ) . correctly folded proteins leave the er after one or two mannose residues have been cleaved (shenkman et al., ) . mannose removal is achieved using er mannosidase i (ermani), the er degradation-enhancing α-mannosidase-like proteins (edems) and/or the golgi-resident protein man c (gonzalez et al., ; hirao et al., ; olivari et al., ; hosokawa et al., ) . several lectins, os- and xtp -b, then interact via their mrh domains with the mannose-trimmed proteins, allowing their association with the retrotranslocon (bernasconi et al., ; christianson et al., ; hosokawa et al., ) . os- and xtp -b also associate with different proteases, lonp and carboxypeptidase vitellogenic-like protein (cpvl), respectively, suggesting that some substrates may be partially degraded prior to dislocation (christianson et al., ; olzmann et al., ) . nonetheless, multiple attempts are made to refold proteins before their triage through erad. the role of chaperones includes recognition of inappropriate glycosylation as well as refolding efforts, but proteins delivered to the retrotranslocon may require unfolding and partial proteolysis to allow their transit through the narrow membrane channel (gogala et al., ) . non-glycosylated proteins can be subjected to erad, but detection of misfolding of these proteins does not involve calnexin and calreticulin (brodsky, ) . notably, the non-lectin chaperone bip is involved in erad targeting of both types of proteins (ushioda et al., ) , yet also serves to prevent leakage of calcium out of the er lumen (schäuble et al., ) . in addition, targeting of unglycosylated proteins to the proteasomes involves edem (shenkman et al., ) , which, like bip, recognizes misfolded glycoproteins, as well as the transmembrane herp protein (usa p in yeast; okuda-shimizu and hendershot, ) . both glycosylated and their non-glycosylated derivatives are recruited to the erderived quality control compartment (erqc) near the nucleus in the presence of a proteasomal inhibitor (shenkman et al., ) . thus, these studies suggest that targeting of misfolded proteins for erad is similar for glycoproteins and non-glycosylated proteins (shenkman et al., ) . interaction of lectin-type and other chaperones with erad substrates allows association with members of the protein disulfide isomerase (pdi) family, which generally are characterized by one or more thioredoxin-like motifs (cxxc; brodsky and skach, ) . interestingly, these proteins can form, break, or rearrange disulfide bonds as well as act as chaperones (benham, ) . the yeast pdi family is composed of five members (pdi , mpd , mpd , eug , and eps ), although only pdi is essential (farquhar et al., ) . in mammalian cells, pdi is one of the best characterized family members, but there are at least such enzymes (benham, ; grubb et al., ) . pdi family proteins are generally confined by a kdel retention sequence (benham, ) to the er, which has an oxidizing environment (costantini et al., ) . the oxidoreductase erp , which is localized near the er-golgi intermediate compartment (ergic), may provide some protection for proteins that might be routed for erad by calnexin (frenkel et al., ) . in addition, some pdi members can escape the secretory system and appear at the cell surface (benham, ) . for example, a disintegrin and metalloproteinase (adam ; also known as tumor necrosis factor alpha-converting enzyme or tace) has been shown to be regulated by an extracellular activity of pdi (bass and edwards, ; willems et al., ; düsterhöft et al., ) . pdi members also have a role in erad, with different requirements for different substrates (grubb et al., ) . in hepatic cells, pdi promotes the folding of apolipoprotein b (apob) figure | the erad process. (a) substrate recognition. many nascent polypeptides (curved line) have one or more high-mannose carbohydrates (shown as a branched structure), which must be recognized and processed in a timely manner to allow exit from the er. binding of these er-luminal proteins to substrates is affected by folding to their native conformations. folding involves formation and breakage of disulfide bonds by members of the pdi family, such as erp and erp , and is facilitated by chaperone proteins, such as bip. specific carbohydrates are bound by different chaperones/lectins in the er lumen. these proteins include ermani, edem, os- , xtp -b, calreticulin, and calnexin. recognition of erad substrates probably results in assembly of the retrotranslocon (shown here as herp and the translocon/bip complex). herp is thought to facilitate oligomerization of the hrd e ligase. bip binds to a number of glycosylated and non-glycosylated erad substrates and provides a barrier on the er luminal side of the translocon. (b) retrotranslocation. recognition of misfolded or misassembled proteins triggers the assembly of the retrotranslocon. current evidence indicates that multiple types of retrotranslocons are possible (see text). a typical retrotranslocon/dislocon is shown containing derlin, the e ligase hrd and its partner sel l, which then recruits the cytosolic atpase p . derlin has transmembrane domains with both the n-terminus and c-terminus in the cytosol. presumably some or all of the recognition components, such as pdi and ermani, disengage as the substrate passes through the translocon. all retrotranslocation events appear to involve p . the retrotranslocon is shown with bip opening the sec channel for substrate passage into the cytosol. (c) ubiquitination of erad substrates. retrotranslocation exposes erad substrates to cytosolic e (unknown), e (shown here as ube g ), and e enzymes (e.g., hrd ). a polyubiquitin chain is produced as the substrate is engaged by the e and e proteins. multiple e s may be responsible for the polyubiquitin chains that then bind to the p partner proteins, ufd and npl . the substrate is shown moving through the translocon into the center of the p hexamer. (d) proteasomal degradation. once the substrate has been retrotranslocated, the bip protein seals the luminal side of the translocon. the retrotranslocon may then be disassembled prior to engagement of a new substrate. the retrotranslocated proteins must be modified by removal of carbohydrate and ubiquitin chains for insertion into the narrow channel of the proteasome. it is possible that p substitutes for the s lid, which provides access to the proteasome channel and the energy for unfolding of substrates. degraded polypeptides are shown emerging from the s lid. this model suggests that there are retrotranslocon-specific proteasomes. www.frontiersin.org through its chaperone activity, whereas erp or erp expression leads to erad (grubb et al., ) . further, various cell types express different pdi proteins, allowing differential regulation of substrates (benham, ; pescatore et al., ) and, presumably, their erad targeting. protein folding involves both formation of disulfide bonds and cis/trans isomerization of peptide bonds preceding proline residues (hebert and molinari, ) . certain erad substrates appear to be dependent on proline isomerization (bernasconi et al., b) , and such refolding events may be necessary for transit through the retranslocon by elimination of turns in substrate secondary structure (määttänen et al., ) . erad requirements for peptidyl-prolyl cis/trans isomerases (ppis) depend on whether the substrate is strictly in the er lumen or is tethered to the er membrane (bernasconi et al., b) . the ppi protein cyclophilin b was needed for erad of a luminal target, but not the same target with a transmembrane domain (bernasconi et al., b) . requirement for ppis during erad may depend on proline residues in the cis configuration (bernasconi et al., b) , potentially by conversion into trans peptidyl-prolyl bonds, thus eliminating secondary structures that hinder retrotranslocation (määttänen et al., ) . mammalian cells have erad factors that are not present in yeast. as observed for other pathways (tsai and weissman, ) , erad components identified in yeast have multiple family members in higher eukaryotes; e.g., instead of a single derlin in yeast (der p), mammalian cells have three proteins (derlin- , - , and - ; oda et al., ) . derlins are multiple membranespanning domain proteins that have been proposed to be part of the retrotranslocon channel and/or regulatory factors for retrotranslocation (brodsky, ; figure b ). in addition, derlin- has a cell-type specific distribution (oda et al., ) , suggesting that recognition of certain substrates may be involved in its function. derlins are related to rhomboid proteases, such as rhbdl , which is an er-resident transmembrane protein that cleaves unstable single-membrane-spanning or polytopic membrane proteins (fleig et al., ) . rhbdl also is upregulated by er stress and binds to the cytosolic aaa atpase p (see below; fleig et al., ) . in contrast to the rhomboid proteases, the derlins lack proteolytic activity, suggesting that these proteins bind to erad substrates and target them to e ligases for ubiquitination and to p for membrane extraction (brodsky, ) . cleavage of erad substrates by rhbdl (fleig et al., ) , sp peptidase (spp; loureiro et al., ) , or proteases associated with os- and xtp -b (olzmann et al., ) may occur prior to retrotranslocation of some substrates (tsai and weissman, ) . on the other hand, it has been proposed that derlins form a six-transmembrane structure with a gate that allows association and unfolding of substrates or access to other retrotranslocon components, such as p (see below; olzmann et al., ) . the p atpase (cdc in yeast) is bound to derlin- and derlin- through their shp domains (greenblatt et al., ) . suppressor/enhancer of lin -like (sel l) appears to link luminal factors that recognize misfolding and inappropriate glycosylation, such as os- , xtp -b, edems, erdj , and the pdi protein erp , to components of the retrotranslocon (olzmann et al., ; williams et al., ) . the transmembrane sel l protein (hrd p in yeast) also participates in regulation of erad by sequestering edem and os- into er-derived vesicles known as edemosomes (bernasconi et al., a) . inducible knockout of sel l in mice leads to death of adult mice from acute pancreatic atrophy (sun et al., ) . sel l expression is required for stability of the e ligase hydroxymethylglutaryl reductase degradation protein (hrd ), and its loss leads to er stress and attenuates translation, leading to cell death. other proteins have been described, such as erlins and and tmub , which may act as adapters between polytopic membrane substrates and e ligases (olzmann et al., ) . the ubiquitin ligases (e s) have been proposed to be a structural part of the retrotranslocon channel (brodsky, ) , but their role is considerably more complex ( figure c ). several e ligases associated with erad are multiple membranespanning proteins with cytosolic ring domains (smith et al., ; ruggiano et al., ) . in yeast, where erad has been studied most extensively, a prototypical transmembrane e , such as hrd p (also called syvn ; nadav et al., ; kikkert et al., ) , can promote erad of a luminal substrate (erad-l). the erad process also involves hrd p (sel l in metazoans) as well as usa p and der p (carvalho et al., ) . herp may assist with hrd oligomerization (carvalho et al., ) , nevertheless, the other components appear to be dispensable if hrd p is overexpressed, consistent with a role for hrd p in erad substrate transfer across the membrane (carvalho et al., ) , although such overexpression may be toxic due to inappropriate protein degradation (denic et al., ) . thus, protein adapters appear to be necessary to achieve substrate specificity (smith et al., ) . hrd p-mediated erad requires oligomerization and transmembrane domains as well as ubiquitin ligase activity (carvalho et al., ) . overexpression of a dominant-negative ring mutant of the hrd ligase prevented erad of a non-glycosylated substrate, but a dominant-negative fbs mutant (a component of scf e ligases) did not (shenkman et al., ) . dependence on hrd also is affected by tethering of the substrate to the er membrane. splice variants of the human beta-site amyloid precursor cleaving enzyme (bace) with the same deletion mutation in the ectodomain are degraded through hrd if they are luminal (erad-l s substrates), but disposal occurs in a hrd independent manner if the variant has a transmembrane domain (erad-l m substrates; bernasconi et al., a) . therefore, hrd recognizes substrates for ubiquitination and, perhaps, modifies the translocon in the er membrane. multiple e ligases participate in erad. these ligases include the transmembrane proteins gp /amfr (fairbank et al., ), trc (stagg et al., ) , rma /rnf (el khouri et al., ) , march /teb (doa in yeast; kreft and hochstrasser, ; olzmann et al., ) , and chip (matsumura et al., ). an additional − membrane-spanning e s may be involved in erad (stagg et al., ) . other e ligases associated with erad frontiers in microbiology | virology are localized to the cytosol, where they recognize misfolded glycoproteins that already have been retrotranslocated (yoshida et al., ; shenkman et al., ) . these ubiquitin ligases are members of the cytosolic scf (s-phase kinase-associated protein (skp )-cullin (cul )-f-box) family, where the f-box components of the scf complex recognize the n-glycans of the retrotranslocated substrate, e.g., fbs and fbs (yoshida, ) . furthermore, e s may work together to direct substrates for degradation (olzmann et al., ) . the p protein (cdc in yeast) is a member of the aaa atpase family (erzberger and berger, ) that functions during erad in a complex with several cofactors that have a ubiquitin-x (ubx) or ubx-like domain (schuberth and buchberger, ; figure ). these cofactors include the heterodimer nuclear protein localization homolog (npl )-ubiquitin fusion degradation (ufd ; meyer et al., ; wolf and stolz, ) , p , ubxd , ubxd , ufd /plaa, vcip , and ataxin- (meyer et al., ) . the ufd l and npl proteins are believed to form a heterodimer, where npl is needed to stabilize ufd l (nowis et al., ) . the heterodimer acts as a substrate adapter to the p atpase associated with the retrotranslocon (bays and hampton, ) . ufd l and npl bind to k -linked and k -linked polyubiquitin chains, respectively, which have been added by e ligases associated with the retrotranslocon (ye et al., ; komander et al., ) . in yeast, the cdc atpase binds to the hrd e ligase in a ring-dependent manner (hampton and sommer, ) , and the transmembrane ubx (sel ) protein acts as an adapter using a uba domain (neuber et al., ; schuberth and buchberger, ) . several other ubiquitin ligases bind p directly or through cofactors (alexandru et al., ) . the p cofactors act as ubiquitin-binding proteins, although p also has ubiquitin-binding activity (ye et al., ; meyer et al., ) . the adapter-p complexes may recognize different substrates and perform independent functions, such as membrane protein segregation and trafficking, as well as directing substrates to the proteasome (ritz et al., ) . alternatively, other models suggest that derlins are involved in unfolding of substrates as well as providing contacts with p and its associated factors (greenblatt et al., ) . the p atpase binds ubiquitin chain editors that can extend shorter chains as well as deubiquitinating enzymes (dubs; jentsch and rumpf, ; sowa et al., ) . two atpase domains (d and d ; meyer et al., ) within p form two stacked hexameric rings that provide the energy for protein remodeling and substrate extraction from the membrane or through the retrotranslocon (hampton and sommer, ) . mutations in the d domain result in dominant-negative proteins that bind, but fail to release, substrates (pye et al., ) . mutant proteins have been widely used to study p function in erad and its myriad other activities (meyer et al., ) . cytosolic chaperones, such as hsp , also may provide energy for extraction of membrane proteins with misfolded cytoplasmic domains (erad-c substrates; taxis et al., ; hrizo et al., ) . once extraction from the er membrane has occurred, p recruits peptide n-glycanase (pngase) to cleave n-linked glycans from glycosylated substrates (hirsch et al., ; li et al., ; figure d ). in addition, p binds to a deubiquitinating enzyme yod , presumably so that polyubiquitin chains will not interfere with insertion into the proteasome (ernst et al., ) . the proteasome is a highly complex structure with a s lid that has an atpase activity very similar to that of p (lipson et al., ; matouschek and finley, ) . these enzymes may function synergistically to deliver substrates to the s core (hampton and sommer, ) . alternatively, p may deliver certain substrates directly to the proteasome core (matouschek and finley, ) . the proteasome core is composed of subunits arranged into four rings, each composed of seven subunits (bhattacharyya et al., ) . proteolytic activity is sequestered in the center of a narrow chamber formed by the rings and, therefore, only unfolded proteins can enter the chamber (groll et al., ) . the s lid, p , or other activators provide docking for substrates and substrate modifying proteins as well as regulated opening of the chamber to allow access of unfolded proteins for degradation in the s core (bhattacharyya et al., ) . many questions remain about erad components and how they identify and interact with different substrates. similar to our analysis of other cellular and molecular biological processes through virology, studies of viruses that use erad are likely to prove insightful. the ability of viruses to cause persistent infections is a consequence of downregulation or subversion of the immune response. the herpesviruses are known to cause persistent infections. one well-studied example of herpesvirus manipulation of the immune response is reduced cell expression of major histocompatibility complex class (mhc-i) molecules by the viral proteins us and us (wiertz et al., ) . both proteins are transmembrane glycoproteins and bind to newly made mhc-i to initiate retrotranslocation. despite their similar function, us and us use different pathways for mhc-i degradation (figure ) . us mediated degradation of mhc-i is independent of derlin- and involves spp (loureiro et al., ) , which cleaves many sps following their removal from nascent er-bound pre-proteins (voss et al., ) . using an sirna screen, trc was identified as the e ligase involved in mhc-i degradation by us , but knockdown of this transmembrane ring-type e had no effect on us mediated destruction of mhc-i (stagg et al., ). the us cytosolic tail interacts with spp and the p atpase (chevalier and johnson, ; loureiro et al., ) , whereas trc and us bind through their transmembrane domains (stagg et al., ; figure a) . unlike the derlin-independent mechanism proposed for us , studies of the us protein facilitated identification of derlin- and sel l as erad components (figure ; lilley and ploegh, ; ye et al., ; mueller et al., ) . us does not require spp for mhc-i degradation (loureiro et al., ), but appears to interact with the e ligase marchvii/axotrophin (flierman et al., ) . the cytosolic domain of mhc-i is required for us mediated erad targeting (story et al., ; barel et al., ) , and deletion of the c-terminal valine of mhc-i reduced interaction with derlin- (cho et al., a) . the er luminal domain www.frontiersin.org also affects degradation (barel et al., ) . in addition, mhc-i substituted with the transmembrane domain of us caused interaction with derlin- and proteasomal degradation (cho et al., b) . the p atpase does not appear to interact directly with mhc-i, but requires the interaction of mhc-i cytosolic domain with the c-terminal domain of derlin- (cho et al., a) . cho et al. speculated that us recognizes mhc-i through its cytosolic domain and transfers it to derlin- , which then interacts with the p atpase for membrane dislocation (cho et al., a ; figure b ). therefore, studies of the herpesvirus us and us proteins revealed that the same substrate does not always use the same erad pathway, and presumably these viral proteins act as adapters that recognize different parts of mhc-i for targeting to the dislocon. herpesviruses use another mechanism to decrease levels of mhc-i. the mouse gammaherpesvirus (mhv ) encodes an e ligase (mk ) that ubiquitinates newly made mhc-i heavy chains for proteasomal degradation (boname and stevenson, ) . the mk ligase also is associated with the transporterassociated with antigen processing (tap) as well as p and derlin- (wang et al., ) . polyubiquitination of mhc-i did not require lysines , but could occur on serine and threonine residues in the heavy chain c-terminal tail via the recruitment of the ube j e enzyme (see figure ; wang et al., wang et al., , herr et al., ) . these data indicate that multiple erad mechanisms can be used by viruses to diminish the adaptive immune response. like the herpesviruses, retroviruses also manipulate the immune system through erad. early studies indicated that human immunodeficiency virus type (hiv- )-infected cells had decreased levels of both cd mrna and protein (hoxie et al., ) . cd acts as the receptor for binding the viral envelope (env) protein (mcclure et al., ) . furthermore, cd participates in t-cell activation by binding to both the t-cell receptor and mhc class ii molecules on antigen-presenting cells. cd + t cells secrete cytokines that control antibody production, phagocytic cell function, and cytotoxic t-cell responses, making them crucial for adaptive immune responses (tubo and jenkins, ) . hiv- encodes a number of accessory proteins, including vpu, which are not required for virus replication in tissue culture, but contribute to viral pathogenesis (strebel, ) . expression of vpu and cd by transient transfection showed dramatic decreases in cd levels, and cd depletion was dependent on serines and in vpu (magadán et al., ) . vpu-induced cd degradation has been shown to involve the erad system. knockdown of both β-trcp and β-trcp largely prevented vpu-mediated cd loss (magadán et al., ) . β-trcp and β-trcp (also known as fbw a, fbxw , fbxw a, or fwd ) are f-box proteins containing wd domains, which are associated with the scf family of ubiquitin ligases (figure ) . these protein complexes are linked to regulation of multiple pathways involving cell cycle checkpoints, nfκb, and wnt (skaar et al., ) . in addition, knockdown of p , ufd l (also called ufd ) or npl (see figure c ) blocked depletion of cd (magadán et al., ) . mutations that prevented atp binding or hydrolysis by p failed to affect cd levels (magadán et al., ) . these experiments indicated that vpu uses erad to degrade cd , but also prevents cell surface expression by retaining cd in the er, probably through transmembrane domain interactions (magadán et al., ) . moreover, vpu used an atypical e ligase to induce erad (margottin et al., ) , and this process involved scf β−trcp ubiquitination of the cd cytosolic tail on lysine, serine, and threonine residues (magadán et al., ) . thus, vpu may act as an adapter between cd , retrotranslocon components, and a cytosolic e ligase. cd degradation promotes hiv- infection by preventing re-infection, facilitating virus release by avoiding env-cd interactions during their trafficking to the cell surface, and minimizing adaptive immune responses (lanzavecchia et al., ; willey et al., ; argañaraz et al., ) . hiv- vpu also targets another cellular protein, tetherin/bst- , for erad (neil et al., ; mangeat et al., ) . tetherin is an unusual type ii membrane protein with an n-terminal frontiers in microbiology | virology knockdown of both β-trcp and β-trcp (shown to be contacting vpu) can prevent cd degradation, suggesting that either f-box protein can provide a functional scf complex for ubiquitination (magadán et al., ) . another e ligase (e ?) also may be involved. the p atpase with the adapters ufd and npl are required for cd degradation, but the ufd l protein recognizes polyubiquitinated cd . lysine and serine/threonine residues in the cd cytosolic tail are needed for ubiquitination (magadán et al., ) . transmembrane segment and a c-terminal gpi anchor (kupzig et al., ; sauter, ) . moreover, two tetherin monomers are bound together by disulfide bonds (ishikawa et al., ; kupzig et al., ) . using a unique method that only allows biotinylation of retrotranslocated molecules by cytosolic bira protein, recent experiments indicate that both cd and tetherin remain glycosylated and retain disulfide bonds during retrotranslocation (petris et al., ) . these data suggest that the typical sec channel used for translocation is insufficiently wide to accommodate retrotranslocation substrates modified with these structures (petris et al., ) , but an alternative model involving lipid droplet formation has not been confirmed . given the large number of proteins that have been implicated, a single mechanism for retrotranslocation is unlikely. despite common delivery of substrates to the proteasome via the p atpase, each of the previous examples of viral erad targeting involves different e ligases. recent evidence suggests that erad can target the retrovirus hiv- env (zhou et al., ) , a glycosylated transmembrane protein. studies of a human cd + t-cell line cem.nkr indicated that hiv- replication is restricted in these cells, which also are resistant to natural killer cell-mediated lysis (howell et al., ) . surprisingly, these cells overexpressed a mitochondrial translocator protein, tspo (braestrup and squires, ; papadopoulos et al., ) , and knockdown or knockout of this protein rescued env and hiv- production (zhou et al., ) . further experiments indicated that drugs inducing erad led to recovery of env levels and viral titers. these results suggested that the er and mitochondria communicate through juxtaposition of their membranes, so that conditions in the mitochondria influence protein folding and erad. in support of this conclusion, gp is an erad-associated e ligase (fang et al., ) localized to mitochondria-er membrane contacts (fu et al., ) . thus, mitochondria proteins may influence erad and modulate hiv- env presentation to the immune system. triggering of an innate immune response to viruses is affected by the erad process. some anti-viral signaling is controlled through mitochondria, which also cooperates with the er for lipid synthesis and calcium-controlled processes at the mitochondrialassociated membrane (mam; jacobs et al., ) . mitochondrial antiviral signaling protein (mavs; also called ips- , visa, or cardif) binds to different retinoic acid-inducible gene-i (rig-i)-like receptor (rlr) proteins, which sense cytosolic viral rnas (kawai et al., ; meylan et al., ; seth et al., ; xu et al., ) . the mavs protein is present in the mitochondrial and peroxisomal membranes, and viral rna triggers both interferondependent or independent responses, respectively, (jacobs and coyne, ; jacobs et al., ) . the levels of mavs are affected by gp , an e ubiquitin ligase that is localized to the ermitochondrial interface (mam; jacobs et al., ) . the gp ligase was detected by a high throughput rnai screen to identify genes that restricted enterovirus replication (coyne et al., ) . downregulation of gp was shown to decrease yields of vesicular stomatitis virus (vsv) and to increase type i interferon responses. some viruses, such as those inducing hepatitis b (hbv) or c (hcv), use erad to reduce the amounts of glycoproteins and particles produced. interestingly, both viruses partially induce the unfolded protein response (upr; li et al., li et al., , saeed et al., ) , which then increases the levels of certain erad components. hbv, a member of the hepadnaviridae, triggers upregulation of the glycoside hydrolase family enzymes, edem and . increased edem levels appear to bypass normal er folding of hbv glycoproteins to result in erad (lazar et al., ) . hcv, a member of the flaviviridae, induces primarily edem through the upr and splicing of x-box binding protein . further experiments suggested that elevated levels of edem and increase binding to sel l, an adapter to the retrotranslocon (figure ) . inhibition of edem binding to sel l interfered with ubiquitination of hcv env protein, e (saeed et al., ) . interestingly, infections by another member of the flaviviridae, japanese encephalitis virus, did not result in edem binding to the env proteins, indicating that not all viral family members control env proteins by this mechanism. overall, manipulation of edem levels appears to be a common mechanism to reduce viral glycoprotein levels. lowered amounts of env proteins and virus particles then contribute to avoidance of innate and adaptive immunity, leading to chronic infections (saeed et al., ; lazar et al., ) . a number of pathogens harness the erad process to facilitate various replication strategies. the best known examples are the bacterial ab toxins, particularly cholera toxin, which is thought to hijack the erad machinery for delivery to the cytosol (hazes and read, ) . cholera toxin has a catalytic a chain divided into two subunits (cta and cta ) inside a pore composed of five receptor-binding b subunits (spangler, ) . the holotoxin www.frontiersin.org binds to the ganglioside gm on the surface of gut epithelial cells, which then triggers toxin internalization and trafficking through the golgi to the er (fujinaga et al., ) . the a subunits are bound to the b subunits by disulfide bonds, and the toxin complex interacts with the er-resident enzyme pdi (figure ) . pdi is a redox-dependent chaperone that unfolds the toxin, which is then released in the oxidized state (tsai et al., ) . this unfolding event appears to be required for the ability of cta to retrotranslocate to the cytosol, where it induces the adp-ribosylation of the gαs protein and, ultimately, opening of chloride channels leading to massive diarrhea (muanprasat and chatsudthipong, ) . as noted above, retrotranslocation of erad substrates is preceded by a recognition step. the chaperone bip, which is known to be involved in identification of non-glycosylated erad substrates, and an er-resident atpase (torsin a) promote cta retrotranslocation (tsai et al., ; winkeler et al., ; forster et al., ; moore et al., ) . sel l and erdj , a co-chaperone of bip, also facilitate cta retrotranslocation, where the j domain of erdj is required (williams et al., ) . erdj also binds to sel l, likely providing interaction with the hrd e ligase (see figure ) . torsin a may provide the link to the membrane-resident derlin- protein (nery et al., ) . cta retrotranslocation appears to involve derlin- (bernardi et al., ) and the transmembrane ubiquitin ligases, hrd and gp . thus, multiple low affinity interactions are likely involved in the identification of cta as a substrate and its delivery to the retrotranslocon. similar to other retrotranslocated substrates, the cytosolic p atpase participates in cta extraction from the er membrane (abujarour et al., ; kothe et al., ) . nevertheless, cta subverts the normal erad process by avoiding polyubiquitination (rodighiero et al., ) . the hypothesis that cta avoids ubiquitination through the absence of lysines targeted for polyubiquitination was not substantiated by mutational analysis (rodighiero et al., ) . these results indicate that cta employs many of the typical components used for erad targeting, including the e ligase, but it is unclear how polyubiquitination and degradation of the substrate are avoided. therefore, retrotranslocon targeting and substrate extraction from the er membrane is not necessarily coupled to ubiquitination, although ubiquitination may be required for proteasomal degradation. viral pathogens also use erad. mouse mammary tumor virus (mmtv) is a betaretrovirus that subverts the erad process to complete its viral replication cycle. all retroviruses synthesize an unspliced viral rna that requires export from the nucleus to the cytosol for translation or packaging into virus particles (cullen, ) . the unspliced rnas of simple retroviruses have a highly structured cis-acting sequence, such as the constitutive transport element (cte) of mason-pfizer monkey virus (mpmv; bray et al., ) . the cte facilitates rna export through the typical tap/nxf -mediated pathway used by cellular mrnas (grüter et al., ) . in contrast, the complex retroviruses encode an adapter protein, such as the rev protein of hiv- (hanly et al., ) , which binds to a structured rna element near the end of the genome (daly et al., ; zapp and green, ) . mmtv also produces a rev-like protein, rem, for export of unspliced rna (mertz et al., ) , but rem binding to viral rna has additional translation-associated functions (mertz et al., b) . unlike other complex retroviruses, rem is made from an internally deleted form of the env protein, and the export function resides in a long sp of amino acids (indik et al., ; mertz et al., ) . interestingly, rem is a precursor protein that is directed to the er membrane for translation, where it appears to be cleaved by signal peptidase into the rev-like rem-sp and a c-terminal glycosylated product (rem-ct) of unknown activity (byun et al., ) . recent evidence indicates that rem-sp uses retrotranslocation for extraction from the er membrane, but, like cholera toxin, avoids proteasomal degradation (byun et al., (byun et al., , . dultz et al. ( ) first reported that rem is directed to the er membrane for translation and cleavage by signal peptidase. they also suggested that the rem precursor (the uncleaved protein) could be detected in the nucleus by fluorescence microscopy (dultz et al., ) . byun et al. ( ) showed that mutation of the predicted signal peptidase cleavage site prevented the appearance of rem-sp as detected by both western blotting and a highly sensitive reporter assay for rev-like function (mertz et al., ; byun et al., ) . this assay requires binding to a specific rna element in viral rna (müllner et al., ; mertz et al., a) . fluorescence experiments indicated that only the cleaved rem-sp enters the nucleus, whereas the uncleaved form was highly unstable and localized to the cytosol (byun et al., ) . furthermore, rem-sp activity was inhibited by expression of a dominant-negative form of the p atpase required for retrotranslocation (byun et al., ) . rem-sp function also was reduced by the expression of a dominant-negative derlin- , but not derlin- protein (byun et al., in preparation) . these results strongly suggest that rem must be cleaved by signal peptidase prior to sp retrotranslocation to the cytosol and import into the nucleus for rna binding (figure ) . experiments indicate that an altered conformation of either the n-terminal rem-sp in the cytosol or the er-luminal portion of rem affect folding and accessibility to signal peptidase, which is associated with translocons (falk and gilula, ) . first, rem tagging at the c-terminus with green fluorescent protein (rem-gfp) resulted in a stable protein that was inefficiently cleaved and had little fluorescence (mertz et al., ; byun et al., ) . rem-gfp also had very low functional activity in reporter assays (mertz et al., ) . in contrast, rem tagged at the n-terminus with gfp was cleaved normally, and gfp-rem-sp localized to the nucleoli, a result typical of other rev-like proteins (cullen, ; mertz et al., ) . second, deletion mutations of the rem c-terminus greatly affected stability of the protein (byun et al., ) . removal of the c-terminal amino acids had little effect on the cleavage or stability of the protein, but deletion of or amino acids produced a highly unstable precursor that could be rescued by the proteasomal inhibitor mg- (byun et al., ) . reduced cleavage of the precursor also was observed. surprisingly, further deletion to give only the sp (rem-sp) again yielded a stable protein (byun et al., ) . third, substitution of the leucine at position in the sp gave a stable precursor protein that was poorly cleaved by signal peptidase (mertz et al., a; byun et al., ). an independent report indicated that residues frontiers in microbiology | virology rem is a precursor protein that has an n-terminal signal peptide (rem-sp) that directs translation to the er membrane. the rem-ct enters the er lumen, where it is modified by n-glycosylation on two different sites. rem recognition for retrotranslocation is not understood, but appears to involve derlin- and, potentially, an e ligase, although ubiquitinated rem has not been observed. full-length rem is cleaved by signal peptidase, and rem-ct is released into the er lumen. similar to other retrotranslocation substrates, rem-sp is extracted from the er membrane using the p atpase. despite its dislocation into the cytosol, rem-sp escapes the proteasome and translocates into the nucleus for binding of mmtv rna. this figure is adapted from byun et al. ( ) . through act as the hydrophobic membrane anchor sequence, suggesting that position is localized in the cytosol (dultz et al., ) . recognition of rem c-terminal sequences in the er lumen, presumably by their interaction or lack of interaction with specific chaperone proteins, prevent degradation by erad. the er-luminal chaperone bip has repeatedly been detected after purification and proteomic analysis of rem-binding proteins (gou et al., manuscript in preparation) . our preliminary data indicate that rem-sp is not ubiquitinated, and it is possible that this feature protects rem-sp from proteasomal degradation. since the rem precursor and c-terminal deletion mutants are subject to erad, cleavage and association with specific cellular proteins appear to be critical for avoidance of the degradative process. the idea that viral proteins manipulate e enzymes to form alternative complexes (olzmann et al., ) would be consistent with rem-sp escape from erad. the polyomaviruses have a unique entry method that uses retrotranslocation, while avoiding erad. the bk polyomavirus (bkv) first binds to the ganglioside receptors gt b and gd b and enters through caveolae (neu et al., ) , which are composed of membrane microdomains/lipid rafts that are enriched for sphingolipids and signaling molecules (head et al., ; figure ) . particle delivery to the cytosol occurs through a phdependent step involving endosomal trafficking via microtubules to the er (eash and atwood, ; moriyama and sorokin, ; jiang et al., ) . other members of the polyomaviridae use caveolae-independent entry for er delivery (neu et al., ) . er localization of these viruses is necessary to access specific retrotranslocation components. the vp capsid proteins of polyomaviruses form pentamers during assembly that are held together by disulfide bonding (li et al., ) . each pentamer is associated with one molecule of either the minor capsid protein vp or vp (barouch and harrison, ) , which become accessible to antibodies after exposure to the unique environment of the er (norkin et al., ) . particle delivery into the er allows reduction and isomerization of disulfide bonds using erp (mouse polyomavirus; magnuson et al., ) or erp and pdi (sv ; schelhaas et al., ) to allow partial uncoating (jiang et al., ; tsai and qian, ) . the partially uncoated virion then engages the retrotranslocation machinery to allow cytosolic entry similar to cholera toxin (neu et al., ) . interestingly, different polyomaviruses use distinct derlin family members for retrotranslocation. sv uses derlin- and sel l (schelhaas et al., ) , whereas mouse polyoma virus uses derlin- ; figure ) . additional experiments indicate that exposure of vp hydrophobic sequences tethers virus particles to the er membrane, and that both bip and bap are needed for dislocation of sv to the cytosol (geiger et al., ) . bap may serve as a shuttle to the erqc that has been associated with enriched erad components (kamhi-nesher et al., ; wakana et al., ) . furthermore, use of epoxomicin or eeyarestatin , inhibitors of the proteasome or p atpase, respectively, blocked early events of bkv infection (bennett et al., ) . epoxomicin treatment of cells allowed accumulation of bkv in the calnexin-rich, bip-deficient erqc (bennett et al., ) . these results are consistent with erad extraction of polyomaviruses from the er to the cytosol, although it is has been suggested that www.frontiersin.org figure | use of erad for polyomavirus uncoating. many polyomaviruses enter through caveosomes that are enriched for viral entry receptors, triggering particle uptake through endosomes. using the microtubule network, vesicles traffic the virus to the er, where the unique environment allows structural changes to the icosahedral capsids. studies of jcv, bkv, and sv indicate that viral particles interact with pdi and erp in the er lumen to rearrange capsid proteins. in contrast, the related mouse polyomavirus (pyv) uses the pdi family member, erp , presumably for a similar function. the altered particles then appear to engage different retrotranslocons (dependent on either derlin- or derlin- ) to induce retrotranslocation to the cytosol, where the reduced calcium environment produces further capsid rearrangements. these particles then bind to the nuclear pore where uncoating occurs to allow passage of viral dna into the nucleus. this figure is adapted from neu et al. ( ). there are cell-type and virus-specific differences and that direct er to nuclear transport may occur (bennett et al., ) . low levels of calcium in the cytosol lead to further capsid destabilization and exposure of the nuclear localization signals on capsid proteins. the partially uncoated capsid then transits through the nuclear pores for initiation of viral dna replication (neu et al., ) . the preceding experiments indicate that erad is used by viruses to allow trafficking events that promote replication. mmtv rem trafficking through the er allows access to signal peptidase and cleavage of rem precursor into functional n-and c-terminal proteins. in contrast, the polyomaviruses use erad to partially uncoat virions on their path to the nucleus. importantly, both types of viruses avoid proteasomal degradation during erad, although the mechanisms remain unclear. erad may be regulated or "tuned" through the rapid turnover of specific components through the proteasomes or autophagosomes/vesicular trafficking to lysosomes (merulla et al., ) . normal secretory vesicles released from the er are - nm in diameter and have coatamer proteins, such as copii, whereas the er-derived tuning vesicles (edemosomes) lack coatamers and are - nm in diameter (bernasconi et al., b) . tuning vesicles contain sel l, edem , and os- , which are transmembrane or luminal proteins involved in erad (figure ; olzmann et al., ) . edemosomes are believed to reduce erad by disposal in acidic organelles (bernasconi et al., b) , favoring the correct folding of polypeptides (calì et al., ) . the coronaviruses are known to take advantage of erad tuning (reggiori et al., ) . many plus-stranded rna-containing viruses manipulate cellular membranes to further rna replication (paul and bartenschlager, ) . these membrane structures have been divided into invaginated vesicle/spherule type and double-membrane vesicle (dmv) type (two lipid bilayers). such vesicles allow viruses to concentrate their replication components, to separate distinct viral processes (e.g., translation, transcription, and replication), and to avoid immune detection (paul and bartenschlager, ) . severe acute respiratory syndrome coronavirus (sars-cov) and mouse hepatitis virus (mhv) induce dmvs for targeting their replication and transcription (reggiori et al., ) . the dmvs originate from er membranes and contain the non-structural transmembrane proteins nsp and nsp and viral double-stranded rna (stertz et al., ; reggiori et al., ) . nevertheless, dmvs lack markers typical of the ergic or the golgi (oostra et al., ) . recent experiments indicate that dmvs are coated with microtubule-associated protein light chain [lc ; atg in yeast (reggiori et al., ) ], which is a ubiquitin-like modifier (van der veen and ploegh, ). lc can exist in a lipidated form (covalent linkage to phosphatidylethanolamine; also known as lc -ii) or a predominantly cytosolic non-lipidated form (lc -i). lc -ii is believed to be involved in fusion of autophagosomes to lysosomes (van der veen and ploegh, ), but coronavirus dmvs display the non-lipidated lc -i (reggiori et al., ) . these ubiquitinlike modifiers recognize specific receptors that target associated vesicles to particular cellular locations (van der veen and ploegh, ). the coronaviruses appear to be redirecting vesicles destined for autophagosomes to sequestered locations in the cytosol where replication will occur. the autophagy machinery is not required for coronavirus replication, and no colocalization of viral non-structural proteins was observed with lc -ii-coated autophagosomes (reggiori et al., ) . coronavirus-induced dmvs and edemosomes both are coated with the non-lipidated lc -i protein (calì et al., ; reggiori et al., ) , which is not covalently attached to membranes like lc -ii (kabeya et al., ) . induction of autophagy in coronavirus-infected cells with rapamycin decreased the levels of edem and coronavirus (reggiori et al., ) . the viruscontaining dmvs had both edem and os- , but not other erad-associated chaperones, and virus infection interfered with erad tuning by hijacking the edemosomes. nevertheless, lc -i, but not edem and os- , is necessary for coronavirus infection, and the hijacked edemosome cargo is not degraded by proteases in the endosomes/lysosomes (reggiori et al., ) . further, the erad transmembrane adapter protein, sel l, is needed for dmv formation, capturing the er-resident edem and os- proteins (and possibly xtp -b and edem ), while using its proline-rich cytosolic domain to bind to lc -i. as expected, sel l knockdown impairs coronavirus replication (bernasconi et al., a) . the organizationally similar arterioviruses (classified with coronaviruses, toroviruses, and roniviruses into the order nidovirales; gorbalenya et al., ) subvert edemosome trafficking for their replication, although the size of the vesicles is smaller (monastyrska et al., ) . the mechanism for altering edem containing vesicular trafficking is unclear, but likely involves expression of viral non-structural proteins that span the erderived membranes (monastyrska et al., ) , perhaps through their interaction with sel l. these experiments indicate that viruses hijack edemosomes to sequester their double-stranded rna from cytosolic sensors that will trigger interferon production and innate immunity (zinzula and tramontano, ) . other components of the erad system, particularly chaperone proteins, also participate in the replication and transmission of both plant and mammalian viruses (verchot, ) . the erad system is a complex and highly regulated process controlling the disposal of misfolded or misassembled proteins that are directed to the er for translation. deregulation of this process results in pathogenic conditions, including infectious diseases. viruses exploit erad to decrease overall viral levels and allow establishment of chronic infections by minimizing antigen presentation to the immune system. trafficking of specific viral proteins or entire virion particles may involve erad for refolding or processing in the unique er environment. alternatively, viruses can use erad-associated components to form isolated lipid vesicles for replication and shelter from immune detection. virus-mediated subversion of erad can lead to degradation of molecules that are involved in innate or adaptive immunity. continued studies of viruses are certain to provide additional insights into both the erad process and the components that regulate it. further experiments may identify targets for viral therapeutics. p is in a complex with cholera toxin and influences the transport of cholera toxin and related toxins to the cytoplasm n-glycan structures: recognition and processing in the er ubxd binds multiple ubiquitin ligases and implicates p in hif alpha turnover intracellular folding of tissue-type plasminogen activator. effects of disulfide bond formation on n-linked glycosylation and secretion enhanced cd down-modulation by late stage hiv- nef alleles is associated with increased env incorporation and viral replication functional and genomic analyses of blocked protein o-mannosylation in baker's yeast a network of cytosolic factors targets srp-independent proteins to the endoplasmic reticulum signal peptidase i: cleaving the way to mature proteins amino acid composition of alpha /alpha domains and cytoplasmic tail of mhc class i molecules determine their susceptibility to human cytomegalovirus us -mediated down-regulation interactions among the major and minor coat proteins of polyomavirus adams and protein disulfide isomerase: the key to regulated cell-surface protein ectodomain shedding? biases and complex patterns in the residues flanking protein n-glycosylation sites the protein disulfide isomerase family: key players in health and disease role of cell-type-specific endoplasmic reticulum-associated degradation in polyomavirus trafficking derlin- facilitates the retro-translocation of cholera toxin the e ubiquitin ligases hrd and gp bind to and promote cholera toxin retro-translocation stringent requirement for hrd , sel l, and os- /xtp -b for disposal of erad-ls substrates cyclosporine a-sensitive, cyclophilin b-dependent endoplasmic reticulumassociated degradation role of the sel l:lc -i complex as an erad tuning receptor in the mammalian er unconventional roles of nonlipidated lc in erad tuning and coronavirus infection a dual task for the xbp -responsive os- variants in the mammalian endoplasmic reticulum: inhibiting secretion of misfolded protein conformers and enhancing their disposal regulated protein turnover: snapshots of the proteasome in action mhc class i ubiquitination by a viral phd/lap finger protein specific benzodiazepine receptors in rat brain characterized by high-affinity ( h)diazepam binding a small element from the mason-pfizer monkey virus genome makes human immunodeficiency virus type expression and replication rev-independent n-linked protein glycosylation in the endoplasmic reticulum cleaning up: er-associated degradation to the rescue protein folding and quality control in the endoplasmic reticulum: recent lessons from yeast and mammalian cell systems substrate-specific mediators of er associated degradation (erad) requirements for mouse mammary tumor virus rem signal peptide processing and function retroviral rem protein requires processing by signal peptidase and retrotranslocation for nuclear function segregation and rapid turnover of edem by an autophagy-like mechanism modulates standard erad and folding activities retrotranslocation of a misfolded luminal er protein by the ubiquitin-ligase hrd p the molecular basis of coupling of translocation and n-glycosylation human cytomegalovirus us chimeras containing us cytosolic residues acquire major histocompatibility class i and ii protein degradation properties the c-terminal amino acid of the mhc-i heavy chain is critical for binding to derlin- in human cytomegalovirus us -induced mhc-i degradation forced interaction of cell surface proteins with derlin- in the endoplasmic reticulum is sufficient to induce their dislocation into the cytosol for degradation defining human erad networks through an integrative mapping strategy os- and grp deliver mutant alpha -antitrypsin to the hrd -sel l ubiquitin ligase complex for erad cysteineless non-glycosylated monomeric blue fluorescent protein, secbfp , for studies in the eukaryotic secretory pathway comparative rnai screening reveals host factors involved in enterovirus infection of polarized endothelial monolayers nuclear mrna export: insights from virology specific binding of hiv- recombinant rev protein to the rev-responsive element in vitro a portrait of the get pathway as a surprisingly complicated young man a luminal surveillance complex that selects misfolded glycoproteins for er-associated degradation the signal peptide of the mouse mammary tumor virus rem protein is released from the endoplasmic reticulum membrane and accumulates in nucleoli membrane-proximal domain of a disintegrin and metalloprotease- represents the putative molecular switch of its shedding activity operated by protein-disulfide isomerase involvement of cytoskeletal components in bk virus infectious entry o-mannosylation precedes and potentially controls the n-glycosylation of a yeast cell wall glycoprotein rnf is a novel e ligase of endoplasmic reticulum-associated degradation (erad) that targets cystic fibrosis transmembrane conductance regulator (cftr) the otubain yod is a deubiquitinating enzyme that associates with p to facilitate protein dislocation from the er evolutionary relationships and structural mechanisms of aaa+ proteins the complex biology of autocrine motility factor/phosphoglucose isomerase (amf/pgi) and its receptor, the gp /amfr e ubiquitin ligase connexin membrane protein biosynthesis is influenced by polypeptide positioning within the translocon and signal peptidase access the tumor autocrine motility factor receptor, gp , is a ubiquitin protein ligase implicated in degradation from the endoplasmic reticulum protein disulfide isomerase is essential for viability in saccharomyces cerevisiae ubiquitin-dependent intramembrane rhomboid protease promotes erad of membrane proteins e - k mediates us -triggered retro-translocation of mhc class i heavy chains in a permeabilized cell system protein disulfide isomerase-like proteins play opposing roles during retrotranslocation create and preserve: proteostasis in development and aging is governed by cdc /p /vcp separate roles and different routing of calnexin and erp in endoplasmic reticulum quality control revealed by interactions with asialoglycoprotein receptor chains regulation of mitophagy by the gp e ubiquitin ligase gangliosides that associate with lipid rafts mediate transport of cholera and related toxins from the plasma membrane to endoplasmic reticulm bap and bip are essential for dislocation of sv from the endoplasmic reticulum to the cytosol structures of the sec complex engaged in nascent peptide translocation or membrane insertion identification, expression, and characterization of a cdna encoding human endoplasmic reticulum mannosidase i, the enzyme that catalyzes the first mannose trimming step in mammalian asn-linked oligosaccharide biosynthesis nidovirales: evolving the largest rna virus genome derlin- is a rhomboid pseudoprotease required for the dislocation of mutant α- antitrypsin from the endoplasmic reticulum a gated channel into the proteasome core particle protein disulfide isomerases contribute differentially to the endoplasmic reticulum-associated degradation of apolipoprotein b and other substrates tap, the human homolog of mex p, mediates cte-dependent rna export from the nucleus the delicate balance between secreted protein folding and endoplasmic reticulum-associated degradation in human physiology finding the will and the way of erad substrate retrotranslocation comparative analysis of the htlv-i rex and hiv- rev trans-regulatory proteins and their rna response elements accumulating evidence suggests that several ab-toxins subvert the endoplasmic reticulum-associated protein degradation pathway to enter target cells interaction of membrane/lipid rafts with the cytoskeleton: impact on signaling and function: membrane/lipid rafts, mediators of cytoskeletal arrangement and cell signaling in and out of the er: protein folding, quality control, degradation, and related human diseases role of the ring-ch domain of viral ligase mk in ubiquitination of non-lysine and lysine mhc i residues edem , a soluble edem homolog, enhances glycoprotein endoplasmic reticulum-associated degradation and mannose trimming a role for n-glycanase in the cytosolic turnover of glycoproteins mechanism and components of endoplasmic reticulum-associated degradation human xtp -b forms an endoplasmic reticulum quality control scaffold with the hrd -sel l ubiquitin ligase complex and bip stimulation of erad of misfolded null hong kong alpha -antitrypsin by golgi alpha , -mannosidases natural killing target antigens as inducers of interferon: studies with an immunoselected, natural killing-resistant human t lymphoblastoid cell line alterations in t (cd ) protein and mrna synthesis in cells infected with hiv the hsp molecular chaperone stabilizes apolipoprotein b from endoplasmic reticulum-associated degradation (erad) a novel, mouse mammary tumor virus encoded protein with rev-like properties molecular cloning and chromosomal mapping of a bone marrow stromal cell surface gene, bst , that may be involved in pre-b-cell growth mechanisms of mavs regulation at the mitochondrial membrane regulation of mitochondrial antiviral signaling (mavs) expression and signaling by the mitochondria-associated endoplasmic reticulum membrane (mam) protein gp cdc (p ): a "molecular gearbox" in the ubiquitin pathway? early events during bk virus entry and disassembly the er translocon and retrotranslocation: is the shift into reverse manual or automatic? post-translational translocation into the endoplasmic reticulum trc can deliver short secretory proteins to the sec translocon lc , a mammalian homologue of yeast apg p, is localized in autophagosome membranes after processing a novel quality control compartment derived from the endoplasmic reticulum ips- , an adaptor triggering rig-i-and mda -mediated type i interferon induction human hrd is an e ubiquitin ligase involved in degradation of proteins from the endoplasmic reticulum expression and degradation of the cystic fibrosis transmembrane conductance regulator in saccharomyces cerevisiae molecular discrimination of structurally equivalent lys -linked and linear polyubiquitin chains role of p aaa-atpase in the retrotranslocation of the cholera toxin a chain, a non-ubiquitinated substrate an unusual transmembrane helix in the endoplasmic reticulum ubiquitin ligase doa modulates degradation of its cognate e enzyme bst- /hm . is a raft-associated apical membrane protein with an unusual topology t cells can present antigens such as hiv gp targeted to their own surface molecules activation of erad pathway by human hbv modulates viral and subviral particle production a window of opportunity: timing protein degradation by trimming of sugars and ubiquitins hepatitis b virus x protein (hbx) activates atf and ire -xbp pathways of unfolded protein response the aaa atpase p links peptide n-glycanase to the endoplasmic reticulum-associated e ligase autocrine motility factor receptor subversion of cellular autophagy machinery by hepatitis b virus for viral envelopment characterization of self-assembled virus-like particles of human polyomavirus bk generated by recombinant baculoviruses murine polyomavirus requires the endoplasmic reticulum protein derlin- to initiate infection a membrane protein required for dislocation of misfolded proteins from the er a proteasomal atpase contributes to dislocation of endoplasmic reticulum-associated degradation (erad) substrates protein o-mannosyltransferases associate with the translocon to modify translocating polypeptide chains signal peptide peptidase is required for dislocation from the endoplasmic reticulum protein quality control in the er: the recognition of misfolded proteins multilayered mechanism of cd downregulation by hiv- vpu involving distinct er retention and erad targeting steps erp triggers a conformational change in polyomavirus to stimulate membrane binding hiv- vpu neutralizes the antiviral factor tetherin/bst- by binding it and directing its beta-trcp -dependent degradation a novel human wd protein, h-beta trcp, that interacts with hiv- vpu connects cd to the er degradation pathway through an f-box motif cell biology. an ancient portal to proteolysis endoplasmic reticulum protein quality control is determined by cooperative interactions between hsp/c protein and the chip e ligase hiv infection of primate lymphocytes and conservation of the cd receptor mapping of the functional boundaries and secondary structure of the mouse mammary tumor virus rem-responsive element rev and rex proteins of human complex retroviruses function with the mmtv rem-responsive element mouse mammary tumor virus encodes a self-regulatory rna export protein and is a complex retrovirus specificity and regulation of the endoplasmic reticulum-associated degradation machinery emerging functions of the vcp/p aaa-atpase in the ubiquitin system cardif is an adaptor protein in the rig-i antiviral pathway and is targeted by hepatitis c virus an autophagy-independent role for lc in equine arteritis virus replication the ero alpha-pdi redox cycle regulates retro-translocation of cholera toxin intracellular trafficking pathway of bk virus in human renal proximal tubular epithelial cells cholera: pathophysiology and emerging therapeutic targets sel l, the homologue of yeast hrd p, is involved in protein dislocation from the mammalian er identification of the rem-responsive element of mouse mammary tumor virus a novel mammalian endoplasmic reticulum ubiquitin ligase homologous to the yeast hrd tetherin inhibits retrovirus release and is antagonized by hiv- vpu torsina participates in endoplasmic reticulum-associated degradation the polyomaviridae: contributions of virus structure to our understanding of virus receptors and infectious entry ubx links the cdc complex to er-associated protein degradation caveolar endocytosis of simian virus is followed by brefeldin a-sensitive transport to the endoplasmic reticulum, where the virus disassembles destabilization of the vcp-ufd -npl complex is associated with decreased levels of erad substrates derlin- and derlin- are regulated by the mammalian unfolded protein response and are required for er-associated degradation characterization of an erad pathway for nonglycosylated bip substrates, which require herp edem regulates er-associated degradation by accelerating de-mannosylation of folding-defective polypeptides and by inhibiting their covalent aggregation lipid droplet formation is dispensable for endoplasmic reticulum-associated degradation the mammalian endoplasmic reticulum-associated degradation system localization and membrane topology of coronavirus nonstructural protein : involvement of the early secretory pathway in replication translocator protein ( kda): new nomenclature for the peripheral-type benzodiazepine receptor based on its structure and molecular function architecture and biogenesis of plus-strand rna virus replication factories protein disulfide isomerase is required for platelet-derived growth factor-induced vascular smooth muscle cell migration, nox nadph oxidase expression, and rhogtpase activation cd and bst- /tetherin proteins retro-translocate from endoplasmic reticulum to cytosol as partially folded and multimeric molecules going through the motions: the atpase cycle of p protein translocation across the eukaryotic endoplasmic reticulum and bacterial plasma membranes coronaviruses hijack the lc -i-positive edemosomes, er-derived vesicles exporting short-lived erad regulators, for replication endolysosomal sorting of ubiquitylated caveolin- is regulated by vcp and ubxd and impaired by vcp disease mutations role of ubiquitination in retro-translocation of cholera toxin and escape of cytosolic degradation quality control: er-associated degradation: protein quality control and beyond role of the endoplasmic reticulum-associated degradation (erad) pathway in degradation of hepatitis c virus envelope proteins and production of virus particles counteraction of the multifunctional restriction factor tetherin bipmediated closing of the sec channel limits ca + leakage from the er simian virus depends on er protein folding and quality control factors for entry into host cells membrane-bound ubx recruits cdc to ubiquitin ligases and their substrates to ensure efficient er-associated protein degradation ubx domain proteins: major regulators of the aaa atpase cdc /p identification and characterization of mavs, a mitochondrial antiviral signaling protein that activates nf-kappab and irf a shared endoplasmic reticulum-associated degradation pathway involving the edem protein for glycosylated and nonglycosylated proteins mechanisms and function of substrate recruitment by f-box proteins road to ruin: targeting proteins for degradation in the endoplasmic reticulum defining the human deubiquitinating enzyme interaction landscape structure and function of cholera toxin and the related escherichia coli heat-labile enterotoxin the trc e ligase ubiquitinates mhc class i molecules before dislocation from the er the intracellular sites of early replication and budding of sarscoronavirus the cytosolic tail of class i mhc heavy chain is required for its dislocation by the human cytomegalovirus us and us gene products hiv accessory proteins versus host restriction factors sel l is indispensable for mammalian endoplasmic reticulum-associated degradation, endoplasmic reticulum homeostasis, and survival use of modular substrates demonstrates mechanistic diversity and reveals differences in chaperone requirement of erad the endoplasmic reticulum-associated degradation pathways of budding yeast cellular entry of polyomaviruses protein disulfide isomerase acts as a redox-dependent chaperone to unfold cholera toxin a ubiquitin-binding rhomboid protease aimed at eradication cd + t cells: guardians of the phagosome glycosylation-independent erad pathway serves as a backup system under er stress ubiquitin-like proteins one step at a time: endoplasmic reticulumassociated degradation the er quality control and er associated degradation machineries are vital for viral pathogenesis mechanism, specificity, and physiology of signal peptide peptidase (spp) and spp-like proteases bap is an itinerant protein that moves between the peripheral endoplasmic reticulum (er) and a juxtanuclear compartment related to erassociated degradation requirements for the selective degradation of endoplasmic reticulum-resident major histocompatibility complex class i proteins by the viral immune evasion molecule mk ubiquitination of serine, threonine, or lysine residues on the cytoplasmic tail can induce erad of mhc-i by viral e ligase mk ube j ubiquitinates hydroxylated amino acids on erassociated degradation substrates the viral e ubiquitin ligase mk uses the derlin/p endoplasmic reticulum-associated degradation pathway to mediate down-regulation of major histocompatibility complex class i proteins sec -mediated transfer of a membrane protein from the endoplasmic reticulum to the proteasome for destruction thiol isomerases negatively regulate the cellular shedding activity of adam human immunodeficiency virus type vpu protein regulates the formation of intracellular gp -cd complexes the erdj -sel l complex facilitates cholera toxin retrotranslocation bip-dependent export of cholera toxin from endoplasmic reticulum-derived microsomes the cdc machine in endoplasmic reticulum associated protein degradation visa is an adapter protein required for virus-triggered ifn-beta signaling function of the p -ufd -npl complex in retrotranslocation from the er to the cytosol: dual recognition of nonubiquitinated polypeptide segments and polyubiquitin chains recruitment of the p atpase and ubiquitin ligases to the site of retrotranslocation at the endoplasmic reticulum membrane a membrane protein complex mediates retro-translocation from the er lumen into the cytosol f-box proteins that contain sugar-binding domains glycoproteinspecific ubiquitin ligases recognize n-glycans in unfolded substrates sequence-specific rna binding by the hiv- rev protein the mitochondrial translocator protein, tspo, inhibits hiv- envelope glycoprotein biosynthesis via the endoplasmic reticulum-associated protein degradation pathway strategies of highly pathogenic rna viruses to block dsrna detection by rig-i-like receptors: hide, mask, hit we thank dr. jon huibregtse for helpful comments and suggestions on the manuscript and marianna grenadier for the figures. this work was supported by nih grants r ca and r ai . the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. key: cord- - dofkxh authors: levine, beth; mizushima, noboru; virgin, herbert w. title: autophagy in immunity and inflammation date: - - journal: nature doi: . /nature sha: doc_id: cord_uid: dofkxh autophagy is an essential, homeostatic process by which cells break down their own components. perhaps the most primordial function of this lysosomal degradation pathway is adaptation to nutrient deprivation. however, in complex multicellular organisms, the core molecular machinery of autophagy — the 'autophagy proteins' — orchestrates diverse aspects of cellular and organismal responses to other dangerous stimuli such as infection. recent developments reveal a crucial role for the autophagy pathway and proteins in immunity and inflammation. they balance the beneficial and detrimental effects of immunity and inflammation, and thereby may protect against infectious, autoimmune and inflammatory diseases. t here is only one known mechanism that eukaryotic cells possess to dispose of intracellular organelles and protein aggregates that are too large to be degraded by the proteasome. it is therefore not surprising that this mechanism -the lysosomal degradation pathway known as autophagy -is also used to degrade microorganisms (such as viruses, bacteria and protozoa) that invade intracellularly , . indeed, the mutation of autophagy genes increases susceptibility to infection by intracellular pathogens in organisms ranging from plants to flies to worms to mice, and possibly to humans. perhaps less apparent, but teleologically as intuitive, the autophagy pathway or unique functions of autophagy proteins also have a central role in controlling other diverse aspects of immunity in multicellular organisms. the autophagy machinery is thought to have evolved as a stress response that allows unicellular eukaryotic organisms to survive during harsh conditions, probably by regulating energy homeostasis and/ or by protein and organelle quality control. the same machinery might therefore be expected to diversify functionally in complex metazoan organisms, so as to regulate new layers of defences used by multicellular organisms to confront different forms of stress. a plethora of genetic, biochemistry, cell biology, systems biology and genomic studies have recently converged to support this notion. the autophagy machinery interfaces with most cellular stress-response pathways , including those involved in controlling immune responses and inflammation. this interface is not only at the level of the autophagy pathway, but also entails direct interactions between autophagy proteins and immune signalling molecules . there is a complex reciprocal relationship between the autophagy pathway/proteins and immunity and inflammation; the autophagy proteins function in both the induction and suppression of immune and inflammatory responses, and immune and inflammatory signals function in both the induction and suppression of autophagy. moreover, similar to cancer, neurodegenerative diseases and ageing , defects in autophagy -through autophagy gene mutation and/or microbial antagonism -may underlie the pathogenesis of many infectious diseases and inflammatory syndromes. in this review, we describe recent advances in our evolving comprehension of the interface between autophagy, immunity and inflammation. we discuss how emerging concepts about the functions of the autophagy pathway and the autophagy proteins may reshape our understanding of immunity and disease. this set of proteins not only orchestrates the lysosomal degradation of unwanted cargo, but also exerts intricate effects on the control of immunity and inflammation. thus, the autophagy pathway and autophagy proteins may function as a central fulcrum that balances the beneficial and harmful effects of the host response to infection and other immunological stimuli. autophagy is a general term for pathways by which cytoplasmic material, including soluble macromolecules and organelles, is delivered to lysosomes for degradation . there are at least three different types of autophagy, including macroautophagy, chaperone-mediated autophagy and microautophagy. macroautophagy, usually referred to simply as autophagy, is the subject of this review (fig. ). in this pathway, a portion of cytoplasm (usually . - μm in diameter) is engulfed by an isolation membrane, or 'phagophore' , resulting in the formation of a double-membrane structure known as the autophagosome. the outer membrane of the autophagosome fuses with the lysosome to become an autolysosome, leading to the degradation of autophagosomal contents by lysosomal enzymes. autophagosomes can also fuse with endosomes or multivesicular bodies and major histocompatibility complex (mhc)class-ii-loading compartments . autolysosomes become larger as more autophagosomes and lysosomes fuse, but at a termination phase lysosomes are tubulated and fragmented for renewal . the membrane dynamics of autophagosome formation involve complex processes, which are not completely understood. nonetheless, the molecular dissection of autophagy membrane dynamics, stimulated by the discovery of atg (autophagy-related) genes in yeast , has shed considerable light on this topic (table ) . several recent studies suggest that the endoplasmic reticulum (er) is crucial for autophagosome formation. the er cisternae often associate with developing autophagosomes, and electron tomography analysis has demonstrated direct connections between the er and autophagosomal membranes , . moreover, the function of several key autophagy proteins seems to be at the level of the er (fig. ) . autophagy is induced by nutrient starvation through the inhibition of mammalian target of rapamycin (mtor), resulting in translocation of the mtor substrate complex (ulk / , atg , fip (also known as rb cc ) and atg ) from the cytosol to certain domains of the er or closely attached structures , . this leads to the recruitment of the class iii phosphatidylinositol- -oh kinase (pi( )k) complex, which includes at least vps (also known as pik c ), vps (pik r and p ), beclin and atg , to the er , . the pi( )k complex produces phosphatidylinositol- -phosphate (ptdins( )p), which recruits effectors such as double fyve-containing protein (dfcp ) and wdrepeat domain phosphoinositide-interacting (wipi) family proteins. dfcp is diffusely present on the er or the golgi, but translocates to the autophagosome formation site in a ptdins( )p-dependent manner to generate er-associated Ω-like structures termed omegasomes . among the four wipi isoforms, wipi is the major form in most cell types and functions downstream of dfcp , and it may promote the development of omegasomes into isolation membranes or autophagosomes . an er-associated multispanning membrane protein, vmp , is also important for autophagosome formation. although vmp interacts with beclin and is present at the autophagosome formation site at an early stage, it seems to function at a late stage in autophagy overview of the autophagy pathway. the top right box shows a model of our current understanding of the molecular events involved in membrane initiation, elongation and completion of the autophagosome. the major membrane source is thought to be the endoplasmic reticulum (er), although several other membrane sources, such as mitochondria and the plasma or nuclear membrane, may contribute. after induction of autophagy, the ulk complex (ulk -atg -fip -atg ) (downstream of the inhibitory mtor signalling complex) translocates to the er and transiently associates with vmp , resulting in activation of the er-localized autophagy-specific class iii phosphatidylinositol- -oh kinase (pi ( ) this is perhaps consistent with other evidence that beclin -class iii pi( )k complexes may function in autophagosomal maturation (in addition to vesicle nucleation), a process that can be regulated by other beclin- -interacting partners such as rubicon (table ) . at the final step of autophagosome formation, elongation of the isolation membrane and/or completion of enclosure require two ubiquitin-like conjugates. the first is the atg -atg conjugate, which is produced by the atg (e -like) and atg (e -like) enzymes, and functions as a dimeric complex together with atg l (ref. ) . the second is the phosphatidylethanolamine (pe)-conjugated atg homologues -lc , gate and gabarap -which are produced by the atg and atg (e -like) enzymes , . although the proteins involved in autophagosome membrane formation have been characterized as discrete complexes (table ) , several potential interconnections between components of the different complexes were identified by a recent proteomics study . such interconnections may function in autophagosome membrane formation or other distinct cellular functions. for example, the atg -atg conjugate is implicated in mitochondrial homeostasis but not in autophagosome membrane formation . in addition to the er, other membranes may be involved in autophagosome formation (fig. ) . atg , another multispanning membrane protein, is essential for autophagy and traffics between the trans-golgi network, endosomes and autophagosome precursors . studies suggest that mitochondria, the plasma membrane and the nuclear membrane could also be membrane sources for autophagosome formation [ ] [ ] [ ] . however, the lack of detection of specific protein markers for these structures on the autophagosomal membrane leaves the decades-old question of the membrane source of the autophagosome unanswered. it is possible that cells may use different membrane sources to form the autophagosome in different contexts, thereby permitting specialization of membrane dynamics in a manner that allows divergent autophagyinducing signals to stimulate the capture of spatially distinct cargo. autophagy was originally considered to be a non-selective bulk degradation process, but it is now clear that autophagosomes can degrade substrates in a selective manner . in addition to endogenous substrates, autophagy degrades intracellular pathogens in a selective form of autophagy, termed xenophagy. similar to bulk autophagy (such as that induced by nutrient deprivation) and other forms of selective autophagy (such as degradation of damaged mitochondria, peroxisomes, aggregate-prone proteins or damaged er), the precise membrane dynamics and specificity determinants of xenophagy are not fully understood. nonetheless, considerable advances have been made, and interesting similarities and differences are beginning to emerge between cellular recognition and degradation of self versus foreign microbial components through autophagy-like pathways (figs and ) . the vacuoles used for the engulfment of intracytoplasmic bacteria are similar to autophagosomes, and their formation requires the core autophagy machinery. but one apparent difference is the vacuole size; for example, the diameter of group a streptococcus-containing autophagosome-like vacuoles (gcav) can be as big as μm. these large gcavs are generated by the rab -dependent fusion of small isolation membranes . by contrast, the formation of starvation-induced autophagosomes requires rab later in the autophagy process, at the autophagosome-lysosome fusion step. a more complex question is how autophagosomes (or components of the autophagy pathway) capture pathogens that are inside vacuolar compartments (fig. ) . there are at least four general pathways that may be used for autophagy-protein-dependent targeting of bacteria to the lysosome. these include autophagy-protein-facilitated fusion of bacteria-containing phagosomes with lysosomes, the envelopment of bacteria-containing phagosomes or endosomes by autophagosomal membranes, the fusion of bacteria-containing phagosomes or endosomes with autophagosomes, or the xenophagic capture of bacteria that have escaped inside the cytoplasm. in some cases, the route of autophagy-dependent targeting to the lysosome has been well defined, such as for group a streptococcus that escapes from endosomes . for several bacteria, however, the precise route is unclear. many studies define bacterial autophagy as the co-localization of bacteria and lc , but we now know that lc can decorate membranous compartments other than autophagosomes (including phagosomes). several lines of evidence suggest that autophagy proteins function more broadly, not only in classical macroautophagy, but also in the process of phagolysosomal maturation during antigen presentation and microbial invasion. autophagy proteins are required for the fusion of phagosomes that contain toll-like receptor (tlr)-ligand-enveloped particles with lysosomes in macrophages , and for the fusion of phagosomes that contain tlr-agonist-associated apoptotic cell antigens with lysosomes in dendritic cells during mhc class ii antigen presentation . the self ligand and cell-surface receptor slam functions as a microbial sensor that recruits the beclin -class iii pi( )k complex to phagosomes containing gram-negative bacteria, facilitating phagolysosomal fusion and activation of the antibacterial nadph oxidase (nox ) complex . in addition, the engagement of tlr or fcγ receptors during phagocytosis recruits lc (and atg ) to the phagosome through nox dependent generation of reactive oxygen species (ros) . thus, in bacterial infections, a paradigm is emerging in which the coordinated regulation of microbial sensing, phagolysosomal maturation and antibacterial activity involves the recruitment of autophagy proteins to the phagosome. as a corollary, an interesting speculation is that impaired recruitment of autophagy proteins to the phagosome may contribute to the pathogenesis of chronic granulomatous disease, a genetic disorder caused by mutations in the nox gene (also known as cybb) and characterized by recurrent bacterial and fungal infections and inflammatory complications, such as inflammatory bowel disease. another autophagosome-independent function of autophagy proteins in pathogen destruction has been described in interferon-γ (ifn-γ)treated macrophages infected with the parasite toxoplasma gondii. the parasite-derived membrane, termed the parasitophorous vacuole, undergoes destruction through a mechanism that involves atg -dependent recruitment of the immunity-related gtpase proteins to the parasitophorous vacuole , , leading to the death of the parasite in the infected cell , . together, these studies suggest that autophagy proteins have diverse roles in membrane dynamics to benefit the host in the removal of invading pathogens ( fig. ), through xenophagy, phagolysosomal maturation, the recruitment of molecules that damage pathogen-derived membranes, and presumably, many other as yet undiscovered mechanisms. the mechanisms that cells use to target intracellular bacteria (and probably viruses) to autophagosomal compartments are notably similar to those used for selective autophagy of endogenous cargo. cellular cargo is commonly targeted to autophagosomes by interactions between a molecular tag (such as polyubiquitin), adaptor proteins such as p (also known as sqstm or sequestome ) or nbr (which recognize these tags and contain an lc -interacting region (lir) characterized by a wxxl or wxxi motif), and lc (ref. ). these adaptor molecules enable autophagy to target designated cargo selectively to nascent lc -positive isolation membranes. as reviewed elsewhere , a similar mechanism involving ubiquitin and p seems to be involved in the targeting of intracellular bacteria, such as salmonella enterica serotype typhimurium (s. typhimurium), shigella flexneri and listeria monocytogenes, to autophagosomes. after escape into the cytoplasm or in vacuolar membrane compartments damaged by type iii secretion system (t ss) effectors, bacteria or bacteria-containing compartments, respectively, may become coated with ubiquitin and associate with p and nascent lc -positive isolation membranes. the autophagosomal targeting of salmonella also requires another cellular factor, ndp (nuclear dot protein ), an autophagy adaptor protein that, like p , contains an lir and ubiquitin-binding domains and restricts intracellular bacterial replication. a ubiquitinindependent pathway (that does not involve p or ndp ) could also function in targeting damaged salmonella-containing vacuoles (scvs) to the autophagosome. in this pathway, a lipid second messenger, diacylglycerol, acts as a signal for the co-localization of scvs with lc -positive autophagosomes by a mechanism that involves protein kinase c and its downstream targets, jnk and nadph oxidase . the autophagic targeting of a cytoplasmic positive-strand rna virus, sindbis virus, also occurs in a ubiquitin-independent manner, but involves the interaction of p with the viral capsid protein . thus, diverse molecular strategies, including ubiquitin-dependent and -independent mechanisms, may be used to target microbes inside the cytoplasm or vacuolar compartments to the autophagosome. beyond targeting intracellular pathogens for degradation, p may have further beneficial effects in infected host cells. for example, shigella vacuolar membrane remnants generated by bacterial t ss-dependent membrane damage are targeted by polyubiquitination, p and lc for autophagosomal degradation (fig. ) . these membrane remnants also accumulate numerous molecules involved in sensing and transduction of pathogen-associated molecular pattern (pamp) and danger-associated molecular pattern (damp) signals, and there is an increase in nuclear factor-κb (nf-κb)-dependent cytokine production, ros production and necrotic cell death in autophagy-deficient cells. thus, the ubiquitin-p -dependent autophagic targeting of pathogen-damaged membranes could help to control detrimental downstream inflammatory signalling during bacterial invasion into host cells. another emerging concept is that selective autophagy of viral proteins, similar to selective autophagy of aggregate-prone toxic cellular proteins, may protect post-mitotic cells such as neurons against cell death. for example, in sindbis-virus-infected mice with neuron-specific inactivation of atg , there is an accumulation of sindbis virus antigens (without increased levels of infectious virus), increased neuronal cell death and increased animal mortality . moreover, p is required for starvation and ifn-γ-induced targeting of fau (and perhaps other ubiquitylated protein complexes) to mycobacteria-containing phagosomes, resulting in the generation of antimycobacterial fau-derived peptides .the role of p in innate immunity is probably evolutionarily ancient, as the drosophila p orthologue ref( )p was originally identified in a screen for modifiers of sigma virus replication . we speculate that p , as well as the other known lc -interacting adaptor proteins (nbr and ndp ), may represent the tip of the iceberg in terms of cellular adaptor proteins that bind to ubiquitin (or other molecular tags) and target microbial substrates and cytosolic material to autophagosomes to coordinate innate immune responses. a recent proteomics study showed that the mammalian atg family, which includes lc , gate and gabarap, has high-confidence interactions with other cellular proteins . some of these new atg -familymember-interacting partners may have an as yet undiscovered role in innate immunity. another open question is whether the known proteins involved in selective autophagy of mitochondria (called mitophagy), such as nix (also known as bnip l) and parkin , also function in microbial autophagy. the autophagy pathway and/or autophagy proteins have a crucial role in resistance to bacterial, viral and protozoan infection in metazoan organisms. the genetic deletion or knockdown of autophagy genes to the proteins shown in the cargo-recognition box in fig. ; however, as yet undiscovered adaptors may be involved in pathogen recognition, and pathogen targeting may involve ubiquitin-dependent or -independent mechanisms. protects plants from viral, fungal and bacterial infection by preventing the uncontrolled spread of programmed cell death during the plant innate immune or hypersensitive response . in other organisms, autophagy proteins function in a cell-autonomous manner to control infection by intracellular pathogens. in drosophila, autophagy gene mutation increases susceptibility to viral (vesicular stomatitis virus) and bacterial (l. monocytogenes) infection. in dictyostelium and caenorhabditis elegans, autophagy gene mutation increases susceptibility to lethal s. typhimurium infection . in mice, knockout of atg in macrophages and neutrophils increases susceptibility to infection with l. monocytogenes and the protozoan t. gondii , and neuron-specific atg knockout increases susceptibility to central nervous system sindbis virus infection . as noted in the next section, the autophagy pathway and proteins may also have 'proviral' or 'probacterial' effects in in vitro studies; however, in vivo evidence for such effects is so far lacking. the mechanisms by which autophagy genes mediate in vivo resistance to infection are not fully understood, but are likely to involve a combination of xenophagy, other autophagy-protein-dependent effects on microbial replication or survival, activation of innate and adaptive immune responses, and/or alterations in pathogen-induced cell death (fig. ). an important question is whether this function of autophagy in broad resistance to infection with intracellular pathogens extends to humans. recent human genetic studies provide some clues. the immunityrelated gtpase human irgm, which regulates autophagy-dependent clearance of mycobacteria in vitro , was identified as a genetic risk locus for tuberculosis in a west african population . numerous studies have shown a crucial role for autophagy in defence against mycobacterial infection in human cells , and a genome-wide analysis of host genes that regulate mycobacterium tuberculosis replication demonstrated that a predominance of factors were autophagy regulators . thus, it is possible that autophagy has a central role in resistance to one of the most important global infectious diseases -tuberculosis. mutations in nod , which encodes an intracellular pathogen-recognition receptor (nucleotide-binding oligomerization-domain-containing protein ) that functions in bacterial autophagy , , are also associated with susceptibility to infection with another mycobacterial agent, mycobacterium leprae, the aetiological agent of leprosy . an exciting future venture will be to confirm whether irgm, nod and other autophagy-related genes are involved in resistance to infection with mycobacteria and other infections in further human populations and, if so, whether this resistance is mediated by autophagy. microbes undergo strong selective pressure to develop strategies to block host defence mechanisms; the number of such strategies is a surrogate measure of the importance of the host defence mechanism in immunity. as reviewed elsewhere , , viruses and intracellular bacteria have evolved several ways to adapt to host autophagy. they can antagonize autophagy initiation or autophagosomal maturation, evade autophagic recognition, or use components of the autophagy pathway to facilitate their own replication or intracellular survival. an emerging theme is that microbial antagonism of autophagy not only blocks the xenophagic degradation of intracellular pathogens, but also blocks the functions of autophagy in innate and adaptive immunity. a relatively unexplored yet crucially important frontier is how microbial antagonism may contribute more broadly to the role of microbes in diseases characterized by defective autophagy, such as cancer, neurodegenerative diseases, ageing and, potentially, autoimmune and inflammatory diseases. viral strategies to shut off autophagy include the blockade of positive upstream regulators of autophagy (such as the ifn-inducible rna-activated eif α protein kinase (pkr) signalling pathway), the activation of negative upstream regulators of autophagy (such as the nutrient-sensing tor kinase signalling pathway) or direct antagonism of the autophagy machinery . the overlapping functions of the eif α kinase signalling pathway in stress-induced general translational arrest, transcriptional activation of stress-response genes and stress-induced autophagy enable viruses to disarm several facets of the cellular stress response to infection by one mechanism -that is, antagonism of eif α kinase signalling. the mtor signalling pathway has a central role in the control of cell growth and metabolism, and interestingly, many of the viruses that activate mtor are oncogenic (for example, epstein-barr virus, kaposi's sarcoma-associated herpesvirus, hepatitis b virus and retroviruses). this suggests another type of pluripotent viral weapon -one that can promote oncogenesis by simultaneously inactivating autophagy and promoting cell growth through tor activation. hiv envelope protein-dependent activation of mtor signalling is also proposed to be a mechanism for hiv evasion of innate and adaptive immune responses in dendritic cells, including the degradation of incoming virions by lysosomes, blockade of hiv transfer to cd + t cells, stimulation of tlr and tlr ligand responses, and presentation of hiv gag antigen to cd + t cells . it will be important to determine whether these effects of hiv-mediated mtor activation and autophagy inhibition contribute to impaired dendritic cell function during hiv infection in vivo. several viral proteins target the core autophagy protein beclin . autophagosome initiation is blocked by interactions between beclin and the herpes simplex virus (hsv- ) neurovirulence factor icp . or the oncogenic γ-herpesvirus-encoded viral bcl -like proteins, whereas autophagosome maturation is blocked by interactions between beclin and the hiv accessory protein nef or the influenza virus matrix protein (ref. ). the interactions between beclin , hsv- icp . and viral bcl are probably physiologically important in vivo; a mutant hsv- virus lacking the beclin- -binding domain of icp . is attenuated in mouse models of encephalitis (presumably through its failure to control xenophagy and innate immunity) and of corneal disease (through its failure to control adaptive immunity) . moreover, a mouse γ-herpesvirus that encodes a mutant viral bcl unable to bind to beclin demonstrates impaired ability to maintain chronic infection review insight beclin is preferentially targeted by viral virulence proteins because of its central role in autophagosome formation, or more likely, whether we are just beginning to identify pairs of viral proteins and their autophagy pathway targets. in support of the latter, viral flice-like inhibitors encoded by kaposi's sarcoma-associated herpesvirus and molluscum contagiosum virus suppress autophagy by interacting with the atg e -like enzyme, thereby preventing it from binding and processing lc (ref. ) . bacteria possess diverse strategies to avoid degradation by autophagolysosomal pathways. as reviewed elsewhere , , many bacteria that reside in phagosomes or other vacuolar compartments have methods to inhibit lysosomal fusion or maturation, which, in the case of mycobacteria, can be partially overcome by treatments that stimulate autophagy. another possible mechanism for bacterial evasion of autophagy has emerged from a genome-wide screen to identify host factors that regulate the intracellular survival of m. tuberculosis . according to bioinformatics analyses, m. tuberculosis infection of human macrophage-like cells activates cellular pathways that inhibit autophagy. intracellular bacteria that escape into the cytoplasm, such as s. flexneri and l. monocytogenes, use strategies to camouflage themselves to avoid autophagic recognition. the shigella t ss effector icsb competitively binds to virg, thereby preventing the interaction between atg and virg, a bacterial surface protein required for actin-based motility and shigella targeting to autophagosomes . the listeria protein acta interacts with cytosolic actin polymerization machinery (arp / , vasp and actin), which blocks bacterial association with ubiquitin, p recruitment and autophagic targeting . the precise mechanism of this block is unknown, but it has been proposed that the acta-dependent recruitment of host cell cytoskeletal proteins may enable the bacterium to disguise itself as a host cell organelle . this concept sheds light on the autophagy pathway in a fundamental aspect of immunology -the basis for discrimination between self and non-self. microbes have evolved not only to antagonize autophagy (as a cellular defence mechanism that threatens their survival), but also to exploit its components and functions in membrane trafficking for their own self-serving purposes , . an early concept in the field is that autophagosomes may serve as a protected niche for intracellular bacteria (provided fusion with acidic compartments is blocked) and/or serve as a source of nutrients for intracellular pathogens (which would require intact autophagolysosomal fusion) . trafficking of the intracellular bacteria yersinia pseudotuberculosis to acidic compartments was recently shown to be enhanced by genetic inhibition of autophagy . this seemingly contradicts other evidence that the autophagy proteins promote phagosomal maturation, but is consistent with the concept that autophagosomes function as a protected intracellular niche for bacteria. the role of the autophagy machinery in promoting and/or inhibiting vacuolar acidification -and the counter effects of microbes that reside in vacuolar compartments -needs to be explored further. the function of autophagy proteins in membrane formation and/ or trafficking is exploited by numerous viruses, including poliovirus, rota virus, hiv, coronaviruses, dengue virus, and the hepatitis b and c viruses . autophagosomes (defined as lc -positive membranes, see caveat below) may act as a scaffold for intracellular membrane-associated replication of certain cytoplasmic rna viruses . autophagy may assist in hiv biogenesis, because the processing of the hiv envelope precursor protein gag and extracellular viral release are enhanced by the autophagy machinery . similarly, autophagy proteins are required for maximal levels of poliovirus egress . another newly defined proviral function of autophagy is its role in productive hepatitis c virus replication; several different autophagy proteins (such as beclin , atg b, atg , atg and atg ) assist in the translation of incoming, but not progeny, viral rna . atg and class iii pi( )k activity also enhance hepatitis b virus dna replication . the mechanisms by which autophagy proteins facilitate the replication and/or egress of certain viruses are not yet understood. some observations may relate to the role of autophagy proteins in remodelling the er (vis-à-vis viral replication) or the role of autophagosomes in fusing with multivesicular bodies (vis-à-vis viral egress). it is possible that autophagy proteins function to provide membrane for viral replication complexes or translation machinery. this may be true for viruses such as hepatitis c virus, for which genetic knockdown of several different autophagy genes decreases productive replication . however, for other viruses such as coronaviruses, the biogenesis of double-membrane, er-derived vesicles used for replication proceeds through a pathway that involves the nonlipidated form of lc (lc -i) but not the general autophagy machinery . thus, caution must be exercised in interpreting the significance of the co-localization (or biochemical interaction) of viral proteins and lc , as lc may have autophagy-independent roles in membrane dynamics. the central importance of autophagy in immunity is further underscored by the multitude of immune-related signalling molecules that regulate autophagy. as reviewed in detail elsewhere [ ] [ ] [ ] , autophagy is induced by different families of pathogen-recognition receptors (such as tlrs, nod-like receptors and the double-stranded rna-binding protein pkr), damps (such as atp, ros and misfolded proteins), pathogen receptors (such as cd ), ifn-γ and downstream immunityrelated gtpases, and dap kinase, jnk, cd , tumour necrosis factor-α (tnf-α), inhibitor of nf-κb (ikk) and nf-κb ( fig. ) . high mobility group box (hmgb) proteins have also been shown to function as both universal sensors of nucleic acids in innate immune signalling and inducers of autophagy . autophagy is inhibited by bcl , nf-κb, t helper (t h ) cytokines and the canonical nutrient-sensing insulin-akt-tor pathway. inactivation of this nutrient-sensing pathway may contribute to vesicular stomatitis virus stimulation of autophagy in drosophila , and autophagy activation in c. elegans with loss-of-function mutations in this pathway may mediate pathogen resistance in long-lived mutant nematodes . thus, both 'immune-specific' and more general nutrient-response signals control autophagy in response to infection. studies with vitamin d have uncovered a possible link between nutrition, innate immunity and the control of autophagy during mycobacterial infection. low vitamin d levels are associated with increased susceptibility to tuberculosis. vitamin d generates an antimycobacterial peptide, cathelicidin, and induces autophagy and mycobacterial killing in human monocytes through cathelicidin-dependent effects . although cathelicidin is required for vitamin-d -dependent transcriptional upregulation of autophagy genes such as becn and atg , and vitamin d enhances the recruitment of cathelicidin to autophagosomes, it is not yet clear how cathelicidin promotes autophagy. nonetheless, these observations may begin to provide some insight into the century-old nobel prize award (niels ryberg finsen, ) for the use of ultraviolet-light therapy (which generates active vitamin d ) in the treatment of diseases such as tuberculosis. in most instances, the mechanisms of autophagy control by immunerelated signalling molecules are not understood. however, there are some examples of specific interactions between immune signals and autophagy proteins that may be relevant to these mechanisms. for example, the interaction between beclin and bcl (which inhibits its activity) is thought to be disrupted by the tlr adaptors myd and trif, as well as by hmgb , which bind to beclin and displace bcl (refs , ) . two intracellular sensors responsible for inducing autophagy in response to bacterial infection, nod and nod , interact with atg l and recruit it to the plasma membrane, resulting in enhanced association of invasive bacteria (s. flexneri) with lc (ref. ). interestingly, a nod mutation associated with crohn's disease impairs atg l plasma membrane recruitment and bacterial co-localization with lc (ref. ). the identification of other possible protein-protein interactions between core autophagy proteins and immune signals by a large proteomics screen has the potential to foster further advances in understanding how different immune signals regulate the autophagy machinery. for example, tectonin proteins with multivalent j a n u a r y | v o l | n a t u r e | review insight β-propeller folds are known to function in pathogen recognition and innate immunity in invertebrates . thus, the interactions between previously uncharacterized human proteins of this tectonin family, tecpr and tecpr , with the atg -atg -atg l complex and atg family members, respectively , may contribute to pathogen-induced autophagy stimulation or selective autophagic targeting of pathogens in mammals. further links between immune signalling molecules and autophagy regulation were suggested by a genome-wide short interfering rna screen . the analysis identified genes that suppressed basal autophagy, largely in a mammalian tor complex (mtorc )independent fashion. these included several cytokines such as clcf , lif, igf , fgf and the chemokine sdf (also known as cxcl ), as well as cellular signalling molecules regulated by cytokines such as stat . these findings raise the possibility that cytokines may have a broader role in the control of autophagy than previously thought. moreover, because these cytokine signalling pathways are important in immune cells, another central question is to what extent cytokinemediated regulation of autophagy governs immune cell function. given the general function of autophagy in cellular homeostasis , and the more specific functions in regulating immune and inflammatory signalling (discussed in 'regulation of immune signalling by autophagy proteins'), cytokine-mediated changes in autophagy levels in immune cells may have a central role in immunity and inflammation. autophagy proteins function in adaptive immunity, including in the development and homeostasis of the immune system and in antigen presentation (table and fig. ) . the knockout of different autophagy genes in specific lymphocyte populations in mice has shown a crucial role for autophagy proteins in the maintenance of normal numbers of b a b cells, cd + t cells, cd + t cells and fetal haematopoietic stem cells , , . in t cells, in which mitochondrial numbers are developmentally regulated during the transition from thymocyte to mature circulating t cell, the developmental defect in autophagy-deficient cells may be related to the defective clearance of mitochondria . another crucial function of autophagy in the development and homeostasis of the immune system is the elimination of autoreactive t cells in the thymus . high levels of autophagy are present in thymic epithelial cells, in which autophagy participates in the delivery of self-antigens to mhc class ii loading compartments. genetic disruption of atg in thymic epithelial cells leads to the altered selection of certain mhc class ii restricted t-cell specificities and autoimmunity . beyond these functions in lymph ocyte survival and thymic negative selection, autophagy may exert other functions in lymphocyte differentiation, perhaps, in part, indirectly through effects on cytokine expression (see the next section). it is not yet known whether autophagy is involved in the development and/or homeostasis of immune cell populations other than lymphocytes and haematopoietic stem cells. autophagy proteins may participate in different facets of antigen presentation, including the delivery of endogenous antigens for mhc class ii presentation to cd + t cells , , the enhancement of antigen donor cell cross-presentation to cd + t cells , dendritic cell crosspresentation of phagocytosed antigens to cd + t cells and, in one report, mhc class i presentation of intracellular antigens to cd + t cells . the discovery that autophagosomes could deliver endogenous antigens to mhc class ii loading compartments sheds light on one of the central mysteries of antigen presentation -how the immune system elicits cd + t-cell responses to antigens that originate in all parts of the cell. the autophagic delivery of endogenously synthesized antigens for mhc class ii presentation has been demonstrated in vitro for certain viral antigens , and probably explains the essential role of atg in vivo in negative thymic selection . however, the relative importance of this pathway in antigen presentation during infection in vivo is not yet known. there is nonetheless interest in exploiting this pathway for optimizing vaccine-elicited cd + t-cell responses, by either pre-treating dendritic cells with autophagy-inducing agents in cell-based vaccine strategies or fusing antigens with lc to enhance their targeting to autophagosomes . of note, autophagy proteins are required for antigen cross-presentation during infection in vivo . dendritic-cell-specific deletion of atg in mice results in defects in priming cd + t-cell responses after hsv and listeria infections, and mice succumb more rapidly to lethal disease after intravaginal hsv infection. atg -deficient dendritic cells have normal migration, innate responses, endocytic and phagocytic activity and cross-presentation of peptides on mhc class i molecules. however, they exhibit defects in phagosome-to-lysosome fusion and in cross-presentation by mhc class ii molecules of phagocytosed antigens containing tlr ligand. thus, the interior of the phagosome, like that of the autophagosome, is a cellular compartment that autophagyprotein-dependent antigen presentation accesses to generate peptides for presentation to cd + t cells. a potential evolutionary advantage of this autophagy-protein-dependent cross-presentation is that, by delegating antigen presentation duties to uninfected dendritic cells, the host can bypass the blockade of antigen presentation that may result from microbial antagonism of autophagy in infected cells. in response to infection, the host must activate those arms of the innate and adaptive immune system (including autophagy-dependent functions; fig. ) that help to control infection while, in parallel, triggering specific responses that limit detrimental, uncontrolled immune activation and inflammation. an exciting new frontier in autophagy research is the growing recognition of the function of autophagy proteins in achieving this balance (fig. ) . autophagy proteins function in both the activation and inactivation of innate immune signalling . the autophagy pathway activates type i ifn production in plasmacytoid dendritic cells by delivering viral nucleic acids to endosomal tlrs . by contrast, autophagy proteins negatively regulate rig-i-like receptor (rlr)-mediated induction of type i ifn production through the autophagic elimination of damaged mitochondria (and reduction of ros) , and by the binding of atg -atg to caspase recruitment domains of rlr signalling molecules . moreover, the autophagy protein atg a, but not atg , negatively regulates the activation of sting, a transmembrane protein that is required for efficient activation of type i ifn and pro-inflammatory cytokine production in response to stimulatory dna . thus, it seems that autophagy proteins can negatively regulate ifn production by both autophagy-dependent and -independent mechanisms. with respect to the latter, different autophagy proteins may be specialized to target different innate immune signalling molecules. the autophagy pathway and/or proteins also have a crucial role in the control of inflammatory signalling. a major effect is on the regulation of inflammatory transcriptional responses. increased levels of the adaptor protein p , which accumulates in autophagy-deficient cells, activate the pro-inflammatory transcription factor nf-κb through a mechanism involving traf oligomerization . the accumulation of p in atg -deficient hepatocytes results in enhanced activity of the stress-responsive transcription factor nrf and nrf -dependent liver injury . in addition, paneth cells (intestinal immune epithelial cells) from mice hypomorphic for atg l (atg l hm ) show enhanced transcription of pro-inflammatory cytokines and adipokines . a second important effect of autophagy proteins on inflammatory signalling is at the level of the inflammasome. this complex contains nod-like receptor cryopyrin proteins, the adaptor protein asc and caspase , and is activated by cellular infection or other stress to promote the maturation of pro-inflammatory cytokines such as interleukin- β (il- β) and . atg l -or atg deficient mouse macrophages produce increased levels of mature il- β and il- after tlr stimulation by endotoxin . in addition, mouse chimaeras engrafted with atg l −/− fetal liver haematopoietic progenitors have increased serum concentrations of il- β and il- | n a t u r e | v o l | j a n u a r y after treatment with dextran sodium sulphate (dss), which contributes to increased dss-induced colitis . the mechanism(s) by which autophagy proteins negatively regulate inflammasome activation are not yet understood. mutually nonexclusive possibilities include direct interactions between autophagy proteins and inflammasome components, indirect inhibition of inflammasome activity through autophagic suppression of ros accumulation, or autophagic degradation of danger signals that activate the inflammasome. in line with the latter model, the autophagic degradation of amyotrophic-lateral-sclerosis-linked mutant superoxide dismutase has been proposed to limit caspase activation and il- β production . in addition to regulating inflammatory signalling, the autophagy pathway may prevent tissue inflammation through its role in apoptotic corpse clearance. the efficient clearance of apoptotic corpses during development and tissue homeostasis prevents secondary necrosis, which releases danger signals (damps) that trigger inflammation. autophagy genes are essential for the heterophagic clearance of dying apoptotic cells during developmental programmed cell death (by the generation of atp-dependent engulfment signals) , and the retinas and lungs of embryonic mice lacking atg have a defect in apoptotic corpse engulfment that is associated with infiltration of inflammatory cells . on the basis of growing evidence that autophagy proteins function in tlrmediated phagolysosomal pathways, it is possible that autophagy also functions in phagocytes to facilitate apoptotic corpse clearance. thus, in tissues such as the intestine, in which physiological regeneration involves continuous shedding or apoptosis of epithelial cells, autophagydependent functions in dying cells and/or phagocytic cells may promote efficient corpse clearance, thereby limiting inflammation. perturbations in autophagy-protein-dependent functions in immunity may contribute not only to increased susceptibility to infection, but also to chronic inflammatory diseases and autoimmune diseases. the only well-characterized link thus far is between mutations in autophagy regulators and crohn's disease, a chronic inflammatory disorder of the small intestine, in which a breakdown in clearance or recognition of commensal bacteria, as well as altered mucosal barrier function and cytokine production, is thought to lead to intestinal inflammation (fig. ) . other emerging links include the autoimmune disease systemic lupus erythematosus (sle), inflammation-associated metabolic diseases such as obesity and diabetes, and inflammation associated with cystic fibrosis lung disease (fig. ) . the role of autophagy proteins in crohn's disease was not suspected until genome-wide association studies identified three crohn's disease susceptibility genes, irgm, nod and atg l , that are involved in autophagy . the irgm risk allele contains a deletion in the promoter region of the gene that may be associated with changes in irgm protein expression and may contribute to crohn's disease, given irgm's role in autophagy-dependent control of bacterial infection . however, this hypothesis has not yet been tested. the three major crohn's-diseaseassociated nod variants (a frameshift mutant and two missense mutants) may be loss-of-function mutants, with impaired muramyl dipeptide (mdp)-induced inflammatory signalling . how the loss of function of a pro-inflammatory signal mechanistically contributes to an inflammatory disorder has been unclear, but the recently discovered links between nod and autophagy may solve this conundrum. in primary immature human dendritic cells, nod is required for mdpinduced autophagy, a process that is essential for the mhc class ii presentation of bacterial antigens to cd + t cells and for bacterial targeting to lysosomes . dendritic cells expressing crohn's disease nod risk variants are defective in both of these functions . thus, in patients with crohn's disease and nod risk variants, aberrant autophagy-dependent bacterial clearance and immune priming could act as a trigger for intestinal inflammation. a mechanistic link may also exist between atg l mutation and crohn's disease pathogenesis. similar to findings with nod variants, dendritic cells from patients with the crohn's-disease-associated atg l (t a) risk variant are defective in presenting bacterial antigen to cd + t cells . however, it is not yet known how the t a mutation affects the function of the mammalian atg l protein. this mutation resides in the carboxy-terminal wd-repeat domain that is absent in yeast atg and is dispensable for autophagy. although some studies have suggested that the atg l (t a) variant has reduced autophagic clearance of enteric pathogens such as adherent-invasive escherichia coli or s. typhimurium , it remains controversial whether the risk versus protective alleles of atg l have differences in stability or antibacterial autophagic activity . despite the uncertain nature of the effects of the t a mutation on atg l function, atg l mutation (null or hypomorphic alleles) in mice results in abnormalities that are relevant to crohn's disease pathogenesis. as noted earlier, loss of atg l function in mice results in enhanced tlr-agonist-induced pro-inflammatory cytokine production by macrophages , enhanced dss-induced colitis , and altered inflammatory gene transcriptional profiles in paneth cells , . in addition, the paneth cells of mice expressing low atg l levels (atg l hm ) show defects in the packaging and extrusion of antimicrobial granules into the gut lumen; paneth cells from patients with crohn's disease and the atg l (t a) risk variant show similar defects . this suggests that, in addition to the overlapping functions of nod and atg l in a common bacterial-sensing pathway that promotes bacterial antigen presentation, atg l may have unique protective functions, including paneth cell antimicrobial peptide release and the negative regulation of pro-inflammatory cytokine production. to connect the striking phenotypes in atg l -mutant mice and the pathogenesis of crohn's disease in humans with the atg l (t a) risk allele, the precise effects of the t a mutation on atg l protein function need to be uncovered. a new dimension in understanding the multifactorial basis of chronic inflammatory diseases such as crohn's disease has emerged from the discovery that a virus trigger is required to observe intestinal figure | autophagy/autophagy proteins act to achieve a balance between activation and inactivation of innate immune signalling. a general model in which the levels of autophagy and autophagy proteins control disease in response to stressors. normal autophagy protein function (green) contributes to balanced inflammatory and metabolic responses, resulting in protection against disease. altered autophagy protein function (red) results in maladaptive inflammatory and metabolic responses, increased inflammation and more severe disease. abnormalities in atg l hm mice . in mice raised in a pathogen-free facility, only atg l hm mice (and not wild-type mice) infected with a virus found in routine conventional animal facilities, a murine norovirus, showed abnormal paneth cell granule secretion, paneth cell proinflammatory gene-expression profiles, and intestinal inflammation in response to dss treatment . this mucosal inflammation depended on the presence of the microbiome and pro-inflammatory cytokines, as it was reversed by antibiotic treatment or by tnf-α or ifn-γ inhibition. thus, variations in a host autophagy gene, exposure to a specific virus and the microbiome can act together to trigger intestinal inflammation in mice that is similar to that in patients with crohn's disease. although environmental factors, including the gut microbiome, have long been suspected to contribute to crohn's disease in genetically susceptible individuals, formal proof of this concept was lacking, and viruses were a previously unsuspected trigger. another implication of this work is the concept that autophagy proteins, through their diverse roles in immunity and the control of inflammation, may serve as a central rheostat that prevents inflammatory diseases triggered by environmental stress (fig. ). an important unanswered question is whether perturbations in autophagy may also result in inflammatory autoimmune disease. genome-wide association studies have linked several single nucleotide polymorphisms (snps) in atg to sle susceptibility [ ] [ ] [ ] . sle is a multifactorial, heterogeneous disease characterized by autoimmune responses against self-antigens generated from dying cells. although the effects of these snps on atg expression and function are not known, the lack of atg -dependent negative thymic selection generates autoimmunity and multi-organ inflammation in mice . loss of other atg dependent effects, including regulation of ifn and pro-inflammatory cytokine secretion , , clearance of dying cells figure | the link between mutations in autophagy regulators and the chronic inflammatory disorder crohn's disease. an overview of the many possible mechanisms by which defects in autophagy and autophagy protein function may contribute to the pathogenesis of a type of inflammatory bowel disease, crohn's disease. a micrograph of a human small intestine from a patient with crohn's disease is shown (centre), demonstrating the severe transmural inflammation that is characteristic of this disease. the postulated mechanisms by which defects in autophagy protein function might contribute to the development or perpetuation of intestinal inflammation are based on studies in vitro and animal models. there is no direct evidence that autophagy defects contribute to human crohn's disease, although mutations in three autophagy-related genes, atg l , nod and irgm, are known to enhance risk of the disease. e, epithelium; igm, immunoglobulin m; l, lumen; la, lymphoid aggregates; tm, thickened muscle. scale bar, µm. antigen presentation , might also contribute to the autoimmunity and inflammation associated with sle. thus, a link between atg mutation (or mutation of other autophagy genes) and sle pathogenesis is biologically plausible, although not yet proven. defects in autophagy may contribute to inflammation-associated metabolic diseases such as diabetes and obesity, which are both linked to insulin resistance. the metabolic inflammasome -a complex composed of signalling molecules such as pkr, eif α, jnk, irs and ikkmay act as a link between er stress and more global stress responses, inclu ding inflammation and metabolic dysfunction (as observed in insulin resistance and obesity) . although most components of the metabolic inflammasome promote autophagy, the induction of autophagy by this signalling complex would be expected to serve as a negativefeedback mechanism that limits er stress and disease progression. consistent with this postulated protective effect of autophagy, hepatic suppression of the autophagy gene atg in mice results in increased er stress and insulin resistance , and mice deficient in the autophagy adaptor protein p develop mature-onset obesity and insulin resistance . furthermore, obesity is associated with the accumulation and activation of macrophages and subsets of t cells in adipose tissue and the production of cytokines such as tnf-α and il- (ref. ) . thus, the failure of autophagy-dependent control of er stress, immune cell homeostasis, immune cell activation and/or pro-inflammatory cytokine secretion may contribute to inflammation-associated responses that underlie the pathogenesis of metabolic diseases. another potential link between autophagy deficiency and chronic inflammation is in cystic fibrosis , a life-threatening genetic disorder caused by mutations in the gene encoding the cystic fibrosis transmembrane conductance regulator (cftr). mutations in cftr lead to autophagy inhibition in lung epithelial cells through a mechanism that may involve ros-mediated sequestration of the beclin -class iii pi( )k complex in perinuclear aggregates (redirecting it from its site of autophagy action at the er). restoration of beclin and autophagy in cystic fibrosis epithelial cells rescues the disease phenotype, and antioxidants reverse the airway inflammation in a cystic fibrosis mouse model by a mechanism postulated to involve autophagy. the first series of studies demonstrating that the autophagy machinery is used to attack invading intracellular bacteria was published in (refs , , ) . although autophagy had been observed at the ultrastructural level in cells infected with intracellular bacteria and viruses decades earlier, these studies were a seminal advance. for the first time, pharmacological and genetic manipulation of autophagy, which built on the discoveries of the yeast screens that identified the autophagy machinery, challenged the very notion of autophagy as an 'auto'-(self), 'phagy' (eating) pathway. indeed, we learned that the same genes that are used to orchestrate the degradation of self-constituents, either for nutritional/energy homeostasis or cellular damage control, are also used to orchestrate the degradation of foreign invaders, termed xenophagy. in the past few years, research in the field has uncovered new layers of complexity and functional diversity in terms of how this set of genesoriginally characterized in the context of macroautophagy -may function to protect multicellular organisms against not only the threats of infection but also the threats of the host's own response to infection. the autophagy machinery does much more than form autophagosomes to engulf microbes -it somehow allows microbes in phagosomes and vacuoles to be targeted to the lysosome; it enables crucial cells in the immune system to develop properly and perform some of their 'normal' functions (such as produce ifn, secrete antimicrobial peptides or present antigens to stimulate adaptive immunity); and it ensures that these responses do not become out of control by functioning in central immunological tolerance and the negative regulation of innate and inflammatory signalling. thus, recent advances may not only modify our understanding of immunity (in terms of understanding new roles of the autophagy machinery in immune regulation) but also reshape our understanding of the pathogenesis of inflammatory diseases (in terms of understanding how perturbations in autophagy protein function may contribute to such diseases). clearly, our understanding of the molecular mechanisms of the plethora of functions of autophagy proteins in immune-related processes is still quite primitive. we speculate that, similar to the way in which the initial genetic screens in yeast transformed autophagy research, current proteomic and genomic screens have the potential to transform research on autophagy and immunity. such a transformation would include facilitating a much deeper understanding of the molecular mechanisms of the existing known immunological functions of autophagy through the use of the tools of modern systems biology to understand autophagy protein-protein interaction and signalling regulatory networks on a broad scale. perhaps more exciting is the possibility that such a transformation will uncover new ways in which this ancient self-defence machinery can function in immunity. ■ autophagy, immunity, and microbial adaptations autophagy genes in immunity autophagy and the integrated stress response regulation of innate immune responses by autophagyrelated proteins autophagy in the pathogenesis of disease methods in mammalian autophagy research this paper provides a concise and critical review of current methods to monitor and modulate autophagy in mammalian cells antigen-loading compartments for major histocompatibility complex class ii molecules continuously receive input from autophagosomes termination of autophagy and reformation of lysosomes regulated by mtor dynamics and diversity in autophagy mechanisms: lessons from yeast a subdomain of the endoplasmic reticulum forms a cradle for autophagosome formation d tomography reveals connections between the phagophore and endoplasmic reticulum the role of the atg /ulk complex in autophagy regulation characterization of autophagosome formation site by a hierarchical analysis of mammalian atg proteins two beclin -binding proteins, atg l and rubicon, reciprocally regulate autophagy at different stages autophagosome formation from membrane compartments enriched in phosphatidylinositol -phosphate and dynamically connected to the endoplasmic reticulum mammalian atg (wipi ) localizes to omegasome-anchored phagophores and positively regulates lc lipidation the pancreatitis-induced vacuole membrane protein triggers autophagy in mammalian cells c. elegans screen identifies autophagy genes specific to multicellular organisms the atg l complex specifies the site of lc lipidation for membrane biogenesis in autophagy lc and gate- /gabarap subfamilies are both essential yet act differently in autophagosome biogenesis network organization of the human autophagy system atg conjugation to atg regulates mitochondrial homeostasis and cell death atg a controls dsdna-driven dynamic translocation of sting and the innate immune response new insights into the function of atg mitochondria supply membranes for autophagosome biogenesis during starvation plasma membrane contributes to the formation of pre-autophagosomal structures autophagy enhances the presentation of endogenous viral antigens on mhc class i molecules during hsv- infection selective autophagy: ubiquitin-mediated recognition and beyond an initial step of gas-containing autophagosome-like vacuoles formation requires rab autophagy defends cells against invading group a streptococcus together with reference , provides the first evidence that autophagy has a key role in bacterial infection. atg is shown to be essential for controlling the replication of group a streptococci that escape into the cytoplasm toll-like receptor signalling in macrophages links the autophagy pathway to phagocytosis in vivo requirement for atg in antigen presentation by dendritic cells this study shows that the autophagy machinery is necessary for dendritic cells to process and present extracellular microbial antigens for mhc class ii presentation in vivo, which protects mice against lethal viral infection slam is a microbial sensor that regulates bacterial phagosome functions in macrophages activation of antibacterial autophagy by nadph oxidases autophagosome-independent essential function for the autophagy protein atg in cellular immunity to intracellular pathogens coordinated loading of irg resistance gtpases on to the toxoplasma gondii parasitophorous vacuole disruption of the toxoplasma gondii parasitophorous vacuole by ifnγ-inducible immunity-related gtpases (irg proteins) triggers necrotic cell death autophagy and innate immunity: triggering, targeting and tuning a diacylglycerol-dependent signaling pathway contributes to regulation of antibacterial autophagy autophagy protects against sindbis virus infection of the central nervous system shigella phagocytic vacuolar membrane remnants participate in the cellular response to pathogen invasion and are regulated by autophagy delivery of cytosolic components by autophagic adaptor protein p endows autophagosomes with unique antimicrobial properties contrôle génique de la multiplication du virus de la sensibilité héréditaire au co chez drosophila melanogaster. caryologia (suppl autophagy in mammalian development and differentiation autophagy regulates programmed cell death during the plant innate immune response autophagy is an essential component of drosophila immunity against vesicular stomatitis virus autophagic control of listeria through intracellular innate immune recognition in drosophila autophagy genes protect against salmonella typhimurium infection and mediate insulin signaling-regulated pathogen resistance human irgm induces autophagy to eliminate intracellular mycobacteria autophagy gene variant irgm - t contributes to protection from tuberculosis caused by mycobacterium tuberculosis but not by m. africanum strains genome-wide analysis of the host intracellular network that regulates survival of mycobacterium tuberculosis nod stimulation induces autophagy in dendritic cells influencing bacterial handling and antigen presentation nod and nod direct autophagy by recruiting atg l to the plasma membrane at the site of bacterial entry genomewide association study of leprosy viruses and the autophagy machinery human immunodeficiency virus- inhibition of immunoamphisomes in dendritic cells impairs early innate and adaptive immune responses hsv- icp . confers neurovirulence by targeting the beclin autophagy protein interaction of icp . with beclin modulates herpes simplex virus type pathogenesis through control of cd + t cell responses viral bcl- -mediated evasion of autophagy aids chronic infection of γherpesvirus flip-mediated autophagy regulation in cell death control listeria monocytogenes acta-mediated escape from autophagic recognition autophagosomes can support yersinia pseudotuberculosis replication in macrophages autophagy pathway intersects with hiv- biosynthesis and regulates viral yields in macrophages the autophagy machinery is required to initiate hepatitis c virus replication this study shows that many autophagy proteins are essential for the initial stages of hepatitis c viral rna translation, illustrating that autophagy proteins may be subverted to enhance the replication of intracellular pathogens the early autophagic pathway is activated by hepatitis b virus and required for viral dna replication coronaviruses hijack the lc -i-positive edemosomes, er-derived vesicles exporting short-lived erad regulators, for replication hmgb proteins function as universal sentinels for nucleic-acidmediated innate immune responses endogenous hmgb regulates autophagy vitamin d induces autophagy in human monocytes/ macrophages via cathelicidin a novel human tectonin protein with multivalent β-propeller folds interacts with ficolin and binds bacterial lps a genome-wide sirna screen reveals multiple mtorc independent signaling pathways regulating autophagy under normal nutritional conditions fip is required for the cell-autonomous maintenance of fetal hematopoietic stem cells autophagy in thymic epithelium shapes the t-cell repertoire and is essential for tolerance this study provided the first evidence that the autophagy machinery functions in mhc class ii antigen presentation in vivo, specifically in shaping the cd + t-cell repertoire during negative thymic selection, and thereby preventing autoimmunity and multi-organ inflammation endogenous mhc class ii processing of a viral nuclear antigen after autophagy this study provided the first evidence that the autophagy machinery can deliver endogenously synthesized antigens for presentation on mhc class ii molecules to cd + t cells antigen processing via autophagy-not only for mhc class ii presentation anymore? autophagydependent viral recognition by plasmacytoid dendritic cells absence of autophagy results in reactive oxygen speciesdependent amplification of rlr signaling the atg -atg conjugate associates with innate antiviral immune responses m. t. p at the crossroads of autophagy the selective autophagy substrate p activates the stress responsive transcription factor nrf through inactivation of keap a key role for autophagy and the autophagy gene atg l in mouse and human intestinal paneth cells this study demonstrates that autophagy proteins negatively control endotoxin-induced inflammasome activation. references and also show that autophagy gene deficiency increases susceptibility to experimentally induced inflammatory bowel disease mutant superoxide dismutase -induced il- β accelerates als pathogenesis autophagy gene-dependent clearance of apoptotic cells during embryonic development this paper shows that autophagy genes are necessary for apoptotic cells to generate engulfment signals required for successful apoptotic corpse clearance and the prevention of tissue inflammation genome-wide association defines more than distinct susceptibility loci for crohn's disease nod -mediated autophagy and crohn disease crohn's disease-associated adherent-invasive e. coli are selectively favoured by impaired autophagy to replicate intracellularly impaired autophagy of an intracellular pathogen induced by a crohn's disease associated atg l variant differential involvement of atg l in crohn disease and canonical autophagy: analysis of the organization of the atg l complex in fibroblasts this study shows that both hypomorphic expression of an autophagy protein and a viral-infection trigger are necessary for experimentally induced inflammatory bowel disease, suggesting that the interaction between host defects in autophagy and environmental stressors such as infection may be crucial for the pathogenesis of certain inflammatory diseases genome-wide association scan in women with systemic lupus erythematosus identifies susceptibility variants in itgam, pxk, kiaa and other loci a large-scale replication study identifies tnip , prdm , jazf , uhrf bp and il as risk loci for systemic lupus erythematosus genome-wide association study in a chinese han population identifies nine new susceptibility loci for systemic lupus erythematosus double-stranded rna-dependent protein kinase links pathogen sensing with stress and metabolic homeostasis defective hepatic autophagy in obesity promotes er stress and causes insulin resistance mature-onset obesity and insulin resistance in mice deficient in the signaling adapter p inflammation and metabolic disorders defective cftr induces aggresome formation and lung inflammation in cystic fibrosis through ros-mediated autophagy inhibition autophagy is a defense mechanism inhibiting bcg and mycobacterium tuberculosis survival in infected macrophages autophagy induction by ifn-γ, rapamycin or starvation results in the conversion of mycobacterial phagosomes into phagolysosomes, thereby enhancing mycobacterial killing escape of intracellular shigella from autophagy the work in the authors' laboratories was supported by national institutes of health (nih) grants ro ca and u ai (b.l.); by grants-in-aid for scientific research from the ministry of education, culture, sports, science and technology, japan, and by the takeda science foundation (n.m.); and by nih grants ro ai , u ai , ro ai and ro ca and the broad medical foundation (h.w.v.). we thank t. stappenbeck for discussions, and a. diehl and m. harstein for scientific illustration. we apologize to those authors whose work could not be cited owing to space limitations. key: cord- - gnf j authors: cheung, ying-kit; cheng, samuel chak-sum; sin, fion wan-yee; chan, kin-tak; xie, yong title: induction of t-cell response by a dna vaccine encoding a novel hla-a* severe acute respiratory syndrome coronavirus epitope date: - - journal: vaccine doi: . /j.vaccine. . . sha: doc_id: cord_uid: gnf j the severe acute respiratory syndrome coronavirus nucleocapsid protein (sars-cov n) is one of the major targets for sars vaccine due to its high potency in triggering immune responses. in this study, we have identified a novel hla-a* restricted epitope, n (lalllldrl), of the sars-cov n-protein through bioinformatics analysis. the n-protein peptide n shows a high binding affinity towards human mhc class i in t -cells, and is capable of activating cytotoxic t-cells in human peripheral blood mononuclear cells (pbmcs). the application of using the n peptide sequence with a single-chain-trimer (sct) approach to produce a potential dna vaccine candidate was investigated in hla-a . k(b) transgenic mice. cytotoxicity assay clearly showed that the t-cells obtained from the vaccinated animals were able to kill the n-protein expressing cells with a cytotoxicity level of % in an effector cells/target cells ratio of : one week after the last vaccination, which is significantly higher than other n-protein peptides previously described. the novel immunogenic n-protein peptide revealed in the present study provides valuable information for therapeutic sars vaccine design. severe acute respiratory syndrome (sars) is caused by a novel coronavirus known as sars-associated coronavirus (sars-cov) and the investigation of the sars-cov immunity has garnered significant attention since the worldwide outbreak spreaded to countries in [ ] [ ] [ ] . the sars-cov is an enveloped positive-stranded rna virus encoding four major structural proteins, known as a spike glycoprotein (s), a small envelope protein (e), an integral membrane glycoprotein (m), and a nucleocapsid rna-binding protein (n) [ ] . upon viral infection, the viral proteins expressed within the infected cells are degraded in the cytosol by proteasomes. peptides generated by the proteasomes are transported by the transporter associated with antigen processing (tap) into the lumen of endoplasmic reticulum (er), where an er aminopeptidase produces the mature mhc class i-peptide complex by trimming the peptide to eight or nine amino acids [ ] . the resulting mhc class i-peptide complex expressed on the surface of professional antigen presenting cells, such as dendritic cells, plays important roles in the activation of specific cytotoxic t-cells for infected cell killing. on the other hand, the mhc class i-peptide complexes expressed on the virus infected cells is a "marker" for being killed. therefore, formation of stable mhc class i-peptide complexes is critical to elicit cytotoxic t-cell response to eliminate the infected cells. immunogenicity of a peptide sequence for the cytotoxic t-cell is determined by its binding affinity towards the human mhc class i molecule and its availability upon proteasome digestion of the parental protein. subunit vaccines containing hla-a* restricted peptides is highly effective for the induction of strong cytotoxic t-cell response against infectious virus [ ] . among the four structure proteins of the sars-cov, the s and n proteins are the major targets for vaccine research studies due to their potency in triggering immune responses [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . the s protein contains amino acids and is involved in viral entry through - x/$ -see front matter © elsevier ltd. all angiotensin-converting enzyme receptor on host cell surface [ , ] and the n protein contains amino acids and is involved in the viral rna packaging [ , ] . previous studies suggested that hla-a* restricted sars s and n protein peptides can trigger specific human cytotoxic tcell response against sars in vitro [ , ] . in the present study, we have identified a noval hla-a* restricted nprotein peptide (n : lalllldrl). the binding affinity of the n peptide towards human mhc class i molecule is comparable to the n peptide and is higher than the n and n peptides previously described [ ] . moreover, in order to stablize the mhc class i-peptide complex for antigen presentation, the n sequence was genetically linked to the cdna of human ␤ -microglobulin and the alpha- and alpha- domains of the human mhc class i heavy chain to form a single-chain-trimer (sct) in our dna vaccine design. the sct approach makes the mhc class i molecules less dependent on chaperone assistance for peptide loading and hence a stable mhc class i-peptide complex for antigen presentation is produced [ ] [ ] [ ] . the potency of the n dna vaccine to trigger cytotoxic t-cell response against n-protein expressing cells was demonstrated in a hla-a . k b transgenic mouse model and our results indicated that the sars protein n peptide is a good vaccine candidate for sars vaccine development. the potential sars n-protein peptide sequence for human mhc class i binding was searched by a hla peptide binding prediction program, syfpeithi (http://www.syfpeithi.de) [ ] . seven nine-amino acid peptides with high scores for human mhc class i binding (n - alntpkdhi, n - lqlpqgttl, n - lalllldrl, n - llldrlnql, n - rlnqlesky, n - gmsrigmev, n - illnkhid) were synthesized by a solid-phase strategy using the fmoc-based protocol on an automated peptide synthesizer. the crude products were purified on a reverse-phase preparative high performance liquid chromatography (hplc) column and the homogeneity of the final products was analyzed by reverse-phase hplc of purity of %. the peptides were stored in dimethyl sulfoxide (dmso, sigma) at − • c until use. the dna sequence encoding the n-protein was cloned into a expression vector pet b (novagen) and the recombinant n-protein is expressed in a escherichia coli system bl -condonplus (de )-ril (stratagene) followed by purification with ni-nta his-bind resin (novagen). after protein purification, the purity and the quantity of the recombinant protein was analyzed by sds-page analysis. to establish a n-protein expressing cell-line for the cytotoxicity assay, lung tissues from the hla-a . k b transgenic mice were isolated, minced and treated with collagenase (gibco) at u/ml for min. tissue samples were seeded on a plate and cultured for week, fibroblasts were then collected and immortalized by transduction of a recombinant retrovirus containing the sars n gene and the hpv e -e genes [ ] . expression of the nprotein in the lung fibroblast cell-line was confirmed by rt-pcr (with primers: n: sense: -atgtctgataatgg- , antisense: -ttatgcctgagttg- ; e e : sense: -atgtataaaactaaggg cgtaacc- , antisense: -ttatggtttctgagaacagatgg- ), and the cell-line is named as n/e e /a . k b . the t -cell ( × cem.t ), which are hla-a positive, but deficient for the tap protein for endogenous antigen presentation, were cultured according to the procedure stated in the atcc manual. to determine whether the selected n-protein peptides could bind to the human mhc class i molecule, the t -cells ( × ) were pulsed with each of the n-protein peptides ( g/ml) in the presence of g/ml human ␤ -microglobulin (sigma) for h. after peptide pulsing, the t -cells were washed twice with cold pbs containing % fbs and then stained with a mouse anti-human hla-a antibody (bb . ) followed by a goat anti-mouse igg fitc antibody (zymed). the peptide bound t -cells were fixed with % paraformaldehyde (sigma) and subjected to flow cytometry to measure the relative amount of mhc class i/peptide complexes formed. the results were presented as the fluorescent index (fi) calculated by the mean fluorescence intensity (mfi) of t cells using the formula: fresh human hla-a* positive peripheral blood mononuclear cells (pbmcs) were isolated by ficoll-hypaque density gradients (amersham), followed by enrichment of cd + t-cells by human cd microbeads (macs). the purified human cd + t cells were then subjected to autologous dendritic cells mediated t-cell activation in vitro. in brief, the pbmcs were seeded on dishes for h and the non-adherent cells were removed. the adherent monocytes were then cultured in aim-v medium (invitrogen) supplemented with % human ab serum, u/ml recombinant granulocyte-macrophage colony-stimulating factor (gm-cff) and u/ml recombinant human interleukin (il- ) to obtain dendritic cells. on day , the dendritic cells were matured by addition of u/ml recombinant human interleukin beta (il- ␤), u/ml recombinant human interleukin (il- ), ng/ml recombinant human tumor necrosis factor-alpha (tnf-␣) and g/ml prostaglandin e (pge ). on day , the mature dendritic cells were loaded with the selected n-protein peptides in serum free medium for h. the peptide-loaded dendritic cells were then cocultured with the purified cd + t cells in the presence of u/ml recombinant human interleukin (il- ) and ng/ml recombinant human interleukin (il- ) for t-cell stimulation. the stimulation procedure was repeated once a week and totally three times of t-cell stimulation by the peptide-loaded dendritic cells was conducted. activation of t-cell was investigated by an ifn-␥ elispot assay as previously described with minor modifications [ ] . briefly, a -well elispot plate (multiscreen-ha, millipore) coated with anti-human ifn-␥ antibodies ( g/ml, ebioscience) was blocked with aim-v medium containing % human ab serum. the stimulated cd + t cells ( × ) were then added into the wells together with autologous n-protein loaded b-cells ( × ) and incubated for h. subsequently, the plate was washed and followed by incubation with . g/ml biotinylated ifn-␥ antibody (ebioscience) at • c for another h. after washing, g/ml streptavidin-ap (zymed) was added and incubated at room temperature for min. spots were developed by adding a bcip/nbt solution (invitrogen) and digitally recorded with an elispot plate reader (service provided by beijing swan tech co., china). the dna sequence containing the human ␤ -microglobulin and the chimeric mhc heavy chain, was synthesized by connecting the mouse ␤ -microglobulin cdna, human hla-a* ␣- cdna, hla-a* ␣- cdna and mouse h -k b ␣- domain cdna together by overlapping pcr to construct an mhk gene (m = mouse ␤ -microglobulin, h = human hla-a* ␣- and ␣- , k = mouse h -k b ␣- ) using two templates. the ova ␤ mk b sct gene which was a generous gift from prof. hansen was the template for mouse ␤ -microglobulin and mouse h -k b ␣- domain [ ] . the hhd gene, which was provided by prof. lemonnier, was another template for the human hla-a* ␣- and hla-a* ␣- [ ] . the dna fragments encoding the selected n-protein peptides were then linked to the n-terminal of the mhk gene by pcr using the primers with the corresponding dna sequence at the region to generate single-chain-trimers. the constructed single-chain-trimer dna fragments were cloned into agei and xhoi sites of pvax (invitrogen) to construct n-protein peptide-expressing plasmids, n mhkpvax , n mhkpvax , n mhkpvax , and n mhkpvax for vaccination purpose. ovamhkpvax was also constructed as a control plasmid expressing a siinfekl peptide of the ovalbumin (ova ). [ ] were bred and maintained under pathogen-free conditions (animal care center, hkust, hong kong). the dna vaccine was delivered into - weeks old transgenic mice with a gene gun device as previously described with some minor modifications [ ] . briefly, cartridges were prepared by precipitating the plasmid dna on m gold particles dissolved in . m spermidine and m cacl . the microcarrier/dna suspension was coated on plastic tubing in the presence of . mg/ml polyvinylpyrrolidone (sigma). the coated tubing was cut into . inch cartridges so that each cartridge contained g of dna. dna-coated gold particles were delivered to the shaved abdominal region of mice using a helios gene gun (bio-rad) with a discharge pressure of psi. each mouse was administrated with g dna per injection for a total of three vaccinations within a -week interval. to investigate the cell mediated cytotoxic response triggered by the sct-dna vaccine, mice were sacrificed week after the last vaccination. splenocytes were obtained and cultured in imdm medium (gibco) containing g/ml of the corresponding target peptides and units of interleukin- (peprotech) in a -well plate (nunc) at • c for days. t-cell cells from spleen were harvested by ficoll-hypaque (amersham) density gradient centrifugation and used as effector cells in a cytotoxicity assay. the n-protein transduced n/e e / . k b cells were used as target cells. in the cytotoxicity assay, the effector cells and the target cells were co-cultured in the ratios of : , : , : , : and : . after h incubation, the culture plates were centrifuged and the medium was collected for further analysis using a lactate dehydrogenase (ldh) cytotoxicity detection kit (roche) according to the procedures stated by the manufacturer. the absorbance of the samples was measured by elisa reader at nm with nm as reference wavelength. the spontaneous release of ldh by target cells or effector cells the full-length amino acid sequence of the sars n-protein was subjected to bioinformatic analysis to search for hla-a* restricted nine-amino acid peptides. the scores of the peptides are within the range from − to , and the amino acid sequence of the seven peptides selected that have the high scores are listed in the middle column. a flu m peptide (gilgfvftl) (which is used as a positive control) shows a score of is listed at the bottom. was assessed by incubation of target cells in the absence of effector cells and vice versa. the maximum release of ldh was determined by incubation of the target cells in % triton x- in assay medium. the percentage of specific cell mediated cytotoxicity was determined by the following equation: specific cytotoxicity (%) = experimental value − effector cell spontaneous release − target cell spontaneous release maximum target cell release × in order to search for the hla-a* restricted peptide sequence of the sars n-protein, the syfpetithi program was employed to compare the binding affinity of the nprotein peptides towards the human mhc class i molecule through a bioinformatic analysis. seven high score peptides were selected (table ) and the binding activity of these peptides towards the human mhc class i molecules was investigated by comparison of their ability to stabilize the empty mhc class i molecules expressed on the cell surface in the presence of ␤ -microglobulin. the presence of the stable mhc class i-peptide complex was then detected by the antibody (bb . ) through facs. the results clearly show that among the seven n-protein peptides, n and n display the highest binding affinity towards the human mhc class i and the resulting fluorescence intensities are higher than the positive control flu m peptide ( - , gilgfvftl) [ ] ( fig. ) , suggesting a high binding affinity of the n and n peptides towards the human mhc class i. the t-cell immunogenicity of the seven selected peptides was further tested by their ability to stimulate human cd + t cells isolated from healthy donor pbmcs. the purified cd + t-cells were primed three times with autologous mature peptide-loaded dendritic cells and the level of tcell activation was then investigated by the release of ifn-␥ through ifn-␥-elispot in the presence of autologous bcells loaded with the recombinant n-protein. if the target peptide of the n-protein can be generated and presented by the target cells, the primed cd + t-cells could specifically recognize the peptide-mhc complex and release ifn-␥. the elispot results show that t-cells primed with n (lll-drlnql) and n (lalllldrl) produce the highest number of spots that are six to seven times higher than that found in the irrelevant flu peptide primed t-cells and is significantly higher than that of the other selected n-protein peptides (fig. ) . the results of the t-cell stimulation assay demonstrated that the novel n-protein peptide revealed in the present study is able to trigger specific cytotoxic t-cell response in human pbmcs. the four most immunogenic peptides (n , n , n and n ) selected in the t -cell binding assay and the human t-cell stimulation assay were further tested for their potency in triggering immune response against the sars n-protein expressing cells in an animal model. to facilitate antigen presentation, the dna sequence of the target peptides was genetically linked to the cdna sequence of the mouse ␤ -microglobulin and the chimeric mhc class i heavy chain domains (fig. ) that the peptide can be translated as a peptide-spacer-␤ -microglobulin-space-h-chain complex for t-cell activation. under this covalently linked mhc class i-peptide construction, the linked mhc molecule is at least -fold less accessible to exogenous peptides than normal mhc loaded with endogenous peptide and is more potent in the stimulation of cytotoxic t-cells [ ] . the cytotoxic tcell response triggered by the dna vaccine was investigated by the activity of the spleen t-cells to kill the target cells n/e e /a . k b , which are immortalized primary a . k b fibroblasts expressing the sars n-gene (fig. ), week after the last vaccination. the cytotoxicity level was measured by the amount of lactate dehydrogenase (ldh) released from the target cells. the results demonstrated that the dna vaccines encoding the n-protein peptides n and n trigger the highest t-cell cytotoxicity towards the n-protein expressing cells with and % cytotoxicity level, respectively, in effector cells/target cells ratio of : compared to the cytotoxicity level of the previously described peptide n , which shows only % cytotoxicity, and n , which is similar to the negative control using an irrelevant ova peptide for vaccination (fig. ). the worldwide outbreak of sars in caused a severe economical loss and the development of sars vaccine is one of the most effective ways to prevent the outbreak in the future. an ideal vaccine against infectious virus should be able to trigger both the neutralizing antibody production and the cytotoxic t-cell responses that play a critical role in the elimination of virus infected cells in controlling viral pathogensis. during sars-cov infection, the n-protein is reported to be highly immunogenic [ ] and it has been the y-axis indicates the percentage of cytotoxicity. four groups of mice were vaccinated with the n-protein peptide plasmids, n mhkpvax ( ); n mhkpvax ( ); n mhkpvax ( ); and n mhkpvax ( ). mice vaccinated with an irrelevant plasmid, ovamhkpvax ( ), was used as a negative control. the cytotoxicity level was calculated as previously described. the differences in cytotoxicity level between all peptides with various effector cells: target cells ratios were calculated with t-test (p < . ). shown that n vaccine can trigger specific t-cell response in mice [ ] . however, only six t-cell specific epitopes of the n-protein have been reported up-to-date [ , , ] . the objective of this study is to identify immunogenic n-protein peptides that can serve as cytotoxic t-cell epitope in sars vaccine. a peptide sequence useful for inducing the cytotoxic t-cell response should be presented as endogenous peptide epitope through proteasome digestion and have a high binding affinity towards the human mhc class i molecules. in contrast to the conventional method of screening numerous amino acid sequences manually from synthetic peptide library that is costly and time consuming, bioinformatics analysis was employed to search for the most immunogenic peptide sequences of the sars n-protein. although the efficiency of the bioinformatics analysis in searching the immunogenic peptide is high, discrepant results could also be obtained from different programs online. for instance, although the novel immunogenic n peptide revealed in the present study is the third potent peptide found in the syfpetithi program, it is ranked according to the hla peptide motif search provided by the national institutes of health and was totally ignored in the previous study about searching the t-cell epitopes in the n-protein [ ] . to investigate whether the peptide predicted from the computer program has a high binding affinity to human mhc, a t -cell binding assay was conduced that is based on the binding affinity of the peptide to the empty mhc class i molecules on the cell surface and to stabilize the mhc class i-peptide complex formed. the results clearly show that the novel peptide, n , identified in the present study has a high binding affinity towards the human mhc class i molecule hla-a . that is comparable to the n peptide and is significantly higher than that of the n and n peptides previously described [ ] . previous studies suggested that a peptide sequence with a high binding affinity to hla-a . is not able to trigger t-cell response if the peptide sequence is not generated from endogenous process through the proteasome system. for instance, although the ny-eso- peptide - has a high affinity towards the hla-a molecules, it is not able to elicit cytotoxic t-cell response since the peptide is not processed naturally from the parental ny-eso- protein by the proteasome [ , , ] . to address the question concerning with intracellular protein processing, a t-cell activation assay with n-protein loaded b-cells, which is able to cross-present protein antigen through the proteasome system was preformed [ , ] . the t-cell activation assay demonstrated that when the n-protein loaded b-cells were used as target cells, the n primed t-cells were induced for ifn-␥ production and the level of ifn-␥ produced is comparable to the n peptide and is significantly higher than that of the n and n peptides previously described [ ] , that is consistent to the results obtained in the t -cell binding assay. although a study has previously mapped the three hla-a* restricted n-protein peptides (n , n and n ) in vaccinated transgenic mice [ ] , and the vaccine potency has not been addressed. in the present study, the potency of the novel n peptide together with the three previously described peptides [ ] , vaccine was investigated in a transgenic mouse model expressing the human hla-a . . in contrast to the conventional method using expensive synthetic peptides for injection [ ] that is infeasible for practical used in a large vaccination program, a sct display dna vaccine mechanism was employed. a dna vaccine is much less costly compared to the synthetic peptides and when couple with the sct display system, the immunogenic peptide is translated together with the ␤ -microglobulin and the mhc class i heavy chain molecule as a complex. after translation, the whole covalently linked mhc class i-peptide complex can be transferred from the er to the cell surface for antigen presentation. the high stability of the covalently linked mhc class i-peptide complex produced the sct system excludes competing peptides and is a potent stimulator for t-cells [ ] . thus, it eliminates the uncertainty of the antigen processing in the professional antigen presenting cells and the cytotoxic t-cells can be primed more directly to ensure that the peptide encoded in the vaccine can be presented for vaccination. in our study, the dna vaccine was injected into the transgenic mice for vaccination and the cytotoxicity assay demonstrated that vaccination of the n peptide is able to trigger the cytotoxic t-cell response against the n-protein expressing cells as early as week after the last vaccination even in the absence of any adjuvants. comparative results obtained from cytotoxicity assay among the tested peptides indicated that the n peptide represents as one of the most potent amino acid sequences of the n-protein that is able to trigger the cytotoxic t-cell response. interestingly, all the reported immunogenic t-cell epitopes of n-protein, including the n revealed in the present study, are mostly clustered between the amino acid residues and . therefore it will be interested to investigate whether a vaccine composed of this amino acid peptide (n to n ) coupled with the known sars b-cell epitopes previously described [ , ] is sufficient to trigger a protective immune response against the sars-cov in human. in summary, we have identified a novel hla-a . specific sars-cov n protein epitope (n -n lalllldrl) which can activate cytotoxic t-cells in vitro and when used with the sct system, it is sufficient to elicit cytotoxic tcell response against n-protein expressing cells in the hla-a . k b transgenic mouse model. the findings of the novel cytotoxic t-cell epitope presented in this study provide worth information in sars vaccine design that may contribute to the sars controlling program in the future. sars: clinical presentation, transmission, pathogenesis and treatment options the comparative pathology of severe acute respiratory syndrome and avian influenza a subtype h n -a review the aetiology, origins, and diagnosis of severe acute respiratory syndrome characterization of viral proteins encoded by the sars-coronavirus genome cellular mechanisms governing 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established tumors with a novel vaccine that enhances major histocompatibility class ii presentation of tumor antigen transduction of dendritic cells with recombinant adenovirus encoding hca activates autologous cytotoxic t lymphocytes to target hepatoma cells cutting edge: single-chain trimers of mhc class i molecules form stable structures that potently stimulate antigen-specific t cells and b cells perarnau b. hla-a . -restricted education and cytolytic activity of cd (+) t lymphocytes from ␤ -microglobulin (␤ m) hla-a . monochain transgenic h- db ␤ m double knockout mice analysis of the hla-restricted influenza-specific cytotoxic t lymphocyte response in transgenic mice carrying a chimeric human-mouse class i major histocompatibility complex the minimum peptide epitope from the influenza virus matrix protein. extra and intracellular loading of hla-a induction of th type response by dna vaccinations with n, m, and e genes against sars-cov in mice long-lived memory t lymphocyte responses against sars coronavirus nucleocapsid protein in sars-recovered patients multiepitope cd (+) t cell response to a ny-eso- peptide vaccine results in imprecise tumor targeting cross-presentation of hla class iu epitopes from exogenous ny-eso- polypeptides by nonprofessional apc cpg-dna aided cross-priming by cross-presenting b cells b lymphocytes participate in crosspresentation of antigen following gene gun vaccination antigenicity analysis of different regions of the severe acute respiratory syndrome coronavirus nucleocapsid protein we thank the hong kong research fund for the control of infectious diseases for funding this project. key: cord- - xk w d authors: flower, darren r; macdonald, isabel k; ramakrishnan, kamna; davies, matthew n; doytchinova, irini a title: computer aided selection of candidate vaccine antigens date: - - journal: immunome res doi: . / - - -s -s sha: doc_id: cord_uid: xk w d immunoinformatics is an emergent branch of informatics science that long ago pullulated from the tree of knowledge that is bioinformatics. it is a discipline which applies informatic techniques to problems of the immune system. to a great extent, immunoinformatics is typified by epitope prediction methods. it has found disappointingly limited use in the design and discovery of new vaccines, which is an area where proper computational support is generally lacking. most extant vaccines are not based around isolated epitopes but rather correspond to chemically-treated or attenuated whole pathogens or correspond to individual proteins extract from whole pathogens or correspond to complex carbohydrate. in this chapter we attempt to review what progress there has been in an as-yet-underexplored area of immunoinformatics: the computational discovery of whole protein antigens. the effective development of antigen prediction methods would significantly reduce the laboratory resource required to identify pathogenic proteins as candidate subunit vaccines. we begin our review by placing antigen prediction firmly into context, exploring the role of reverse vaccinology in the design and discovery of vaccines. we also highlight several competing yet ultimately complementary methodological approaches: sub-cellular location prediction, identifying antigens using sequence similarity, and the use of sophisticated statistical approaches for predicting the probability of antigen characteristics. we end by exploring how a systems immunomics approach to the prediction of immunogenicity would prove helpful in the prediction of antigens. vaccines are agentseither molecular or supramolecular -which can stimulate protective immunity against microbial pathogens and the diseases they cause. protective immunity is a specific and enhanced adaptive immune response to subsequent re-infection or, when luck holds, infection by related organisms. such augmented immunity is mediated by the exacerbation of immune memory, which militates against the effects of infectious organisms. the word vaccine itself is derived from vacca (latin for cow). [ ] [ ] [ ] . it is a thing of near universal agreement that mass vaccination -synergising as it does with the herd immunity it helps engender -is the most effective and efficacious prophylactic treatment currently available for contagious disease. humankind is commonly affected by over seventy infectious diseases, many of which are or will be targets for vaccines. there are in excess of fifty licensed vaccines, half of which are deemed to be in common use. most vaccines prevent childhood infections or are used by travellers to tropical or subtropical regions; only a minority combat disease in third-world countries. as recently as the late s, there were over million cases of smallpox spread through countries, with about two million deaths a year, yet now smallpox is wholly and totally eradicated. poliomyelitis or polio is the other key global disease close to eradication. in , the pan american health organization eliminated polio from the western hemisphere. in the first world, the annual death rate arising for contagious diseases such as polio, diphtheria, or measles is less than one in a thousand. the global polio eradication program has now greatly reduced the prevalence of polio in the rest of the world. only cases of polio were reported in . nevertheless, polio remains endemic in nigeria, afghanistan, pakistan, and india. despite such outrageous and egregious success, many major issues persist. no licensed vaccines exist for hiv and malaria, two of the world health organization (who)'s three big global killers, and there are no realistic hopes for such vaccines appearing in the short to medium term. bacille calmette guérin (bcg), the only vaccine licensed currently for the third major world disease, tuberculosis, has only limited efficacy [ ] . add to this the new, previously unknown infectious diseases identified in the past years: hiv, marburg's disease, sars, dengue, west nile, and over human infections with the potentially pandemic h n influenza. it is commonly believed that new contagious diseases will continue to emerge in the st century. the world of the st century is threatened by parasitic diseases and emerging zoonotic infections; antibiotic-resistant bacteria; and bioterrorism [ ] the vaccine arena has long been neglected, partly as a consequence of the extraordinary success just adumbrated, but activity within it is now feverish and febrile [ , ] . dozens of vaccine candidates have passed through phase ii clinical trials, and during the past decade, vaccines in late development have numbered over . unlike antibiotics, resistance to vaccines is negligible. in the same way that vaccines target many kinds of disease, themselves caused by microbial pathogens of all types, so there are many types of fundamentally distinct vaccine. see figure . these include attenuated or inactivated whole pathogens, subunit vaccines, peptide vaccines, and vaccines based on carbohydrates, amongst others. historically, the most successful and prevalent types vaccines have been those based on attenuatedthat is "weaken" or noninfective -whole pathogen vaccines, for example bcg for tb or sabin's polio vaccine. safety concerns have fomented other vaccine strategies to develop, which focus on antigens and latterly epitopes as the intrinsic component of single or composite vaccines. hepatitis b vaccine is an antigen -or subunitbased vaccine. while many epitope-based vaccines have now entered clinical trials, they are yet to fulfil their potential, medically or commercially. antigens, immunogenicity, and subunit vaccines subunit vaccines are typically but not exclusively protein molecules and their discovery is often based around a somewhat haphazard, essentially empirical search for antigenic or immunogenic protein antigens. the word antigen has several meanings in immunology and thus in vaccinology. the oxford english dictionary defines an antigen as a "…foreign substance which, when injected into an organism, stimulates antibody production." they trace its first use to , and by the middle of the century the word had found its way into common technical dictionaries. an immunogen is on the other hand: "any substance capable of eliciting an immune response", although the word had other, earlier definitions. its first use in its modern rendering is traced to , but was doubtless in use before then. figure a schema summarising the components of a generic vaccine. the immunogenic component will typical be an attenuated or chemically-inactivated microbe, a protein antigen, or a poly-epitope or conjugate. this component will be combined with an appropriate delivery mechanism and an adjuvant. delivery mechanisms and adjuvants overlap in their ability to increase the immunogenicity of a weaklyactive vaccine. dictionary definitions seldom capture the scientific meaning of scientific terms particularly well. so, to be more precise, an immunogen, that is a moiety exhibiting immunogenicity, is any substance able to elicit a specific immune response, while an antigen -a moiety exhibiting antigenicity -is a substance which is recognized by an existing immune response, and its associated underlying molecular moieties such as t cells or antibodies, in a recall response. immunogenicity is the most important and interesting property for vaccine design and discovery. it is this property that allows a molecular moiety to induce a significant immune response. for the purposes of this review, our use of the terms antigen and immunogen shall be restricted to a single meaning; that of a protein, specifically one from a pathogenic microorganism, that evokes a measurable immune response. currently, the prophylactic responses of almost all effective vaccineswith the exception of bcg which is mediated by the quadrille of interacting antigen presenting cells (apcs), t-cells, and other phagocytotic cells that characterise cellular immunityare mediated by humoral immunity and antibodies. the pathogenesis of most diseases for which vaccines are not currently available are typically if not exclusively mediated by cellular immune mechanisms. thus, when seeking to identify immune responses for untreated disease, the immunogenicity sought need not be mediated by antibodies; mediation by cellular mechanisms or a combination of humoral and cellular mechanisms is equally valid. in what follows, we will endeavour to explore the availability of informatic tools and techniques for the identification of candidate subunit vaccines. even today, in the early years of the st century, many experimental scientists still retain an atavistic distrust of any and all computational approaches. the very suggestion that algorithms can help them, saving effort and time is an anathema to them and their way of thinking, undermining all that they hold dear. they are deeply distrustful of the reliability of computer methods, preferring what they perceive as the infallible reliability of the experiment. not that they would admit to views so philosophically -and practicallynaive; but deep down this is how they think and how they feel. yet things have changed and changed dramatically, and things will continue so to change. slowly but surely, reverse vaccinology is becoming a discernibly more prevalent means of identifying subunit vaccines; and slowly but surely the contribution made by computation to the practice of reverse vaccinology is becoming ever more significant and ever more obvious [ ] . see figure . conventional laboratory-based, experimental microbiological approaches to antigen identification typically start with the cultivation of the target pathogenic micro-organism under laboratory conditions, then dissecting them into their component proteins, assaying these in some cascade of in vitro and in vivo assays, leading ultimately to the identification of proteins which display requisite protective immunity. it would indeed be wonderful if the identification of candidate vaccines was really and consistently this simple and systematic? unfortunately, it is often so much more complex, confusing, and confounding. it is not always possible to cultivate a particular pathogen outside of the host organism. many proteins are only expressed transiently during the course of infection. nor are all proteins easily expressed in sufficient quantities in vitro. thus many potential candidate vaccines may be missed. reverse vaccinology [ ] [ ] [ ] [ ] is, by contrast, able to analyze a genome to identify potential antigens. initially, the pathogenic genome is scanned for "open reading frames" or orfs. once all orfs have been identified, proteins are selected on the basis that they will be accessible to immune system surveillance, usually using some form of informatic-based prediction methodology or, more likely, set of methdologies. reverse vaccinology was established by a group studying neisseria meningitidis, which is responsible for sepsis and meningococcal meningitis. vaccines are available for all serotypes except subgroup b. potential orfs in n. meningitidis genome were identified [ ] [ ] ; proteins were identified, expressed in vitro, and were surface exposed. seven proteins conferred immunity over a broad range of strains. another good example is streptococcus pneumoniae, a major cause of sepsis, pneumonia, and meningitis [ ] [ ] . potential orfs were identified, of which could be expressed; six proteins were found to induce protection. another example is porphyromonas gingivalis, a gram-negative anaerobic bacterium found in subgingival plaques present in chronic adult inflammatory gum disease periodontitis. of the orfs identified in the genome [ ] , protein sequences were predicted to be open to surveillance by the immune system, were positive for one or more sera. when used to vaccinate mice, two antigens exhibited clear protection. this very brief survey highlights the potential for vaccine discovery using this approach. however, the number of potential antigens is still high, particularly when we explore all potential difficulties in expressing and characterising them. both experimental and computational methodologies may thus omit potentially important antigenic proteins from analysis, albeit for different reasons, and both require continual optimisation. in particular, bioinformatics support for preclinical vaccine discovery has yet to flourish; but, as interest in vaccines waxes, this situation is changing: expanding from the nursery to at least the kindergarten. there are two main forms of support for vaccine discovery provided by bioinformatics. the first is technically indistinguishable from support for more general target discovery, and includes genomic annotation for both host and pathogen sequences and immunotranscriptomics, the application of microarray analysis to the immune system. the other type kind of support is focussed on immunoinformatics: it addresses problems such as the accurate prediction of epitopes. currently, prediction of t cell epitopes is essentially confined to predictions of varying accuracy of peptide binding to major histocompatibility complex. binding of peptides to class i mhc are reasonably accurate, at least for well characterised alleles [ ] . however, several comparative studies have shown recently that the prediction of class ii mhc binding prediction t-cell epitopes is typically poor [ ] [ ] [ ] , and likewise for structure-driven prediction of mhc-mediated epitopes [ ] [ ] . similarly, both structure- [ ] and data-driven [ ] prediction of antibody-mediated epitopes is suboptimal. while the prediction of b cell epitopes remains primitive, or depends on an oft elusive knowledge of protein structure, methods for t cell epitopes prediction displaying not inconsiderable algorithmic sophistication have been and continue to be developed. despite this, and for different reasons, reliable and consistent prediction remains somewhat elusive; this regrettable decision will doubtless continue so for some time. ultimately, all methods, however sophisticated, are severely circumscribed by the data used to propagate and parameterise them. no data-driven method can go beyond the data used to train it; all methods are likewise much superior in their ability to interpolate than their ability to extrapolate. it is only by compiling extensive, high-quality data that we can aspire to create excellent models of general applicability. what we require are sets of data that are complete, thorough, and properly thought through. such sets would be able to explore both efficiently and effectively the complex and confounding multi-dimensional phase space of properties evinced by the molecules we target. iedb [ ] [ ] [ ] [ ] [ ] [ ] is a step-change in this direction. iedb is the dominant force in current immunoinformatics and stands as one of the principal achievements of the field. the number and quality of epitopes within it are an enormous improvement over all that has gone before. alex sette and his co-workers are to be congratulated on their achievement, but, like all dominant things, iedb has tended to choke most other efforts in the field, and even iedb has its limitations. data is usually multi-dimensional, and each dimension will typically be correlated, to a greater or lesser extent, with each other dimension. together, these many dimensions chart a space: a space of structural variation or variation of properties. if the data we use is itself of sufficient quality and provides a good enough coverage figure whole antigen discovery. taking a reverse vaccinology dynamic, the process of discovering candidate subunit vaccines begins with a microbial genome, perhaps newly sequence, progresses through an extensive computational stage, ultimately to deliver a shortlist of antigens which can be validated through subsequent laboratory examination. the computational stage can be empirical in nature; this is typified by the statistical approach embodied in vaxijen [ ] . or this stage can be bioinformatic; this involves predicting subcellular location and expression levels and the like. or, this stage can take the form of a complex mathematical model which uses immunoinformatic models combined with mathematical methods, such as metabolic control theory [ ] , to predict cell-surface epitope populations. of the space, then straightforward methods drawn from, say, computer science -of which there are indeed very many -are now of satisfactory accuracy to build models of high predictive accuracy. as we have said, there is an on-going need for the quality, quantity, and availability of data to improve and increase. prediction is enthral to its underlying data. bias within the data places strict limitations upon the interpretability and generality of models from which it is derived. in general, for mhc-peptide binding experiments, the sequences of peptides studied are very biased in terms of amino acid composition, often favouring hydrophobic sequences. this arises, in part, from preselection processes that result in self-reinforcement. binding motifs are often used to reduce the experimental burden of epitope discovery. very sparse sequence patterns are matched and the corresponding subset of peptides tested, with an enormous reduction in sequence diversity. irrespective of their poor performance in prediction, several other problems exist, albeit different for t-and b-cell epitope prediction. for t-cell prediction, the key issue is the availability of data. it has recently been shown that t-cell epitopes, which were previously thought to be short peptides of - amino acids, can be up to amino acids or perhaps even more. the existence of these longmer epitopes has greatly expanded the repertoire of peptides open to inspection by t-cells [ ] [ ][ ] [ ] . problematic as this seems, the situation is made worse by the fundamental logistic constraints of sampling within the specificity exhibited by a single allele. the number of possible sequences that a nonameric peptide might possess is in the region of billion; considering that a single model is built from at most a few hundred peptides, the sampling undertaken is infinitesimally small. similarly, the + different mhc alleles known to exist in the global human population indicates the extraordinary potential for distinct peptide specificities within the potential patient cohort. for b-cell prediction, explaining the poor performance of b-cell epitope prediction algorithms may point to a fundamental misinterpretation of extant epitope data. pepscan is perhaps the most abundant data available currently but may not be what it seems. experimentally derived epitopes are identified by being assayed against pre-existing antibodies with affinity for whole antigens. however, if such "epitopes" are mapped on to their parent antigen structure they are randomly located throughout the protein and do not equate to clear surface patches, as one might expect if they simply mimic discontinuous epitopes identified by crystallography; in situ these antigenic regions can be completely buried, and thus inaccessible to antibody binding, rather than exposed. if we compare the conformation of antibodybound peptides with those from the intact antigens, they are usually quite different. however, b-cell epitopes in intact antigen and in whole antigen-antibody complexes are similar. it may be that the preformed antibody recognizes denatured antigen in vivo or that the isolated peptide adopts a conformation able to mimic the surface features of a discontinuous epitope. moreover, there is also evidence that the responsiveness of the immune system to pathogenic proteins is only poorly correlated with the possession of t cell epitopes, and that many potential epitopes have been deleted in proteins regularly accessible to immune surveillance, perhaps as an evolutionary counter measure in the war between host and pathogen [ ] . taken together, these factors suggest that methods which rely solely on the possession of epitopes are unlikely to be fully effective when tasked with identifying antigens or immunogens. this conjecture is confirmed by what information there is, which indicates that there is little simple correspondence between antigens selected on this basis and experimentally verified antigenic or protective proteins. thus we must seek alternative methods for selecting, and prioritising within that selection, proteins likely to be antigenic and protective. we shall below examine three key approaches: subcellular location prediction, sequence similarity, and empirical statistical approaches, typified by vaxijen. for a protein to be accessible to surveillance by the immune system, it is often assumed to be physically external to the microbial organism or at least present on its surface rather than being sequestered away far from the roving eye of the immune system. for bacteria this means it must be secreted or located on -or in -the outer membrane surface. in this context, immune surveillance is undertaken by a variety of cellular and molecular actors, but chiefly by neutralising antibodies. thus the goal of identifying antigens through the use of subcellular location prediction is principally to identify surface proteins accessible to binding by neutralizing antibodies. unlike the relatively straightforward and well-studied task of identifying orfs, selecting the subcellular location of proteins can be challenging to the point of confusion. nonetheless, and notwithstanding the obvious caveats inherent in this strategy, this has become a favoured approach taken by many when tasked with selecting vaccine candidates. we should remember, however, that antigens are not epitopes, and epitopes are not antigens. so possession of t-cell and b-cell epitopes is in certain senses independent of sub-cellular location, and they may in principle be possessed by any protein. as we have said, there is evidence for t-cell epitope depletion in pathogenic proteins [ ] ; or it may be that just picking immune-accessible proteins naturally favours proteins rich in antibody and helper t-cell epitopes, as opposed to cytotoxic t-cell epitopes, at least for membrane proteins. as data accumulates on the specific responses made to differently located accessible proteins we can build these recondite subtleties into prediction strategies. there are two basic kinds of prediction method: manual construction of rules of what determines subcellular location and the application of data-driven machine learning methods, which determine factors that discriminate between proteins from different known locations. accuracy differs markedly between different methods and different compartments, mostly due to a paucity of data. data used to discriminate between compartments include the amino acid composition of the whole protein; sequence-derived features of the protein, such as hydrophobic regions; the presence of certain specific motifs; or a combination thereof. databases, such as prodom [ ] , pfam [ ] , and pro-site [ ] , contain within them the capacity to identify sequence motifs characteristic of certain protein families; this can in turn help us to predict if a protein belongs to an family of proteins which are typically extracellular. however, gross sequence similarity alone is neither a necessary nor a sufficient condition for a protein to share a common sub-cellular location. very similar sequences may be located quite differently, while lack of similarity is no guarantee that proteins will not be co-located. moreover, many proteins can and do exist in several locations within the cell, and often evince different if related functions in these different locations [ ] . likewise, different organisms, and specifically eukaryotes versus prokaryotes, have quite different locations, both in number and in physical nature. the number of such compartments used in prediction studies varies considerably, and few papers address the full rich complexity of cell biology. for example, a common schema reduces eukaryotic cells to compartments (cytoplasmic, extracellular, mitochondrial, and nuclear) and prokaryotic to compartments (cytoplasmic, extracellular, and periplasmic), though some papers use over compartments for eukaryotes. in fact, compartments is an under-estimate, and the richness and the complexity of sub-cellular location is daunting, since any attempt to predict it must account for transient and permanent location, multiple locations, and both membrane organelles and multi-protein complexes as potential locales. among binary approaches, arguably the best method is signalp, which employs neural networks and predicts n-terminal secretion signals cleaved by signal peptidase-i-and their associated cleavage site [ ] [ ] [ ] . the signal predicted is the type ii signal peptide common to both eukaryotic and prokaryotic organisms, for which there is a wealth of data, in terms of both quality and quantity. a recent enhancement to signalp is the implementation of a hidden markov model (hmm) which seeks to discriminate uncleaved signal anchors from cleaved signal peptides. one of the limitations of signalp is overprediction, as it cannot reliably discriminate between several very similar yet distinct signal sequences, regularly predicting membrane proteins and lipopteins as type ii signals. many other kinds of signal sequence exist. a number of methods have been developed to predict lipoproteins, for example. the prediction of proteins that are translocated via the tat-dependent pathway is also important but has yet to be addressed in any depth. many programs and servers have been devised and developed able to predict the subcellular location of gene products and proteins. psort is a well-known, well-used example; it is knowledge-based, multicategory prediction method, composed of several programs [ ] [ ] [ ] [ ] . psort i predicts different subcellular compartments, while psort ii predicts locations. there are several specialized versions of psort. ipsort deals specifically with secreted, mitochondrial, and chloroplast locations. psort-b only predicts bacterial sub-cellular locations. it reports precision values of . % and recall values of . %. another effective program is hensbc [ ] . this constructs a hierarchical ensemble of classifiers by applying a series of if-then rules. hensbc is able to assign proteins to one of four different types (cytoplasmic, mitochondrial, nuclear, or extracellular) with approximately % accuracy for gram-negative bacterial proteins. another program is subloc [ ] , a client/server suite which offers an interface for the prediction of prokaryotic subcellular location in three compartments. another still is gpos-ploc [ ] , which fuses many basic classifiers, engineered using the optimized evidence-theoretic k-nearest neighbours rule. other programs include tatp . [ ] , lipop . [ ] , and phobius [ ] . this list goes ever on and on. a comparison of a subset of these programs, which used a test set of mycobacterial proteins [ ] , indicated that subcellular localization methods generally had high predictive specificity and recognised true negatives reliably. we have also developed a range of programs specifically aimed at addressing the prediction of subcellular location within bacteria. building on a set of algorithms able to predict membrane proteins, tat secretion, and lipoproteins [ ] [ ] [ ] [ ] [ ] [ ] [ ] , a set of bayesian networks, which were able to make individual predictions for specific subcellular locations was implemented in three pipelines with different architectures: a parallel implementation with a confidence level-based decision engine and two serial implementations with a hierarchical decision structure [ ] . these two hierarchies were rooted differently, one by prediction between membrane types and the other by soluble versus membrane prediction. the parallel pipeline outperformed the serial pipeline, but took twice as long to execute. the soluble-rooted serial pipeline outperformed the membrane-rooted predictor. assessment using genomic test sets was more equivocal, as many more predictions are made by the parallel pipeline, yet the serial pipeline identifies more of the proteins of known location. this work is indicative of a clear direction that the development of future subcellular prediction strategies might take. currently, the richness of subcellular structures is yet to be integrated into location prediction. many cellular organelles are not included in such studies, nor are proteins with multiple locations, nor are the different routes by which proteins can reach the same compartment. the list of such caveats continues. clearly, if we can develop high specificity predictors for each such compartment, then combining them appropriately [ ] , must be the way forward. there is then a need for a more sophisticated phase of supervised learning, where better and more complete annotation is built into any approaches taken. many difficulties remain however, most notably the lack of validated, high quality datasets where the location of proteins has been established unambiguously. this paucity is particularly acute for certain important but less well-studied kinds of secreted protein, such as those secreted by the type iii secretion system. while databases such as dbsubloc [ ] have been developed, their utility is open to question. so too is the extraction of datasets directly from swiss-prot. here the inherent uncertainty in some of the available annotation can be confounding or at least frustrating. likewise, much more work on the prediction of the subcellular location of viral proteins is required; while some studies have been undertaken [ ] [ ] , the complexity of their interactions with their host organism must be factored into future analyses. generally, we can make the observation that ignoring this complexity is, in trying to simplify matters, more likely to render any analysis simplistic. fortunately, subcellular location prediction, useful though it can be, and quibbles and cavils notwithstanding, is not the only method available to us. there are other weapons in our arsenal, many with an equal provenance and of equal utility. one of the most obvious ways to identify new potential antigens in newly sequenced microbial genomes is through similarity searching. assuming that we know the sequence of one or more extant antigens, we can make use sequence searching programs of various complexity and sophistication, such as blast or fasta, to identify similar sequences in the target genome. this set of selected candidate antigens can then be prioritised for further theoretical and ultimately experimental validation. obviously, all the same caveats that exist for any sequence search hold here also: which thresholds are appropriate? are apparently high-scoring matches real or an artefact of search methodology? this process also presupposes that enough known antigens are available so that such searches can be comprehensive and thus effective. such compilation is the role played by the database. in the last decade, factory-scale experimentation allied to extensive literature mining has generated many functional immunology databases. databases, such as syf-peithi [ , ] , which focus on properties of cellular immunology, and look primarily at data relevant to mhc processing, presentation, and t-cell recognition have existed since the mid s. arguably, the best such database is the hiv molecular immunology database [ ] , although clearly the depth of the database is at the expense of breadth and generality. it archives cd + and cd + t-cell and b-cell epitopes derived from the virus. it also includes detailed biological information regarding the response to the epitope, including its impact on long term survival, common escape mutations, whether an epitope is recognized in early infection, and curated alignments summarizing the epitope's global variability. other recent databases include mhcbn [ , ] , which contains , mhc-binding peptides, , mhc nonbinding peptides, , tap binders and nonbinders, and , t-cell epitopes. epimhc [ ] is a relational database of naturally occurring mhc-binding peptides and t-cell epitopes. presently, the database includes distinct peptide sequences from various sources, including tumor antigens. two databases in particular, warrant special attention, albeit for different reasons. they are antijen [ ] , formerly known as jenpep [ , ] , and iedb [ ] . antijen seeks to integrate a wider range data than is archived by other databases. implemented as a relational postgresql database, antijen is sourced from the primary literature and contains over , entries; it includes quantitative kinetic, thermodynamic, functional, and cellular data within the context of immunology and vaccinology. as well as t-cell and mhc binding data, antijen holds over , entries for linear and discontinuous b-cell epitopes, and includes measurements of peptide interactions with tap transporter and peptide-mhc complex interactions with t-cell receptors (tcr), as well as immunological protein-protein interactions. iedb is a database lavishly funded by the nih, which addresses issues of biodefence. as we have said, it is on a much larger scale than any other similar database, and benefits significantly from the input of dedicated epitope sequencing projects. these exist, in part at least, to populate the database. iedb has largely eclipsed other efforts in functional immune databases. however, it does not, as a priority, address antigenicity at the whole protein level. at this point it is worth discussing the distinction between functional annotation and the objective discussed here. generally speaking, the function that a protein performs within the context of its organism of origin is irrelevant to its status as an antigen. here the ubiquity and multiple meanings of the word antigen are of little if any help. a protective antigen is a protein which is recognised and recalled by the host. this characteristic does not seem to be linked to the fact that a protein is an enzyme or a dna binding protein, nor does logic require such a link. thus identifying function is not a necessary condition for a protein to be an antigen, though the unequivocal identification of certain functions, such as being a virulence factor for example, greatly increases the probability that it will be such. concerning antigens, however, these databases, although replete with information concerning individual b cell epitopes, t cell epitopes, and major histocompatibility complex (mhc) binding peptides, remain otherwise partial and incomplete. their focus is on the epitope, not the antigen. there are many antigens for which specific epitope or mhc binding information is not currently available, yet many such antigens are known experimentally to induce either or both innate or adaptive immune responses. fortunately for the future of vaccine design and discovery specific antigen-orientatedrather than epitope-orientated -databases are now becoming available. arguably, the clearest and most unequivocal example of an antigenic protein is the so-called virulence factor (vf). these proteins are able to undertake the colonization of a host organism and/or induce disease. they are the front-line weapons in the pathogenic arsenal. analysis of known pathogenic species, such as vibrio cholerae or streptococcus pyogenes, has enabled the recognition of recurrent "systems" of vfs and toxins that may total or more distinct proteins. these may exist as discrete pathogenicity islands or be spread more widely in the genome. traditionally, classification of vfs has categorised them as belonging to several thematic groups: adherence/colonization factors, invasions, exotoxins, transporters, iron-binding siderophores, and miscellaneous cell surface factors. a broader definition groups vfs into three classes: ( ) "true" virulence factors; ( ) vfs associated with the expression and regulation of class vf genes; and ( ) vfs required for the colonization of the host [ ] . a number of databases that archive vfs have been reported. the virulence factor database (vfdb; url: http://www.mgc.ac.cn/vfs/) contains characterized bacterial genomes with an emphasis on functional and structural biology and can be searched using text, blast, or functional queries [ , ] . tvfac (los alamos national laboratory toxin and virulence factor database; url: http://www.tvfac.lanl.gov/) contains genetic information on over organisms and separate records for thousands of virulence genes and associated factors. the fish pathogen database (url: http://www. fishpathogens.eu/vhsv/index.php), set up by the bacteriology and fish diseases laboratory, has identified over virulence genes using fish as a model system. pathogens studied include aeromonas hydrophila, edwardsiella tarda, and many vibrio species. candida albicans virulence factor (candivf) is a small species-specific database that contains vfs, which may be searched using blast or a hla-dr hotspot prediction server [ ] . phi-base is a noteworthy development as it integrates vfs into a cohesive whole a variety of pathogens of plants and animals [ ] . obviously, antigens need not be vfs, they need only be accessible to immune surveillance and need not be directly or indirectly involved in infectivity. because of this, other types of database are required, able to capture and contain a wider tranche of relevant data. in the recent past, another database has been developed: anti-gendb [ ] contains a compilation of over antigens drawn from the literature and other immunological resources. it marks a new beginning in immunoinformatics, signalling a switch away from the peptide epitope and toward the whole protein antigen. these antigens come from important pathogenic species. in antigendb, a database entry contains information regarding the sequence, structure, origin, etc. of an antigen with additional information such as b and t-cell epitopes, mhc binding, function, gene-expression and post translational modifications, where available. anti-gendb also provides links to major internal and external databases. antigendb will be updated on a rolling basis, with the regular addition of antigens from other organisms. this database will form the core of future attempts to predict antigens both by sequence similarity and using more recondite analysis. at this point it is worth mentioning the issue of thresholds. clearly, when one runs a sequence search, using blast for example, one might generate huge lists of near-identical proteins or get no hits at all; and, for that matter, we could also obtain almost any intermediate result. the issue is to judge which result is useful and which is not. this typically equates to setting a threshold, above which we anticipate usefulness and below which we might expect a lack of utility. setting such thresholds is however no easy task. they are dependent on the nature of the family in question. for the lipocalin family [ ] [ ] [ ] , for example, hits are still valid in terms of structure and function at levels that would simply be noise for most other protein families. thus thresholds are family dependent, as well as problem dependent. empirically-selected cut-offs are thus the order of the day, but much thought and experimentation is needed in order to select appropriate values. as well as seeking similarity to known antigens, there is another, quite prevalent, idea that is deserving of comment: that the immunogenicity of protein is determined solely by its lack of similarity to the host. what we search for is some quantitatively-meaningful measure of the "foreignness" of a protein which correlates highly with its immanent immunogenicity. in this context, what we mean by the word "foreignness" is the evolutionary distance between the hosta man or a cow or mouse -and the candidate antigen, or the organism that produced it, or both. some consider this to be the prime factor determining the potential immunogenicity of a protein [ , ] . clearly, since we are dealing with proteins and carbohydrates and the like, this evolutionary distance must be specified in terms of their molecular structures, or more likely their sequences, rather than in terms of morphological features. the potential importance of such a concept is supported by the observation that immune systems are actively educated to lack reactivity when presented with self-proteins [ ] , a processoften called immune tolerancewhich is generated via epitope-specific mechanisms including clonal anergy, receptor editing, and clonal deletion [ ] [ ] . but how can we progress beyond this rather inexactly-specified philosophical standpoint to something which is practically useful when we select vaccine candidates? a potentially more useful way to express this conjecture is that the likelihood that a protein is immunogenic is solely a function of that protein's dissimilarity to the whole host proteome at the sequence level. or, to be more precise, how close in terms of sequence similarity is the candidate to the closest or most similar member of the host proteome. most sequence software is well suited to solving this problem, since it is precisely this problem that they were written to address. more difficult is assessing average dissimilarity to the whole proteome, a problem compounded when we use the similarity of overlapping peptide fragments rather than looking at the similarity at the level of global sequence alignment. in terms of choosing the length of such fragments, the epitope would seem to be the most logical choice, since this immunological quantum is the moiety most likely to be recognised during the immune response. yet even at the level of the epitope, a peptide of say amino acids, even one mismatch in an otherwise perfect match can be significant, since such sequence differences, comprising a single amino acid, may exacerbate or abrogate neutralizing antibodies binding to a particular antigenic protein. moreover, the crossreactivity of a single high-affinity monoclonal antibody is rather different in nature to the cross-reactivity of large set of less affine polyclonal antibodies, and so too their ability to tolerate amino acid mismatches. it will also vary between individuals, since the immunization history of each organism will dictate to a large extent the recognition of epitopes. our understanding of epitopes can inform our understanding of mismatch tolerance, since the affinity of t-cell epitopes is more dependent on the possession of suitable anchor residues than it is on the possession of non-anchor residues. having said, the dogma of anchordependent affinity was long ago debunked, since all residues make some kind of contribute to affinity, entropic or enthalpic, although generally it is right to say that socalled anchors do make more significant contributions. our understanding of the structural-basis that determines the affinity of antibody-mediated epitopes is much less assured and complete, and the underlying thermodynamic causes of affinity, if strict causes they are, typically only become clear when high resolution structural data combines with measured thermodynamic metrics. likewise, when one looks not at a representative individual, but at the whole population, then the deletion of a single protein, within one host versus another, can render candidates previously valid and immunogenic as suddenly neither. again, these are difficult issues; as yet, they remain unresolved. given the hypothesis that immunogenicity is in some sense mediated by the level of similarity between a pathogenic protein and the host proteome, we have, in as yet-unpublished work, sought to bench-mark sequence similarity analysis as a means of quantifying the differences between populations of antigens and non-antigen. to that end, we identified sets of known antigenic and non-antigenic protein sequences derived from a variety of sources: allergens, bacteria, fungi, parasites, tumours and viruses. these were compared to the human and mouse genomes using standard sequence similarity searching protocols. whole pathogen proteomes were also aligned to these host proteomes. most antigenic and non-antigenic sequences were observed to be non-redundant; this implies a lack of clear homologues between pathogens and the human or mouse proteomes, although a number of parasite antigens were found to have a much higher level of similarity. these proteins comprised heat shock proteins, catalases, and enzymes involved in hydrolysis. these protein families are structurally conserved, though they might display significant functionality diversity. we also used statistical approaches such as the nonparametric mann-whitney test to assess if comparisons between the two populations were significant. the statistical null hypothesis was accepted in most cases, signifying that the effect presumably resulted solely from chance. the mann-whitney test supported the observations from sequence similarity analysis. we were unable to determine a threshold or cut-off based on the hypothesis of non-redundancy to the host's proteome. these results suggest that this is not in itself a solution to the identification of antigens. a variant based on fragments may be more successful, and this is clearly an issue crying out for further, deeper research. there are, of course, many other ways to approaches identifying antigenic proteins. one notable way, is looking for the horizontal transfer of so-called pathogenicity islands, clusters of pathogenic proteins acquired by transfer between micro-organisms. detection of such islands, which are typically large gene clusters with an atypical yet characteristic g + c content, can in turn lead to the identification of antigenic proteins [ ] [ ] [ ] [ ] . analysis at the nucleic acid level rather than at the protein level can facilitate the discovery of virulence proteins, perhaps using similar approaches to that used to identify cpg islands [ ] [ ] [ ] [ ] . however, rather than look at nucleic acid sequences, or at protein sequences directly, a new approach, based upon alignment-free techniques, has been developed which shows significant potential; we examine this next. most in silico approaches to predicting antigens make use of bioinformatics tools of one kind or another. such tools can identify membrane proteins, signal peptides, or lipoproteins with some success, yet most algorithms still rely on sequence alignment to identify characteristic sequence relationships or motifs characteristic of antigens. this may prove problematic in several ways. some proteins formed through divergent or convergent evolution lack obvious sequence similarity, although they may share similar structures and/or biological properties [ , ] . in such a situation, alignment-based approaches may produce ambiguous results or fail spectacularly. many methods presuppose a direct sequence relationship such as that which can be revealed by simple sequence search techniques, such as blast. this is not always the case. immunogenicity, as a property, may instead be encoded within the sequence and structure of a protein in such a cryptic and subtle manner as to go beyond the conventional limitations imposed when one seeks direct identification by sequence alignment protocols. likewise, the discovery of truly novel antigens will be frustrated by their lack of similarity to antigens of known provenance. as a departure from alignment-dependent techniques, which dominate immunoinformatics as much as they do bioinformatics, we have implemented an approach which seeks to discriminate between candidate vaccines and nonantigens, using an alignment-free sequence representation [ , ] . rather than concentrate on epitope and nonepitope regions, the method uses data on immunoprotective antigens derived from pathogenic sources to derive statistical models for predicting wholeprotein antigenicity. in attempting to overcome the limitations imposed by alignment-dependent sequence similarity methods, we have implemented a novel alignment-independent method for antigen identification based on auto cross covariance (acc) transformation of protein sequences into uniform equal-length vectors. the acc transform is a protein sequence mining method originally devised by wold et al. [ , ] , which has found much application to the quantitative structure-activity relationships (qsar) study of peptides and also for the classification of proteins [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . the principal properties of the amino acids were represented by z descriptors [ ] [ ] [ ] , which describe amino acid hydrophobicity, molecular size and polarity. the acc transformation accounts for neighbour effects, i.e. the lack of independence between different sequence positions. initially, we sought to develop models able to distinguish immunoprotective proteins from the general microbial proteome. we applied acc pre-processing to sets of known bacterial, viral and tumour antigens and developed alignment-independent models for identifying antigens. in a separate paper, extra models were added which address fungal and parasite antigens. for bacterial, viral and tumour antigens, models had prediction accuracies in the % to % range [ , ] [ ] . for the parasite and fungal antigens, models had good predictive ability with % to % accuracy under internal cross validation in groups. under external validation, they gave % sensitivity ranking the true immunoprotective proteins in the first % of their proteomes. the models were implemented in a server for the prediction of protective antigens and subunit vaccines, which we have christened vaxijen [ ] (url: http:// www.darrenflower.info/vaxijen). the accuracy values noted above are indicative of an approach that is more accurate than has been seen before; for example, for b-cell epitope prediction. vaxijen is an imperfect beginning; future research will yield significantly more insight as the number of known protective antigens increases [ ] . there are several bioinformatics problems unique to immunology: the foremost and greatest is the challenge of accurately predicting immunogenicity. the successful computational prediction of immunogenicity may manifest itself at one extreme in the identification of a t-cell or b-cell epitope. this is mediated by straightforward if not uncomplicated molecular recognition events, which can be understood through properly exploring relationships between fundamental physical propertieshydrogen bonding and lipophilicity for exampleand apparent biological activity. establishing such structureactivity or property-activity relationships is a considerable concern of current immunoinformatics. at the other extreme, we encounter an altogether more complex phenomenon, where we see immunogenicity made manifest in the accurate estimation of antigenicity at the whole protein level. this is a system property; that is a property of the whole immune system rather than an individual molecular recognition event. at present, this is very much a secondary aspect of modern day immunoinformatics. the task of predicting whole protein immunogenicity is probably several orders of magnitude more complex and demanding than say predicting the binding of peptides to an mhc or even of predicting the binding of a pmhc complex to a tcr. this is not to say that such calculations are in any way trivial or lacking difficulty, but rather than that such complex prediction exercises are themselves subsumed in the task of predicting immunogencity at the system level. in our view, and in the view of other commentators, the clinical manifestation of the immunogenicity of a vaccine antigen, as opposed to the immunogenicity of an isolated epitope, arises as the very complex amalgam of many intertwining intrinsic and extrinsic factors, operating at various length-scales and at various different rates. such factors include host-side properties and pathogen-side properties, as well as protein-side properties that arise more or less exclusively from the protein itself. see figure . so-called host-side properties are properties intrinsic to the immune system of the host. they include the possession of b-cell epitopes or t-cell epitopes, as recognised by the adaptive immune system, or the possession of pathogen-associated molecular patterns or pamps, which are recognised by pattern recognition receptors, such as toll-like receptors (tlrs), part of the innate immune system. pathogen-side properties are properties intrinsic to the pathogen as a whole organism. they include the expression level evinced by an antigen, the time-course of its expression and secretion, and also its subcellular location. protein-side properties include the state of aggregation exhibited by a candidate vaccine and any post-translational danger signals that the protein might possess. some of these properties have been discussed before. thus one would expect, at least naively, that a bona fide vaccine antigen would be both highly expressed and available for immune surveillance, as well as possessing epitopes that the host can recognise, where as a protein which is not immunogenic would lack such characteristics. identifying and predicting this diverse tranche of properties is a problem and thus a challenge. indeed, each component part is a challenge unto itself. consider the prediction of a t cell epitope: the best understood and most accurate of immunoinformatic prediction problems. such an epitope needs to be processed properly into a population of different peptides, and for the processed peptides to exist in a reasonable amount, and then be bound by an mhc with reasonable affinity, before being recognized appropriately within the context of a pmhc complex by a tcr. this then is a complex and contingent process, similar to a generic markov process, comprised of many stages which are themselves dependent on preceding steps. in terms of both mechanism and what steps-within-steps there may be, many of these steps are less than adequately understood. however, each step remains amenable to statistical evaluation. all stages are inherently predictable given an appropriate accumulation of relevant data. the situation is very similar but not identical for biopharmaceuticals. in this case, we can dispense with the pathogen and replace it with several classes of extrinsic factors which include amongst others product manufacture, patient health, and medication strategy. acute illness, particularly systemic illness, or latent bacterial or viral infections such hcv, can have a most profound effect on the manifestation of immunogenicity. likewise, a patient's genetics may alter profoundly the immune reaction to biopharmaceuticals, most obviously, the mhc haplotype will affect host t-cell responses. the nerve program is an expert system for vaccine antigen discovery, which addresses in a practical way some of the issues of prioritisation discussed above. the program has been developed to help automate the process of reverse vaccinology [ ] . the identification and prioritising of potential orfs using nerve comprises stages. firstly, the prediction of subcellular localization; then whether the protein is an adhesin; followed by the identification of transmembrane domains; then the protein is compared with both the human and pathogen proteomes; after which the protein is finally assigned a suggestive function. vaccine candidates are thus filtered and ranked, and a list of proteins is produced graded by precedence and the probability that it will be an immunogenic antigen. but nerve and other extant programs, such as dynavacs [ ] , tasked with such a confounding task necessarily fall far short of what is needed or indeed what is possible. there needs to be a concerted and farreaching effort to address this issue. the most direct line of attack is to address first the subcellular location issue, as we discussed above. likewise, we can deconstruct the antigen presentation pathway, building models for each step and then integrating them into a fully functional model. we can develop empirically-based, statistically-grounded approaches -of which vaxijen [ , , ] is merely the vanguardthat have their basis in emerging antigen databases. yet all of this is just the beginning. we need to factor in b-cell and antibody mediated issues using structural data. we need to properly assess the role of aggregation, of expression levels, of post-translational danger signals and the possession of pathogen-derived pamps, as well as the ability of large molecule and small molecule adjuvants to raise the intrinsic immunogenicity of candidates to useful levels. we should note in passing that many hostderived molecules are also labelled pamps; these include fibronectin, hsp , and heparan sulphate, amongst others. however, these molecules are endogenous activators of innate immunity and not relevant to this discussion, though they may figure in auto-immune scenarios. vaccines are a good thing; putting caveats and cavils to one side, no one sensible really doubts the truth of this statement. vaccines have proved their worth time and again, but for them to continue to prove their worth we must find new ways of making them. almost all present-day vaccines are mediated by antibodies, and the majority target viral diseases. unfortunately, we are now running short of target diseases which fit this restrictive bill. many of the pathogens responsible for current recalcitrant and emergent diseases are, and are set to prove, much more demanding and difficult to target. in many senses, the low hanging fruit has been cut down and now the fruit we most desire is well out of reach. many diseases, including the who's big three diseases: hiv, tb, and malaria, are much more complicated functionally and/or structurally, and cellular, as opposed to humoral, immunology is tasked with defence against these dark arts. peptide vaccines and those based on apcs are important new if as yet unspectacular directions for research in vaccine discovery, yet modern strategies for vaccine figure factors underlying immunogenicity as elaborated in the text, the phenomenon of immunogenicity can be explored through the diversity of underlying individual factors contributing to the instigation of the immune response. these factors can be assigned to the host (epitope recognition), the pathogen (location and expression level), and also factors intrinsic to the protein antigen itself, such as the possession of post-translational danger signals. development hinge primarily upon the effective search for vaccine antigens, at least for the proactive search for vaccines targeting long-standing but untreated diseases rather than the reactive response to so-called pandemics. such antigens, once discovered, will, in time, and with careful manipulation and an appropriate delivery system and/or appropriate adjuvant, become first candidate subunit vaccines and, after proper clinical evaluation, the vaccines of tomorrow. there are, as we have outlined, many competing ideas, thoughts, and concepts that can help us in our quest. certain of these hypothesis we have had cause to outline, are indubitably persuasive, even compellingly convincing, yet in execution many such methods fall far short of our desires. no single approach, however promising, is able to deliver on its promise. the reason for this failure is comparatively simple if uncompromisingly unsatisfying; as we are dealing with over simplified abstractions, we cannot hope to capture what is necessary for prediction by looking in a relatively superficial way at a single contributory factor, since protein immunogenicity arises from many, many factors. this is not a bioinformatics problem that is easily solved; instead it is like protein secondary structure prediction which has resisted all attempts over many decades, so obscure and recondite and far removed from direct experience a problem as it is. factors mediating protein immunogenicity are many and include host-side properties -possession of b or t cell epitopes for example -and pathogen-side propertiesprotein expression levels and sub-cellular location -as well as its aggregation state and the possession of posttranslational danger signals. a candidate vaccine should be highly expressed, available for immune surveillance, and possess epitopes that the host recognises. predicting such diverse properties remains challenging, though several contributing factors can be reliably predicted. yet, no one factor is itself enough. what we need is an integrative, systems biology approach to the problem. as the poet maya angelou so neatly puts it: "we all should know that diversity makes for a rich tapestry, and we must understand that all the threads of the tapestry are equal in value no matter what their colour." no one method is universally applicable and successful; rather we need to integrate several equally-valid, equally-partial methods and draw from their synergy, useful, helpful data. this is the as yet unattained goal; yet as these are issues of such importance even a partial solution would be prize enough. computer-aided biotechnology: from immunoinformatics to reverse vaccinology new vaccines against tuberculosis 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'super-bulged' major histocompatibility complex class ibound peptide high resolution structures of highly bulged viral epitopes bound to major histocompatibility complex class i. implications for t-cell receptor engagement and t-cell immunodominance have we cut ourselves too short in mapping ctl epitopes? a long, naturally presented immunodominant epitope from ny-eso- tumor antigen: implications for cancer vaccine design proteins accessible to immune surveillance show significant t-cell epitope depletion: implications for vaccine design are bacterial vaccine antigens t-cell epitope depleted? the prodom database of protein domain families: more emphasis on d the pfam protein families database prosite, a protein domain database for functional characterization and annotation single proteins might have dual but related functions in intracellular and extracellular microenvironments locating proteins in the cell using targetp, signalp and related tools improved prediction of signal peptides: signalp . a comprehensive assessment of nterminal signal peptides prediction methods wolf psort: protein localization predictor secreted protein prediction system combining cj-sphmm, tmhmm, and psort psort-b: improving protein subcellular localization prediction for gram-negative bacteria psort: a program for detecting sorting signals in proteins and predicting their subcellular localization predicting protein subcellular locations using hierarchical ensemble of bayesian classifiers based on markov chains subloc: a server/client suite for protein subcellular location based on soap gpos-ploc: an ensemble classifier for predicting subcellular localization of gram-positive bacterial proteins prediction of twinarginine signal peptides prediction of lipoprotein signal peptides in gram-negative bacteria advantages of combined transmembrane topology and signal peptide prediction-the phobius web server validating subcellular localization prediction tools with mycobacterial proteins toward bacterial protein sub-cellular location prediction: single-class discrimminant models for all gram-and gram+ compartments multi-class subcellular location prediction for bacterial proteins alpha helical transmembrane proteins: enhanced prediction using a bayesian approach beta barrel transmembrane proteins: enhanced prediction using a bayesian approach a predictor of membrane class: discriminating alpha-helical and beta-barrel membrane proteins from non-membranous proteins tatpred: a bayesian method for the identification of twin arginine translocation pathway signal sequences lippred: a web server for accurate prediction of lipoprotein signal sequences and cleavage sites combining algorithms to predict bacterial protein sub-cellular location: parallel versus concurrent implementations dbsubloc: database of protein subcellular localization predicting the subcellular localization of viral proteins within a mammalian host cell virus-ploc: a fusion classifier for predicting the subcellular localization of viral proteins within host and virus-infected cells syfpeithi: database for searching and t-cell epitope prediction syfpeithi: database for mhc ligands and peptide motifs hiv sequence databases mhcbn . : a database of mhc/tap binding peptides and t-cell epitopes mhcbn: a comprehensive database of mhc binding and non-binding peptides epimhc: a curated database of mhc-binding peptides for customized computational vaccinology antijen: a quantitative immunology database integrating functional, thermodynamic, kinetic, biophysical, and cellular data jenpep: a novel computational information resource for immunobiology and vaccinology jenpep: a database of quantitative functional peptide data for immunology bacterial virulence: can we draw the line? vfdb release: an enhanced webbased resource for comparative pathogenomics vfdb: a reference database for bacterial virulence factors candivf -candida albicans virulence factor database phi-base: a new database for pathogen host interactions antigendb: an immunoinformatics database of pathogen antigens the lipocalin protein family: structure and function structure and sequence relationships in the lipocalins and related proteins the lipocalin protein family: structural and sequence overview epitopic peptides with low similarity to the host proteome: towards biological therapies without side effects peptimmunology: immunogenic peptides and sequence redundancy primer: mechanisms of immunologic tolerance recent advances in immune modulation cutting edge: contributions of apoptosis and anergy to systemic t cell tolerance a computational approach for identifying pathogenicity islands in prokaryotic genomes identification and characterization of pathogenicity and other genomic islands using base composition analyses identification and characterization of specific sequences encoding pathogenicity associated proteins in the genome of commensal neisseria species prediction of pathogenicity islands in 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multivariate characterization of amino acids bioinformatic approach for identifying parasite and fungal candidate subunit vaccines. the open vaccine journal nerve: new enhanced reverse vaccinology environment dynavacs: an integrative tool for optimized dna vaccine design fell da: enzymes, metabolites and fluxes computer aided selection of candidate vaccine antigens we should like to thank the referees for their helpful an insightful comments. this article has been published as part of immunome research volume supplement , : computational vaccinology: state-of-the-art assessments. the full contents of the supplement are available online at http://www.immunome-research.com/supplements/ /s . the authors declare no competing financial interests. key: cord- - kr e authors: alcami, antonio; koszinowski, ulrich h. title: viral mechanisms of immune evasion date: - - journal: mol med today doi: . /s - ( ) - sha: doc_id: cord_uid: kr e during the millions of years they have coexisted with their hosts, viruses have learned how to manipulate host immune control mechanisms. viral gene functions provide an overview of many relevant principles in cell biology and immunology. our knowledge of viral gene functions must be integrated into virus–host interaction networks to understand viral pathogenesis, and could lead to new anti-viral strategies and the ability to exploit viral functions as tools in medicine. viruses can exist in two forms: extracellular virion particles and intracellular genomes. virions are more resistant to physical stress than genomes but are susceptible to humoral immune control. virus genomes can be maintained in host cells by limited gene expression and can evade the host immune response. nevertheless, to exist as a species, virus replication and transfer to a new host are essential. these processes are associated with the production of antigenic proteins that make the virus vulnerable to immune control mechanisms 'warning' the host of the presence of an invader. however, viruses have evolved strategies to evade such immune control mechanisms, and the list of these strategies forms the 'who's who' of today's immunology. there are two classes of viral immunoregulatory proteins: those encoded by genes with and those encoded by genes without sequence homology to cellular genes. viral homologs of host genes involved in the immune system are mainly found in large dna viruses (herpesviruses and poxviruses) and their existence suggests that viruses have 'stolen' genes from the host that were subsequently modified for the benefit of the virus. viral genes without sequence similarity to cellular genes might represent a paradigm for co-evolution or could simply be examples of proteins for which the host homologs have not yet been identified. these proteins might possess specific motifs or particular folding properties required for interaction with the host cellular machineries. in this review and the accompanying poster we provide an overview of the different mechanisms that viruses use to evade host immune responses. the basic concepts of virus immune evasion will be discussed, with some examples to illustrate particular points; however, space constraints have not allowed a comprehensive review of all immune-evasion strategies. the strategies are listed in the accompanying tables and are discussed in more detail in the references given throughout the text. antigenic variability was one of the first viral immune-evasion strategies to be identified. because of the low fidelity of rna polymerases, viral rna genomes comprise a collection of rna species (quasispecies) with random mutations. therefore, in rna viruses the generation and selection of variants with different antigenic properties that can evade recognition by neutralizing antibodies is common. genetic viruses must be extremely successful predators as they depend on living cells for replication. almost all living species represent prey for a viral invader. viruses have coevolved with their hosts and therefore have limited pathogenicity in an immunocompetent natural host. in turn, probably as a result of the constant evolutionary pressure from viral invaders, higher vertebrates have developed a complex immune system. only in the last decade have we have caught a glimpse of what viruses do beyond invading cells for replication. for millions of years viruses have 'studied' cell biology and immunology the hard way, to acquire and defend an ecological niche. it is remarkable that, in the process, individual virus families have targeted many common immunological principles. viruses that belong to different families are subject to different constraints. owing to the low fidelity of rna polymerase, the genome size of rna viruses is limited. although this confers the advantage of being able to use mutation to escape immune control, there is little room in the genome to allow immune defences to be encoded by individual genes. the proteins encoded by rna viruses are therefore multifunctional. this particular constraint is less rigid for dna viruses as their genome size allows a larger number of genes to be devoted to host control. in the case of herpesviruses and poxviruses, these genes probably account for Ͼ % of the total genome. variability can also generate variant peptide sequences that are either new antigens or that do not bind to major histocompatibility complex (mhc) molecules at all. the complement system is a major non-specific host defense mechanism - . viruses encode homologs of complement regulatory proteins that are secreted and block complement activation and neutralization of virus particles (table ; box ). the cowpox virus (cpv) complement inhibitor, termed inflammation modulatory protein (imp), blocks immunopathological tissue damage at the site of infection, presumably by inhibiting production of the macrophage chemoattractant factors c a and c a (ref. ) . viruses protect the membranes of infected cells and the lipid envelopes of virus particles from complement lysis by encoding homologs of inhibitors of the membrane-attack complex. viruses such as hiv, human cytomegalovirus (hcmv) and vaccinia virus (vv) utilize a clever strategy, 'borrowing' host cellular factors, including cd , which normally protects cells from complement lysis, and incorporating them into the viral envelope. lastly, some viruses encode fc receptors ( table ) . antibodies bound to infected cells or virus particles might therefore be bound at the fc region, thereby inhibiting fc-dependent immune activation of complement and phagocytes. fc receptors probably have additional functions in vivo . interferons (ifns) were discovered because of their ability to protect cells from viral infection. the key role of both type i (␣ and ␤) and type ii (␥) ifns as one of the first anti-viral defense mechanisms is highlighted by the fact that anti-ifn strategies are present in most viruses [ ] [ ] [ ] ( table ). viruses block ifn-induced transcriptional responses and the janus kinase (jak)/signal transducers and activators of transcription (stat) signal transduction pathways, and also inhibit the activation of ifn effector pathways that induce an anti-viral state in the cell and limit virus replication. this is mainly achieved by inhibiting doublestranded (ds)-rna-dependent protein kinase (pkr) activation, the r e v i e w s phosphorylation of eukaryotic translation initiation factor ␣ (eif- ␣) and the rnase l system, which might degrade viral rna and arrest translation in the host cell. poxviruses encode soluble versions of receptors for ifn-␣ and -␤ (ifn-␣/␤r) and ifn-␥ (ifn-␥r), which also block the immune functions of ifns . the vv-secreted ifn ␣/␤r is also localized at the cell surface to protect cells from ifn (table ) . additionally, several viruses inhibit the activity of ifn-␥, a key activator of cellular immunity, by blocking the synthesis or activity of factors required for its production, such as interleukin (il)- or il- (table ): cpv cytokine response modifier (crm a) inhibits caspase- , which processes the mature forms of il- ␤ and il- (refs , ); various poxviruses encode soluble il- binding proteins (il- bps) [ ] [ ] [ ] ; measles virus (mev) binds cd in macrophages and inhibits il- production ; and herpesviruses and poxviruses express il- homologs that diminish the th response by downregulating the production of il- (refs , , ). cytokines play a key role in the initiation and regulation of the innate and adaptive immune responses, and viruses have learned how to block cytokine production, activity and signal transduction (tables and ). african swine fever virus (asfv) replicates in macrophages and encodes an ib homolog that blocks cytokine expression mediated by nuclear factor (nf)-b and the nuclear factor activated t cell (nfat) transcription factors . many viruses block signal tranduction by ligands of the tumor necrosis factor (tnf) family, whereas others deliberately induce some cytokine pathways; for example, the epstein-barr virus (ebv) latent membrane protein (lmp ) recruits components of the tnf receptor (tnfr) and cd transduction machinery to mimic cytokine responses that could be beneficial for the virus, such as cell proliferation (table ) . one of the most interesting mechanisms identified in recent years is the mimicry of cytokines (virokines) and cytokine receptors (viroceptors) by large dna viruses (herpesviruses and poxviruses) , , , , (table ). the functions of these molecules in the animal host are diverse. soluble viral cytokine receptors might neutralize cytokine activity and cytokine homologs might redirect the immune response for the benefit of the virus. alternatively, viruses that infect immune cells might use these homologs to induce signaling pathways in the infected cell that promote virus replication. the herpesvirus cytokine homologs vil- and vil- might have immunomodulatory activity but might also increase proliferation of cells that are targets for viral replication , . viral semaphorin homologs have uncovered a role for the semaphorin family -previously known as chemoattractants or chemorepellents involved in axonal guidance during development -in the immune system, and have identified a semaphorin receptor in macrophages that mediates cytokine production . secreted cytokine receptors or binding proteins are mainly encoded by poxviruses , , , , . these proteins were originally identified as alcami, unpublished) . binding and activity assays have identified secreted proteins that bind ifn-␣ and -␤, chemokines (cks) or granuloctye-macrophage colony-stimulating factor (gm-csf) and il- , and that have no sequence similarity to cellular counterparts , , . in poxviruses, three distinct secreted il- bps that have recently been identified are homologs of human and mouse secreted il- bps but not of membrane il- rs [ ] [ ] [ ] . inactivation of poxvirus cytokine receptor genes results in virus attenuation in vivo but, interestingly, deletion of the vv il- ␤r enhances virus virulence and the onset of fever, suggesting that the purpose of some immune-evasion mechanisms is to reduce the immunopathology caused by viral infection . herpesviruses and poxviruses modulate the activity of chemoattractant cytokines or cks that regulate leukocyte trafficking to sites of infection , , . virus-encoded cks are either antagonists that block leukocyte recruitment to sites of infection, or agonists that could enhance the recruitment of cells that support viral replication or prevent th anti-viral responses. murine cytomegalovirus (mcmv) chemokine (mck- ) activates monocytes in vitro and increases monocyte-associated viremia in vivo . hiv tat is partially homologous to cks and is a potent monocyte chemoattractant . herpesviruses encode many ck receptors (vckrs) but their function is not clear. kaposi's sarcoma-associated virus [human herpesvirus ] open reading frame (orf) is constitutively activated and induces cell proliferation, which might favor virus replication. vckrs encoded by hcmv and hhv- reduce the amount of the factor regulated upon activation normal t-cell expressed and secreted (rantes) in tissue culture and/or its transcription and might inhibit ck activity locally , . a role for vckrs in vivo has been shown for mcmv. vcks and vckrs might contribute directly to pathology. the angiogenic properties of hhv- macrophage inflammatory protein (vmip- ) could account for the increased vascularization found in hhv- -associated tumors, human cytomegalovirus (hcmv) us mediates vascular smooth muscle cell migration and perhaps vascular disease , and expression of hhv- orf in transgenic mice results in kaposi's-sarcoma-like lesions . three soluble vckbps have been identified that have no sequence similarity to cellular ckrs , , , . vckbp-i is a soluble ifn-␥r encoded by mv, but not vv, which binds the heparin-binding domain of a wide range of cks and might prevent the correct localization of cks in vivo by blocking their interaction with proteoglycans. the poxvirus-secreted vckbp-ii, which has a novel protein structure , binds cc cks with high affinity and blocks their activity. murine gamma-herpesvirus (mhv- ) has recently been shown to encode a distinct secreted protein (vckbp-iii) that sequesters c, cc, cxc and cx c cks . apoptosis or programmed cell death can be triggered by a variety of inducers, including ligands of the tnf family, irradiation, cell-cycle inhibitors or infectious agents such as viruses. apoptosis can be considered an innate cellular response to limit viral propagation, and viruses express proteins that block the death response (table ) ; however, apoptosis might also facilitate virus dissemination, and viral pro-apoptotic mechanisms have been described . in addition, cytotoxic t lymphocytes (ctls) and natural killer (nk) cells kill virusinfected cells by inducing apoptosis via secretion of cytokines such as tnf, the release of perforin and granzymes, or the activation of fas in the target cell. the cellular proteins implicated in the control of apoptosis are targeted by viral anti-apoptotic mechanisms , , , . viruses inhibit activation of caspases, encode homologs of the anti-apoptotic protein bcl- , block apoptotic signals triggered by activation of tnfr family members by encoding death-effector-domain-containing proteins, and inactivate ifn-induced pkr and the tumor suppressor p , both of which promote apoptosis. an alternative mechanism is provided by the glutathione peroxidase of molluscum contagiosum virus (mcv), which provides protection from peroxide-or uv-induced apoptosis, and perhaps from peroxides induced by tnf, macrophages or neutrophils. how to achieve persistence in the face of a vigorous host immune response is a problem that must be solved by viruses that establish lifelong infections. cellular proteins are degraded by the proteasome, the complex major intracellular protease, and the resulting peptides are translocated by transporters associated with antigen processing (tap) molecules into the endoplasmic reticulum (er), where they contribute to the assembly of mhc class i molecules , , [ ] [ ] [ ] transport either specifically or generally (table ). viruses use various mechanisms to modify the maturation, assembly and export of mhc class i molecules. to date, no cellular homologs have been found for the proteins and functions that target peptide processing, transport and mhc maturation. with few exceptions , the viral proteins bind their target molecule directly. there is only limited functional homology and no sequence homology among the different viral effectors. nevertheless, the general outcome of these functions is the same: downregulation of mhc class i molecules or of some mhc class i alleles. the study of mhc class i regulation has revealed additional genes in herpesviruses of different species [ ] [ ] [ ] , which might affect many cell types or only those tissues relevant for virus maintenance. although the downregulation of mhc class i expression prevents cd ϩ t-cell recognition, cells that downregulate these molecules become targets for nk cells , , [ ] [ ] [ ] . nk cells, the first line of cellular defence against viruses, have receptors for certain mhc molecules. some of these receptors silence the cytolytic machinery of nk cells and act as killer cell inhibitory receptors (kir). other receptors, designated leukocyte immunoglobulin-like receptors (lir), are expressed mainly on monocytes and b cells. engagement of an nk receptor can alternatively result in nk activation as not all receptors have immunoreceptor tyrosine-based inhibitory motifs (itims) in their intracellular domains. the hcmv protein ul and the mcmv m protein, which are homologous to mhc class i, could be associated with nk killing, and ul is instrumental in the identification of lir- . in addition, the hcmv ul protein provides a peptide selectively required for the maturation of the hla-e molecule, an nk target , . however, clinical isolates of hcmv confer a much stronger nk resistance than the laboratory strains sequenced and tested so far, and this resistance is unrelated to mhc class i expression and lir- (ref. ) . clinical isolates carry additional genes, and in vitro propagation has probably led to a loss of certain nk-specific gene functions. effects on mhc class ii expression fall into two classes, namely effects on transcription and post-translational effects , , [ ] [ ] [ ] . adenovirus, mcmv and hcmv affect mhc class ii transcription but the target in the signal cascade, although known to be different for these viruses, has not been defined and the viral gene or genes responsible are unknown. at the post-translational level, the hcmv us protein, which affects mhc class i, apparently also translocates the dr␣ and the dm␣ chain into the cytosol for degradation by the proteasome. another target involved in interference with mhc class ii function is the shuttling between endosomal peptide loading and surface expression. human papilloma virus (hpv) and hiv nef affect vesicle traffic as well as the function of the endocytic machinery. accordingly, in addition to mhc class ii, other proteins that use this pathway, for example the cd molecule, are also affected. an understanding of the functions of the viral immunoregulatory genes isolated to date is now emerging. however, we do not yet know whether the list is complete (table ) . additionally, it is unclear when and why a virus deploys one specific function rather than another. many questions therefore remain unanswered, including which genes are needed during primary infection to 'conquer the territory'; which genes are required to support active replication; and which genes are required to ensure transmission to a new host in the face of a vigorous host immune response? moreover, why is there such complexity and functional redundancy? is there a hierarchy in terms of general importance or do some functions operate only in certain tissues? is complexity and redundancy a viral strategy that enables viruses to infect individuals resistant to some functions? are the functions of an individual viral gene modulated by its genetic context, and is there any evidence for cooperativity? to date, we only have limited information because the construction of virus mutants and the in vivo testing of the predicted gene function is still in its infancy and, additionally, owing to the species specificity of many viruses, this information can only be gathered from some animal models. the identification of novel immune-evasion strategies and the analysis of their functions in the context of a viral infection should lead to a better understanding of the immune system and the interaction of viruses with their hosts. this will help us to treat virus-induced pathology, to design safer and more immunogenic virus vectors as vaccines or gene delivery systems, and to identify new strategies for immune modulation. viral subversion of the immune system vaccinia virus immune evasion poxviral mimicry of complement and chemokine system components: what's the end game? virus attenuation after deletion of the cytomegalovirus fc receptor gene is not due to antibody control virus interception of cytokine-regulated pathways poxviruses: interfering with interferons interferons: cell signalling, immune modulation, antiviral responses and virus countermeasures il- binding and inhibition of interferon gamma induction by human poxvirus-encoded proteins ectromelia, vaccinia and cowpox viruses encode secreted interleukin- -binding proteins a poxvirus protein that binds to and inactivates il- and inhibits nk cell response one step ahead of the game: viral immunomodulatory molecules human cytomegalovirus harbors its own unique il- homolog (cmvil- ) a viral mechanism for inhibition of the cellular phosphatase calcineurin signal transduction from the epstein-barr virus lmp- transforming protein immunomodulation by viruses: the myxoma virus story modulating chemokines: more lessons from viruses shared resources between the neural and immune systems: semaphorins join the ranks poxviruses: capturing cytokines and chemokines a third distinct tumor necrosis factor receptor of orthopoxviruses vaccinia virus strains lister, ussr and evans express soluble and cell-surface tumour necrosis factor receptors orf virus encodes a novel secreted protein inhibitor of granulocyte-macrophage colony-stimulating factor and interleukin- abduction of chemokine elements by herpesviruses cytomegalovirus-encoded beta chemokine promotes monocyte-associated viremia in the host hiv- tat protein mimicry of chemokines rantes binding and down-regulation by a novel human herpesvirus- beta chemokine receptor the human cytomegalovirus chemokine receptor us mediates vascular smooth muscle cell migration transgenic expression of the chemokine receptor encoded by human herpesvirus induces an angioproliferative disease resembling kaposi's sarcoma structure of a soluble secreted chemokine inhibitor vcci (p ) from cowpox virus a broad spectrum secreted chemokine binding protein encoded by a herpesvirus apoptosis: an innate immune response to virus infection control of apoptosis by poxviruses a comparison of viral immune escape strategies targeting the mhc class i assembly pathway from sabotage to camouflage: viral evasion of cytotoxic t lymphocyte and natural killer cell-mediated immunity immune evasion by cytomegalovirus -survival strategies of a highly adapted opportunist the luminal part of the murine cytomegalovirus glycoprotein gp catalyses the retention of mhc class i molecules downregulation of major histocompatibility complex class i molecules by kaposi's sarcoma-associated herpesvirus k and k proteins kaposi's sarcoma-associated herpesvirus encodes two proteins that block cell surface display of mhc class i chains by enhancing their endocytosis inhibition of mhc class i-restricted antigen presentation by gamma -herpesviruses surface expression of hla-e, an inhibitor of natural killer cells, enhanced by human cytomegalovirus gpul the human cytomegalovirus ul gene product contains a ligand for hla-e and prevents nk cell-mediated lysis human cytomegalovirus strain-dependent changes in nk cell recognition of infected fibroblasts immune evasion by adenoviruses the equine herpesvirus e open reading frame encodes a functional chemokine receptor a highly selective cc chemokine receptor (ccr) antagonist encoded by the poxvirus molluscum contagiosum a novel vascular endothelial growth factor encoded by orf virus, vegf-e, mediates angiogenesis via signalling through vegfr- (kdr) but not vegfr- (flt- ) receptor tyrosine kinases m l: a novel mitochondria-localized protein of myxoma virus that blocks apoptosis of infected leukocytes ectromelia virus virulence factor p acts upstream of caspase- in response to uv light-induced apoptosis a cytomegalovirus-encoded mitochondria-localized inhibitor of apoptosis structurally unrelated to bcl- retrograde protein translocation: eradication of secretory proteins in health and disease deletion of a cd -like gene, -dr, from african swine fever virus affects viral infection in domestic swine acknowledgements. the work in the authors' laboratories is funded by the wellcome trust and the deutsche forschungsgemeinschaft. key: cord- -eap i authors: lee, seung-hwan; dimock, ken; gray, douglas a; beauchemin, nicole; holmes, kathryn v.; belouchi, majid; realson, john; vidal, silvia m. title: maneuvering for advantage: the genetics of mouse susceptibility to virus infection date: - - journal: trends in genetics doi: . /s - ( ) - sha: doc_id: cord_uid: eap i abstract genetic studies of host susceptibility to infection contribute to our understanding of an organism's response to pathogens at the immunological, cellular, and molecular levels. in this review we describe how the study of host genetics in mouse models has helped our understanding of host defense mechanisms against viral infection, and how this knowledge can be extended to human infections. we focus especially on the innate mechanisms that function as the host's first line of defense against infection. we also discuss the main issues that confront this field, as well as its future. genetic studies of host susceptibility to infection contribute to our understanding of an organism's response to pathogens at the immunological, cellular, and molecular levels. in this review we describe how the study of host genetics in mouse models has helped our understanding of host defense mechanisms against viral infection, and how this knowledge can be extended to human infections. we focus especially on the innate mechanisms that function as the host's first line of defense against infection. we also discuss the main issues that confront this field, as well as its future. viral infections in humans are notable for the diversity of host responses, rates of progression, and disease outcomes. a large body of evidence indicates that these differential responses depend not only on viral factors but also on inherited components affecting host susceptibility. significant advances in our understanding of the host response to infection in humans and other animal species have recently been achieved through the use of mouse models of infection. in laboratory mice, years of inbreeding have fixed deleterious alleles, which can be detected as susceptibility phenotypes. two major breakthroughs advanced this field. one was our ability to manipulate the mouse genome using transgenic and gene knockout technologies. this 'reverse genetics' approach allows direct testing of specific genes for their role in host resistance or susceptibility to infection. the other was our ability to clone genes responsible for natural variation in susceptibility to infection among inbred strains of mice, through molecular genetics. this 'forward genetics' approach can uncover novel mechanisms of host defense that are crucial to effective and protective responses to infection. to date more than mouse loci (table ) and many fewer human genes ( table ) have been associated with the outcome of virus infection [ , ] . each locus controls infection by a single virus family or strain. this is probably related to the vast diversity of virus life cycles and the likelihood that the product of each host resistance gene interacts with unique molecules encoded by individual viruses (fig. ) . in this review we illustrate the contribution of mouse genetics to our understanding of mechanisms of host resistance to virus infection. these mechanisms might manifest themselves as: ( ) barriers to infection at the host cell membrane; ( ) intracellular host responses to infection; or ( ) recognition or destruction of infected cells. viruses used as examples are described in table . barriers to infection at the host cell membrane the first step in the life cycle of a virus is attachment to a receptor on the cell surface and delivery of the viral genome into the interior. usually the virus takes advantage of a host molecule with an unrelated function, such as an adhesion molecule or complement regulator, for its own benefit. receptors are recognized as important determinants of virus host range and tissue tropism; and some host resistance/susceptibility loci encode molecules that are expressed on the cell surface. for example, sjl mice are times more resistant to a lethal dose of mouse hepatitis virus (mhv) -a murine coronavirus -than c bl/ or balb/c mice [ ] . resistance is due to allelic variation in the gene encoding carcinoembryonic antigen-related cell adhesion molecule (ceacam ) [ ] . susceptible strains carry the ceacam a allele, which encodes the principal mhv receptor, cea-cam a. resistant strains, such as sjl, are homozygous for the ceacam b allele, which encodes a -amino-acid substitution in the n-terminal virus-binding domain of ceacam a [ ] . an immunoreceptor tyrosine-based inhibitory motif (itim) (see glossary) is present in the cytoplasmic domain of ceacam , also suggesting a potential immunomodulatory function for this molecule during mhv infection. although no role for human ceacam a has been described during viral infection of humans [ ] , a possible immunomodulatory function is consistent with the observation of a ceacam -mediated suppression of cd þ t-cell activation during infection by the pathogenic bacteria neisseria gonorrhoeae, neisseria meningitidis and haemophilus influenzae. these human pathogens bind to domain of ceacam , very close to the site recognized by mhv [ ] . another example of natural host resistance is the restriction of ecotropic murine leukemia virus (mulv) infection by the mouse fv gene. binding of retroviral env glycoproteins to cellular receptors triggers fusion of viral and cellular membranes, and allows the virus nucleocapsid to enter the cell. the fv (or akvr) locus is a defective endogenous ecotropic provirus that encodes an env protein, which also has a fusion defect [ , ] . it has been proposed that the env protein encoded by fv blocks mulv infection by interacting with the cellular receptor and restricting the amount of receptor available for exogenous retrovirus attachment (box ) [ ] . in humans, resistance to human immunodeficiency virus (hiv) is also mediated by a barrier at the cell surface. cd is the primary or 'attachment' receptor for hiv, but cd is necessary but not sufficient for the productive entry of hiv into target cells. the identification of ccr as a coreceptor for hiv prompted genetic screening of individuals that escape hiv disease despite high-risk behavior [ ] . individuals carrying a homozygous bp deletion in the coding sequence of ccr (ccr d ) are extremely resistant to the 'r 'strain of hiv because the deletion results in a frame shift and generates a nonfunctional receptor [ ] . the ccr d mutation is found predominantly in the caucasian population and is absent in africans, american indians and east asians [ ] . it has been speculated that this distribution is consistent with resistance to an agent that predates hivand caused enormous mortality. one candidate is yersinia pestis, the causative agent of bubonic plague, although other pathogens targeting the macrophage/monocyte lineage cannot be excluded [ ] . delivery of a viral genome to the interior of an infected host cell does not guarantee that an infection will be glossary advanced intercross lines : mouse lines generated by producing an f generation between two inbred strains and then intercrossing in each subsequent generation, but avoiding sibling matings. this provides increasing probability of tightly linked genes recombining. congenic strain : a mouse strain that has been bred to be identical to an inbred strain, except for a selected differential chromosomal segment. congenic strains are derived by backcrossing to a parental inbred strain for at least ten generations while selecting for heterozygosity at a particular locus. consomic strain : a variation on a congenic strain in which recombinants between two inbred strains are backcrossed to produce a strain that carries a single chromosome from one strain on the genetic background of the other. epistasis : the interaction between alleles at different loci that allows an allele at one locus to alter the effects of alleles at a different locus. immunoreceptor tyrosine-based activation motif (itam) : a cytoplasmic tyrosine-containing motif that is the site of tyrosine phosphorylation. these motifs are associated with tyrosine kinases and other phosphotyrosinebinding proteins involved in cellular activation. immunoreceptor tyrosine-based inhibitory motif (itim) : a cytoplasmic tyrosine-containing motif. in contrast to itams, phosphorylation of the tyrosine residues within itims recruits the src-homology- -domain-containing tyrosine phosphatase shp- and/or shp- , transducing a negative signal that inhibits cellular activation. interferons (ifns) : a group of immunoregulatory proteins that are produced by cells infected with a virus and have the ability to inhibit viral growth. ifns are classified as type i (ifn-a/b), which have antiviral properties, and as type ii (ifn-g), which is known as immune interferon. despite the use of different receptors, certain ifn-a/b and ifn-g functions are shared because the signal transduction pathways activated through these receptors partly overlap. murine leukemia virus (mulv) : there are four subgroups of naturally occurring mulvs based on receptor usage on mouse cells: ecotropic mulvs, which are able to infect only mouse cells utilizing the cationic amino acid transporter as a receptor; amphotropic mulvs, which are able to infect mouse cells as well as those of other species (including human) by binding to an inorganic phosphate transporter protein; polytropic mulvs, which can infect cells from mouse as well as nonmurine species using yet another cellular receptor protein, thought to be a g-protein-coupled receptor; and xenotropic mulvs, which use receptors from cells of most species except mice. quantitative trait loci (qtls) : the locations of genes that affect a trait that is measured on a quantitative (linear) scale, such as body weight or blood pressure in animals, as identified by statistical analysis. these traits are typically affected by more than one gene, and also by the environment. recombinant congenic strain : a variation on recombinant inbred strains in which the initial outcross is followed by several generations of backcrossing before inbreeding. retroviral interference : the phenomenon by which prior infection of cells with a retrovirus confers strong resistance to infection of the same cells by a retrovirus that utilizes the same receptor; however, cells remain susceptible to viruses that use a different receptor. this process results from masking or downregulation of the receptor due to interaction with the glycoprotein of the established virus. an important element of resistance to retroviral infection is provided by endogenous retroviral elements. some retroviral elements in the mammalian genome are transcriptionally active but carry deletions and point mutations that render them unable to replicate. some of their gene products, such as the fv and fv proteins, interfere with infection by their exogenous relatives thus providing a selective advantage to their hosts. for example, fv encodes an env protein that interferes with ecotropic murine leukemia virus (mulv) infection (see main text) at the level of viral entry. examples of this phenomenon can be found in mice, chickens and cats, suggesting that env-mediated retroviral interference is commonly used for limiting virus spread [ ] . gene therapy based on the principle of receptor interference has demonstrated that introduction of fv -envtransduced bone marrow cells into a thymectomized host confers resistance to friend leukemia virus-induced leukemogenesis [ ] . these findings suggest that a similar approach could be used as a therapeutic strategy to inhibit infections by other retroviruses in vivo, including immunodeficiency virus. for example, transfer of a gene encoding a normally processed but fusion-defective retroviral env protein into susceptible cells would interfere with viral entry and potentially reduce the infectiousness of viruses emerging from the cell. the fv locus [ , ] encodes a retrovirus capsid-like protein [ ] that restricts mulv infection at a post-entry stage, before integration of the provirus [ ] . because all retroviruses replicate in very similar ways, it is conceivable that similar restriction mechanisms could be found in humans. interestingly, although an fv ortholog was not detected in nonmurine species, a mechanism of preintegration interference, resistance factor (ref ), has been demonstrated in human cell lines [ ] . at the time of writing, the ref gene has not been cloned, but there is speculation that, like fv , ref might consist of endogenous retroviral sequences. a recent study suggests the existence of a ref -like restriction in humans and nonhuman primates that determines lentivirus tropism [ ] . consistent with this proposal, the target for this restriction is within the capsid protein of hiv [ ] . an example of an alternative mechanism of resistance to retroviral infection is provided by fv , which determines the progression of friend virus complex-induced erythroleukemia. fv is identical to ron, which encodes stk, a member of the met family of receptor tyrosine kinases. susceptible mice express a positive-acting truncated version of stk lacking most of the extracellular domain, which is associated with proliferation of sffv-infected erythroblasts [ ] . successful, because potent defense mechanisms act at the intracellular level. one of the initial cellular responses to viral infection is the production of type i interferons (ifns). ifns, in turn, stimulate the expression of several gene products, including the double-stranded rna-activated protein kinase (pkr) and rnase l, which leads to the establishment of an antiviral state in cells, characterized by a general inhibition of protein synthesis. the ifn-induced mx protein is one of the best-studied determinants of innate immune responses to viral infection. the inbred mouse strain a g is resistant to doses of mouse-adapted influenza virus (an orthomyxovirus) that are lethal for other inbred strains [ ] . resistance in these mice is determined by a single dominant locus, mx , whose gene product, mx , rapidly accumulates in the nuclei of cells following influenza virus infection, and blocks virus spread [ ] . susceptible mice produce no functional mx protein due to either a nonsense mutation (cba/j) or gene deletion (balb/c) [ ] . the mx product belongs to the dynamin superfamily of large gtpases conserved in all vertebrates. in mice there are actually two mx gene products, mx and mx . mx protein blocks transcription of influenza virus, probably via interaction with the pb subunit of the influenza virus polymerase [ ] . by contrast, the mx protein is cytoplasmic and has been shown to inhibit viruses that replicate in the cytoplasm, such as vesicular stomatitis virus (vsv) and members of the family bunyaviridae [ ] . mx proteins inhibit virus replication by interfering with the transport of viral nucleocapsids, and by sorting them to locations where they become unavailable for the generation of new virus particles [ ] , possibly to promyelocytic leukemia protein nuclear bodies (pml nbs) [ ] , which are known to be a site of proteasome-mediated degradation. humans also possess mx genes, mxa and mxb, but only the mxa gene product has antiviral properties. however, in contrast to its mouse mx ortholog, the mxa gtpase accumulates in the cytoplasm of ifn-treated cells, where it inhibits not only the replication of orthomyxoviruses, vsv and bunyaviruses but also the replication of other rna viruses such as measles virus, coxsackievirus b and semliki forest virus [ ] . a mechanism of resistance to flaviviruses, which also appears to be ifn-mediated, was recently revealed by the cloning of flv, which confers resistance (flv r ) or susceptibility (flv s ) to flavivirus infection. virus titers in the brains of resistant mice infected with murray valley encephalitis virus, yellow fever virus or west nile virus are orders of magnitude lower than in susceptible animals. viral yields in tissue cultured from resistant or susceptible animals are also dramatically different, indicating that the flv gene product acts intracellularly on flavivirus replication [ ] . genetic mapping localized flv to chromosome [ ] and, more recently, it was demonstrated that flv susceptibility is associated with a nonsense mutation in a member of the , -oligoadenylate synthetase (oas) family, oas b [ , ] . oas proteins are induced by ifns and play a central role in producing the antiviral state by binding double-stranded rna and catalyzing the synthesis of , -oligoadenylates ( - a), which, in turn, interact with and activate rnase l to degrade singlestranded viral and cellular rnas. whereas oas b genes from resistant mice encode full-length proteins, those from susceptible mice encode proteins truncated at the c-terminus that might not form active synthetases. in contrast to the single oas gene found in humans, there are eight closely linked oas genes in the murine genome [ , ] , but only oas b is associated with resistance to flaviviruses. in addition to resistance imparted by barriers to virus entry or by intracellular antiviral defense mechanisms, cytotoxic cells can control the level of viral replication in an animal (box ). host recognition of virus-infected cells is mediated by two players: natural killer (nk) cells and cytotoxic t cells. herpesviruses avoid recognition and activation of the adaptive immune system by downregulating major [ ] . strains of the c bl background carry a dominant resistance allele, cmv r , that restricts viral replication in target organs, whereas other mouse strains carry a recessive susceptibility allele, cmv s , that allows rapid proliferation of the virus [ ] . cmv r encodes an activating nk-cell receptor, ly h [ - ] , which is absent in susceptible strains. mouse strains express different repertoires of ly molecules, which are part of a large family of polymorphic receptors expressed on overlapping populations of nk cells. upon binding of mhc class i ligands, different ly molecules deliver either activating or inhibitory signals that modulate nk-cell function [ ] . recent studies have revealed that ly h recognizes mcmv-infected cells via direct interaction with the viral antigen, m , which shares structural homology with mhc class i molecules and is expressed on the surface of infected cells [ , ] . m also binds to an inhibitory receptor, ly i, expressed in susceptible strain mice, suggesting that m could have evolved as a mechanism to escape nk-cell killing by targeting inhibitory receptors [ ] . in resistant mice, ly h might have evolved as a countermeasure against virus-encoded mhc class i homologs, providing an overriding activating signal to the nk cell, and promoting the elimination of mcmv-infected cells (fig. a) . considering the prevalence of human cytomegalovirus (hcmv) in the human population, we expect that there are likely to be hcmv-specific activating receptors present on human leukocytes that might function in a manner analogous to ly h. the mhc (box ) is a set of genes, present in all vertebrates, with immunological and nonimmunological functions. mhc class i genes play a crucial role in in vertebrates, host defense against virus infection is mediated by innate and adaptive immunity. innate immunity constitutes the first line of defense, providing a rapid response through germ-line encoded proteins that pre-exist or are induced within hours of infection. adaptive immunity is a slower, yet highly specific response mediated by b cells and t cells that confers effective and long-lasting protection against infection, and is characterized by immunological memory. major histocompatibility complex (mhc) molecules are highly polymorphic glycoproteins encoded by mhc class i and ii genes, which are mainly involved in the presentation of peptide antigens to t cells. mhc class i molecules bind peptides derived from proteins synthesized in the cytosol, such as viral proteins, and present them to cytotoxic cd þ t cells. by contrast, mhc class ii molecules are loaded with peptides derived from exogenous antigens engulfed within intracellular vesicles, for presentation to cd þ t cells. mhc class i molecules also play an important role in modulating the cytotoxic activity of natural killer (nk) cells. nk cells are a component of the innate system, so named because of their propensity to kill certain neoplastic and virus-infected cells without prior sensitization. the cytotoxic activity of nk cells is regulated by signals elicited by inhibitory receptors containing immunoreceptor tyrosine-based inhibitory motifs (itims), or stimulatory receptors associated with immunoreceptor tyrosine-based activation motifs (itam)-bearing adaptor molecules. inhibitory receptors interact with mhc class i molecules and prevent cell killing of healthy cells by autologous nk cells. in situations where mhc class i is downregulated, such as during tumorigenesis or viral infection, reduced inhibitory signals result in nk-cell-mediated lysis [ ] . activating nk-cell receptors recognize stress-induced or pathogenencoded mhc class i-like proteins and stimulate killing of cells expressing these molecules [ ] . review trends in genetics vol. no. august combating viral infection in mice (fig. a) . congenic mice with intra-mhc recombinations provide an important tool for dissecting the contribution of mhc class i subregions to virus infection. for example, comparisons of t-cell responses in h congenic mice that differ in their recovery from friend leukemia virus infection were used to localize friend leukemia virus resistance loci rfv and rfv . rfv mapped to the h d subregion and determined t-cell activation. rfv mapped to the ia subregion and determined unresponsiveness of t cells in a proliferation assay [ ] . resistance to acute lethal infection of mcmv is also controlled by genes linked to h , with the k haplotype being more resistant than b or d. resistance was associated with genes of subregions k/ia and d. interestingly in this model, transfection of macrophages with sequences encoding mhc molecules such as h -k d , d d or k b renders these cells, which are major reservoirs for mcmv, sensitive to mcmv infection [ ] , consistent with a role for mhc molecules as mcmv receptors. in humans, association analysis between mhc and specific viral infections has also suggested a differential role of specific alleles in susceptibility to hiv, hepatitis b virus (hbv) and hepatitis c virus (hcv) (fig. b) . for example, hla-drb * is associated with resistance to chronic hbv infection in both african and european populations [ , ] whereas hla-b* -cw* is associated with the rapid progression of aids in caucasian populations [ ] . although polymorphisms in the classical mhc class i and class ii genes are known to be related to antigen presentation, it is important to note that there are other genes within the mhc class iii region, such as components of the complement cascade (c , c ), cytokines -tumor necrosis factor (tnf)-a and -b -and proteins involved in antigen processing (lmp , tap ), which also have an important function in immunity [ ] . therefore, disease resistance or susceptibility that maps to the mhc must be interpreted with caution, to differentiate the role of antigen presentation from the other roles of mhcassociated genes. although advances in genomic analysis have paralleled the discovery of host susceptibility traits that are determined by single alleles, most differences in host susceptibility to viral infection are the result of interactions among different genes with multiple alleles. the study of infectious disease under complex genetic control in humans is complicated by a variety of factors including genetic heterogeneity, low penetrance and environmental factors. the tools currently available to the mouse geneticist make the identification and isolation of disease susceptibility genes much more attainable. an example is the response to theiler's murine encephalomyelitis virus (tmev), a rodent model for multiple sclerosis [ ] . in genetically susceptible mice, certain tmev strains cause persistent infection, inflammation, and a chronic demyelinating disease. one of the loci determining theiler's murine encephalomyelitis virus persistence (tmevp ), maps to the mhc region and corresponds to the h d gene [ ] . mice transgenic for the h d b allele acquired resistance to persistent virus infection, suggesting that the gene effect was at the level of peptide presentation by h d b [ ] . using the amount of viral rna in the spinal cords of persistently infected mice as a phenotype, and a genetic dissection strategy based on genome scans, a second locus, tmevp , was localized near the type ii interferon (ifn-g) gene on distal chromosome [ ] . analysis of a panel of congenic mice that inherited different portions of mouse chromosome revealed that tmevp was actually two distinct loci, now termed tmevp and tmevp , and excluded the ifn-g gene from the candidate region [ ] . recently, a strong candidate for tmevp , named tmevpg , has been identified as a noncoding rna expressed in immune cells infiltrating the central nervous system of resistant mice, where an inverse relationship with ifn-g mrna levels has been observed [ ] , suggesting a role for this rna in regulating ifn-g synthesis. consistent with this observation, the same reciprocal pattern of rna expression was observed for tmevpg , the human counterpart, and ifn-g in human nk cells and t cells. several complementary avenues promise significant acceleration in the identification of genes that determine resistance or susceptibility in complex models of virus infection. the well-defined sets of recombinant congenic strains (rcs) [ ] , consomic (chromosome substitution) strains [ ] and advanced intercrossed lines [ ] that are now available can be used to map a locus of interest and to characterize the specific phenotypic component of disease susceptibility under genetic control. also, traditional genetic mapping techniques are now complemented by high-throughput methods for studying gene function and regulation. for example, high-density oligonucleotide arrays [ ] or cdna arrays [ ] allow for gene expression monitoring on a genome-wide scale and offer an opportunity to establish functional links between genotype and phenotype. a strategy that combines congenic mapping with microarray expression profiling would aid in the identification of novel susceptibility genes and biochemical pathways not previously known to be involved in disease etiology. furthermore, the availability of the human and mouse genome sequences represents the ultimate breakthrough in quantitative trait loci (qtl) studies by placing the identification of candidate genes within a defined genetic interval only a (computer) mouse click away. one of the current limitations of using inbred strains of mice to study susceptibility to viral infection is the limited extent of genetic variation, mainly due to a small number of original progenitor strains [ ] . therefore, although these resources are valuable, they do not permit the identification of all possible resistance or susceptibility loci. wild-derived inbred strains of mice are an important source of additional resistance alleles [ ] that is just beginning to be exploited, as demonstrated by the characterization of flv. chemical mutagenesis, a method that has been successfully applied to the study of other model organisms, from bacteria to drosophila, is another promising strategy for creating novel susceptibility alleles in the mouse. the alkylating agent n-ethyl-n-nitrosourea (enu) introduces point mutations and creates random variation throughout the genome [ ] . this is highly advantageous for defining host susceptibility phenotypes because it represents an unbiased method for identifying previously unrecognized physiological pathways because no assumptions are made about the relevant genes. enu mutagenesis yields models of simple inheritance that are readily accessible to analysis by positional cloning [ ] . the effort to understand the genetic basis of susceptibility to viral disease is driven by three considerations: ( ) the increased public awareness of the toll imposed by viruses on the host; ( ) the increase in susceptible human populations because of longer life expectancy, frequently accompanied by chronic illness, and the consequences of advances in medical technology, including immunosuppressive therapies for organ transplantation or treatment of malignancy; and ( ) the need to develop new therapies for infections caused by multidrug-resistant human killer-cell immunoglobulin-type receptor (kir) is considered to be a functional homolog of mouse ly . an epistatic interaction between kir ds and hla-b delays progression to aids, suggesting that hla-b behaves as a ligand for kir ds [ ] . the peptide presented on hla-b that is responsible for this interaction remains to be identified. because kir ds receptor is also associated with the adaptor molecule dap , the intracellular signaling cascade leading to cellular activation and killing of virus-infected cells seems to be similar to that triggered by mouse ly h. by using inhibitory small molecules, represent promising approaches to antiviral therapy. therapies based on the principle of receptor interference for viruses other than retroviruses (box ) could be explored. type i ifn induces an intracellular antiviral response against many different rna and dna viruses. mouse genetics has provided a starting point for dissecting intracellular mechanisms of host defense, and has revealed that several ifn-inducible gene products have inhibitory effects on a much more restricted number of viruses than expected. the precise nature (sequence or structural) of determinants recognized by effector proteins such as mx or oas b remain to be characterized. the identification of additional effector molecules and viral targets must continue to be an important area of active research, particularly in view of the existence of orthologous human genes. mouse genetics has also demonstrated that recognition and destruction of virus-infected cells by nk cells is mediated by specific interactions between activating nkcell receptors and viral target molecules. viruses pose a particular problem for specific recognition because all the components of the virus are synthesized and assembled in the host cell. important questions to be answered include: ( ) do other activating nk-cell receptors have specialized functions in virus recognition? ( ) is the mhc class i fold a recognition-associated structure? ( ) what is the extent of the repertoire of target molecules for nk-cell-activating receptors? in addition, studies of mouse nk-cell receptors, such as the ly family, have provided a conceptual framework for understanding human nk-cell biology. because no functional human ly counterpart has been identified, prime candidates for the recognition of viral pathogens by human nk cells are members of the killercell immunoglobulin-like receptor (kir) family [ ] . although the ligands of activating kir receptors remain to be identified, it has been proposed that activating kir molecules have evolved to recognize infectious agents (fig. b) [ ] . it is suspected that several common and/or chronic human diseases under complex genetic control are triggered by a viral infection. this idea is supported by experimental evidence derived from mouse models for initiation and exacerbation of atherosclerosis following mcmv infection [ ] , for diabetes [ ] or dilated cardiomyopathy [ ] following coxsackievirus infection, and for multiple sclerosis-like disease following tmev [ ] or mhv [ ] infection. identifying genes that control susceptibility to acute and chronic viral infection in these models is a crucial step in understanding the development of these complex pathologies. comparisons between mouse and human mechanisms of host resistance or susceptibility to viral infection have increased our awareness not only of their differences but also, more importantly, of their similarities. in the future, the use of comparative genomic approaches in animal model systems will provide more comprehensive knowledge of the impact of viruses on human health. forward genetics of infectious diseases: immunological impact genetics of susceptibility to human infectious disease resistance to fatal central nervous system disease by mouse hepatitis virus strain jhm. i. genetic analysis specificity of coronavirus/ receptor interactions human aminopeptidase n is a receptor for human coronavirus e crystal structure of murine sceacam a[ , ]: a coronavirus receptor in the cea family molecular characterization of the akvr- restriction gene: a defective endogenous retrovirus-borne gene identical to fv- r fv- resistance gene: a truncated endogenous murine leukemia virus with ecotropic interference properties fv- : identification of the defect in env and the mechanism of resistance to ecotropic murine leukemia virus chemokine receptors as hiv- coreceptors: roles in viral entry, tropism, and disease global distribution of the ccr gene -basepair deletion dating the origin of the ccr -delta aids-resistance allele by the coalescence of haplotypes resistance of mice to mouse adapted influenza a virus interferon-induced mx proteins: dynamin-like gtpases with antiviral activity influenza virus-susceptible mice carry mx genes with a large deletion or a nonsense mutation function of the mouse mx protein is inhibited by overexpression of the pb protein of influenza virus interferon-induced antiviral mx gtpase is associated with components of the sumo- system and promyelocytic leukemia protein nuclear bodies genetically determined resistance to infection with group b arboviruses. ii. increased production of interfering particles in cell cultures from resistant mice development and characterization of new flavivirus-resistant mouse strains bearing flv(r)-like and flv(mr) alleles from wild or wild-derived mice positional cloning of the murine flavivirus resistance gene a nonsense mutation in the gene encoding - -oligoadenylate synthetase/l isoform is associated with west nile virus susceptibility in laboratory mice gene structure of the murine - -oligoadenylate synthetase family selective rejection of h- -deficient lymphoma variants suggests alternative immune defence strategy inhibition of natural killer cells by a cytomegalovirus mhc class i homologue in vivo cmv- , a genetic locus that controls murine cytomegalovirus replication in the spleen susceptibility to mouse cytomegalovirus is associated with deletion of an activating natural killer cell receptor of the c-type lectin superfamily vital involvement of a natural killer cell activation receptor in resistance to viral infection murine cytomegalovirus is regulated by a discrete subset of natural killer cells reactive with monoclonal antibody to ly h the ever-expanding ly gene family: repertoire and signaling direct recognition of cytomegalovirus by activating and inhibitory nk cell receptors recognition of a virus-encoded ligand by a natural killer cell activation receptor epistatic interaction between kir ds and hla-b delays the progression to aids genetics of susceptibility to theiler's virus infection serial backcross analysis of genetic resistance to mousepox, using marker loci for rmp- and rmp- h- d control of recovery from friend virus leukemia: h- d region influences the kinetics of the t lymphocyte response to friend virus genetic control of sensitivity to moloney leukemia virus in mice. iii. the three h- linked rmv genes are immune response genes controlling the antiviral antibody response are mhc proteins cellular receptors for cmv? hla and hiv- : heterozygote advantage and b* -cw* disadvantage influence of combinations of human major histocompatibility complex genes on the course of hiv- infection hla alleles determine human t-lymphotropic virus-i (htlv-i) proviral load and the risk of htlv-i-associated myelopathy a tumor necrosis factor-alpha (tnf-alpha) promoter polymorphism is associated with chronic hepatitis b infection hla-drb * and * protect against chronic hepatitis b association between an mhc class ii allele and clearance of hepatitis b virus in the gambia class ii hla alleles and hepatitis b virus persistence in african americans influence of mhc class ii genotype on outcome of infection with hepatitis c virus. the hencore group. hepatitis c european network for cooperative research association between mhc class ii alleles and clearance of circulating hepatitis c virus. members of the trent hepatitis c virus study group genes of the major histocompatibility complex class ii influence the outcome of hepatitis c virus infection association between hla class ii genotype and spontaneous clearance of hepatitis c viraemia influence of hla haplotypes on the clinical courses of individuals infected with hepatitis c virus hla drb and dqb alleles and haplotypes influencing the progression of hepatitis c complete sequencing and gene map of a human major histocompatibility complex (mhc) the infection of mouse by theiler's virus: from genetics to immunology fvb mice transgenic for the h- db gene become resistant to persistent infection by theiler's virus mapping loci influencing the persistence of theiler's virus in the murine central nervous system two loci, tmevp and tmevp , located on the telomeric region of chromosome , control the persistence of theiler's virus in the central nervous system of mice tmevpg , a candidate gene for the control of theiler's virus persistence, could be implicated in the regulation of gamma interferon recombinant congenic strains derived from a/j and c bl/ j: a tool for genetic dissection of complex traits analysing complex genetic traits with chromosome substitution strains simultaneous detection and fine mapping of quantitative trait loci in mice using heterogeneous stocks high density synthetic oligonucleotide arrays quantitative monitoring of gene expression patterns with a complementary dna microarray the mosaic structure of variation in the laboratory mouse genome wild mice: an ever-increasing contribution to a popular mammalian model enu mutagenesis: analyzing gene function in mice divergent and convergent evolution of nk-cell receptors does cytomegalovirus play a role in atherosclerosis coxsackieviruses and diabetes the transition from viral to autoimmune myocarditis mouse hepatitis virus infection of the central nervous system: chemokine-mediated regulation of host defense and disease susceptibility to hiv infection and progression of aids in relation to variant alleles of mannose-binding lectin genetic restriction of hiv- pathogenesis to aids by promoter alleles of il mutation of gene of mannose-binding protein associated with chronic hepatitis b viral infection tuberculosis and chronic hepatitis b virus infection in africans and variation in the vitamin d receptor gene host response to ebv infection in x-linked lymphoproliferative disease results from mutations in an sh -domain encoding gene the x-linked lymphoproliferative-disease gene product sap regulates signals induced through the co-receptor slam endogenous retroviruses and the evolution of resistance to retroviral infection a gene therapy model for retrovirus-induced disease with a viral env gene: expression-dependent resistance in immunosuppressed hosts susceptibility to two strains of friend leukemia virus in mice inheritance of susceptibility to friend mouse leukemia virus. . spleen foci method applied to test the susceptibility of crossbred progeny between a sensitive and a resistant strain positional cloning of the mouse retrovirus restriction gene fv physical mapping of the fv- tropism host range determinant of balb/c murine leukemia viruses a conserved mechanism of retrovirus restriction in mammals cellular inhibitors with fv -like activity restrict human and simian immunodeficiency virus tropism fv encodes a truncated form of the stk receptor tyrosine kinase natural killer cells, viruses and cancer we are grateful to christine di donato for critical reading of the manuscript. our laboratories are supported by grants from the canadian institutes of health research (cihr) and natural sciences and engineering research council of canada (nserc). seung-hwan lee is supported by a cihr doctoral scholarship and silvia m. vidal is a junior scientist of cihr. key: cord- - kqrl rv authors: hedger, m.p. title: immunology of the testis and male reproductive tract date: - - journal: comprehensive toxicology doi: . /b - - - - . -x sha: doc_id: cord_uid: kqrl rv a large body of evidence points to the existence of a close, dynamic relationship between the immune system and the male reproductive tract, which has important implications for our understanding of both systems. the testis and the male reproductive tract provide an environment that protects the otherwise highly immunogenic spermatogenic cells and sperm from immunological attack. at the same time, secretions of the testis, including androgens, influence the development and mature functions of the immune system. activation of the immune system has negative effects on both androgen and sperm production, so that systemic or local infection and inflammation compromise male fertility. the mechanisms underlying these interactions have begun to receive the attention from reproductive biologists and immunologists that they deserve, but many crucial details remain to be uncovered. a complete picture of male reproductive tract function and its response to toxic agents is contingent upon continued exploration of these interactions and the mechanisms involved. in the decade since this chapter was first published (hedger ) , there has been considerable progress in the understanding of the regulation of immunity. advances such as the emergence of the innate immune system as a major theme in immunity following the discovery of the toll-like receptor (tlr) family (o 'neill and dinarello ) , the formalization of multiple macrophage phenotypes corresponding to the type and helper t (th) cell subsets (mantovani et al. ) , the rehabilitation of suppressor and regulatory t cells (piccirillo and thornton ) , and the identification of new lymphocyte subsets, including th , th , and natural killer t (nkt) cells (godfrey et al. ; macdonald ; romagnani ) , also have had considerable influence on the concept concerning the immunology of the male reproductive tract. nonetheless, while there have been advances in our understanding of the interface between the male reproductive tract and the immune system, there is still much we simply do not know. the need to understand this interface and the potential implications for reproductive toxicology continues to be just as important as ever. there is no doubt that the testis and the male germ cells in particular are susceptible to immunological damage. clinically significant testicular autoimmunity, most commonly indicated by the presence of antisperm antibodies in the serum or ejaculate, is implicated in the case of - % of all male infertility patients within the developed world (baker et al. ; pattinson and mortimer ) , but the incidence is much higher in populations where access to reproductive health care is limited (ekwere ) . common factors suspected to contribute to testicular autoimmunity include reproductive tract infections, physical trauma, immune system dysfunction, and genetics. the release of spermatogenic antigens from the reproductive tract following vasectomy generally results in autoimmune responses, ranging in severity from sperm antibodies to orchitis, in both humans and experimental animals (alexander and anderson ; flickinger et al. a; kojima and spencer ; tung and alexander ) . the potential for spontaneous testicular autoimmune disease in otherwise normal men remains poorly characterized, although studies in animals clearly indicate the possibility of such reactions in humans (furbeth et al. ; tung et al. ) . complicating matters is the fact that the testis is an immunologically privileged tissue, in the sense that foreign tissue grafts into the testes of experimental animals survive for prolonged periods (barker and billingham ; head et al. a; whitmore and gittes ) . this dichotomy between immune susceptibility and privilege is indicative of a highly specialized interaction between the male reproductive system and the immune system, with potentially important implications for both systems. the objective of this chapter is to provide an overview of the current state of knowledge and to highlight issues for further consideration that may be of relevance to male reproductive toxicology. . . the interface between the immune system and the reproductive tract the immune system is the body's protective arsenal against disease. it operates via the detection of potential aggressors, recognized through conserved motifs found on pathogenic organisms (innate immunity) or confrontation by completely novel (i.e., foreign) molecular structures (adaptive immunity) (vivier and malissen ; zinkernagel ) . immunity involves specialized cells possessing highly developed recognition and activation abilities (macrophages and dendritic cells) and the cells that carry out the protective responses: t and b cells in the case of adaptive immunity and nk cells and mononuclear or polymorphonuclear phagocytes (pmns) in the case of innate immunity. many of these cells play roles in both the recognition and effector arms of the immune response and in both innate and adaptive immunities (figure ) . innate immunity provides the rapid response elements of the immune system, but is limited in its attack repertoire. adaptive immunity is much more flexible in its responses, but requires some time to become effective. once the adaptive arm has been activated toward a particular pathogen, however, it can respond rapidly in the future due to the persistence of memory lymphocytes, which form the basis of immunization (mcghee et al. ; schittek and rajewsky ) . the innate immune system operates upon recognition by specific pattern-recognition receptors of specific motifs found on bacterial, viral, fungal, and protozoan pathogens. functionally, these receptors have evolved to recognize pathogen-associated molecular patterns (pamps), such as bacterial lipopolysaccharides (lpss), lipoproteins, peptidoglycans, and viral nucleic acids (bianchi ; roach et al. ) . unlike classical ligand receptors, these receptors respond to multiple ligands possessing related, but not necessarily identical, structures. the largest and best-studied class of pattern-recognition receptors is the tlrs, which are members of a larger family of proteins that include the interleukin (il) receptor (il r), and which share a conserved cytoplasmic domain called the toll/il r (tir) domain (akira and takeda ; o'neill and bowie ) . in humans, functional tlrs (tlr - ) have been identified (figure ) . mammalian tlrs - are known from laboratory rodent species, but are either pseudogenes or absent in humans (roach et al. ) . although they are particularly associated with cells of the monocyte lineage (i.e., macrophages and dendritic cells), these receptors are also found on certain t and b cells, as well as many fibroblastic and epithelial cells, including those of the male urogenital tract (lauw et al. ; zhang et al. ) . significantly, ligands for some of the tlrs include endogenous molecules of mammalian origin (barrat et al. ; karikó et al. ; tsan and gao ; yasuda et al. ; yu et al. ) . tlr signaling occurs through engagement of crucial adaptor proteins, specifically myeloid differentiation primary response protein (myd ) or tir domain-containing adaptor protein inducing interferon (trif), via tir domain interactions, leading to activation of tumor necrosis factor (tnf) receptorassociated factor and (traf , traf ) (akira and takeda ; o'neill and bowie ; o'neill and dinarello ) . based on the tlr engaged, this results in nuclear translocation of the inflammatory transcription factor, nuclear factor kappa b (nfb), activation of the jun n-terminal kinase (jnk) and p mitogen-activated protein (map) kinases, and induction of type interferon (ifn and ifn) figure polarization in the immune system. the immune system is conceptually divided into innate and adaptive arms, with adaptive immunity further subdivided into type (cell-mediated) and type (antibody-mediated) responses. triggering of the immune response involves the induction of inflammation by various agents, which may include infections, particulates, debris from damaged cells, or noxious agents. innate immunity involves fixed pattern-recognition receptors, such as the tolllike receptors, whereas adaptive immunity depends upon highly polymorphic receptors expressed by t cells (t cell receptors) and b cells (membrane-bound and secreted immunoglobulin). in general, type responses are associated with cellular immunity, autoimmune reactions, and graft rejection. type responses involve antibody production by the b cells, but also play a more complex role in allergy, immunoregulation, and control of tolerance, favoring immune privilege. within these divisions, various cell types, cytokines, chemokines, bioactive lipids, and other molecules interact to integrate or mediate the responses. the direction of the adaptive immune response type is determined primarily by the activity of the antigenpresenting cells (primarily dendritic cells) under the influence of cells with immunoregulatory capacity, such as macrophages, nk cells, and nkt cells, and either type or type cytokines produced locally. several cell types and molecules play multiple roles within different functional divisions. abbreviations: mo, monocyte; nk, natural killer cell; pmn, polymorphonuclear cell; th; helper t cell; b, b cell; t reg , regulatory t cell; m, macrophage; nkt, natural killer t cell; ctl, cytotoxic t cell; apc, antigenpresenting cell, ros, reactive oxygen species; ifn, interferon; pge, prostaglandin e; cox, cyclooxygenase; inos, inducible nitric oxide synthase; tnf, tumor necrosis factor ; il, interleukin; il ra, interleukin receptor antagonist; tgf, transforming growth factor ; gc, glucocorticoid; mif, macrophage migration inhibitory factor. production (hertzog et al. ; malmgaard ; nakano ) . the pattern-recognition receptor family also includes the nucleotide binding and oligomerization domain (nod)-like receptor (nlr) family and the retinoic acid-inducible gene (rig)-like helicases (rlhs) (becker and o'neill ; thompson and locarnini ) . the nlrs detect various bacterial pamps within the cytosol. some nlrs, such as nod and nod , act via nfb, while other nlrs work through induction of the cysteine protease caspase , which activates the proinflammatory cytokines il and il and initiates caspase-mediated proapoptotic pathways within the target cell. the rlhs, which detect the presence of viral rna in the cytosol, activate both nfb and production of type ifns. activation of innate immunity leads to inflammation, characterized by increased blood and lymphatic flow within the affected tissue and recruitment of immune cells to the site through production of chemokines and expression of vascular leukocyte adhesion molecules (moser and loetscher ; picker and butcher ) . this mobilization of immune cells is accompanied by activation of complement, production of proinflammatory cytokines tlr tlr tlr tlr tlr tlr tlr tlr cd tlr tlr cytoplasmic cell surface cpg dna ssrna dsrna lipoproteins lps flagellin/profilin ? irf nfkb ap map kinases il α, tnfα, il , inos, activin a, il , il , cox , ifnγ mal tram tlr tlr tlr apoptosis irf , , ifnα, ifnβ, tnfα myd traf traf traf , myd trif figure the toll-like receptors: major pathogenic ligands, adaptor proteins, signaling, and cytokine production. there are active human toll-like receptors (tlr - ). tlr - are present in rodents, and tlr is specific to the urogenital tract. the tlrs respond to a variety of pathogen-related molecules, usually forming homo-or heterodimers during signaling: for example, tlr can self-associate or combine with either tlr or tlr to mediate responses to various bacterial lipoprotein classes. tlr forms a complex with the receptor cofactor cd , to facilitate binding of bacterial lipopolysaccharide (lps). the tlrs can be subdivided into cell surface receptors, which largely respond to bacterial proteins, lipoproteins, and lipopolysaccharides, and intracytoplasmic receptors, which recognize single-stranded and double-stranded rna (ssrna and dsrna) and cpg-containing dna of viral or bacterial origin. most tlrs signal via the adaptor protein myd (myeloid differentiation primary response protein ), except tlr , which acts through the adaptor protein trif (tir domaincontaining adaptor protein inducing interferon ). uniquely, tlr can interact with either myd or trif, through engagement of the adaptor proteins mal (myd adaptor-like) or tram (trif-related adaptor molecule). downstream signaling involves the tumor necrosis factor receptor-associated factors and (traf and traf ), activation of the transcription factors nuclear factor kappab (nfb) and interferon regulatory factor (irf ), or activation of the mitogenactivated protein kinases (map kinases) jun n-terminal kinase (jnk) and p to produce the fos/jun transcription factor activated protein (ap ). these transcription factors interact to induce the expression of multiple proinflammatory genes, including interleukin (il) , tumor necrosis factor (tnf), inducible nitric oxide (inos), cyclooxygenase (cox ), and interferon (ifn), or the type interferons (ifn, ifn) and tnf. note that many details of the signaling pathways have been omitted or truncated for the sake of simplicity. and acute-phase proteins, and stimulation of phagocytosis and killing of pathogens and infected cells by macrophages, pmns, and nk cells, through production of proteases and other lytic proteins, and cytotoxic reactive oxygen species (ros) and induction of apoptotic pathways (rosenberg and gallin ) . moreover, activation of innate immunity is responsible for initiation of the adaptive immune response, through production of regulatory cytokines and stimulation of the antigen-presenting cell (apc) abilities of dendritic cells, macrophages, and some b cells (kelsall and rescigno ; spörri and reis e sousa ) . among the cell types that mediate the innate immune response, nk cells represent a special case. these cells are of the lymphocytic lineage, but they are capable of spontaneously attacking target cells without prior sensitization by exposure to antigen (mori et al. ; westermann and pabst ) . through a complex of specific receptors on their surface, nk cells are able to recognize and directly target transformed cells, such as virally infected or tumor cells (lanier ; mori et al. ) . in this, nk cells provide a first-line of lymphocyte defense within the context of the innate immune response, but activated nk cells also participate in adaptive immunity, both as regulators and as effector cells of the response (mocikat et al. ; raulet ) . highly specific interactions between apcs and t cells, mediated via the polymorphic proteins of the major histocompatibility complex (mhc; the human leukocyte antigen (hla) complex in humans) and antigen-specific t cell receptors (tcrs), are crucial to the adaptive immune response. the apcs process exogenous foreign proteins into short antigenic peptides, which are then incorporated into a structural groove on the external surface of the mhc protein complex during its assembly within the cell (cresswell et al. ; sant et al. ) . the tcr on the surface of a t cell that has specificity for the antigen binds to the antigen-mhc complex on the apc surface, which results in activation and proliferation of the t cell. circulating t cells express one of two coreceptors, cd and cd , as part of the tcr, which direct them to recognize antigens associated with either mhc class ii or i proteins, respectively (janeway ) . antigens are presented to cd þ t cells by the so-called professional apcs that express mhc class ii antigens (i.e., dendritic cells, macrophages, and b cells) (heath and carbone ; unanue ) . the cd þ t cells recognize antigens presented in the context of mhc class i proteins, which are expressed by almost all cell types in the body. activation of the t cell also requires physical interaction between the apc and t cell involving costimulatory ligand-receptor pairs, particularly cd :b and cd :cd l, as well as the production of either type cytokines (ifn, il , and il ) or type cytokines (il , il , il , and il ) (constant and bottomly ; heath and carbone ; moser and murphy ) . as a consequence, the activated t cell can have a number of different fates depending on the costimulatory molecules engaged and cytokines produced: activated cd þ t cells may become type helper (th ) cells, which direct development of the cellular immune response involving cytotoxic cd þ t cells, or they may become type helper (th ) cells, which promote the development of b cells into antibody-secreting plasma cells following interaction with their antigen (defranco ) . recently, a new th effector cell subset (th ) developmentally related to th cells, but producing il , il , and il has been described (ouyang et al. ; romagnani ) . these th cells direct a number of responses involved in protection against extracellular pathogens, including recruitment and activation of the major circulating pmn subset, the neutrophils, differentiation of b cells, and inflammatory reactions of epithelial cells. however, this cell subset has also been implicated in the development of autoimmune diseases and, along with th cells, in allergy (oboki et al. ; ouyang et al. ) . the absence of appropriate costimulatory molecule interactions and/or the presence of specific regulatory cytokines may also lead to t cell inactivation and deletion or generation of regulatory (i.e., suppressor) t cell subsets (gilliet and liu ; nossal ; piccirillo and thornton ; thompson and thomas ) . finally, at least some activated t and b cell clones persist as memory cells following the resolution of the immune response (dutton et al. ; schittek and rajewsky ) . the initial antigen presentation and lymphocyte activation events generally occur in the secondary immune tissues, the local lymph nodes and spleen. consequently, an effective lymphatic drainage is usually necessary for immune responses to develop in a particular tissue. however, since leukocytes recirculate through tissues, many of the important events, including antigen exposure and processing, antigen recognition by antigen-exposed lymphocytes, and lymphocyte maturation and proliferation, also occur at the reaction site itself (ascher et al. ; gruber ; pedersen and morris ) . moreover, while it is generally accepted that the adaptive immune system is activated by the presence of foreign antigens, there is an alternative activation model that combines elements of both the innate and adaptive immune systems. this is encapsulated by the 'danger hypothesis,' which proposes that apcs respond to substances that cause or signal damage, rather than to those that are simply foreign (matzinger ) . these danger signals include cd l, the early proinflammatory cytokines il and tnf, ifns, and heat-shock proteins as well as substances that are normally found only inside cells (e.g., nucleotides, unmethylated cpg sequences in mammalian double-stranded dna) and hyaluron breakdown products (gallucci and matzinger ) . in this model, activation of the immune system occurs as a response to evidence of an extant threat, rather than toward a specific feature of the threat itself. this mechanism may contribute to the onset of certain autoimmune diseases. while an effective immune response is essential for protection of the host from a range of threats, the immune system also has its dark side. the capacity of the immune system for destructiveness is expressed in chronic inflammatory and autoimmune diseases, including those that affect the male reproductive tract (roper et al. ; schuppe and meinhardt ; schuppe et al. ; suominen ) . these examples of an immune system seemingly out of control highlight the crucial importance of effective immunoregulation. at the center of immunoregulation is the concept of tolerance, or the capacity of the immune system to ignore certain molecular patterns, and the systems that restrain the immune response once its usefulness has expired. tolerance is the ability of the immune system to discriminate self from nonself. it is fundamentally based on the removal, inactivation, or suppression of self-reactive lymphocyte subsets, so that the immune system becomes effectively incapable of recognizing and reacting to these antigens. induction of tolerance comprises both central and peripheral mechanisms. the central mechanism involves clonal deletion of t and b cell subsets that are potentially autoreactive, a process that normally occurs during gestation as part of the early maturation of the immune system (kappler et al. ; nossal ) . many details of this editing process remain incompletely understood, although the process involves broad expression by thymic medullary cells of self-antigens, including many endocrine tissue-specific antigens under the control of the transcription factor autoimmune regulator (aire) (liston et al. ). lymphocytes that recognize these self-antigens within the thymus during this process are targeted for destruction, effectively removing them from the lymphocyte repertoire. in addition, there are a number of complex mechanisms of peripheral tolerance, which involve the inactivation of antigen-specific lymphocytes in peripheral lymphoid organs by weak antigen stimulation in the absence of appropriate costimulation and the production of lymphocytes with immunoregulatory activities (gilliet and liu ; piccirillo and thornton ; thompson and thomas ) . peripheral immunoregulation involves several antigen-specific regulatory or suppressor t cell types, most notably the cd þ cd þ regulatory t cell (t reg ) subset. these cells selectively produce the immunosuppressive cytokines transforming growth factor (tgf) and il , but also appear to act via direct contact with the cell being suppressed (nakamura et al. ; piccirillo et al. ) . other t cell subsets implicated in immunosuppression through their ability to produce tgf and il include th cells, tr (t regulatory ) cells, and t cells, the latter being a minor t cell subset that is generally associated with epithelia (kuhnlein et al. ; macdonald ; seo et al. ) . the nkt cells, which are t cells with nk activity that possess unique restriction to glycolipid antigens presented by the mhc-like molecule cd d, play a role in promoting graft survival and development of cd þ regulatory/suppressor t cells through production of il and il (godfrey et al. ; nakamura et al. ; sonoda et al. ) . the frontline nk cells are able to modulate adaptive immune responses by inducing dendritic cell death through contact-mediated lysis, but are also capable of producing ifn to stimulate the antigen-presenting activity of dendritic cells (laouar et al. ; raulet ) . it is the absence of tolerance that inhibits graft success in the very artificial condition of tissue transplantation. the immune cells of both the graft recipient and the donor tissue respond to one another, particularly with respect to polymorphic differences of the mhc proteins, leading to rejection of tissues that are not antigenically matched (gould and auchincloss ) . even under normal conditions, failure of preexisting tolerance may occur through a number of mechanisms, leading to autoimmune disease. somatic mutation of the antigen receptor expressed by a t or b cell may result in the creation of new self-reactive clones, subverting central tolerance (burrows et al. ) . autoimmunity may also occur when the inflammatory response to an infection damages or overwhelms normal mechanisms of self-tolerance, or where the infection involves organisms that express antigens that may cross-react with self-antigens (molecular mimicry) (merkler et al. ; regner and lambert ) . in addition to lymphocyte-mediated tolerance already discussed, the immune system possesses a repertoire of mechanisms that control and limit inflammatory and immune responses once they have commenced. inflammation triggers the production by the adrenal gland of corticosteroids, which inhibit the production of proinflammatory cytokines and other immune mediators, reduce the expression of leukocyte adhesion molecules, and induce lymphocyte apoptosis (buckingham et al. ; kapcala et al. ) . during inflammation, immune cells themselves produce anti-inflammatory and immunosuppressive molecules, including prostaglandins d and j and the lipoxins (herlong and scott ; levy et al. ) . moreover, activated lymphocytes have a limited lifespan, undergoing a process of activation-induced cell death following upregulation of the extrinsic apoptotic signal mediated by interaction of the fas receptor (cd ) with its ligand (fas ligand: fasl or cd l), eventually leaving behind a relatively small number of long-lived memory cells (dutton et al. ; schittek and rajewsky ) . likewise, there are mechanisms to inhibit antibody activity and promote antibody clearance (isaacs ). classically, the term immune privilege applies to organs or tissues where survival of foreign grafts may be prolonged (barker and billingham ) , but more accurately, the term denotes tissues where immune responses are inhibited or suppressed (hedger ) . privileged tissues include the brain, eye, pregnant uterus, and the testis. initially, privilege was attributed to deficient lymphatic drainage in such tissues, or physical barriers that prevented immune cells from gaining access (barker and billingham ) . several privileged tissues do possess highly specialized epithelial or endothelial cell barriers that may sequester antigens away from the normal immune surveillance: the best characterized of these are the blood-brain barrier and the trophoblast in the pregnant uterus (hunt ; streilein ) . however, physical exclusion is now considered an overly simplistic explanation for immune privilege, given that lymphocyte and antibody responses to introduced antigens occur even in sites where grafts can survive for extended periods (head et al. b; kaplan and streilein ; tafuri et al. ) . although barrier mechanisms and altered lymphatics may be contributory factors in some tissues, immune privilege is a consequence of tissue-specific specialized systems for controlling, subverting, or preventing local inflammatory or immune activation responses (hedger ) . the male reproductive tract comprises the paired testes, associated epididymis and vas deferentia, accessory glands (chiefly the prostate and seminal vesicles), and the urethra. these tissues represent a diversity of structures and, consequently, the immunology of each tissue retains distinctive features (hedger and hales ) . best studied in this context is the testis, which comprises two discrete tissue compartments, the seminiferous tubules and the interstitial tissue. the interstitial tissue contains the blood supply and lymphatics, as well as the innervation of the testis (fawcett et al. ; setchell et al. ) . the epithelium of the seminiferous tubules is entirely avascular. within the seminiferous epithelium, spermatogenesis is supported and maintained by the sertoli cells, which remain in physical contact with the developing germ cells at all times. during spermatogenesis, a stem cell spermatogonium sitting on the basement membrane of the seminiferous tubule becomes committed to a series of mitotic divisions, producing a cohort of spermatocytes. the spermatocytes subsequently move toward the tubule lumen, passing through tight junctions between adjacent sertoli cells, into the highly specialized environment of the adluminal compartment (wang and cheng ) . within this compartment, the spermatocytes divide meiotically to produce haploid spermatids and then undergo differentiation into spermatozoa, which are ultimately released into the lumen of the tubule leaving behind the majority of their cytoplasm to be digested by the sertoli cells as residual bodies. the released spermatozoa are swept by fluid secreted from the sertoli cells toward the rete testis, a collecting structure lined by a simple epithelium that is connected to the adjacent epididymis by a series of efferent ducts. sperm mature and are stored in the epididymis, until they are either released through the vas deferentia at the time of ejaculation or removed by epididymal phagocytes (barratt and cohen ; roussel et al. ) . the process of spermatogenesis is highly organized, with multiple generations of developing germ cells in each region of the epithelium maintaining distinct, progressing cellular associations or stages, which comprise the cycle of the seminiferous epithelium ( stages in the rat, stages in the mouse, stages in man). these stages are arranged in sequence along the length of each seminiferous tubule, creating waves of coordinated spermatogenic development (clermont ) . spermatogenesis is maintained and controlled by follicle-stimulating hormone (fsh) and testosterone, the latter being produced by the leydig cells in the interstitial tissue under the influence of luteinizing hormone (lh). importantly, fsh and testosterone act upon sertoli cells and not directly on the germ cells (mclachlan et al. ) . these hormones determine the efficiency of the spermatogenic process largely by controlling germ cell survival, that is, the balance between differentiation and apoptosis (matthiesson et al. ; ruwanpura et al. ) . in order to maintain the cycle of the seminiferous epithelium, the developing germ cells and sertoli cells are engaged in continuous intercellular communication, although the precise nature of this communication remains very poorly understood. the testis, and spermatogenesis in particular, presents a unique challenge for the immune system. although there are reports of direct drainage of some lymphatic vessels to the thoracic duct, there is no evidence of any structural or functional deficiency in the efferent lymphatics of the testis, and allogeneic cells injected into the rat testis induce typical immune responses in the draining lymph nodes (fawcett et al. ; head et al. b; itoh et al. a; moller ) . nonetheless, the vast majority of spermatogenic differentiation occurs long after central tolerance is established and, while thymic expression of some testicular antigens is under the control of the aire transcription factor, it is obvious from clinical and experimental studies that germ cells express multiple autoreactive antigens and are highly immunogenic when brought into contact with the immune system (itoh et al. ; tung ) . surprisingly, this is a problem for a relatively small cohort of men only, and autoimmune orchitis or epididymitis is rarely reported in humans, except in the context of local infection (krieger ) . even allowing for the possibility that most cases of spontaneous autoimmune reactions against the developing germ cells are never diagnosed, since they would occur early in reproductive life, and would simply present as spermatogenic failure during infertility work-ups (schuppe and meinhardt ; schuppe et al. ) , why are such reactions not more common? it is widely believed that the blood-testis barrier plays a role in protecting testicular antigens from the immune system. however, as a result of a common misunderstanding of the nature of this vitally important structure, its actual role is usually overestimated. critically, the blood-testis barrier involves highly specialized occluding or tight junctions between adjacent sertoli cells, which separate the spermatogonial and early meiotic cells from the majority of meiotic and postmeiotic germ cells (dym and fawcett ; setchell et al. ) . the structure and functions of the barrier do not need to be discussed in detail here, as this topic is covered in much more detail in other chapters in this volume (see chapter . ). by preventing the passage of most molecules between adjacent sertoli cells, the bloodtestis barrier allows the creation of a highly specialized biochemical environment essential for meiotic development (cheng and mruk ; griswold ) . it also blocks access by lymphocytes, complement, and antibody (ben et al. ; yule et al. ) . by contrast, the vascular endothelium and peritubular cells surrounding the tubules play a negligible role in maintaining the blood-testis barrier in practical terms, and immune cells and proteins have relatively free access to the interstitium and basal seminiferous epithelium (mahi-brown et al. ; yule et al. ). thus, this barrier is quite different in properties and function from the blood-brain barrier, located on the vascular endothelium, or the trophoblastic barrier of pregnancy, which entirely segregates the developing fetal tissues from the maternal host. a reinterpretation of the blood-testis barrier as a series of cellular elements that protects the germ cells from harmful influences has been proposed more recently (bart et al. ) . in this model, the bloodtestis barrier consists not only of the structural elements already discussed, but also the efflux pump barrier system involving the drug transport proteins p-glycoprotein and the multidrug resistance-associated protein- expressed on the capillary endothelium, peritubular myoid cells, and the basal aspect of the sertoli cells (bart et al. ; melaine et al. ) . while these elements may certainly be important for protection of the testis against toxic agents, their contribution to immunoregulation is probably marginal. several lines of evidence indicate that the bloodtestis barrier cannot account for all manifestations of immune privilege in the testis. the barrier does not prevent exposure of germ cell antigens to the immune system, as antibodies and lymphocytes specific for spermatogenic antigens appear to be a normal feature of the circulating immune repertoire even under normal conditions (turek and lipshultz ) . spermatogenic cell autoantigens are not confined behind the sertoli cell tight junctions, and are expressed by spermatogonia and the early spermatocytes (mahi- ; yule et al. ). moreover, the barrier does not extend into the rete testis or beyond. significantly, orchitis can be passively transferred to naive mice using lymphocytes obtained from mice with active autoimmune orchitis, with the initial reaction concentrated in the interstitial tissue around the rete testis tung et al. ) . a similar initial pattern of development of orchitis within the rete testis region has been observed in mice actively immunized with viable germ cells (itoh et al. a ). in many seasonally breeding species, annual regression of the bloodtestis barrier occurs without inducing overt inflammation or autoimmunity (pelletier ; tung et al. ) . finally, the blood-testis barrier cannot explain the enhanced survival of grafts within the interstitial tissue (i.e., outside the barrier) and does not explain survival of autoantigenic sperm in the epididymis, or remainder of the male reproductive tract. however, while the blood-testis barrier does not explain immune privilege in the testis, the breach of the tight junctions comprising the barrier remains a critical event in the development of any autoimmune destruction of the seminiferous epithelium during orchitis (mahi- brown and tung ; mahi-brown et al. ) . recognition of antigen in association with mhc is essential to normal adaptive immune responses, and reduced expression of mhc class i (hla-a, hla-b, and hla-c in humans) and class ii proteins (hla-d) appears to be an important feature of immune-privileged tissues and tumors (garrido et al. ; haas et al. ; head and billingham ; wekerle et al. ) . reduction in class i and ii expression reduces the likelihood of immune-activating events involving cd þ helper t cells and cytotoxic cd þ t cells in the tissues. studies indicate a characteristic absence of expression of mhc proteins on the cells of the seminiferous epithelium under normal conditions (haas et al. ; head and billingham ; lustig et al. ; tung et al. ) , although both mhc classes are expressed in human spermatozoa (martín-villa et al. ) . there is evidence from studies on mouse sertoli cells in culture that mhc class ii expression can be upregulated in these cells by ifn (dal secco et al. ) . in contrast to the seminiferous epithelium, both mhc class i and ii proteins are expressed throughout the testicular interstitial tissue: mhc class i is expressed on most interstitial cells, including the leydig cells (haas et al. ; pöllänen and maddocks ) , while testicular macrophages and dendritic cells express mhc class ii (haas et al. ; head and billingham ; hedger and eddy ; mahi-brown et al. ; tung et al. ) . thus, it appears unlikely that a lack of apcs is a contributing factor in testicular immune privilege, although differences in the number and distribution of mhc class ii-expressing cells in the interstitial tissue of different species or strains may help to explain differences in susceptibility to autoimmune orchitis (flickinger et al. b; kojima and spencer ; teuscher et al. ) . the fact that the testis is not isolated from the immune system, yet can tolerate both endogenous (auto-) antigens associated with spermatogenesis and exogenous (allo-or xeno-) antigens expressed by grafts, is evidence of tissue-specific inhibition of the immune response. the question then arises: if immune responses against exogenous and endogenous antigens are inhibited in the testis environment, does the testis display a reduced capacity to deal with infections and greater susceptibility to certain kinds of tumors? there is evidence that immunity may be compromised in some situations, for example, the tendency toward relapsing acute lymphocytic leukemia in the testes (hudson et al. ) , and several systemic viral and mycobacterial infections target the testis, including mumps, human immunodeficiency virus, tuberculosis, and the severe acute respiratory syndrome virus (krieger ; nistal et al. ; xu et al. ) . however, the testis does not appear to be immunodeficient. even in comparison with the rest of the male reproductive tract, orchitis due to ascending infections is relatively rare (krieger ; ness et al. ) . moreover, the extremely variable level of success of experiments by different groups using different transplant models has shown that immune privilege of the testis is both limited and conditional (dobrinski ; head et al. a; maddocks and setchell ; selawry and whittington ; setchell et al. ) . just what are these necessary conditions, however, remains poorly defined. studies indicate that the effector arm of the immune response is intact within the testis, and it is the ability to recognize and react toward foreign antigens and/or mount a normal inflammatory response that is suppressed (head and billingham ; head et al. a; mahi-brown and tung ; mahi-brown et al. ) . in summary, there appears to be a potentially unique functional interaction between the testis and the immune system, which involves control of the mechanisms normally involved in immune activation, in order to limit adaptive immune responses without drastically compromising the ability of the testis to protect itself when required. although their presence and influence is almost universally neglected, macrophages, lymphocytes, and pmns have varying degrees of access to the interstitial tissue of the testis (table ) . they are generally excluded from the seminiferous epithelium under normal conditions. over the past years, studies have started to fill in some of the details concerning these cells. macrophages are by far the most prominent immune cells in the testis. they are found in the testes of all mammalian species so far examined, but are particularly numerous in human, rat, and mouse testes (frungieri et al. ; hume et al. ; mendis-handagama et al. ; miller et al. ; vergouwen et al. ; wang et al. ) . typically, macrophages arise from myeloid precursors in the bone marrow and circulate in the blood as monocytes. they are capable of adopting a broad range of structural and functional phenotypes that are largely dictated by the tissues in which they become resident (mantovani et al. ; van furth ) . characteristically, they are robustly phagocytic and possess a potent arsenal of antimicrobial and cytotoxic activities, such as the ability to generate large amounts of ros. they produce many immunoregulatory products, including cytokines, eicosanoids (prostaglandins, thromboxanes, leukotrienes, and lipoxins), and nitric oxide, all of which are important in establishing and controlling the inflammatory process, but also have important roles in tissue remodeling, healing, and normal homeostasis. all macrophage lineages can express mhc class ii antigens and have the capacity to present to cd þ helper or t reg cells (unanue ) . thus, macrophages are the cells that link innate and adaptive immunity, and dendritic cells are a highly specialized macrophage subset that is particularly effective in this role (heath and carbone ) . testicular macrophages have been most extensively studied in the rat, with a few studies also in the mouse and human. studies in the rat indicate that they are heterogenous in their morphology and functions (bryniarski et al. ; gerdprasert et al. a; itoh et al. b; wang et al. ) . their absolute numbers are primarily under leydig cell control, but evidence suggests that fsh acting via the sertoli cell has a role in regulating their functional activity (duckett et al. a,b; gaytan et al. c; wang et al. ) . macrophages accumulate within the interstitial tissue at the time of puberty, and play critical roles in the normal development of the testis, particularly by promoting the proliferation and maturation of the leydig cell population (cohen et al. ; gaytan et al. a,b; hardy et al. ; itoh et al. ; raburn et al. ; vergouwen et al. ) . through their ability to produce a broad array of growth factors, prostanoids, and vasoactive regulators, other developmental and homeostatic roles within the testis may be predicted as well. data concerning the immunological functions of testicular macrophages are based on a relatively small number of studies in either the rat or the mouse, and are patchy at best. indeed, there is some evidence for distinct differences even between these two closely related species. in general, testicular macrophages have intact phagocytic, cytotoxic, and antimicrobial functions (miller et al. ; wei et al. ) . rat testis macrophages express mhc class ii antigens (head and billingham ; hedger and eddy ; wang et al. ), but display a reduced capacity for lymphocyte activation in vitro . testicular macrophages in the human and mouse also express mhc class ii, although expression in the mouse appears to be more restricted (haas et al. ; itoh et al. b; pöllänen and niemi ; tung et al. ) . a lack of expression of the essential costimulatory molecules, b - (cd ) and b - (cd ), has been reported in the mouse testis as well (sainio-pöllänen et al. ) . studies also indicate that macrophages from the rat testis have a significantly reduced ability to produce proinflammatory cytokines, such as il and tnf, following activation (gerdprasert et al. a; hayes et al. ; meinhardt et al. ) , and stimulated mouse testicular macrophages produce the anti-inflammatory/immunosuppressive cytokines il and tgf in vitro (bryniarski et al. (bryniarski et al. , . while the data suggest that testicular macrophages may have a predominantly anti-inflammatory and/or immunosuppressive phenotype, this has yet to be formally established. furthermore, dendritic cells, which are a highly specialized macrophage subset also found within the testicular interstitium (fijak et al. ; hoek et al. ; itoh et al. b; rival et al. a) , presumably play a more important role in controlling immune responses to antigens within the testis (fijak et al. ; rival et al. a rival et al. , . in the rat testis, macrophages can be discriminated by their differential expression of two molecular markers recognized by the antibodies ed and ed . antibody ed recognizes the lysosomal protein cd , which is associated with antigen processing and presentation (groisman et al. ) , while ed recognizes the scavenger receptor cd (fabriek et al. ) . cd is involved in the clearance of hemoglobin:haptoglobin complexes and the resolution of inflammation (philippidis et al. ) . about - % of rat testicular macrophages express this scavenger receptor and about half of these cells also lack expression of cd (gerdprasert et al. a; meinhardt et al. ; wang et al. ). in the human testis, most macrophages appear to be cd þ , but expression of cd is a feature of a subset of these cells as well (frungieri et al. ) . the heterogeneous expression of these two markers points to the existence of multiple populations of testicular macrophages, and evidence suggests that these phenotypic differences also correspond to functional differences. in the normal and inflamed adult rat testis, cd þ cells lack expression of the key inflammatory mediators il and inducible nitric oxide synthase (inos), as measured by double-label immunohistochemistry, but these molecules are detectable in cd þ cells that do not express cd (gerdprasert et al. a; o'bryan et al. ) . indeed, purification of cd þ macrophages from the rat testis using immunomagnetic beads confirms that these cells have reduced capacity to produce il , il , or no upon stimulation, although they retain the ability to produce prostaglandins (gerdprasert et al. a; hedger, unpublished data) . this suggests that the cd þ macrophages have reduced proinflammatory activities, while the cd -negative testicular macrophage population retains normal proinflammatory functions. this observation is important because macrophages that express cd and produce il , but display reduced expression of cd , cd , inos, tnf, and il , are polarized toward the immunoregulatory m subset (mantovani et al. ; sica et al. ) . the m macrophage subset is analogous to the th subset among t cells, and is associated with late-stage inflammation and its resolution, as well as tumors and other immunologically privileged tissue sites. it would appear that a significant proportion of the resident macrophages of the testis possess an immunoregulatory phenotype that is consistent with immune privilege. the functional roles of this macrophage population, as well as those of the macrophages that appear to retain normal inflammatory (i.e., m subset) properties, demand particular attention if we are to understand the unique immunological environment of the testis. while the functions and regulation of lymphocytes within the testis have yet to be properly characterized, by their presence alone these cells will have significant effects on testicular events. it may also be assumed that, in contrast to the largely resident population of testicular macrophages, lymphocytes circulate through the testis. this may be because they possess specificity for testicular antigens, or because they have been activated during earlier intratesticular inflammatory or infectious events (czerkinsky et al. ; springer ) . quantitative studies in the rat indicate that, at any given time, lymphocytes are approximately % as numerous as the macrophages in the normal testis (hedger and meinhardt ; hedger et al. b; wang et al. ). in the rat and mouse, intratesticular lymphocytes express t cell and nk cell markers, and comprise distinct t cell, nk cell, and nkt cell subsets (tompkins et al. ; hedger, unpublished data) . both t cell and nk cell markers are expressed by lymphocytes in the human testis as well (hedger, unpublished data) . b cells are not usually observed in normal tissues, and this includes the testis. in the rat, many t cells possess a memory phenotype, as would be expected (hedger et al. b; tompkins et al. ). reanalysis of the data for individual animals across several studies suggests that their numbers tend to be proportional to the numbers of macrophages, but gradually increase with the age of the animal (hedger et al. b; hedger and meinhardt ; wang et al. ) . among the t cells of the testis, there is a strong bias toward the cd þ (i.e., mhc class i restricted, largely cytotoxic) subset (hedger et al. b; pöllänen and niemi ; ritchie et al. ; tompkins et al. ; wang et al. ) . the prominence of nk cell subsets in the testis is also intriguing given the reduced mhc class i antigen expression in the seminiferous compartment, which would tend to make these cells more susceptible to nk cells (lanier ) . consistent with the anti-inflammatory/immunosuppressive phenotype of the major testicular macrophage subset, t cells and nkt cells in the rat testis constitutively produce il (hedger, unpublished data) . while much remains to be discovered, the existing data concerning the types and activity of testicular lymphocytes are consistent with maintaining immune privilege (i.e., the presence of immunoregulatory lymphocytes) and increased innate immunity (i.e., cytotoxic cell activity). evidence that pmns are important in testicular function comes from the fact that mast cells and eosinophils are significant elements of the interstitial tissue in many species. in the rat, mouse, dog, cat, bull, and deer, both cell types are largely absent from the testicular parenchyma, but are associated with blood vessels in the testicular capsule (anton et al. ) . mast cells are found throughout the interstitial tissue in equine and human testes (anton et al. ; nistal et al. ; yamanaka et al. ) , and both mast cells and eosinophils are present in porcine species (anton et al. ) . the role of these cells in vascular control and innate immunity can be predicted, but they may also play immunoregulatory roles, as mast cells do in other tissues . in humans, testicular mast cell numbers are increased in infertility (meineke et al. ; nagai et al. ; yamanaka et al. ) , but decline with advancing age (nistal et al. ) . the possibility that they accumulate due to past immune events needs to be considered, but they also appear to be actively regulated. in the adult rat testis, mast cell proliferation is under the control of the leydig cells, suggesting that these cells produce an inhibitor of mast cell activity (gaytan et al. ; wang et al. ) . neonatal estrogen treatment increases mast cell numbers in the rat testis (gaytan et al. (gaytan et al. , , and a relationship with elevated leydig cell aromatase activity is suggested by the prevalence of these cells in boar and equine testes (eisenhauer et al. ; raeside and renaud ) . neutrophils are found in the testis only under conditions of testicular inflammation or damage (gerdprasert et al. a; kohno et al. a; o'bryan et al. b ). the majority of this chapter deals with the testis because it is the primary source of male gametes and hormones, and damage to this organ tends to have more significant consequences for fertility. the remainder of the male reproductive tract comprises the epididymis, vas deferens, accessory glands, and the urethra, which are all epithelial-lined ductal tissues of varying complexity. the immunology of these tissues appears to be quite distinct from that of the testis. while it is true that sperm spend relatively little time within most of these tissues, mature sperm reside within the epididymis for extended periods without provoking an overt immune response, and inflammation and immune cell activity can nonetheless have damaging effects in this organ. significantly, these tissues possess normal interepithelial cell junctions, but lack a completely occluding intercellular structure analogous to the blood-testis barrier (levy and robaire ; pelletier ) . secreted iga, which lacks the ability to bind complement and possesses principally antiinflammatory properties as a result, plays an important role in the male urogenital tract (clifton et al. ; politch et al. ; pudney and anderson ) , and the immunology of these organs tends to more closely resemble that of other elements of the mucosal immune system, such as the gastrointestinal and respiratory tracts (beagley et al. ; clifton et al. ; mestecky et al. ) . in the absence of the specialized mucosal-associated lymphoepithelial tissue normally found in the gastrointestinal and respiratory tracts, it is likely that the epithelial cells themselves, together with the numerous intraepithelial and stromal macrophages and lymphocytes that are normally found within these tissues, mediate local immunoregulatory mechanisms (barratt and cohen ; dym and romrell ; el-demiry et al. ; ritchie et al. ; pudney and anderson ; theyer et al. ; yeung et al. ) . a better understanding of the immunology of these tissues is important. inflammation, infection, and physical damage can lead to immune reactions against the sperm, which in turn may compromise fertility. moreover, many men suffer from chronic pelvic pain, generally manifesting as epididymitis or prostatitis in the absence of a detectable infection, a condition that almost certainly involves chronic inflammatory processes within the male reproductive tract. as outlined in the preceding sections, the testis is able to support immune privilege without sacrificing immune surveillance and protection. the mechanisms responsible for this immunological balancing act are still far from understood, but a number of inflammatory and immunoregulatory systems that operate within the testis have been investigated in varying degrees of detail. it is perhaps not very surprising that many of these regulatory systems also impact upon normal testis physiology, in addition to their immunological and pathological roles. given that the types and properties of leukocytes found within the testis environment are consistent with immunoregulation and enhanced innate immunity, the focus should be on which testicular elements are involved in creating this potentially unique immunological environment. most attention in this regard has been directed toward the highly versatile sertoli cell. evidence that the sertoli cell possesses specialized immunoregulatory properties comes from cotransplantation studies. sertoli cells from immature rat, murine, or porcine testes display extended survival as allografts or xenografts, and cotransplantation of sertoli cells or testis cell mixtures containing these cells confers increased survival on neural cell xenografts, and pancreatic islet allografts and xenografts (sanberg et al. ; selawry and cameron ; suarez-pinzon et al. ) . the ability to form a physical barrier through inter-sertoli tight junctions and the fact that sertoli cells have reduced mhc expression (haas et al. ; kohno et al. b; sanberg et al. ; turek et al. ) undoubtedly enhance their potential to avoid t cell activation both in the intact testis and in engraftment studies. moreover, a soluble form of the nonclassical mhc class ib molecule hla-g is produced by the sertoli cells, as well as spermatocytes, spermatids, and some interstitial cells, in the rhesus monkey testis (ryan et al. ) . this molecule has been implicated in apoptosis of alloreactive cd þ cytotoxic t cells (fournel et al. ) . sertoli cells display lymphocytotoxic activities and produce molecules with immunosuppressive activity, such as tgf (de cesaris et al. ; suarez-pinzon et al. ; wyatt et al. ; yin et al. ) and fasl (bellgrau et al. ; yin et al. ). an active immunoregulatory role has also been suggested: murine sertoli cells treated with ifn increase expression of mhc class ii molecules and the negative costimulatory ligand b -h , and stimulate t reg numbers in culture (dal secco et al. ) . the ability of the sertoli cell to suppress other critical inflammatory events through expression of complement regulatory proteins ) and secretion of inhibitors of lymphocyte cytotoxic activities (sipione et al. ) is also likely to be important. finally, sertoli cells possess an enormous capacity for phagocytosis of senescent cells, cell debris, and other potentially antigenic complexes, which could otherwise activate an immune response. data concerning immunoregulatory roles of other testicular cells are less comprehensive. the evidence clearly indicates a role for leydig cells in recruiting macrophages into the testis, although testicular macrophage function may actually be determined by the sertoli cells (duckett et al. a,b; gaytan et al. c; wang et al. ) . leydig cells also produce various steroids, proteins, and other factors with immunosuppressive activity, and inhibit lymphocyte activity in vitro (born and wekerle ; hedger et al. ). peritubular cells, like sertoli cells, produce immunoregulatory tgf family members (konrad et al. ; mullaney and skinner ; okuma et al. b) . the possibility that the germ cells themselves have a direct effect on immune cells has also been considered (hurtenbach and shearer ) , although it seems more likely that germ cells may influence immune responses indirectly by inducing immunoregulatory activities of the sertoli cells and other somatic cells. the relatively recent discovery of the tlrs has radically expanded our understanding of innate immunity and promises to do the same for testicular immunology. in addition to their predictable expression on testicular macrophages and dendritic cells, tlr mrna, protein, and/or ligand responses have been described in several other testicular cells. rat and mouse sertoli cells express tlr , , , and (bhushan et al. ; riccioli et al. ; starace et al. ; wu et al. ) . tlr , , and mrna expression has been observed in rat sertoli cells (bhushan et al. ) , and low levels of tlr , , and mrna have been detected in mouse sertoli cells (riccioli et al. ; wu et al. ). in the rat, germ cells also produce mrna for tlr , , and in a stage-specific manner, and the peritubular cells express tlr and , along with low levels of tlr , , and (bhushan et al. ) . rat leydig cells express tlr mrna and low levels of tlr (bhushan et al. ) . however, the cytoplasmic receptors tlr and have yet to be observed in any nonmyeloid testicular cell type. of the other pattern-recognition receptors, nod mrna has been detected in rat sertoli cells, peritubular cells, and spermatogonia, while nod was detected in spermatogonia only (bhushan et al. ) . expression of tlrs and other pattern-recognition receptors in the testis is obviously important for responses toward intratesticular pathogens. infections and inflammation, both systemic and localized, have negative effects on testicular function, resulting in reduced androgen production, lowered sperm counts, and temporary loss of fertility (adamopoulos et al. ; baker ; carlsen et al. ) . presumably, tlrs expressed by the sertoli cells and germ cells are necessary to detect pathogens that ascend the reproductive tract and, as a consequence, lie behind the blood-testis barrier. this expression within the seminiferous epithelium also suggests a mechanism whereby inflammation can directly inhibit spermatogenesis. activation of sertoli cells by tlr ligands induces production of inflammatory mediators, such as il , il , and no, which have direct effects on germ cell development and leydig cell steroidogenesis (gérard et al. ; stéphan et al. stéphan et al. , o'bryan and hedger ; riccioli et al. ; wu et al. ) . moreover, while inhibition of leydig cell steroidogenesis during inflammation may involve indirect effects at the level of the pituitary, or the actions of inflammatory mediators produced by the testicular macrophages and sertoli cells, there is evidence that these cells can also respond directly to tlr ligands (bhushan et al. ; xiong and hales a) . although the tlrs have evolved to recognize pathogen-derived molecules, such as lps (tlr ) and bacterial lipoproteins (tlr ), recent data show that they can also be activated by certain endogenous molecules. the ability of preparations of some endogenous molecules, such as heat-shock proteins, to activate tlr signaling is now attributed to contamination of these preparations by bacterial products (tsan and gao ) , but there is convincing evidence that mammalian mrna is a ligand for tlr (karikó et al. ) and mammalian cpg dna can activate tlr (yasuda et al. ) . the high mobility group box chromosomal protein (hmgb ), produced by both spermatogonia and sertoli cells, is a tlr and ligand (yu et al. ; zetterström et al. ) . expression of tlrs by the sertoli cells under normal conditions, therefore, suggests a potential role for these receptors in responding to these endogenous ligands. . . . . the interleukin family il is the best studied of all the cytokines normally expressed in the testis. it is produced in two forms, and , which are encoded by separate genes and share approximately % sequence homology. both forms act through the same receptor complex (il r ), and exert a similar range of proinflammatory and physiological effects (dinarello ; o'neill and dinarello ) . most il signaling occurs through activation of the myd /traf and jnk/p map kinase pathways, thereby regulating many proinflammatory genes through stimulation of the transcription factors nfb and activated protein (ap ) (medzhitov et al. ) . the il s are synthesized as precursor proteins, which are cleaved to produce kda active proteins. in the case of il , the precursor possesses low-level biological activity, but the il precursor protein is inactive (black et al. ; watanabe and kobayashi ) . the precursor of il is cleaved by the il converting enzyme (ice, caspase ) during the process of secretion, whereas il is cleaved by the calcium-dependent membrane-associated cysteine protease (calpain) or by extracellular proteases (thornberry and molineaux ; watanabe and kobayashi ) . il is produced by activated monocyte/macrophages and is the main secreted form during inflammation, whereas il is more commonly found within the cell, where it may act as an autocrine or paracrine growth factor involved in direct cell-to-cell communication. in addition to the prototypical il and il , the il family comprises a number of structurally related proteins, which appear to have arisen by gene duplication (dunn et al. ) . the closest structurally to il / is il , which is processed by caspase activity, but acts via a separate receptor rather than il r (gracie et al. ) . another member of the family, which does bind to il r but lacks the ability to transduce a signal, blocks il / action and is called il receptor antagonist (il ra) (arend ) . production of il is constitutively high in the normal testis, as il is produced by the sertoli cells in a cyclical manner within the mature seminiferous epithelium (jonsson et al. ; söder et al. ) . its production is driven by the developing germ cells and by phagocytosis of the residual cytoplasm cast off by the spermatids at the time of their release into the tubule lumen (gérard et al. ; syed et al. ) . sertoli cell il production is also stimulated by lps, but not by fsh (stéphan et al. ) . production of il by the normal testis is low, but is upregulated in a subset of testicular macrophages and the leydig cells during inflammation (gérard et al. ; jonsson et al. ; o'bryan et al. ) . curiously, the majority of this secreted testicular il exists as the precursor (o'bryan et al. ) . the receptor il r has been localized to germ cells and sertoli cells (gomez et al. ) . il stimulates dna synthesis in spermatogonia (i.e., mitosis) and early spermatocytes (i.e., meiosis), proliferation of immature sertoli cells, and a number of activities of the mature sertoli cell that support spermatogenesis, such as the production of lactate and transferrin (hakovirta et al. b; hoeben et al. ; nehar et al. ; parvinen et al. ; pöllänen et al. ; söder et al. ) . it also regulates the ability of the sertoli cell to maintain contact with other sertoli cells and the developing germ cells, by altering the sertoli cell cytoskeleton (sarkar et al. ). in the interstitium, both il and il suppress lh-stimulated androgen production by adult leydig cells through inhibition of steroidogenic enzyme activity (hales ; hales , ) . il ra is produced by the sertoli cell, and is stimulated by fsh, il , and lps (zeyse et al. ) . presumably, its role is to regulate the activity of il / within the testis. both il and its receptor have been found in the seminiferous epithelium, with il mrna and protein localized to spermatocytes and round spermatids (strand et al. ) . as is the case for il , the majority of il produced within the testis is in the precursor form, indicating that the processing of these cytokines by caspase in the testis is an area requiring more investigation. il stimulates spermatogonial dna synthesis in cultures of rat seminiferous tubules, without influencing germ cell apoptosis (strand et al. ) . another member of the family localized to the testis is il f , although its function there is entirely unknown (kumar et al. ) . . . . . tumor necrosis factor and fas ligand tnf and fasl are transmembrane and secreted cytokines with typically trimeric structures and they interact with specific receptors to mediate a cell death signal (ju et al. ; schütze et al. ) . they are implicated in both immunoregulation and the control of normal spermatogenic function in the testis. tnf is a kda glycosylated polypeptide and is a major early product of activated monocytes and macrophages. it binds to either of two tnf receptor subsets (tnfr and tnfr ) and plays a central role in initiating the inflammatory response by stimulating the production of il and il (basak and hoffmann ; bradley ) . whether tnf exerts proinflammatory or cytotoxic effects depends on the receptor subtype engaged and the expression of specific adaptor proteins within the target cell (basak and hoffmann ; chung et al. ; hsu et al. ; mak and yeh ) . consistent with its name, tnf exerts a cell death signal via tnfr , through interaction with the tnfr-associated death domain protein (tradd) or the fas-associated death domain protein (fadd), and activation of the caspase-dependent apoptotic pathway (schütze et al. ) . however, interaction with tradd can also result in recruitment of other adaptor proteins leading to the binding of traf and activation of the nfb pathway and/or the map kinases jnk or p (chung et al. ) . moreover, tnfr and its related receptors, which include cd , do not contain a death domain, and instead associate with the trafs leading to activation of cell signaling events (bradley ; mak and yeh ) . in the mouse testis, tnf is expressed by round spermatids, pachytene spermatocytes, and testicular macrophages, and mrna for tnfr has been located on both sertoli and leydig cells . in porcine sertoli cells, tnf receptor subunit protein expression is stimulated by fsh (mauduit et al. ) . there is no evidence that tnf is produced by the sertoli cell, but expression of tnf by the germ cells within the seminiferous epithelium, like that of il and il , is cyclical. within the seminiferous epithelium, tnf produced by the germ cells appears to play a complex role in the control of both sertoli cell function and spermatogenesis. tnf reduces spontaneous germ cell degeneration in cultured human and rat seminiferous tubules, suggesting a germ cell survival effect presumably mediated through the sertoli cell (pentikäinen et al. ; suominen et al. ). conversely, tnf disrupts sertoli cell tight junction assembly by inhibiting the production of junction proteins and by regulating matrix metalloprotease and protease inhibitor activity (siu et al. ) . tnf also stimulates plasminogen activator inhibitor expression in rat testicular peritubular cells (le magueresse-battistoni et al. ) . similar to il , tnf stimulates basal lactate production by cultured sertoli cells, but tnf generally antagonizes the actions of fsh on sertoli cell function, including the stimulation of aromatase activity and lactate production (mauduit et al. ; nehar et al. ; riera et al. ). however, delfino et al. ( ) have shown that tnf stimulates androgen receptor expression in sertoli cells via upregulation of nfb, which binds to several enhancer motifs in the androgen receptor promoter. tnf also stimulates the expression of inflammatory cytokines, chemokines, and leukocyte adhesion molecules in both sertoli cells and peritubular cells (aubry et al. ; de cesaris et al. ; stéphan et al. ) . in testicular pathology, tnf has been implicated as a major causative agent in the development of experimental autoimmune orchitis (eao) (yule and tung ) . there is a significant increase in the number of tnf-positive testicular macrophages and the number of tnfr -positive germ cells undergoing apoptosis during eao in rats (suescun et al. ) . within the interstitium, tnf acts as an effective regulator of leydig cell steroidogenesis, by inhibiting lh receptor binding and steroidogenic gene expression (li et al. ; mauduit et al. b mauduit et al. , hales b, ) . no intratesticular cytokine has excited more controversy than the cell death signaling ligand fasl. interactions between fasl and its receptor on activated t cells are important in the regulation of the immune response (ju et al. ) . the death domain in the cytoplasmic region of the fas receptor recruits the fadd adaptor protein and induces t cell death via caspase-dependent apoptosis (schütze et al. ). there is evidence that fasl expression on epithelial cells in immune-privileged tissues, including sertoli cells, may be responsible for deleting activated t cells within the tissue, thereby suppressing adaptive immunity (bellgrau et al. ; griffith et al. ; saas et al. ; stuart et al. ). however, this hypothesis has been challenged by the observation that increased fasl expression on cells such as tumor cell lines and pancreatic islet cells does not increase protection in transplantation studies, but actually provokes a massive inflammatory reaction and rejection (allison et al. ; kang et al. ) . moreover, fasl appears to be abundantly expressed in the epithelia of several human tissues that lack any evidence for immunological privilege (xerri et al. ) , while absence or inhibition of fas or fasl does not automatically cause failure of immune privilege (rogers et al. ; suarez-pinzon et al. ; wahlsten et al. ) . it seems unlikely that fasl expression on its own is a fundamental explanation for immune privilege, although it may contribute through interaction with other immunoregulatory processes . localization of fasl and fas in the testis under normal conditions also has proven controversial, which may be attributed to differences in detection methods, limitations of some of the reagents that have been used, and the fact that these molecules are inducible (d'alessio et al. ; restifo ) . studies have shown fasl to be present in rat, mouse, porcine, and human sertoli cells and absent in most germ cells (bellgrau et al. ; braendstrup et al. ; d'abrizio et al. ; lee et al. ) , but other studies have reported that fasl expression in the rat seminiferous epithelium is confined to the germ cells (celik-ozenci et al. ; d'alessio et al. ) . the receptor fas has been found on isolated mouse sertoli cells (riccioli et al. ) , but in intact testes has been localized to spermatogonia and spermatocytes from the pubertal period onward (celik-ozenci et al. ; lee et al. ; lizama et al. ; ogi et al. ). in germ cells, fas expression is associated with cells that are undergoing apoptosis (lee et al. ; lizama et al. ) , and fas is induced by tnf and ifn in the sertoli cell (riccioli et al. ) . curiously, in one study using fragment cultures of human seminiferous tubules, tnf downregulated fasl expression and inhibited apoptosis of germ cells (pentikäinen et al. ) . less controversially, fas and fasl expression is upregulated in various models of seminiferous epithelium damage, indicating that this mechanism is important in regulating germ cell apoptosis in cases of physical and toxicological insult (lee et al. ; ogi et al. ). members of the il family of cytokines exert their actions via binding to specific receptors that associate with a common membrane signal transducer gp , leading to the activation of the janus kinase/signal transducers and activators of transcription (jak/stat) and map kinase cascades (heinrich et al. ) . in addition to their functions in inflammation and the immune response, these cytokines play crucial roles in hematopoiesis, liver and neuronal regeneration, embryonic development, and fertility. members of this family that have been detected in the testis are leukemia inhibitory factor, oncostatin m, ciliary neurotropic factor, il , and il itself (de miguel et al. (de miguel et al. , du et al. ; jenab and morris ; okuda et al. ; stéphan et al. ) . the principal roles of most of these cytokines in the testis appear to lie in regulating leydig cell development and the onset of spermatogenesis. a role in testicular immunology is most likely for il , which possesses both proinflammatory and anti-inflammatory properties, and regulates the acute-phase response as well as several aspects of immune cell development and activity (kopf et al. ; tilg et al. ) . sertoli cells, leydig cells, and peritubular cells have been shown to produce il in vitro (cudicini et al. b; okuda et al. ; syed et al. syed et al. , . in rat sertoli cells, il production is stimulated by fsh, testosterone, phagocytosis, and other inflammatory stimuli, including il , il , tnf, and lps (cudicini et al. a,b; okuda et al. okuda et al. , stéphan et al. ; syed et al. ) . production of il by cultured mouse sertoli cells is inhibited by inf (riccioli et al. ; stéphan et al. ) . within the seminiferous epithelium, endogenous production of il follows a cyclical pattern that corresponds with the changes in the stages of the spermatogenic cycle, most probably under the influence of il and fsh (hakovirta et al. ; syed et al. syed et al. , . both the il -specific receptor subunit (il r) and the gp mrna are expressed in rat sertoli cells, and are stimulated by il and il , but only the il r subunit is stimulated by fsh (fujisawa et al. ) . il increases basal and fsh-induced transferrin and cyclic gmp secretion by the sertoli cell, and inhibits meiotic dna synthesis in preleptotene spermatocytes (boockfor and schwarz ; hakovirta et al. ; hoeben et al. ) . in models of eao, il appears to play an ameliorative or protective role within the seminiferous epithelium (li et al. ; rival et al. b ). leydig cells are also active producers of il following stimulation by lh, lps, or il in vitro, and evidence suggests that these cells actually may be the major testicular source (boockfor et al. ; cudicini et al. b; okuda et al. okuda et al. , . several studies have implicated il as a key player in testicular immunology. testicular il production is preferentially induced by systemic lps treatment in the adult rat testis (o'bryan et al. ) , and macrophages and t cells isolated from the rat testis constitutively produce il , which may contribute to testicular immune privilege (hedger, unpublished data) . in a mouse model of eao, elevation of endogenous il levels by adenoviral-mediated transfection of human il was found to significantly reduce the incidence of immune activation, orchitis, and spermatogenic damage (watanabe et al. ) . although male mice deficient in il do not show evidence of male fertility problems, intratesticular immune responses have yet to be examined in these animals (kühn et al. ) . . . . . nitric oxide and oxygen metabolites production of ros, particularly by macrophages, is an important component of the early response to infection (takemura and werb ) . significant ros include the superoxide anion (o -), hydrogen peroxide (h o ), the hydroxyl radical (ho?), nitric oxide (no?), and the peroxynitrite anion (onoo À ). most are products of normal cellular metabolism, but their production is stimulated during inflammation through the activity of the enzymes nadph oxidase (nicotinamide adenine dinucleotide phosphate-oxidase) and inos (forman and torres ; nathan ). these small molecules are cytotoxic to many microorganisms, although some play a more complex role in cell signaling processes. they also interact with crucial intracellular macromolecules, such as proteins, lipids, and dna, causing oxidative damage. although there are endogenous repair systems to correct oxidative damage, excessive production of ros contributes to the pathology of many diseases. among the ros, nitric oxide represents a special case. at high levels, no is highly cytotoxic, especially as a precursor of the peroxynitrite anion, but at low levels no acts as an intracellular and extracellular signaling molecule (bogdan ; schmidt and walter ) . it is produced by one of three related enzymes, neuronal nos (nnos or nos ), inducible nos (inos or nos ), and endothelial nos (enos or nos ), which catalyze the conversion of l-arginine to l-citrulline and no (alderton et al. ) . the nos enzymes are homodimeric proteins encoded by three separate genes ( table ) . both nnos and enos are constitutively expressed enzymes whose activity is regulated through a calcium-calmodulin-mediated mechanism, whereas inos is a constitutively activated enzyme that is regulated at the transcriptional and translational levels by various inflammatory mediators, including lps, il , and tnf (wolkow ) . in studies in various species, nos has been found in sertoli cells, leydig cells, peritubular cells, germ cells, testicular macrophages, and vascular endothelial cells (kim et al. ; lee and cheng ) . the nnos gene also produces a testis-specific isoform, tnnos, which has been localized to leydig cells (wang et al. ). in the normal rat seminiferous epithelium, inos is expressed by elongating spermatids and pachytene spermatocytes, particularly during the stages immediately following sperm release, with relatively lower levels of expression in sertoli cells and peritubular cells throughout the (o'bryan et al. a ). in the interstitium, macrophage expression of inos appears to be largely confined to the minority of cd -negative macrophages of the testis, and is not detectable in the majority of resident macrophages even during inflammation (o'bryan et al. a; gerdprasert et al. a) . testicular cell inos expression and no production are increased by inflammatory events induced by lps, testicular torsion, or testicular heating (lue et al. ; o'bryan et al. a; shiraishi et al. ). production of no by germ cells, in particular, is implicated in the control of the formation and disassembly of the sertoli cell junctions that constitute the blood-testis barrier, as well as the junctional complexes involved in sertoli-germ cell adhesion lee et al. ) . moreover, pachytene and round spermatid apoptosis is significantly reduced in inos null mice, leading to an increase in daily sperm output and indicating a key role for inos in limiting germ cell survival and/or the carrying capacity of the sertoli cells (lue et al. ) . no inhibits leydig cell steroidogenesis directly, an action that is probably mediated through oxidative damage, and treatment with nos inhibitors counteracts the normal decrease in testosterone associated with sepsis or stress (del punta et al. ; sharma et al. ; welch et al. ) . similarly, production of other ros has been implicated in the loss of steroidogenic function in various inflammatory models and in leydig cell cultures (georgiou et al. ; quinn and payne ) . last but not least, no is a potent vasodilator, and plays a role in the endogenous control of testicular blood flow and formation of the interstitial fluid (lissbrant et al. ; o'bryan et al. a) . production of no is an important mediator of germ cell death in testicular torsion (moon et al. ; shiraishi et al. ) , and may be involved in the testicular damage associated with varicocele (santoro et al. ; türker köksal et al. ) . it is evident that the major inflammatory signaling pathways mediated via nfb and the p /jnk map kinases play important, albeit complex, intratesticular roles. notably, nfb has been implicated as an apoptosis-inducing signal for germ cells in numerous studies (lysiak et al. ; pentikäinen et al. ; rasoulpour and boekelheide ; starace et al. ) , even though it stimulates androgen receptor expression in the sertoli cell (delfino et al. ) . activation of p /jnk is implicated in the stimulation of proliferation by immature sertoli cells and the regulation of blood-testis barrier dynamics, steroidogenesis, and multiple inflammatory responses, including the production of il , inos, monocyte chemoattractant protein , and leukocyte adhesion molecules, in the mature sertoli cell (de cesaris et al. , ishikawa and morris ; ishikawa et al. ; petersen et al. ; riccioli et al. ) . while these pathways mediate many of the effects of inflammation on the testis, endogenous regulation of these pathways also appears to be involved in the normal functions of the seminiferous epithelium, as will be discussed later. the ifns are functionally related cytokines with antiviral activity and they comprise three main groups (, , and ), based on their structural relationships and major cellular sources (hertzog et al. ; malmgaard ) . the type i ifns (ifn and ) are produced by a variety of cell types in addition to leukocytes, and exert primarily antiviral and antiproliferative effects via the ifn receptor (ifnar). the type ii ifn is produced by nk and nkt cells, activated t cells and, under certain conditions, by macrophages and dendritic cells (schoenborn and wilson ; varma et al. ) . it acts through a separate receptor, and regulates the activity of apcs as part of the adaptive immune response in addition to its antiviral functions. hence, ifn is a type ii ifn, but a type cytokine. type i ifn production is stimulated through tlr , which detects double-stranded viral rna, and tlr , the bacterial lps receptor (hertzog et al. ) . these two tlrs, which are expressed in the testis (bhushan et al. ; riccioli et al. ; starace et al. ; wu et al. ) , are able to induce the ifn transcription factor irf , via engagement of the trif adapter protein (akira and takeda ; hertzog et al. ; o'neill and bowie ) . the regulation of ifn is cell specific and complex, but involves jak/stat signaling and various transcription factors, including nfb and ap (schoenborn and wilson ; varma et al. ) . its production is stimulated by type i ifns and by il , among other cytokines. viral infections stimulate type ifn and, somewhat more surprisingly, ifn production by sertoli cells, peritubular cells, leydig cells, and testicular macrophages, leading to typical antiviral responses in the testis (dejucq et al. (dejucq et al. , a hu et al. ; starace et al. ) . a role for ifn in maintaining testicular immune privilege has been indicated by the observation that mouse sertoli cells upregulate their expression of the negative costimulatory ligand b -h in response to ifn in vitro, but remain devoid of positive costimulatory molecules (dal secco et al. ). on the other hand, in vivo studies have identified ifn as a causative factor in eao in mice (itoh et al. b) . moreover, ifn stimulates sertoli cell production of fas and caspase , and is implicated as the mediator of sertoli cell and germ cell apoptosis under various conditions (kanzaki and morris ; riccioli et al. ) . these disparate observations are indicative of a complex relationship between ifn, the local immune system, and spermatogenesis. ifn production also affects testicular steroidogenesis. normal healthy men treated with human ifn had significantly decreased serum testosterone levels in conjunction with normal serum gonadotropins, and both ifn and ifn inhibit testosterone production in primary cultures of leydig cells (orava et al. ). studies using porcine leydig cells indicate that ifn exerts its inhibitory effect on testosterone production at the level of cholesterol transport into the mitochondria, and inhibits expression of the steroidogenic acute regulatory (star) protein and the p- steroidogenic enzymes, cholesterol side-chain cleavage complex (p scc) and -hydroxylase/c -c lyase (p c ) (orava et al. (orava et al. , . these data indicate that ifns may contribute to the decline in steroidogenic function of patients with viral infections, in particular. however, dejucq et al. ( dejucq et al. ( , a have shown that an increase in ifn and ifn expression in sertoli and leydig cells of rats infected with sendai virus was actually associated with an increase in testosterone production. these results suggest that indirect or secondary stimulatory effects on testicular steroidogenesis may be involved, providing further evidence that ifns play a complex role in testicular function. and activin a the three tgf family members that are expressed in mammals (tgf , tgf , and tgf ) are the archetypes of a much larger superfamily of mostly homo-and heterodimeric proteins, which includes the activins, the bone morphogenetic proteins (bmps), inhibin, and anti-müllerian hormone (lin et al. ) . the tgfs themselves are multifunctional growth and differentiation factors involved in many aspects of tissue remodeling and repair as well as regulation of the immune system (ashcroft ; chang et al. ; licona-limón and soldevila ) . nearly all cells synthesize a form of tgf and possess functional receptors for these cytokines. most tgf family ligands act via dual transmembrane serine/threonine kinase receptors, called type i and type ii, which interact upon ligand binding (cárcamo et al. ; ebner et al. ; massagué et al. ) . signals are transduced from the membrane to the nucleus through phosphorylation of the regulatory smad signaling proteins, with smads , , and responsible for mediating tgf and activin signaling (datto et al. ; de caestecker et al. ) . all three mammalian tgf forms are differentially expressed by sertoli cells, peritubular cells, and leydig cells in the fetal and immature testis, but production declines considerably during sexual maturation gautier et al. ; mullaney and skinner ; olaso et al. ) . in the postpubertal testis, they have also been localized to the germ cells (caussanel et al. ; teerds and dorrington ) , and tgf receptors are found in both somatic and germ cells (caussanel et al. ; goddard et al. ; le magueresse-battistoni et al. ) . the tgfs appear to be involved in testicular development by controlling, among other things, apoptosis of undifferentiated spermatogonia (gonocytes) (olaso et al. ) , seminiferous tubule formation, and leydig cell differentiation (cupp et al. ; dickson et al. ; khan et al. a; konrad et al. ) . in the adult testis, tgf and tgf regulate sertoli cell tight junction dynamics, indicating a role in regulating the permeability of the bloodtestis barrier to facilitate the passage of spermatocytes across the barrier during spermatogenesis (lui et al. ; xia et al. ) . however, the tgfs are also potent inhibitors of the activity of immune cells, especially t cells, b cells, and nk cells (ahmad et al. ; ashcroft ; cousins et al. ; licona-limón and soldevila ; wahl et al. ) , suggesting a prominent role in creating testicular immune privilege. accordingly, tgf contributes to the lymphocyte inhibitory activity of testis extracts (pöllänen et al. ) and to the immunoprotective properties of sertoli cells in cotransplantation studies (suarez-pinzon et al. ) . activins are homodimers or heterodimers of homologous subunits, designated a -e , which are structurally related to the tgfs (chang et al. ; de kretser et al. ) . homodimers of a form activin a, which is widely expressed and has been extensively studied. relatively less is known about the other activin forms, which appear to be both less abundant and less widely distributed. activin a is a multifunctional growth factor and inflammatory regulator (phillips et al. ) , although activins were originally named for the ability of activin a and b, in particular, to stimulate pituitary fsh secretion (corrigan et al. ; ling et al. ). heterodimers of either a or b subunits with a homologous subunit, are called inhibin a and inhibin b, respectively, and act as feedback inhibitors of fsh secretion from the pituitary (robertson et al. ). unlike the activins, inhibins are not widely produced, and their chief source is the sertoli cell, the cellular target of fsh action meunier et al. ) . activin functions are highly regulated. like the tgfs, they act via specific type i and type ii transmembrane serine/threonine kinase receptors, linked to the smad signaling pathway (ethier and findlay ; vale et al. ) . the inhibins are competitive inhibitors of activin receptor binding, but activin homodimers and heterodimers comprising the b and c subunits also appear to act as weak competitive agonists of activin a (brown et al. ; makanji et al. ; mellor et al. ) . these interactions are modulated and/or facilitated by specific inhibitory coreceptor proteins, such as betaglycan and the bmp and activin membrane-bound inhibitor (bambi) (lewis et al. ; makanji et al. ; onichtchouk et al. ) . moreover, activin bioactivity can be effectively neutralized in the circulation by the high-affinity activin binding protein, follistatin (nakamura et al. ) . activin a is abundantly produced in the testis, particularly during early testicular development (barakat et al. ; buzzard et al. ) . even in the adult rat, intratesticular fluid levels of activin a are - times higher than normal circulating concentrations (o'bryan et al. ) . the sertoli cells appear to be the main source in the adult testis, although the a subunit is expressed in most germ cells kaipia et al. ) , and activin a protein is present in the resident macrophages and mast cells (okuma et al. b (okuma et al. , . activin a is expressed at low levels throughout the cycle of the seminiferous epithelium, but there is a distinct peak of production immediately following spermiation, which is driven by the surge of il produced by the sertoli cell at this time (kaipia et al. ; okuma et al. ) . activin receptors are expressed by most, if not all, of the somatic and germ cells in the testis (cameron et al. ; de winter et al. ; feng et al. ; kaipia et al. ) . activin a exerts a complex regulation of germ cell, sertoli cell, and leydig cell proliferation and/or differentiation during development and in the adult (barakat et al. ; buzzard et al. ; mather et al. ; mauduit et al. a; meehan et al. ; meinhardt et al. ) , and disorders of activin signaling are implicated in the onset of testicular cancer (dias et al. ) . activin a is produced by activated monocytes, macrophages, dendritic cells, bone marrow stromal cells, and some lymphocytes (erämaa et al. ; ogawa et al. ; robson et al. ; yamashita et al. ) , and plays a facilitating role in early inflammation responses (jones et al. ) . consistent with its homology to the tgf proteins, however, activin a also possesses a repertoire of anti-inflammatory and immunoregulatory activities, and displays characteristics of a type cytokine (hedger et al. ; ogawa et al. ; wang et al. ; yu et al. ) . in various experimental systems, activin a has been found to antagonize the production and actions of il and il , to inhibit critical t cell and b cell activation responses, and to induce macrophages to polarize toward the m phenotype (brosh et al. ; gribi et al. ; ogawa et al. ; russell et al. ) . both sertoli cells and testicular macrophages in culture respond to lps by increasing activin a production (okuma et al. a ) (hedger, unpublished data) , and il is a very potent stimulus for activin a production by the sertoli cell (okuma et al. b (okuma et al. , . the actual role of activin a in testicular inflammation remains unclear, since the high intratesticular levels of activin a are not affected by lps treatment in vivo (o'bryan et al. ) , but a role for activin a in modulating testicular immune responses can be predicted. androgens produced by the leydig cells have immunosuppressive properties that contribute to differences in immunity between the sexes (cutolo et al. ; miller and hunt ) . the majority of these effects are believed to be exerted at the level of the immune tissues, rather than by direct effects on circulating leukocytes, which lack classical androgen receptors (grossman et al. ; sasson and mayer ) . recently, however, it has been discovered that steroids can interact with membrane-bound g-protein-coupled receptors to trigger nongenomic responses (braun and thomas ; rahman and christian ) . studies have shown that androgens alter [ca] fluxes in lymphocytes and macrophages via such membrane-mediated interactions, affecting gene expression and function in the target cells (benten et al. ; walker ; wunderlich et al. ) . although studies of the role of androgens in suppressing immune responses, such as graft rejection, in the testis have been somewhat equivocal (cameron et al. ; selawry and whittington ; whitmore and gittes ) , the observation that immune cells can respond directly to androgens has resurrected the intriguing possibility that androgens, and possibly other testicular steroids, may directly regulate immune cell activity within the testis and adjacent draining lymph nodes. glucocorticoids play an important role in modulating both testicular immunity and steroidogenesis via a complex extratesticular regulatory loop. during inflammation, proinflammatory cytokines activate the hypothalamic-pituitary-adrenal axis leading to increased secretion of glucocorticoids, which act through the ubiquitously expressed glucocorticoid receptors (buckingham et al. ; sapolsky et al. ) . glucocorticoids suppress innate and acquired immunity at multiple levels and are an essential control in the inflammatory/immune response. they inhibit the inflammatory response primarily by repression of nfb, suppressing the production and actions of the proinflammatory cytokines, reducing adhesion molecule expression, and stimulating the production of anti-inflammatory cytokines (auphan et al. ; brack et al. ; kapcala et al. ) . glucocorticoids also exert inhibitory effects at all levels of the hypothalamic-pituitary-leydig cell axis (bambino and hsueh ; hales and payne ; hardy et al. ; monder et al. ), thereby contributing to the inhibition of androgen production that accompanies local and systemic inflammatory events. prostaglandins (pgd, pge, and pgf), prostacyclins (pgi), and thromboxanes (tx), collectively called the prostanoids, are fundamental to many physiological processes, including inflammation and immunity. prostanoids arise from hydrolysis of membrane glycerophospholipids to release the free fatty acid arachidonic acid through the action of one of the more than enzymes with phospholipase a (pla ) activity (kudo and murakami ; schaloske and dennis ) . arachidonic acid is in turn directed down the prostanoid synthetic pathway by the action of the cyclooxygenase (cox; also called prostaglandin-endoperoxide synthase) enzymes (figure ) . there are two cox enzymes, which are the products of different genes: the constitutively expressed cox and an inflammation-induced form, cox (table ) (smith et al. ; tanabe and tohnai ) . while conversion of arachidonic acid to the prostanoid precursor pgh by cox is the rate-limiting step, production of specific prostanoids depends on the activity of the prostaglandin synthases, that is, pge synthase (pges), pgis, pgds, pgfs, and thromboxane a synthase (txas) (helliwell et al. ; ueno et al. ) . each prostanoid acts via one or more specific receptors to regulate cell growth, vascular smooth muscle constriction or relaxation, vascular permeability, and immune cell activity (bos et al. ; hata et al. ) . several prostanoids exert both proinflammatory and immunosuppressive actions through different receptor interactions, or through metabolism to other active immunoregulatory metabolites, such as -deoxy-pgj , a ligand for the peroxisome proliferator-activated receptor (ppar) (jiang et al. ). an alternative synthetic pathway, catalyzed by the lipoxygenases, converts arachidonic acid to the related leukotrienes and lipoxins, which also possess diverse metabolic, vascular, and inflammation-regulating actions (samuelsson et al. ; takano et al. ) . most of the enzymes responsible for prostanoid production are ubiquitously expressed, and this includes the male reproductive tract (gerena et al. ; lazarus et al. ; takahashi et al. ; winnall et al. ) . in fact, the levels of e series prostaglandins in human seminal plasma are remarkably high (cooper and kelly ) . most testicular cells express both cox forms, and possess the capacity to produce prostaglandins in vitro (winnall et al. ) , although there appear to be cell type-specific and species-specific differences in the relative levels of expression (balaji et al. ; frungieri et al. ; lazarus et al. ). in the normal rat testis, cox is responsible for the majority of prostaglandin production (winnall et al. ), although intratesticular pge levels are only marginally affected by acute inflammation (winnall et al. ) . this is probably due to the fact that macrophages in the rat testis express cox at very low levels, but are the only testis cell type to respond to an inflammatory stimulus with a significant increase in cox activity (winnall et al. ). these data point toward a previously unanticipated maintenance role for the socalled inducible cox enzyme in testicular function, as well as an anomalous inflammatory response of testicular cox , consistent with an altered capacity of this organ to initiate and support inflammatory reactions. fertility is retained in male mice lacking either cox or cox , whereas double deletions are lethal (langenbach et al. ; lim et al. ; sales and jabbour ) . studies on male fertility and spermatogenesis using prostaglandins, or nonsteroidal anti-inflammatory drugs such as aspirin and indomethacin, which act primarily via inhibition of cox activity, have produced conflicting conclusions (abbatiello et al. ; biswas et al. ; sanyal et al. ; winnall et al. ) . these diverse outcomes indicate the complexity of the roles of prostanoids in controlling various aspects of testicular function. prostaglandins, particularly pge and pgf , have been implicated in the control of leydig cell development in the immature testis, production of proinflammatory cytokines by the leydig and sertoli cells, autoregulation of steroidogenesis in the adult, and the decline in leydig cell function that occurs during aging (baker and o'shaughnessy ; cooke et al. ; haour et al. ; ishikawa et al. ; walch and morris ; wang et al. ) . production of pge and pgf by cox also mediates the effects of il on protein and lipid regulation by the sertoli cell involved in supporting figure prostanoid and lysophosphatidylcholine synthesis and targets. synthesis of the prostanoids commences through the action of phospholipase a (pla ) on arachidonyl-containing phospholipids (usually a -acyl- -arachidonylglycerophospholipid) to produce the free fatty acid arachidonic acid and the corresponding lysophospholipid. arachidonic acid is converted into the prostanoid precursor prostaglandin h (pgh ) by the action of cyclooxygenases and this is further converted into the various prostanoid subtypes, prostaglandins (pgd, pge, and pgf), prostacyclins (pgi), and thromboxanes (tx), by the action of specific prostaglandin and thromboxane synthases. synthesis of lipoxins and leukotrienes from arachidonic acid occurs via the activity of a separate lipoxygenase enzyme (not shown). the prostanoids interact with their various receptor subtypes: there are two pgd receptors (dp - ) and four pge receptors (ep - ). it is the cellular expression of these receptor subtypes that ultimately determines the eventual biological responses; for example, different prostanoid and receptor combinations may produce either proinflammatory or anti-inflammatory responses, or may have opposing vasodilatory and vasoconstrictive effects. when the phospholipid is a phosphatidylcholine, a lysophosphatidylcholine (lpc) is formed, which may interact with cells via g-protein-coupled receptor-mediated or direct membrane interactions, or may undergo further enzymatic modification to form other bioactive lipids. spermatogenesis and inflammation in the testis (ishikawa and morris ; ishikawa et al. ). in addition, pge and pgf produced by mature spermatozoa play a role in the acrosome reaction (breitbart et al. ; joyce et al. ) . products of the lipoxygenase pathway, such as leukotriene b , are also produced by the testis, where they intervene in the regulation of leydig cell steroidogenesis by lh (papadopoulos et al. ; sullivan and cooke ) , but other functions can be expected. direct evidence for the anticipated roles of prostanoids or lipoxygenase products in testicular inflammation and immunity is lacking. initially, there was some evidence to suggest that pge might be responsible for the immunosuppressive phenotype of resident testicular macrophages . this now seems unlikely, because testicular macrophages produce little pge under normal conditions, and blocking cox activity in vivo or in vitro does not affect the production of proinflammatory cytokines in the testis in response to lps (gerdprasert et al. a; winnall et al. winnall et al. , . in the rat, chronic inhibition of cox inhibited interstitial fluid formation in normal testes, but ameliorated the loss of fluid that usually occurs during lps-induced inflammation (winnall et al. ) . the latter data would seem to indicate roles for products of cox in the control of vascular tone in the normal and inflamed testis and this deserves further exploration. when phosphocholine (pc)-containing phospholipids are used as the substrate for pla action, lysophosphatidylcholines (lpcs) are also produced (aoki et al. ; snyder ) . lpcs usually comprise a glycerophosphocholine backbone, a single long-chain fatty acid, and a free hydroxyl group (khaselev and murphy ) . the platelet-activating factors (pafs) are a subset of lpcs with an acetyl group in place of the hydroxyl group, which facilitates binding to the paf receptor (snyder ). the effective concentrations of these phospholipidderived molecules in biological fluids are normally tightly controlled by binding to albumin and lipoproteins (croset et al. ) , since some lpcs, particularly those derived from the saturated c and c fatty acids, cause cell lysis at high concentrations (weltzien ) . at physiological concentrations, however, lpcs induce specific responses in various cell types, including most immune cell types. nonacetylated lpcs are chemotactic toward macrophages, t cells, and nk cells, and stimulate cox expression by macrophages, cytokine production by t cells, and the cytotoxic activity of nk cells (légrádi et al. ; radu et al. ; whalen et al. ; yang et al. ) . in addition, lpcs induce apoptosis in t cells, indicating a role in immunosuppression as well (kabarowski et al. ; takeda et al. ) . the details of the mechanisms involved are poorly understood. while there is evidence for involvement of specific g-protein-coupled receptors (flemming et al. ; lin and ye ; radu et al. ; soga et al. ; yang et al. ) , lpcs may interact with g-proteins, membrane kinases, and ion channels directly, thereby bypassing the need for any specific receptor-ligand interactions. the interstitial fluid of the testis has long been known to possess potent lymphocyte-inhibiting activity in vitro (hedger et al. a; pöllänen et al. ) . the molecules responsible for this activity have now been isolated and identified as the specific lpcs: -palmitoyl-sn-glycero- -phosphocholine ( : a-lpc), -oleoyl-sn-glycero- -phosphocholine ( : a-lpc), a : a-lpc (putatively, -linoleoylsn-glycero- -phosphocholine), and a : a-lpc (putatively, -arachidonyl-sn-glycero- -phosphocholine) (foulds et al. ). these molecules inhibit t cell proliferation in response to activation and induce apoptosis of these cells in a time-and dose-dependent manner at physiologically relevant concentrations. although paf activity has also been found in the testis (muguruma et al. ) , these acetylated lpcs did not appear to contribute significantly to the t cell inhibitory activity. the emergence of lpcs as regulators of critical immune events in the testis opens up new avenues of inquiry into the origins of autoimmune infertility and more general mechanisms of peripheral immunoregulation. these molecules may also have other roles in testis biology that demand consideration. . . . . cell surface regulatory molecules: complement inhibitors and nonclassical mhc inhibition of complement activation is an important mechanism for reducing transplant rejection, particularly in the early phase. membrane complement inhibitors, such as cd , cd , cd , and the complement receptor-related protein (crry), are expressed in several immune-privileged tissues and have been implicated in suppression of inflammation in the eye and placenta (bora et al. ; bulla et al. ) . cd and cd appear to be confined to spermatids in the adult rat testis, crry is expressed on early spermatocytes and interstitial cells, but cd is widely expressed throughout the seminiferous epithelium and interstitium . a role for cd expression by the sertoli cells in prolonging the survival of these cells in transplantation studies has been suggested . moreover, complement regulators both in seminal plasma and on the surface of the spermatozoa (cd , cd , and cd ) may contribute to complement evasion by the spermatozoa in the female tract . classical mhc class i molecules (hla-a, hla-b, and hla-c) are expressed on nearly all cell types, and facilitate antigen presentation by these cells and activation of cytotoxic t cells (cresswell et al. ; janeway ; sant et al. ) . by contrast, the nonclassical mhc class ib molecules (hla-e, -f, and -g) are much less widely expressed, and are associated with suppression of the adaptive immune response. membrane-bound and soluble variants of these mhc class ib molecules have been implicated in the apoptosis of alloreactive cytotoxic t cells, inhibition of nk cell cytolytic activity, suppression of t cell proliferation, and deviation of type to type responses, most notably in pregnancy (bainbridge et al. ; fournel et al. ; ishitani et al. ; kapasi et al. ; kanai et al. ; riteau et al. ) . hla-g has been found in the rhesus monkey testis (ryan et al. ; slukvin et al. ) , and hla-e is expressed on germ cells in the human testis (fiszer et al. ) , indicating that these molecules may contribute to immune privilege in the testis. from the evidence outlined in the previous sections, it should be evident that proinflammatory and immunoregulatory processes are involved in both normal testicular function and testicular pathophysiology. in the normal testis, roles in the maintenance of immune privilege, the control of leydig cell development, and the fine regulation of spermatogenesis are indicated (figure ) . contributions to pathology include the development of autoimmunity within the inflammatory and immunoregulatory signaling in the control of intratesticular interactions. sertoli cells are primarily regulated by the hormones follicle-stimulating hormone (fsh) and testosterone, the latter being produced by the leydig cells on stimulation by luteinizing hormone (lh). sertoli cells and germ cells interact through production of various inflammatory mediators, including interleukin (il) , il , activin a, tumor necrosis factor (tnf), and no, to facilitate and coordinate critical events throughout the cycle of the seminiferous epithelium. the development and activity of the leydig cells and the resident macrophages involve a close mutual interaction, mediated by mechanisms that remain to be completely defined. the sertoli cell and the resident macrophages, in turn, influence the activity of the antigen-presenting cells (apc; e.g., dendritic cells) and lymphocytes within the interstitial tissue to control local immune events, such as the maintenance of an immunologically privileged environment. this communication involves the immunoregulatory cytokines transforming growth factor (tgf), activin a, and il , although other mechanisms may also be involved. male reproductive tract, disruption of steroidogenesis and spermatogenesis during systemic and local inflammatory events, and the response to vascular disturbances. . . . . normal testicular function direct evidence that immune privilege is due to a specialized immune environment originally comes from studies involving the central nervous system. injection of soluble antigen into the compartments of the eye or brain produces antigen-specific suppression of cell-mediated immunity, specifically type reactions (kaplan and streilein ; wenkel et al. ) . this phenomenon is called acquired immune deviation (aid), and it is possible to elicit similar inhibition of antigen-specific immune responses by prior injection of antigens into the testis (ditzian-kadanoff ; li et al. ; veräjänkorva et al. ) . the sertoli cell and leydig cell are almost certainly central to this process, although contributions from other testicular cells are to be expected. the mechanisms responsible presumably involve the recruitment and functional deviation of macrophages (and possibly dendritic cells) within the testis (hedger ) , the subsequent recruitment of immunoregulatory lymphocyte subsets (tompkins et al. ) , the production of immunoregulatory cytokines, such as tgf family members and il (o'bryan et al. ; suarez-pinzon et al. ) , local high concentrations of androgenic steroids, prostanoids, and other bioactive lipids (foulds et al. ; winnall et al. ) , complement inhibitors mizuno et al. ) , and testicular expression of inhibitory mhc and costimulatory molecules (dal secco et al. ; fiszer et al. ; ryan et al. ) . macrophages play an important role in leydig cell development and function. there is a close temporal link between the maturation of the adult leydig cell population and the increase in the number of testicular resident macrophages during puberty (hardy et al. ; raburn et al. ; vergouwen et al. ) . reductions in intratesticular macrophages due to specific depletion methods or in the macrophage-deficient (op/op) mouse lead to disorders of leydig cell development and mature function (cohen et al. ; gaytan et al. a,b) , and testicular macrophage-conditioned medium stimulates leydig cell steroidogenesis under certain conditions (nes et al. ; yee and hutson ) . these supportive functions of the testicular macrophages may involve production of cytokines and other secreted mediators (khan et al. b; warren et al. ) , as well as the distinctive intercytoplasmic specializations that develop between the two cell types (hutson ; miller et al. ). it has also been shown that testicular macrophages support basal steroidogenesis in the leydig cells by providing -hydroxycholesterol as a substrate for testosterone biosynthesis (nes et al. ) . endogenous regulation of inflammatory pathways within the seminiferous epithelium appears to be involved in the fine control of spermatogenesis. studies in the rat and mouse indicate that nuclear localization (i.e., activation) of the key proinflammatory transcription factor nfb, and expression of the inflammation-responsive genes, il , tnf, il , activin a, and inos, in the sertoli cell and the germ cells describe cyclical patterns within the seminiferous epithelium that coincide with critical events in the cycle of the seminiferous epithelium (o'bryan and hedger ) . most notably, release of spermatozoa from the epithelium (spermiation) in the rat testis is followed by an increase in the production of il , il , and activin a by the sertoli cells and of tnf and inos by the spermatocytes hakovirta et al. ; kaipia et al. ; söder et al. ; syed et al. ; o'bryan et al. a; okuma et al. ). in the mouse, spermiation is accompanied by a period of elevated nuclear nfb levels in spermatocytes (delfino and walker ) . these responses coincide with a peak of dna synthesis by preleptotene spermatocytes and type a spermatogonia prior to meiotic and mitotic division, and reorganization of sertoli cell tight junctions to allow the meiotic cells to enter the adluminal compartment (o'bryan and hedger ) . given that il , il , and activin a are regulators of spermatogonial proliferation and meiotic progression (hakovirta et al. a (hakovirta et al. , mather et al. ; meehan et al. ; meinhardt et al. ; parvinen et al. ; söder et al. ) , while tnf and inos/ no induce the disassembly of the intercellular tight junctions lee et al. ; li et al. a; siu et al. ) , this concurrence of events is unlikely to be a coincidence. it would appear that release of the spermatozoa and/or phagocytosis of the residual cytoplasm triggers elements of the inflammatory machinery within the seminiferous epithelium. a role for tlrs or other patternrecognition receptors in this process may be postulated, and similar regulatory networks may also operate throughout the remainder of the cycle. many questions regarding the details of this functional regulation await clarification, and it must be noted that there is a curious lack of concordance between the content of nfb in the sertoli cell nucleus observed across the cycle in the mouse (delfino and walker ) and inflammatory cytokine production by these same cells in the rat hakovirta et al. ; kaipia et al. ; o'bryan et al. a; okuma et al. ; söder et al. ; syed et al. ) . regardless of the questions that remain, however, key elements of the inflammatory response appear to play essential roles in the physiological regulation of the seminiferous epithelium, which are entirely separate from their roles in pathology. although it is a common assumption that male reproductive failure caused by local or systemic illness, infection, and chronic inflammatory disease is due to the negative effects of raised body temperature on spermatogenesis, there is little clinical or experimental evidence to support this belief (o'bryan et al. b) . there is much more convincing evidence that the damage is due to activation of local inflammatory pathways and the activity of immune cells recruited during inflammatory events. as already outlined in the previous sections, inflammatory mediators such as il , ros, tnf, and no, as well as glucocorticoids produced in response to inflammation, have mostly negative effects at all levels of the hypothalamic-pituitary-leydig cell axis (bambino and hsueh ; del punta et al. ; hales ; hales and payne ; hardy et al. ; li et al. ; mauduit et al. b mauduit et al. , monder et al. ; sharma et al. ; welch et al. ; xiong and hales b . the subsequent reduction in androgen production may lead to reduced spermatogenic capacity, but may also exacerbate the effects of inflammation on spermatogenesis itself. expression of tlrs on the sertoli cells is obviously important in protective responses against pathogens, but also provides a mechanism whereby pathogens can directly inhibit spermatogenesis. thus, tlr activation and the production of inflammatory mediators almost certainly alter sertoli cell functions, and this will affect the ability of these cells to support and regulate spermatogenesis, as well as activate signaling pathways that promote germ cell apoptosis (lysiak et al. ; pentikäinen et al. ; rasoulpour and boekelheide ; starace et al. ) . inflammatory events play a role in testicular damage due to vascular disturbance. spermatogenesis is an energy intensive process, and the seminiferous epithelium is not vascularized. consequently, unobstructed blood flow to the testis is essential and even minor anomalies in the testicular vasculature, such as varicocele, have cumulative negative effects on sperm production (jarow ) . the deleterious effects of cadmium on fertility through disruption of the testicular vasculature are well known (schrag and dixon ) . in experimental animals, transient torsion of the spermatic cord that renders the testis ischemic followed by reperfusion causes an increase in tnf and il expression, activation of the stress-related jnk/p map kinase signaling pathway, neutrophil recruitment and infiltration into the testis, increased oxidative stress, germ cell apoptosis, and significantly decreased serum testosterone levels (lysiak ; lysiak et al. ; rodriguez et al. ) . a specific role for testicular nitric oxide in mediating damage in both the testicular torsion and human varicocele models has been implicated (moon et al. ; santoro et al. ; shiraishi and naito ; shiraishi et al. ; türker köksal et al. ) . administration of high doses of human chorionic gonadotropin (hcg), a treatment used to correct delayed testicular descent in young boys, causes a hyperstimulation syndrome in rats comprising increased testicular blood flow and pressure, increased vascular permeability, and accumulation of interstitial fluid (hjertkvist et al. ; setchell and sharpe ; van vliet et al. ). these vascular changes are accompanied by accumulation of intravascular and interstitial neutrophils in the testis (bergh et al. ; widmark et al. ) , and spermatogenic damage (kerr and sharpe a,b) . the responses to hcg can be eliminated by depletion of either the leydig cells or the neutrophils (setchell and rommerts ; widmark et al. ) , and il is able to replicate most of the effects (bergh and söder ; bergh et al. ) , suggesting that the response involves an inflammatory event mediated via il secreted by the leydig cells. thus, it appears that disruption of the seminiferous epithelium in both the ischemia/reperfusion model and hcg hyperstimulation models is directly linked to local recruitment of neutrophils. exactly how these cells exert damage on the germ cells is not yet known, but increased oxidative stress is obviously involved. the responses are quite different from those observed in lps-induced inflammation, which paradoxically causes a large reduction in interstitial fluid volume and an increase in mononuclear cells in the rat testis (o'bryan et al. b; gerdprasert et al. a,b) , although the germ cell damage is similar in both models. in summary, different testicular inflammation models have direct effects on spermatogenic development, in addition to causing damage to the steroiodogenic function of the leydig cells. it should also be noted that, as in most other tissues, inflammation responses within the testis do not automatically lead to autoimmune complications, but damage to various testicular functions that may be less readily apparent may lead to longer-term consequences for reproductive health (schuppe and meinhardt ; schuppe et al. ; suominen and soderstrom ) . . . . the epididymis, vas deferens, accessory glands, and urogenital tract as already noted above, the immune environment of the remainder of the male tract is quite distinct from that of the testis, or even from that of other mucosal tissues. the mechanisms whereby sperm within the male reproductive tract are protected from the immune system remain largely unknown, but an intact tract is obviously important. in humans, vasectomy leads to sperm antibody formation in approximately % of cases, and sperm antibodies are also associated with obstructive azoospermia and congenital absence of the epididymis and/or vas (alexander and anderson ; de kretser et al. ; hellema et al. ; patrizio et al. ) . the incidence of antibody formation in humans appears to be directly related to the distance of the lesion from the testis, implying a limited role for elements of testicular immune privilege in preventing immune reactions. there is evidence from patients and animal models that vasectomy can have deleterious effects on the testis and epididymis as well, and that at least some of these effects may have an immunological basis (aitken et al. ; bigazzi et al. ; flickinger et al. a; raleigh et al. ) . in certain strains of rats, mice, and rabbits, vasectomy rapidly leads to orchitis and sterility, so it is not clear why this degree of damage is so rare in humans. in reality, the immunology of the male reproductive tract has yet to be investigated in depth, but two areas of recent interest are worth highlighting: the tlrs and the defensins. given the exposure to the external environment, it is not surprising that the tlrs as well as many essential tlr-related genes, such as myd , are expressed throughout the male reproductive tract, including the epididymis, vas deferens, and accessory glands (nishimura and naito ; palladino et al. ; quintar et al. ; rodrigues et al. ) . expression is found on both epithelial cells and leukocytes in these tissues. moreover, weak expression of tlr , originally localized to the urinary tract of experimental rodents, has been detected in the epididymis and vas deferens of the rat (lauw et al. ; palladino et al. ; zhang et al. ) . the importance of these molecules in acute and chronic inflammation, and the response to infection, in the male reproductive tract is an important area for future studies. moreover, changes in susceptibility to inflammation through polymorphisms in the tlrs have been associated with differences in susceptibility to the onset of cancer in the prostate (sun et al. ; zheng et al. ) . defensins are small ( - kda) positively charged peptides that are able to disrupt bacteria, fungi, parasites, and some enveloped viruses by forming multimeric pores in the pathogen membrane (selsted and ouellette ) . the -defensins are produced by most mucosal epithelial tissues, including the testis and epididymis (com et al. ) , and their production is stimulated via tlr activation and cytokines. a number of epididymal-specificdefensins have been identified in the mouse and the rat (li et al. ; jalkanen et al. ; yamaguchi et al. ; yenugu et al. ; zhou et al. ) , and a role for androgen in their regulation has been indicated (jalkanen et al. ; yenugu et al. ) . apart from their obvious role in protection against pathogens, defensins may have other functions in the reproductive tract. the novel -defensin, bin b, which is exclusively produced and secreted by the rat caput epididymis, binds to sperm (li et al. ; zhou et al. ) , and blocking bin b reduces sperm motility in vivo, suggesting a novel role for this molecule in sperm maturation (zhou et al. ) . in humans, semen is the most accessible window into the health of the male reproductive system, as it is the number and quality of sperm in the semen that provide the principal foci for male reproductive toxicity outcomes. however, semen is a complex secretion, comprising many components, including leukocytes, immunoglobulins, cytokines, prostaglandins, and various immunosuppressives. changes in the number or activity of these immunological components of the semen are also potential indicators of toxic actions within the tract. elevated numbers of leukocytes in the semen are generally considered to be an indication of infection, but leukocytes are present even in the semen of men with normal fertility barratt et al. ) . the origin of these cells is somewhat obscure. evidence suggests that the epididymis or vas may be a major source (anderson et al. ; schwartz ), but the main leukocyte subset present in most semen samples are neutrophils, which are not a normal feature of the tissues of the genital tract el-demiry et al. ; wolff and anderson ) . the impact of these cells on fertility is also poorly understood. some studies have shown a relationship between leukocytospermia and impaired sperm function (auroux ; aziz et al. ; berger et al. ; wolff et al. ) , and some data suggest that these cells might be a cause of sperm damage due to production of ros and cytokines (hill et al. ; tomlinson et al. a ). moreover, leukocytes may attack the sperm directly, particularly in the presence of sperm autoantibodies, and may provoke or mediate hostile responses in the female tract. other studies, however, have failed to confirm a clear link between seminal leukocytes and infertility or sperm antibody formation barratt et al. ; tomlinson et al. ) , and it has even been suggested that leukocytes might play a beneficial role by removing abnormal sperm or by assisting in sperm capacitation (tomlinson et al. b (tomlinson et al. , . molecules with immunomodulatory properties in seminal plasma include immunoglobulins, cytokines, and immunosuppressives. both igg and iga are the major constituents of seminal plasma, and there appears to be a negative correlation between iga concentrations and lymphosuppressive activity in seminal plasma (eggert-kruse et al. ; imade et al. ; moldoveanu et al. ) . these antibodies may play either protective roles in the control of immunity and infection, or destructive roles, when directed against functional elements of the sperm. proinflammatory cytokines such as il , il , il , il , il , il , tnf, and ifn are found in all seminal plasma samples, even those from men with apparently normal fertility (eggert-kruse et al. ; huleihel et al. ; koumantakis et al. ; maegawa et al. ; naz and evans ; politch et al. ). this is not particularly surprising, as these cytokines are present in most biological fluids and are modulated by the general health status of the individual. soluble il and il receptors, molecules that modulate the activity of their respective cytokines, have also been found in seminal plasma (huleihel et al. ) . numerous studies have shown that proinflammatory cytokines are dramatically elevated in patients with inflammation and/or leukocytospermia (eggert-kruse et al. ; krause et al. ; maegawa et al. ; miller et al. ; rajasekaran et al. ) . however, specific positive and negative associations with other forms of infertility have also been reported (eggert-kruse et al. ; gruschwitz et al. ; huleihel et al. ; loras et al. ; naz and evans ) . seminal plasma has profoundly immunosuppressive properties, as defined by the ability to inhibit various t cell and nk cell activities in vitro. this inhibitory activity has been attributed to a number of seminal factors, including prostasomes (kelly et al. ) , oxidized polyamines (allen and roberts ) , prostaglandins of the e series (skibinski et al. ) , nonspecific lymphocyte-suppressing proteins (maccioni et al. ; veselský et al. ) , and immunoregulatory cytokines (anderson et al. ; huleihel et al. ; loras et al. ; miller et al. ; nocera and chu ; rajasekaran et al. ; srivastava et al. ) . the main cytokines with immunosuppressive activity in human seminal plasma are tgf , tgf , il , and activin a. immunosuppressives are believed to play a role in preventing lymphocyte responses against sperm autoantigens in the male and female reproductive tracts ochsenkühn et al. ). in the current state of understanding of the interactions between the male reproductive tract and the immune system, many questions still remain. nonetheless, it is clear that the mammalian testis in particular displays a special, if not unique, relationship with the immune system, involving distinctive cell-cell interactions and shared cytokines. recently, microarray analysis of testicular gene expression signatures in human spermatogenic failure were found to correspond with gene sets usually associated with inflammation and autoimmune disease (spiess et al. ). this relationship between reproduction and immunology impacts upon normal function, since perturbations in one system caused by toxicants will almost certainly impinge upon the other system. this extends into the characteristic responses of the testis to toxins and various pathologies (figure ) . the complexity and potential consequences of these interactions may be appreciated by consideration of the following propositions: . if leukocytes play a role in maintaining normal testicular function, then drugs and toxicants that reduce macrophage and lymphocyte function or numbers may also have an indirect effect on testicular function. the effect of dichloromethylene diphosphonate on macrophages in the rat provides a good experimental example of this possibility, by demonstrating that the loss of functioning macrophages leads to a decline in androgen levels and retarded leydig cell development gaytan et al. a ). inhibition of macrophage and lymphocyte functions or numbers may also compromise the immune-privileged status of the testis, potentially leading to testicular autoimmune disease. a related subset would involve chemicals that have direct action on both the immune system and testicular function, such as cyclosporin and cyclophosphamide (cavallini et al. ; meistrich ; seethalakshmi et al. ; wetzels ) , which are commonly used to suppress graft rejection response and in the treatment of some autoimmune diseases. fere with normal testicular function. the serious consequences for testicular function associated with immune activation and inflammation are illustrated by the experimental model of lps administration, which leads to leydig cell dysfunction and testicular failure (christeff et al. ; wallgren et al. ; o'bryan et al. b) . any drug or toxicant that induces macrophage activation (e.g., bacterial toxins, carbon monoxide, sulfur dioxide) has the potential to induce testicular production of inflammatory cytokines, such as il , tnf, and ifn, as well as ros, which may interfere with leydig cell function and androgen deficiency figure model of toxicant actions and potential immunological consequences in the testis. toxicants may damage or alter the function of somatic cells (in particular the sertoli and leydig cells), immune cells, or germ cells, thereby inhibiting androgen production and spermatogenesis, leading to increased germ cell death. this damage may lead to an overt inflammatory event (orchitis) through activation of the proinflammatory activities of local macrophages, which may exacerbate or prolong the testicular dysfunction. these events may be followed by a full or partial recovery of normal function, but intratesticular immune cell numbers and activity may become permanently altered, with possible implications for future toxic or inflammatory episodes. in certain circumstances, which may depend upon the severity of the initial damage caused or the genetic background of the patient, the antigen-presenting activity of intratesticular dendritic cells or macrophages may also be activated. this may lead to recruitment and activation of testicular antigen-specific t cells, development of cytotoxic t cells (ctl), and b cell antibody (ig) production. this subsequent autoimmunity may have a more permanent effect on testis or sperm function (epithelial destruction or sperm antibody formation). spermatogenic development, or local immunoregulation. the danger hypothesis model of autoimmune disease may be particularly relevant in this instance (matzinger ) . at least one of the putative 'danger' molecules (i.e., tnf) is endogenously produced in the testis, potentially presenting a continuous challenge to the testicular immune system. . drugs or toxicants that induce autoimmune disease or allergy may also induce testicular autoimmune disease. there are many drugs that induce polysystemic autoimmune disease and this may also include reactions against the germ cells. an example here is polyarteritis nodosa, a necrotizing vasculitis of small arteries, which frequently involves the testis (fauci et al. ) . sulfonamides, penicillin, phenytoin, arsenicals, thiouracil, iodides, and thiazides are frequently suspected as causes of this condition (nusinow et al. ) . likewise, mutagens and other toxins that may alter t and b cell receptor repertoires to create self-reactive lymphocytes could also contribute to the onset of autoimmune disease in the male reproductive tract. . drugs or toxicants that affect testicular cell development or viability may also induce immune responses. there are drugs or toxicants that affect sertoli-sertoli and sertoli-germ cell junction stability, as well as agents that damage the sertoli cell or germ cells directly, resulting in massive germ cell death, which may overwhelm the normal immunoregulatory capacity of the testis. examples of this interaction are seen after testicular heating, serotonin administration, or treatment with ethane dimethane sulfonate (eds) or other testicular toxicants, which are frequently followed by transient immune and/or inflammatory events (creasy et al. ; hedger et al. ; padmanabhan and singh ; wang et al. ) . even if the epithelium recovers fully, there is the potential for sperm antibody development, leading to reduced fertility. increased exposure to estrogenic compounds is associated with activation of immune cells in the testis, most notably the macrophages, providing evidence for a mechanism whereby environmental estrogens may affect male fertility indirectly via effects on local immunity (li et al. b ). . testicular toxicants that alter steroidogenic cell function will have a direct effect on the immune system. toxicants that damage leydig cells and reduce steroid production possibly could alter thymus function, leading to an enhanced susceptibility to autoimmune disease. balanced against this is the likelihood that other androgen-deficiency symptoms are far more likely to be manifest and treated. all these interactions, however, illustrate to one degree or another the difficulty of separating direct effects of toxic agents on the immune cells and on the testis. there is a dynamic relationship between the male reproductive tract and the immune system that should be considered when assessing the effects of various toxicants on male fertility and reproductive development. the effects of toxicants on male reproductive functions, most notably the production of androgenic steroids, will in turn impinge upon the functions of the immune system. many mechanisms underlying these interactions have begun to emerge in recent years, but a complete picture remains hidden. further studies will no doubt lead to important new concepts concerning this relationship and its implications for reproductive toxicology. proc. natl. acad. sci cellular transplantation from laboratory to clinic; halberstadt in knobil and neill's physiology of reproduction proc. natl. acad. sci proc. natl. acad. sci tight junctions sainio-pö llä nen, s.; sö der aderem, a. proc. natl. acad. sci fundamental immunology pö llä nen inflammation: basic principles and clinical correlates key: cord- -wzndpcq authors: albagi, sahar obi abd; al-nour, mosab yahya; elhag, mustafa; abdelihalim, asaad tageldein idris; haroun, esraa musa; essa, mohammed elmujtba adam; abubaker, mustafa; hassan, mohammed a. title: a multiple peptides vaccine against ncovid- designed from the nucleocapsid phosphoprotein (n) and spike glycoprotein (s) via the immunoinformatics approach date: - - journal: biorxiv doi: . / . . . sha: doc_id: cord_uid: wzndpcq due to the current covid- pandemic, the rapid discovery of a safe and effective vaccine is an essential issue, consequently, this study aims to predict potential covid- peptide-based vaccine utilizing the nucleocapsid phosphoprotein (n) and spike glycoprotein (s) via the immunoinformatics approach. to achieve this goal, several immune epitope database (iedb) tools, molecular docking, and safety prediction servers were used. according to the results, the spike peptide peptides sqcvnlttrtqlppaytnsftrgvy is predicted to have the highest binding affinity to the b-cells. the spike peptide ftisvttei has the highest binding affinity to the mhc i hla-b allele. the nucleocapsid peptides ktfpptepk and rwyfyylgtgpeagl have the highest binding affinity to the mhc i hla-a allele and the three mhc ii alleles hla-dpa * : /dpb * : , hla-dqa * : /dqb - * : , hla-drb , respectively. furthermore, those peptides were predicted as non-toxic and non-allergen. therefore, the combination of those peptides is predicted to stimulate better immunological responses with respectable safety. human coronaviruses (hcovs) including the severe acute respiratory syndrome coronavirus (sars-cov- , covid- ) are enveloped, positive-sense, single-stranded polyadenylated rna viruses belong to the coronaviridae family. they cause systemic and respiratory zoonotic diseases [ ] . the sars-cov- is a novel strain detected firstly in the city of wuhan, the republic of china in december [ ] . it causes fever, cough, dyspnea, bilateral infiltrates on chest imaging and may progress to pneumonia [ ] . the covid- is characterized by rapid spreading; "as the feb, it is reported in countries, causing over , infections with , deaths" [ ] and till the th may, more than . million positive cases and . million deaths have been identified globally [ ] . unfortunately, until now covid- has no effective antiviral drug for the treatment or vaccine for the prevention, hence extensive researches should be conducted on the development of safe and effective vaccines and antiviral drugs [ ] . to develop a safe and effective covid- vaccine rapidly, the who recommended that "we must test all candidate vaccines until they fail to ensure that all of them have the chance of being tested at the initial stage of development". ensuing this point, recently, there are over proposed vaccines. six of them in the clinical evaluation and in pre-clinical evaluation [ ] . the vaccine development is achieved by multiple approaches including the inactivated, live-attenuated, non-replicating viral vector, dna, rna, recombinant proteins, and peptide-based vaccines. "as of april , the global covid- vaccine r&d landscape confirmed active candidate vaccine" [ ] . consistent with global efforts, this study aims to predict potential covid- peptide-based vaccine utilizing the nucleocapsid phosphoprotein (n) and spike glycoprotein (s) via the immunoinformatics approach. due to the respectable antigenicity of the nucleocapsid and spike glycoprotein, they are appropriate targets for vaccine design [ ] . the peptide vaccines are sufficient to stimulate cellular and humoral immunity without allergic responses [ ] . they are" safe, simply produced, stable, reproducible, cost-effective" [ ] , and permits a broad spectrum of immunity [ ] , consequently, they are the targets for this study as well as they utilized in multiple studies concerning covid- vaccine [ ] [ ] [ ] [ ] . a total of covid- nucleocapsid phosphoprotein (n) and spike glycoprotein (s) were retrieved from the national center for biotechnology information (ncbi) database [ ] as fasta format in march . the sequences with their accession number are listed in the supplementary file s . the protein sequences of mhc i alleles hla-a* : , hla-b : , hla-c* : , and mhc ii alleles hla-dpa * : /dpb * : , hla-dqa * : /dqb * : , hla-drb were obtained from the immuno polymorphism database (ipd-imgt/hla) [ ] . the retrieved covid- nucleocapsid phosphoprotein (n) and spike glycoprotein (s) sequences were aligned using the clustalw algorithm [ ] on the bioedit software version . . [ ] to identify the conserved regions between sequences. the b-cells peptides were predicted from the conserved regions using the linear epitope prediction tool "bepipred-test" on the immune epitope database (iedb) [ ] at the default threshold value - . . to predict the epitopes accurately, a combination between the hidden (parker and levitt) method and the markov model (hmm) [ ] was used. the surface accessibility of b-cells peptides was predicted via the emini surface accessibility tool [ ] on the iedb [ ] at the default threshold holding value. to identify the antigenic sites within the nucleocapsid phosphoprotein and spike glycoprotein, the kolaskar and tongaonker method's on the iedb [ ] at the default threshold value. to predict the interaction with different mhc i alleles, the major histocompatibility complex class i (mhc i) binding prediction tool on the iedb [ ] was used. all peptide length was set as amino acid. to predict the binding affinity, the artificial neural network (ann) prediction method was selected with a half-maximal inhibitory concentration (ic ) value of less than . in contrast, to predict the interaction with different mhc ii alleles, the major histocompatibility complex class ii (mhc ii) binding prediction was used. to predict the binding affinity, the nn align algorithm was selected with an ic value of less than . the human allele reference sets (hla dr, dp, and dq) were included in the prediction. to predict the percentage of peptides binding with various mhc i and mhc ii alleles that cover the world population, the population coverage tool on the iedb [ ] was used. to predict the peptides' allergicity, the allergenfp v. . [ ] and allercatpro v. . [ ] servers were used. in contrast, to predict the peptides' toxicity, the toxinpred server [ ] was used. to model the d structure of the nucleocapsid, spike, and mhc molecules, the swiss-model server [ ] , and the phyre web portal [ ] were used. to visualize the modeled structures, the uscf chimera . software [ ] was used. the predicted peptides were docked with mhc i alleles hla-a* : , hla-b : , hla-c* : , and mhc ii alleles hla-dpa * : /dpb * : , hla-dqa * : /dqb * : , hla-drb . the modeled mhc structures were prepared for the docking via cresset flare software [ ] at the normal type calculation method. to dock the predicted peptides, the mdockpep [ ] and hpepdock [ ] [ ] [ ] [ ] [ ] servers were used. the predicted peptides were submitted as amino acid sequences. the d and d interactions were visualized using the poseview [ ] at the proteinplus web portal [ ] and cresset flare viewer [ ] , respectively. according to the iedb [ ] prediction, the average binding score for the nucleocapsid phosphoprotein and spike glycoprotein were . , . , respectively. all values equal to or greater than the default threshold were predicted as potential b-cell binders. regarding the cytotoxic t-lymphocyte peptides, the mhc i binding prediction tool predicts peptides from the nucleocapsid and peptides from the spike glycoprotein could interact with the different mhc i alleles. the most promising peptides were listed in table . in contrast, the mhc ii binding prediction tool predicts peptides from the nucleocapsid and peptides from the spike glycoprotein could interact with the different mhc ii alleles. the most promising peptides were listed in table according to the mdockpep [ ] and hpepdock [ ] servers prediction, the spike peptide (ftisvttei) has the highest binding affinity to the mhc i hla-b allele and the spike peptide (miaqytsal) has the highest binding affinity to the mhc i hla-c allele. the nucleocapsid peptide (ktfpptepk) has the highest binding affinity to the mhc i hla-a allele ( table ) according to the allergenfp v. . [ ] , allercatpro v. . [ ] , and toxinpred servers [ ] , all the predicted peptides except the spike peptide (evfnatrfasvyawn) were nonallergen and non-toxin (tables and ) . the hydrophobicity scores of the spike glycoprotein peptides were not available. due to the current covid- pandemic, the rapid discovery of a safe and effective vaccine is an essential issue [ ] . since the successful vaccine relies on the selection of the most antigenic parts and the best approaches [ ] , covid- nucleocapsid phosphoprotein (n) and spike glycoprotein (s) were selected to design a peptide vaccine. the antigenicity of the nucleocapsid and spike is well predicted [ ] , and the advantages of the peptide vaccines are well established [ , ] . the peptide design via the immunoinformatics approach is achieved through multiple steps including the prediction of; b-cells and t-cell peptides, the surface accessibility, antigenic sites, and the population coverage. after the selection of the candidate peptides, their interaction with the mhc molecules is simulated and their safety is predicted [ ] . regarding the b-cells peptides prediction, the successful candidates must pass the threshold scores in the bepipred, parker hydrophilicity, kolaskar and tongaonkar antigenicity, as well as emini surface accessibility tests [ ] . the iedb bepipred test [ ] on the nucleocapsid showed that eleven peptides were predicted, however, the peptide dayktfpptepkkdk-kkkadetqalpqrqkkqqtvtllpaadldd was the only one that passed all the tests. in contrast, the iedb bepipred test [ ] on the spike showed that forty-two peptides were predicted, but the peptides sqcvnlttrtqlppaytnsftrgvy and lgky the only two that passed all the tests. as the length of effective b-cell peptides varies from - amino acids [ ] , the peptide lgky is too short and the peptide dayktfpptepkkdk-kkkadetqalpqrqkkqqtvtllpaadldd is too long. consequently, the spike peptide peptides sqcvnlttrtqlppaytnsftrgvy is predicted to have the highest binding affinity to the b-cells (table ) . concerning the t-cells peptides prediction, the test measures the peptides' binding affinity to the mhc molecules [ ] . the available mhc i alleles hla a, hla b, hla c, hla e, and mhc ii alleles hla-dr, hla-dq, and hla-dp were used. the mhc i iedb tests [ ] the results of collective the iedb tests [ ] revealed that the spike glycoprotein peptides ftisvttei, miaqytsal, and the nucleocapsid peptide ktfpptepk are the most promised mhc peptides. on the other hand, the spike peptides evfnatrfasvyawn, vfrssvlhstqdlfl, and the nucleocapsid peptides aalalllldrlnqle, alall-lldrlnqles, prwyfyylgtgpeag, rwyfyylgtgpeagl are the most promised mhc ii peptides. to stimulate better immunological responses by the predicted peptides, they must interact and bind effectively with the mhc and mhc ii molecules [ ] , therefore we must study their interaction with the mhc molecules. the simulation and prediction of the interaction between the predicted peptides and the mhc molecules are conducted using molecular docking studies that rely on the calculation of the binding free energy. the lowest binding energy scores of the mhc-peptide complex will indicate the best interaction and the highest stability [ ] . to validate the results of molecular docking, mdockpep [ ] and hpepdock [ ] [ ] [ ] [ ] [ ] servers were used. the mdockpep server predicts the mhc-peptides interaction by "docking the peptides onto the whole surface of protein independently and flexibly using a novel the conformation restriction in its novel iterative approach. it ranks the docked peptides via the itscorepep scoring function that uses the known protein-peptide complex structures in the calculations" [ ] . in contrast, hpepdock uses "a hierarchical flexible peptide docking approach" to predict the mhc-peptides interaction [ ] . the mhc i, hla-a , hla-b , hla-c was predicted to present the highly conserved sars-cov- peptides more effectively [ ] , hence, they were used in molecular docking study. the molecular docking results showed that the spike peptide ftisvttei has the lowest docking energy score with the mhc i hla-b allele, hence it is predicted to have the highest binding affinity. the spike peptide miaqytsal showed the lowest docking energy score with the mhc i hla-c , consequently, it is predicted to have the highest binding affinity to the mhc i hla-c allele. in contrast, regarding the mhc i hla-a ; the results of the mdockpep [ ] server showed that the nucleocapsid peptide ktfpptepk has the lowest docking energy score, but the results of hpepdock [ ] server showed the spike peptide miaqytsal has the lowest docking energy score ( table ). to illustrate the mhc-peptide interaction, the poseview [ ] at the proteinplus web portal [ ] that illustrate the d interactions and cresset flare viewer [ ] that illustrate the d interaction were used. the spike peptide ftisvttei interacts with the mhc i hla-b allele by forming hydrogen bonds with the amino acids thr , ser , ser a, asn a, tyr a, thr a, lys a, trp a, glu a and hydrophobic bonds with the amino acids thr , thr , trp a, ser a, trp a, ala a, leu a. the spike peptide miaqytsal interacts with the mhc i hla-c allele by forming hydrogen bonds with the amino acids tyr a, arg a, lys a, gln a, thr a and hydrophobic bonds with the amino acids ile , gln , gln a, ala , arg a, tyr a, trp a. in comparison, it interacts with the mhc i hla-a allele by forming hydrogen bonds with the amino acids thr , glu a, arg a, trp a, and hydrophobic bonds with the amino acids ile , trp a, tyr a, trp a. it forms six hydrogen bonds and seven hydrophobic bonds with the mhc i hla-c allele that is more than its bonds with the mhc i hla-a allele. this finding indicates the higher binding affinity of spike peptide miaqytsal to the mhc i hla-c allele and supports the mdockpep [ ] server score (table and figures , ). the nucleocapsid peptide ktfpptepk interacts with the mhc i hla-a allele by forming hydrogen bonds with the amino acids pro , glu a, lys a, asp a, trp a and hydrophobic bonds with the amino acids pro , pro , leu a, thr a, trp a, ala a, val a (figures and ). among the reported mhc i alleles, the hla-b allele was predicted to have "the greatest ability to present the highly conserved sars-cov- peptides" [ ] , therefore, the spike peptide ftisvttei is predicted to make the highest response, since the binding with the mhc i stimulates the natural killer and the cytotoxic t cells [ ] . regarding the interaction with the mhc ii molecule, the spike peptide evfnatrfasvyawn showed the lowest docking energy score with the three mhc ii alleles hla-dpa * : /dpb * : , hla-dqa * : /dqb * : , and hla-drb , hence it is predicted to have the highest binding affinity to the three alleles. hence, it predicted to stimulate the cd + (helper) t cells more effectively, since the mhc ii molecule presents the antigenic peptides to the cd + (helper) t cells [ ] . on the contrary, the nucleocapsid peptide (rwyfyylgtgpeagl) showed lower docking energy scores with the mhc ii allele hla-dqa * : /dqb * : . the results of the mdockpep [ ] server showed that the peptide (prwyfyylgtgpeag) has the lowest docking energy score, however, the results of hpepdock [ ] server differed from it in the mhc ii alleles hla-dpa * : /dpb * : and hla-drb (table ) . spike peptide (evfnatrfasvyawn) interacts with hla-dpa * : /dpb * : allele by forming hydrogen bonds with the amino acids val , ser , tyr , tyr a, glu a, hydrophobic bonds with the amino acids val , phe , tyr a, ala a, ala a, asn a, leu a, leu a, trp , tyr a, and pi-pi bonds with the aromatic amino acid phe , ser , tyr , trp , tyr a. it interacts with the hla-drb allele by forming hydrogen bonds with the amino acids val , thr , ser , phe b, tyr b, tyr b, gly b, hydrophobic bonds with the amino acids val , asn , thr , phe , tyr , trp , glu b, cys b, phe b, asn b, tyr b, pro b, trp b, and pi-pi bonds with the aromatic amino acid phe , tyr , trp . its interaction with the hla-dqa * : /dqb * : allele was not obtained from the server. the nucleocapsid peptide (rwyfyylgtgpeagl) interacts with hla-dpa * : /dpb * : allele by forming hydrogen bonds with the amino acids glu , leu a, hydrophobic bonds with the amino acids trp , phe , tyr , gly , glu , gln a, leu a, leu a, leu a, phe a, pro a, pro a, tyr a, and pi-pi bonds with the aromatic amino acid trp , phe , tyr , phe a, tyr a. it interacts with the hla-dqa * : /dqb * : allele by forming hydrogen bonds with the amino acids glu , asn a, asn a, leu a, thr a, asn a, hydrophobic bonds with the amino acids trp , phe , tyr , gly , glu , gly , gln a, phe a, ala a, tyr a, thr a, ala a, ala a, and pi-pi bonds with the aromatic amino acid trp , phe , tyr , tyr a. it interacts with the hla-drb allele by forming hydrogen bonds with the amino acids glu , val b, cys b, tyr b, tyr b, asn b, gln b, hydrophobic bonds with the amino acids trp , phe , tyr , gly , glu , lys b, his b, tyr b, asn b, tyr b, trp b, and pi-pi bonds with the aromatic amino acid trp , phe , tyr . the nucleocapsid peptide (rwyfyylgtgpeagl) interacts most effectively with the hla-dqa * : /dqb * : allele, hence it showed the highest binding affinity to it (figures and ) . concerning the two nucleocapsid peptides (rwyfyylgtgpeagl) and (prwyfyylgtgpeag) in the interaction with the mhc ii alleles hla-dpa * : /dpb * : and hla-drb , the d and d interaction results showed that the peptide (prwyfyylgtgpeag) more effectively with the hla-dpa * : /dpb * : allele and the peptide (rwyfyylgtgpeagl) more effectively with the hla-drb allele, however, they differ only in the first and last amino acid (figures and ). in comparison between the spike and nucleocapsid peptides, the spike peptide (ftisvttei) showed a higher binding affinity to the mhc i hla-b allele. the nucleocapsid peptides (ktfpptepk) and (rwyfyylgtgpeagl) showed a higher binding affinity to the mhc i hla-a allele and the three mhc ii alleles hla-dpa * : /dpb * : , hla-dqa * : /dqb * : , hla-drb , respectively, however, the total population coverage of the peptides ftisvttei and ktfpptepk is not high (table ) . joshi a, et al. in their predictive covid- peptide-based vaccine study found that the orf- a protein's peptide (itlcftlkr) binds most effectively with the mhc i hla alleles hla-a* : , hla-a* : [ ] . enayatkhani m, et al. in their predictive study included nucleocapsid n, but they studied its interaction with the mhc i hla-a* : allele [ ] . kalita p, et al. included the nucleocapsid protein and spike glycoprotein. they used the predicted peptides from nucleocapsid, spike, and membrane glycoprotein to design a subunit vaccine [ ] . singh a, et al. used the nucleocapsid protein to design multi-peptides vaccine [ ] . since we didn't include the two alleles hla-a* : , hla-a* : in our study and didn't design subunit or multi-peptides vaccine, the logical comparison will not be applied. besides the binding with the mhc molecules, the predicted peptide must be non-toxic and non-allergen, hence, their safety was predicted using the allergenfp v. . [ ] , allercatpro [ ] v. . , toxinpred [ ] servers. the result showed that all the peptides were non-toxic. the allercatpro v. . [ ] server results showed there is no evidence about the allergicity of all peptides, however, the allergenfp v. . [ ] server predicts spike peptide (evfnatrfasvyawn) as an allergen (tables and ). a potential covid- peptide-based vaccine was predicted from the nucleocapsid phosphoprotein (n) and spike glycoprotein (s) via the immunoinformatics approach. the spike peptide peptides sqcvnlttrtqlppaytnsftrgvy is predicted to have the highest binding affinity to the b-cells. the spike peptide ftisvttei has the highest binding affinity to the mhc i hla-b allele. the nucleocapsid peptides ktfpptepk and rwyfyylgtgpeagl have the highest binding affinity to the mhc i hla-a allele and the three mhc ii alleles hla-dpa * : /dpb * : , hla-dqa * : /dqb -* : , hla-drb , respectively. furthermore, those peptides were predicted as non-toxic and non-allergen. therefore, the combination of those peptides is predicted to stimulate better immunological responses. since the study is an in silico predictive work, further experimental studies are recommended to validate the obtained results. a comparative sequence analysis to revise the current taxonomy of the family coronaviridae novel coronavirus ( -ncov) : situation report, coronavirus disease (covid- ): epidemiology, virology, clinical features, diagnosis, and prevention sars-cov- : an emerging coronavirus that causes a global threat covid- situation update worldwide who solidarity trial -accelerating a safe and effective covid- vaccine the covid- vaccine development landscape genomic characterization of the novel human-pathogenic coronavirus isolated from a patient with atypical pneumonia after visiting wuhan peptide vaccine: progress and challenges peptide-based vaccine successfully induces protective immunity against canine visceral leishmaniasis reverse vaccinology approach to design a novel multi-epitope vaccine candidate against covid- : an in silico study design of a peptide-based subunit vaccine against novel coronavirus sars-cov- epitope based vaccine prediction for sars-cov- by deploying immuno-informatics approach designing a multi-epitope peptide-based vaccine against sars-cov- database resources of the national center for biotechnology information ipd-imgt/hla database clustal w: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties, and weight matrix choice bioedit: a user-friendly biological sequence alignment editor and analysis program for windows / /nt the immune epitope database (iedb) . improved method for predicting linear bcell epitopes induction of hepatitis a virusneutralizing antibody by a virus-specific synthetic peptide allergenfp: allergenicity prediction y descriptor fingerprints allercatpro-prediction of protein allergenicity potential from the protein sequence in silico approach for predicting toxicity of peptides and proteins swiss-model: an automated protein homology-modeling server the phyre web portal for protein modeling, prediction, and analysis ucsf chimera--a visualization system for exploratory research and analysis a. molecular field extrema as descriptors of biological activity: definition and validation mdockpep: an ab-initio protein-peptide docking server hpepdock: a web server for blind peptide-protein docking based on a hierarchical algorithm hhblits: lightning-fast iterative protein sequence searching by hmm-hmm alignment improved tools for biological sequence comparison comparative protein structure modeling of genes and genomes the protein data bank poseview--molecular interaction patterns at galance proteinsplus: a web portal for structure analysis of macromolecules developing covid- vaccines at pandemic speed vaccines: from empirical development to rational design immunoinformatics approach for epitope-based peptide vaccine design and active site prediction against polyprotein of emerging oropouche virus antibody epitope prediction -tutorial present yourself! by mhc class i and mhc class ii molecules relative binding free energy calculations in drug discovery: recent advances and practical considerations human leukocyte antigen susceptibility map for sars-cov- ," medrxiv major histocompatibility complex (mhc) class i and mhc class ii proteins: conformational plasticity in antigen presentation function and regulation of mhc class ii molecules in t-lymphocytes: of mice and men the authors declare that have no conflict of interest. key: cord- -fp d if authors: malone, karen e.; stohlman, stephen a.; ramakrishna, chandran; macklin, wendy; bergmann, cornelia c. title: induction of class i antigen processing components in oligodendroglia and microglia during viral encephalomyelitis date: - - journal: glia doi: . /glia. sha: doc_id: cord_uid: fp d if glia exhibit differential susceptibility to cd t cell mediated effector mechanisms during neurotropic coronavirus infection. in contrast to microglia, oligodendroglia are resistant to cd t cell perforin‐mediated viral control in the absence of ifnγ. kinetic induction of mhc class i expression by microglia and oligodendroglia in vivo was thus analyzed to assess responses to distinct inflammatory signals. flow cytometry demonstrated delayed class i surface expression by oligodendroglia compared with microglia. distinct kinetics of class i protein upregulation correlated with cell type specific transcription patterns of genes encoding class i heavy chains and antigen processing components. microglia isolated from naïve mice expressed high levels of these mrnas, whereas they were near detection limits in oligodendroglia; nevertheless, class i protein was undetectable on both cell types. infection induced modest mrna increases in microglia, but dramatic transcriptional upregulation in oligodendroglia coincident with ifnα or ifnγ mrna increases in infected tissue. ultimately mrnas reached similar levels in both cell types at their respective time points of maximal class i expression. expression of class i on microglia, but not oligodendroglia, in infected ifnγ deficient mice supported distinct ifn requirements for class i presentation. these data suggest an innate immune preparedness of microglia to present antigen and engage cd t cells early following infection. the delayed, yet robust, ifnγ dependent capacity of oligodendroglia to express class i suggests strict control of immune interactions to avoid cd t cell recognition and potential presentation of autoantigen to preserve myelin maintenance. © wiley‐liss, inc. in the quiescent central nervous system (cns), glia express little, if any, detectable major histocompatibility complex (mhc) molecules on their surface until exposed to inflammatory stimuli (dorries, ; hickey, ; wong et al., ) . mhc class i antigen presentation is critical for cd t cell mediated control of viral infections, as secretion of anti-viral cytokines and perforinmediated cytolysis require t cell receptor: mhc molecule interactions (slifka et al., ) . t cell effector func-tions are also associated with pathologies and neurological abnormalities (dorries, ; huseby et al., ; ip et al., ) . consequently, t cell interactions with cns glial cells require stringent regulation to balance the positive aspects of antiviral activity with the potential for detrimental immunopathological damage. class i surface expression is regulated at multiple levels including proteolysis, peptide transport, and chaperone-aided complex assembly. the majority of peptides presented by class i molecules are derived from proteasomal degradation of ubiquitin-tagged proteins in the cytoplasm. proteasomal specificities are conferred by three b subunits localized in the catalytic s core complex (strehl et al., ) . the constitutive proteolytic subunits involved in homeostatic protein turnover can be replaced by interferon (ifn) inducible proteosomal subunits type , and (psmb , psmb , psmb ; also known as lmp , lmp , and mecl- , respectively) to form the immunoproteasome. immunoproteosomes release peptides with increased class i binding affinities (strehl et al., ) . peptides are selectively shuttled from the cytoplasm into the endoplasmic reticulum (er) by the heterodimeric transporter associated with antigen processing (tap), formed by the tap and tap subunits (garbi et al., ) . in the er, chaperones aid in assembly of class i heavy chains, peptides and b -microglobulin (b m) to form a stable complex capable of trafficking to the cell surface (garbi et al., ) . the major antigen processing components, immunoproteosomes and tap are encoded in the mhc class i locus and are inducible by both ifna/b and ifng (jamaluddin et al., ; paulsson, ) . consistent with sparse class i expression, the cns expresses minimal antigen processing associated pro-teins compared with peripheral organs and lymphoid tissues (fruh et al., ; stohwasser et al., ) . however, inflammatory stimuli rapidly induce class i expression on microglia in vitro and in vivo, coincident with upregulation of antigen processing components (bailey et al., ; stohwasser et al., ) . similar to microglia, ifng stimulates class i expression on oligodendroglia in vitro and in vivo (popko and baerwald, ; sedgwick and hickey, ) . by contrast, oligodendroglia specific transgenic expression of class i heavy chains leads to er accumulation and severe myelination defects (baerwald et al., ; power et al., ; turnley et al., ) . this suggests basal levels of the antigen processing machinery in oligodendroglia are insufficient to deliver peptides for class i assembly and egress from the er. the regulation of class i antigen processing components by oligodendroglia is not only of interest in elucidating their role as targets of anti-microbial cd t cells, but also of self reactive cd t cells in autoimmune diseases, such as multiple sclerosis (huseby et al., ; sun et al., ) . stringently regulated class i expression is also important in prolonging survival of oligodendroglia grafts for therapeutic remyelination approaches (tepavcevic and blakemore, ) . the present study investigated the regulation of class i antigen presentation components in oligodendroglia and microglia in a model of neurotropic coronavirus induced encephalomyelitis . the sublethal jhm strain of mouse hepatitis virus (mhv-jhm) infects astrocytes, microglia and oligodendroglia, spreading rapidly from the brain into the spinal cord (wang et al., ) where it establishes a persistent infection (marten et al., ) . acute viral replication in the cns is controlled by cd t cells, consistent with class i expression on all glia (bergmann et al., ; hamo et al., ; ramakrishna et al., ; redwine et al., ) . microglia and astrocytes, but not oligodendroglia are susceptible to perforin-mediated cd t cell viral control in the absence of ifng. by contrast, ifng is required to control virus in oligodendroglia (gonzalez et al., ; lin et al., ; parra et al., ) . whether ifng has a direct antiviral effect or enhances class i antigen presentation by oligodendroglia is unclear. delayed class i upregulation on astrocytes compared with microglia in this viral model presents a precedence for differential regulation of class i antigen presentation (hamo et al., ) . class i antigen presentation by oligodendroglia and microglia was compared using flow cytometry and gene expression analysis of cells isolated from the cns of infected mice. despite undetectable class i protein na€ ıve microglia expressed significantly higher basal levels of mrnas encoding class i antigen presentation components compared with oligodendroglia. following infection, delayed class i expression by oligodendroglia compared with microglia correlated with a requirement for ifng and de novo transcription of mhc genes required for antigen presentation. by contrast, microglia only moderately upregulated mrnas encoding antigen presentation machinery, yet expressed mhc class i protein earlier and independent of ifng. these results are the first to demonstrate more stringent regulation of the class i antigen presentation pathway by oligodendroglia compared with microglia during cns inflammation. mice, viruses and cell lines c bl/ mice were purchased from the national cancer institute (frederick, md). mice expressing green fluorescent protein (gfp) under control of the proteolipid protein (plp) promoter (plp-gfp) (fuss et al., ) were backcrossed six times with c bl/ mice prior to use. mice deficient in ifng (ifng / ) on the c bl/ background were previously described (parra et al., ) . all mice were housed and bred at accredited animal facilities at the keck school of medicine, university of southern california, los angeles, ca, or the cleveland clinic, cleveland, oh. mice, - weeks of age, were injected intracranially (i.c.) with ll of endotoxin-free pbs containing plaque forming units of the . v- monoclonal antibody (mab)-derived variant of mhv-jhm (fleming et al., ) . all procedures were carried out in compliance with institutional animal care and use committee approved protocols. the antigen presentation sufficient human b lymphoblastic cell line c r-l d and the antigen processing deficient cell line t -l d stably transfected with the murine h -l d gene were kindly provided by peter creswell, yale university medical center, new haven, ct (anderson et al., ) . t -l d cells have a deletion in the mhc locus comprising tap and tap resulting in intracellular accumulation of unassembled class i in the er (grandea et al., ) . the t -l d and c r-l d cells and the h- d j a. macrophage cell line (american type culture collection, manassas, va) were all maintained in dulbeccos' modifed mem containing % fcs. for phenotypic analysis, brains and spinal cords (n - per time point) were digested with a . % trypsin solution and glia enriched by centrifugation at g for min at °c on %/ % percoll (pharmacia, uppsala, sweden) gradients as previously described (gonzalez et al., ; hamo et al., ) . cns derived cells were stained with anti-cd -apc or percp (clone -f , bd biosciences, san jose, ca) and pe conjugated anti-mhc class i (clone - - , ebioscience, san diego, ca) as previously described (gonzalez et al., ; ramakrishna et al., ) . oligodendroglia from plp-gfp mice were defined as gfp cd and microglia as cd lo . oligodendroglia from ifng / mice were identified by expression of the o surface marker (fuss et al., ; gonzalez et al., ) using biotinylated anti-o mab, followed by streptavidin-apc (bd biosciences) as described . cells were fixed in % paraformaldehyde prior to analysis on a facscalibur flow cytometer (becton dickinson, mountain view, ca) using flowjo . software (tree star, ashland, or). for pcr analysis glial cells were released by trypsin digestion of brains or spinal cords from plp-gfp mice (n - per time point) as described earlier. following percoll gradient enrichment, nonspecific antibody binding was blocked with % fcs and anti-fciii/iir (clone .g , bd biosciences) in rpmi supplemented with mm hepes (ph . ). cells were stained with anti-cd apc and suspended in rpmi %fcs at a concentration of - cells/ml. gfp cd oligodendroglia and cd lo microglia were purified using a facsvantage se. a minimum of , cells was collected per sample for detection of rare transcripts. to detect intracellular class i heavy chains, brain cells derived from uninfected plp-gfp mice or continuous cell lines were incubated with golgi-plug ( ll/ml media; bd biosciences) in dmem % fcs for h at °c. nonspecific antibody adsorption was blocked by incubation with anti-fciii/iir ( .g ) and % serum mixture consisting of equal parts; mouse, human, rat, and rabbit serum. cells were then incubated with lg anti-class i mab (clone - - ) for min at °c to block subsequent surface class i detection. brain derived cells were stained with anti-cd (apc) to identify microglia. cells were fixed and permeabilized with cytofix/cytoperm reagents (bd biosciences). permeabilized cells were treated with anti-fciii/iir and serum to prevent nonspecific antibody binding as described earlier, prior to incubation with anti-mhc class i-pe (clone - - ) or anti-mhc class i-pe (h -k k , clone - - ) (bd biosciences) as an isotype control. cells were analyzed on a facscalibur flow cytometer as described above. facs purified cells were collected by centrifugation ( g for min, °c) and lysed by the addition of . ml trizol (invitrogen, carlsbad, ca). rna was precipitated in the presence of glycogen (ambion, foster city, ca) before resuspension in ll of rnase-free water. rna was digested with dnase i (roche applied science, indianapolis, in) for min, followed by heat inactivation. rna was reversed transcribed using amv reverse transcriptase (promega, madison, wi) with lg oligo-dt primers (promega) in a total volume of ll. cdna samples were diluted -fold in tris-edta (ph . ). all reactions were carried out in the presence of rnasin (promega). spinal cords from individual mice were homogenized in ml of trizol (invitrogen) using tenbroeck tissue homogenizers. rna integrity was verified by electrophoresis on . % formaldehyde agarose gels. rna ( lg) from individual spinal cords was dnase i treated and reverse transcribed as described earlier. exon junction spanning real-time pcr primers for the murine (h b ) psmb , psmb (lmp ), psmb (lmp ), psmb (mecl- ), tap , tap , irf- , irf- , ifng and gapdh genes were designed by perlprimer v . . (marshall, ). h -d /l and b m primers were designed using primer express (abi, foster city, ca). pan-ifna primers were designed using generunner v . (hastings software, hastings-on-hudson, ny) by aligning reference sequences for all ifna genes. sequences for all primers are listed in supplementary table . quantitative real time pcr was carried out using ll of cdna and syber green pcr (abi) in duplicate on an mj dna engine opticon (biorad, hercules, ca). specificity for each gene was confirmed by examining melting curves. transcript levels are calculated relative to gapdh using the formula ^( ct gapdh ct target gene) and compared with peak expression levels for each cell type over the course of infection. transcript levels were calculated for na€ ıve glia or untreated j . cells relative to gapdh for three independent experiments. significant differences between each group were determined by a two-tailed distribution analysis of an unpaired student's t-test. p values less than . were considered statistically significant. spinal cords of uninfected and mhv-jhm infected plp-gfp mice were analyzed by flow cytometry to compare temporal expression of class i molecules by oligodendroglia and microglia. in na€ ıve mice - % of spinal cord derived cells comprised cd gfp mature oligodendroglia (fig. a) , compared with - % of brainderived cells (data not shown). no mhc class i expression was detected on na€ ıve microglia or oligodendroglia (fig. b) . by day post infection (p.i.), greater than % of microglia expressed class i (fig. b) . by stark contrast, only % of oligodendroglia expressed class i. however, class i expression on oligodendroglia increased rapidly thereafter reaching % by day p.i. the population of class i expressing microglia and oligodendroglia declined by day and remained elevated throughout day p.i. the kinetics of class i expression on oligodendroglia and microglia in the brain followed similar patterns (data not shown). these data implicated microglia as primary glial targets for cd t cells early during infection. delayed class i surface expression by oligodendroglia during viral encephalomyelitis could be due to an initial absence or low abundance of antigen processing components relative to microglia. to assess basal transcript levels of genes required for mhc class i peptide presentation, oligodendroglia and microglia were facs purified from spinal cords of na€ ıve mice. both glial populations expressed similar levels of gapdh transcripts per cell when compared with j a. macrophages ( fig. a) . furthermore, the ratio of gapdh mrna levels per number of purified cells was consistent throughout infection (data not shown). these data confirmed gapdh as a stable reference gene, whose mrna levels were not affected by alterations in the cns environment due to inflammation. although microglia from na€ ıve mice did not express detectable class i protein, class i transcripts were expressed at levels similar to j a. macrophages (fig. b) , which constitutively express surface class i molecules. by contrast, oligodendroglia within the na€ ıve cns expressed times lower levels of these transcripts compared with microglia or j a. cells. similarly, na€ ıve microglia expressed comparable levels of tap and psmb encoding mrna as j a. cells, whereas these transcripts were barely detectable in na€ ıve oligodendroglia. the relative levels of b m, tap , and psmb mrnas were similar to class i, tap , and psmb mrnas in each glial cell type, respectively (data not shown). transcripts encoding the constitutive proteasomal subunit psmb , not encoded at the mhc locus, were analyzed to evaluate the overall proteasomal degradation capacities in the glial subsets. basal levels of psmb transcripts were present at similar levels in na€ ıve microglia and oligodendroglia, but levels were reduced compared with j a. cells (fig. b) . intracellular class i protein mhc class i and antigen processing proteins are barely detectable in na€ ıve brains by flow cytometry, western blot (fruh et al., ) or immunohistochemistry (fuss et al., ) . the relative abundance of these transcripts in microglia suggests post-transcriptional repression of class i antigen presentation. to confirm a block in translation rather than er retention of class i in na€ ıve microglia, intracellular retention of h -d b was examined using a d b and l d cross-reactive mab (see fig. ). antigen processing deficient t -l d cells were used as a control for intracellular accumulation of unassembled class i in the er and negligible surface staining. j a. cells were used as a positive control for fig. ). accumulation of intracellular class i was analyzed following monensin treatment to block the secretory pathway. detection of surface expression was blocked by pre-incubation with unlabeled mab to maintain similar conditions (see fig. ). under these conditions, intracellular class i remained undetectable in na€ ıve microglia. by contrast t -l d cells exhibited robust intracellular accumulation of class i molecules. j a. cells also accumulated intracellular class i, albeit at lower levels compared with t -l d cells. similar results were obtained in two additional independent experiments in the absence of monensin treatment. these data suggest that the inability of na€ ıve microglia to express class i complexes (see fig. ), despite abundant transcripts encoding all the necessary components (see fig. ), is the result of translational repression rather than a post-translational block. to assess a correlation between class i surface expression and transcriptional upregulation of genes required for class i assembly and transport, microglia and oligodendroglia were purified from spinal cords over the course of infection. microglia exhibited a clear increase in mrnas encoding class i, b m, tap and inducible proteasomal subunits psmb and psmb at day p.i. (see fig. ). the respective mrna levels dropped thereafter, but remained elevated through day p.i. tap transcripts did not change more than twofold in microglia throughout infection. although levels of mrnas encoding class i and the antigen processing components were only increased by -to -fold at their peak relative to na€ ıve microglia, peak mrna accumulation at day p.i. coincided with maximal class i surface expression (fig. b) . these data suggest rapid class i expression by microglia is due to both a release from translational repression and transcriptional activation. oligodendroglia exhibited drastically different patterns of mrna upregulation over the course of infection (see fig. ). the first transcriptional increase was observed for b m mrna. at day p.i., b m mrna levels were similar to those in na€ ıve microglia. although b m mrna did not peak until day p.i. in oligodendro-glia, maximal levels relative to gapdh mrna were similar to those in microglia. transcripts encoding class i, tap / and psmb / in oligodendroglia were not notably increased until day p.i. and also peaked at day p.i. similar to b m mrna, individual transcripts encoding class i processing components reached comparable maximal levels in both cell types, with the exception of tap mrna. in two independent experiments peak expression of the respective mrnas relative to gapdh differed less than threefold between the two cell types. after day p.i., all mrnas declined in oligodendroglia, but remained elevated relative to basal levels through day p.i. (see fig. ). relative to basal levels, virus-triggered inflammation induced at least -to , -fold overall increases in the mrna populations in oligodendroglia compared with modest -to -fold increases in microglia. the most potent increase was observed for class i mrna, reaching , -and , -fold values relative to baseline levels in two separate experiments. this dramatic increase in rna accumulation coincided with expression of class i molecules on the surface of oligodendroglia (fig. b) and suggests de novo transcriptional activation of genes encoding class i presentation components. although the kinetics and magnitude of mrna induction were vastly different between oligodendroglia and microglia, overall levels of mrnas encoding class i and antigen processing components, with the exception of tap , were similar at their peak comparing the two cell types. the strikingly different patterns of class i antigen processing gene expression in microglia compared with oligodendroglia suggests distinct responsiveness to inflammatory events. type i and ii ifn were investigated as prime candidates promoting expression of mhc genes. ifna transcripts were detectable at low levels in spinal cords of na€ ıve mice, whereas ifng transcripts were undetectable (fig. a) . in the majority of infected mice, ifna transcripts were up regulated approximately -fold at day p.i. but these levels rapidly declined by day p.i. (fig. a) . ifng transcripts were largely undetectable at days and p.i., but increased dramatically by days and p.i. induction of ifna mrnas preceded ifng mrna by at least days. by day p.i. ifng transcripts declined markedly and were no longer detectable in the majority of animals by day p.i. to further examine the responsiveness of microglia and oligodendroglia to ifn signaling, transcripts encoding ifn regulatory factor (irf- ) and its antagonist, ifn regulatory factor (irf- ) were measured in purified glia populations. irf- is associated with amplifying the effects of initial ifn signaling as well as promoting expression of its antagonist, irf- (kroger et al., ) . irf- mrna was detectable in microglia from na€ ıve mice, but near detection limits in oligodendroglia, fig. . cell-type specific regulation of mhc class i antigen processing genes. microglia and oligodendroglia were purified by facs from na€ ıve or infected mice at different days p.i. and the levels of mrna encoding mhc class i (h -d), b -microglobulin (b m), peptide transporter units (tap and tap ) and inducible proteasomal subunits (psmb and psmb ) were calculated relative to gapdh. peak transcript levels were set to % for each cell type in two independent timecourse studies. bars represent the average percentage of peak expression for each cell type with standard deviation. whereas both na€ ıve glia expressed relatively higher levels of irf- transcripts. (figs. b,c) . following infection irf- transcripts increased to peak levels at days and p.i. in microglia and oligodendroglia, respectively. whereas the irf- mrna increases were -fold in microglia and greater than , -fold in oligodendroglia relative to basal levels, irf- levels in both cell types only changed two to threefold (figs. b,c) . as a result irf- mrna increased three to fourfold over irf- levels in both cell types. peak increases in irf- mrna levels coincided with peak levels of transcripts encoding antigen processing components. these data suggest oligodendroglia preferentially amplify ifng mediated signals via the irf- pathway. the parallel kinetics of class i expression on oligodendroglia and induction of ifng during infection suggested a direct relationship. to assess if ifng is required for class i expression by oligodendroglia, ifng / mice were infected with mhv-jhm. in the absence of ifng oligodendroglia did not express class i on their surface (see fig. ). by contrast, microglia were still capable of up regulating class i as early as day p.i. (see fig. ). although the percentage of microglia expressing class i increased to % by day p.i., it declined more rapidly thereafter compared with ifng competent mice (see fig. ). concordant results were obtained using ifng / mice on the balb/c background, in which infection induced class i d d expression on %, % and % of spinal cord microglia at days , and p.i., respectively, but not on oligodendroglia. these data demonstrate that oligodendroglia require ifng for class i expression and support an ifng-independent signal in regulating class i antigen presentation by microglia (bergmann et al., ) . the response of glia to infection, specifically their interaction with infiltrating t cells, determines how t cell effector functions contribute to microbial control and pathogenesis. the present data demonstrate more stringent regulation of class i expression by oligodendroglia compared with microglia during viral induced inflammation. rapid class i expression by microglia suggests they are the initial infected cells that interact with early infiltrating virus specific cd t cells to trigger ifng secretion. this local ifng in turn induces class i expression by oligodendroglia, which peaks coicident with maximal ifng transcript levels and prominent t cell infiltration into spinal cords (marten et al., ) . the timing of these interactions is thus critical in establishing a balance between viral spread and cd t cell mediated control, as cytolysis is downregulated during the transition to viral persistence and ifng is crucial in controlling mhv-jhm replication specifically in oligodendroglia (bergmann et al., ; gonzalez et al., ; parra et al., ) . the cell type specific class i expression patterns were associated with distinct levels of basal transcription of mhc genes, as well as differences in the timing and magnitude of transcriptional upregulation. the high basal levels of class i transcripts in purified microglia, suggest they are a major source of class i transcripts previously detected at low levels in the na€ ıve adult brain (fahrner et al., ) . class i expression is predominantly regulated at the transcriptional level with conserved promoter elements determining both tissue specific expression as well as responsiveness to cytokines and hormones (howcroft and singer, ) . coordinated transcriptional upregulation of mhc genes in both oligodendroglia and microglia is consistent with similarities in their core promoter elements (arons et al., ; brucet et al., ) . the tap and psmb genes even share a bi-directional promoter. ifna/b and ifng directly activate class i transcription via jak/stats or indirectly via irf- induction, which binds the ifn-stimulated response element. irf- also binds the shared promoter of tap /psmb to drive transcription (brucet et al., ) . the critical role of irf- in controlling the class i presentation pathway is demonstrated by low levels of class i expression in irf- / mice (hobart et al., ) . following mhv-jhm infection, increased irf- to irf- mrna ratios support irf- as a primary candidate in enhancing transcription of these genes. ifna mrna was associated with upregulation of irf- mrna and class i expression by microglia, whereas ifng mrna expression correlated with accumulation of mrnas encoding irf- and antigen presentation components in oligodendroglia. ifng dependent class i expression by oligodendroglia, but not microglia, was confirmed by class i expression patterns in infected ifng / mice. the mhv-jhm model suggests the more rapid class i upregulation by microglia resides in an inherently increased responsiveness to ifna/b signaling and earlier transcriptional activation compared with oligodendroglia. delayed class i expression by microglia in mhv-jhm infected mice deficient in ifna/b signaling, confirm ifna/b as a primary inducer of early class i presentation by microglia (ireland et al., ) . in addition, the modest increases in mrnas encoding antigen presentation components in microglia support a partial contribution of translational regulation. post-transcriptional repression of mhc genes has been observed in a variety of cancers, yet the mechanisms are unclear (luo et al., ; ponzoni et al., ) . analysis of proteasomes from primary microglia cultures derived from neonatal mice revealed low, yet detectable protein levels of the immunoproteasome subunits psmb and psmb (stohwasser et al., ) , which were significantly increased following ifng treatment. although ifna/b stimulation was not analyzed in the latter studies, the data support constitutive transcription of class i related genes in na€ ıve microglia. our data suggest that translational repression of mhc associated transcripts, potentially released by ifna/b signaling, may constitute a provocative new mechanism regulating mhc class i expression by microglia. the modest overall increase of mhc encoded mrnas in microglia relative to oligodendroglia may reflect cell type specific integration of both ifna/b and ifng signals, or additional responses to other inflammatory mediators in the cns environment. the fact that microglia strongly upregulate class ii expression in an ifng dependent manner (bergmann et al., ) , even in the absence of ifna/b signals (ireland et al., ) , suggest that ifng responsiveness is not impaired by ifna/b pre-conditioning. the apparent lack of additional ifng mediated transcription of genes encoding class i antigen presentation components may be due to saturation at the promoter sites in microglia. class i expression by oligodendroglia required ifng stimulated de novo transcription. the negligible levels of mhc class i related transcripts in oligodendroglia derived from the na€ ıve cns are consistent with active repression of class i gene transcription by oligodendroglia-specific dna binding factors (mavria et al., ) . low mrna levels and undetectable protein expression were also observed in unstimulated neonatal oligodendroglia cultures (massa et al., ) . although ifna/b does not appear to play a role in regulating class i anti- fig. . ifng dependent class i expression on oligodendroglia. glial cells derived from spinal cords of infected ifng / (h- b ) mice (pooled from n per time point) were analyzed for class i expression by flow cytometry at the indicated days p.i. cells were gated on cd lo microglia (left panel) and cd o oligodendroglia (right panel). histograms depict microglia and oligodendroglia expressing class i (shaded lines); isotype control indicated by thin lines. numbers represent percentage of glia expressing class i. data are from a single experiment. gen presentation by oligodendroglia, other effects on oligodendroglia biology including induction of antiviral pathways have been demonstrated in the poliovirus model (delhaye et al., ) . the delayed, yet prominent increase in class i related genes may result from synergistic stimulation by ifng and tnfa (agresti et al., ) . this strict regulation suggests deviation of cellular resources from myelin maintenance to host defense, a notion consistent with ifng mediated induction of er stress in oligodendroglia and perturbation of remyelination (lin et al., ) . overall the stringent induction of mhc genes by oligodendroglia implies a tendency to avoid cd t cell cytolysis and preserve myelin maintenance and neuronal function. nevertheless, as oligodendroglia are myelin factories, it is feasible to assume they present myelin derived epitopes during inflammation associated with ifng secretion. indeed, mbp specific cd t cells can recognize and lyse oligodendroglia in the absence of exogenous peptide in vitro (huseby et al., ) . similarly, cd t cell inflammation induced by overexpression of plp by oligodendroglia is associated with class i expression on oligodendroglia and cns damage (ip et al., ) . activation and clonal expansion of cd t cells in brain biopsy samples and csf of multiple sclerosis patients supports a role of local cns autoantigen presentation (babbe et al., ; jacobsen et al., ; skulina et al., ) . furthermore, although class i and irf- was prominently expressed by activated microglia/macrophages in multiple sclerosis lesions, both proteins were also localized to oligodendroglia (gobin et al., ) . however, in vivo interactions between cd t cells and class i peptide complexes presented by oligodendroglia have not been formally proven. the potent transcriptional upregulation of class i antigen presentation components by oligodendroglia presented in this report clearly demonstrates a potential for oligodendroglia to present myelin antigens. however, the exquisite sensitivity of microglia to early inflammatory cytokines implicates these cells as targets for initial cd t-cell interactions. synergistic stimulation of mhc class i, irf- gene expression by ifn-g and tnf-a in oligodendrocytes intracellular transport of class i mhc molecules in antigen processing mutant cell lines organization and functional analysis of the mouse transporter associated 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qa genes in embryonic and adult mice pathogenicity of antigenic variants of murine coronavirus jhm selected with monoclonal antibodies alternative exon usage and processing of the major histocompatibility complex-encoded proteasome subunits normal cns myelination in transgenic mice overexpressing mhc class i h- l(d) in oligodendrocytes purification and analysis of in vivo-differentiated oligodendrocytes expressing the green fluorescent protein accessory molecules in the assembly of major histocompatibility complex class i/peptide complexes: how essential are they for cd ( ) t-cell immune responses? upregulation of transcription factors controlling mhc expression in multiple sclerosis lesions expression of a dominant negative ifng receptor on mouse oligodendrocytes inhibition of interferon-g signaling in oligodendroglia delays coronavirus clearance without altering demyelination dependence of peptide binding by mhc class i molecules on their interaction with tap distinct regulation of 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neuroblastoma cells oligodendroglial response to the immune cytokine interferon g major histocompatibility complex class i expression in oligodendrocytes induces hypomyelination in transgenic mice vaccine-induced memory cd t cells cannot prevent central nervous system virus reactivation in vivo expression of major histocompatibility complex molecules on oligodendrocytes and neurons during viral infection antigen presentation in the central nervous system multiple sclerosis: braininfiltrating cd t cells persist as clonal expansions in the cerebrospinal fluid and blood rapid on/off cycling of cytokine production by virus-specific cd t cells biochemical analysis of proteasomes from mouse microglia: induction of immunoproteasomes by interferon-g and lipopolysaccharide molecular cloning of the mouse proteasome subunits mc and mecl- : reciprocally regulated tissue expression of interferon-gmodulated proteasome subunits interferon-g, the functional plasticity of the ubiquitin-proteasome system, and mhc class i antigen processing myelin antigen-specific cd t cells are encephalitogenic and produce severe disease in c bl/ mice haplotype matching is not an essential requirement to achieve remyelination of demyelinating cns lesions dysmyelination in transgenic mice resulting from expression of class i histocompatibility molecules in oligodendrocytes sequential infection of glial cells by the murine hepatitis virus jhm strain (mhv- ) leads to a characteristic distribution of demyelination inducible expression of h- and ia antigens on brain cells the authors thank emanuel dimacali and the usc norris cancer center flow cytometry core for assistance with flow cytometry. they also thank maria ramirez for maintenance of the plp-gfp mouse colony. key: cord- -ey kc zj authors: degauque, nicolas; brouard, sophie; soulillou, jean-paul title: cross-reactivity of tcr repertoire: current concepts, challenges, and implication for allotransplantation date: - - journal: front immunol doi: . /fimmu. . sha: doc_id: cord_uid: ey kc zj being able to track donor reactive t cells during the course of organ transplantation is a key to improve the graft survival, to prevent graft dysfunction, and to adapt the immunosuppressive regimen. the attempts of transplant immunologists have been for long hampered by the large size of the alloreactive t cell repertoire. understanding how self-tcr can interact with allogeneic mhc is a key to critically appraise the different assays available to analyze the tcr vβ repertoire usage. in this report, we will review conceptually and experimentally the process of cross-reactivity. we will then highlight what can be learned from allotransplantation, a situation of artificial cross-reactivity. finally, the low- and high-resolution techniques to characterize the tcr vβ repertoire usage in transplantation will be critically discussed. being able to track donor reactive t cells during the course of organ transplantation is a key to improve the graft survival, to prevent graft dysfunction, and to adapt the immunosuppressive regimen. the attempts of transplant immunologists have been for long hampered by the large size of the alloreactive t cell repertoire. understanding how self-tcr can interact with allogeneic mhc is a key to critically appraise the different assays available to analyze the tcr vβ repertoire usage. in this report, we will review conceptually and experimentally the process of cross-reactivity. we will then highlight what can be learned from allotransplantation, a situation of artificial cross-reactivity. finally, the low-and high-resolution techniques to characterize the tcr vβ repertoire usage in transplantation will be critically discussed. keywords: tcr repertoire, transplantation, cross-reactivity, alloreactivity, tcr, mhc, t cell understanding the cross-reactivity shaping the t lymphocyte receptor repertoire through evolution, numerous processes have been selected to generate a diverse repertoire of tcrαβ able to protect mammalian from pathogenic insults (figure ) . highly similar genes recombine to form functional genes and generate a highly diverse tcr repertoire. tcrβ chains are encoded by distinct variable (v; trbv), diversity (d; trbd), and joining (j; trbj) genes, whereas tcrα chains are encoded by distinct sets of v and j genes (trav and traj). junctional diversification further extends the combinatorial diversity by either trimming gene ends or adding nucleotides between the recombining genes ( ) . in contrast to the ighv (v genes of immunoglobulin heavy chain) germline dataset compiled by the immunogenetics (imgt) group that greatly benefit from the advanced of deep-sequencing technologies, the human tcr germline has been only minimally changed since the complete sequencing of the tcr gene loci in ( , ) . the functional genes, orfs, and pseudogenes have been reported for the trbv, for the trav and for the trbd dataset. the analysis of the tcr cdr is still a very challenging process. the identification of the trbd genes figure | understand the cross-reactivity of a highly diverse tcr repertoire. a highly diverse tcrαβ repertoire is generated by iterative processes selected through evolution. combinatory diversity results from the selection of variable (v; trav and trbv), diversity (d; trbd) and joining (j; traj and trbj) genes. junctional diversification further extends the combinatorial diversity by either trimming gene ends or adding nucleotides between the recombining genes. finally, the association of the tcrα and tcrβ chain constitutes the final steps of the numerous iteration processes that lead to the generation of a highly diverse tcr repertoire, which is able to efficiently protect individuals from pathogenic stimulations. tcrαβ adopts a stereotype docking geometry atop the mhc/peptide complex. this orientation leads to a spatial interaction between the germline-encoded cdr and cdr of the tcrα and β chains and the edges of the peptidegroove of mhc. the accumulation of reported crystallographic structures has challenged the stereotypic view of the angle of the tcr docking. however, the recognition of conserved motifs on the side of mhc molecules by cd / cd co-receptor constrained the tcr docking geometry. despite the high diversity of the tcr repertoire, a high degree of cross-reactivity has been reported that could be explained by the "natural" ability of tcr to interact with mhc molecules (mhc focus model) as well as the interaction of tcr to a limited number of amino acids of the peptide bound to the mhc peptide groove. march | volume | article frontiers in immunology | www.frontiersin.org cannot be performed due to the high degree of similarities of the trbd at their ′ ends, the short length of the two genes, and the presence of g-rich n nucleotides at the ′ ends that could be also added by the tdt enzyme. it is misleading to estimate the combinatory diversity by simply multiplying together the number of v, d, and j genes ( ). rather than a random combination of the tcr genes, studies have shown that tcr genes are highly biased in their usage, and that only part of the theoretical diversity is selected ( , ) . chromosomal recombination patterns can be explained by variations in enhancers and recombination signal sequences (rss) and organization of the trbj genes (a block of six and seven genes located respectively downstream from the trdb and trdb gene) that leads to a bias in d-j pairing. the diversity of the tcr repertoire is further broaden during the rearrangement process first by the addition of p nucleotides (palindromic nucleotides) thanks to recombination activating gene- and - (rag and rag ) ( ) that form hairpin loops at the gene end and then by the addition of n nucleotides (with a biased toward g nucleotides) by the terminal deoxynucleotidyl transferase (tdt) ( ) . insertions of nucleotides have a profound impact on the diversity of the complementary-determining regions (cdr ) sequences and contribute to most ( %) of this diversity ( ) . the coding ends of the genes can be also trimmed by exonucleases. however, given the limited number of amino acids, the removal of nucleotides by exonucleases is constrained to generate a productive codon and therefore limits the contribution of exonuclease trimming to the diversity of the tcr repertoire. finally, the association of the tcrα and tcrβ chain constitutes the final steps of the numerous iteration processes that lead to the generation of a highly diverse tcr repertoire, which is able to efficiently protect individuals from pathogenic stimulations. six cdr will engage the peptide/mhc complexes, endogenous and exogenous peptides being presented respectively by mhc class i and ii molecules. mhc class i grooves constrain the length of the presented peptides ( - amino acids length) while the open nature of peptide-binding cleft of mhc class ii molecules allow a broader range of peptides to be presented. the hla locus is the most polymorphic region of the human genome, with more than , variant alleles ( , hla class i alleles and , hla class ii alleles according to the imgt/hla). the high diversity of hla conferring an almost unique signature of hla for mankind is further extended by the combinatory diversity resulting from the association of six hla class i (two alleles of hla-a, -b, and cw) and six hla class ii molecules (two alleles of hla-dr, -dp, and -dq). the high mutation level of the hla loci is preferentially focused on the peptide-binding cleft that clustered most of the variability of the amino acid sequence. the focus of mutations underlines the function of the hla molecules, namely being able to display a very large array of peptides. garcia et al. were the first to report the crystallographic structure of a murine tcr c bound to peptide/mhc class i (h- k b -dev ). the cytotoxic t cell clone c is one of the most well-characterized tcr and has been initially isolated from a balb/b mouse as an allospecific t cell that recognized l d on the mastocytoma p . beside its primary antigen (peptide p c), the c tcr can bind to different antigens, including the dev ( ) and siyr ( ) . they also showed that tcrαβ adopts a ° diagonal orientation to the long axis of the peptide ( ) . this orientation leads to a spatial interaction between the germlineencoded cdr and cdr of the tcrα and β chains and the edges of the peptide-groove of mhc (figure ) . the highly diverse cdr region is facing the central portion of the bound peptide. the multiple crystallographic structures of tcr/peptide mhc complexes [more than of crystallographic structures tcr repertoire in transplantation frontiers in immunology | www.frontiersin.org have been obtained ( ) ] have revealed that the docking angle of the tcr is conserved with a stereotype position of a ° diagonal orientation to the long axis of the peptide ( ) . the conserved binding model has lead to the concept that tcr and mhc are hardwired to interact, resulting from a coevolution selection of conserved regions (codons) to lock in tcr onto mhc molecule. the stereotyped orientation of tcr atop mhc molecule is however more flexible than initially proposed, with the accumulation of crystal structures. the median docking angle of tcr is . ° (min-max - °) with mhc class i and . ° (min-max - °) with mhc class ii ( ) (figure ) . different theories have been postulated to explain the hardwire of tcr to mhc molecules ( ) , including the key role of co-receptors of cd and cd that imposed steric requirements for concurrent associations of tcr, cd , cd /cd , and mhc complexes allowing the appropriate signaling events to occur. indeed, the main role of co-receptor cd and cd is to recruit the src tyrosine kinase p lck (lck) via its association with the cytoplasmic tail of cd or cd . lck concentration promotes phosphorylation of immunoreceptor tyrosine activation motifs (itams) in the cytoplasmic tails of cd subunits and then initiates the cascade of signaling events leading to the full activation of the t lymphocyte. given the key role of co-receptor cd and cd in process, it was assumed that their ability to bind, respectively, the membrane-proximal α and β domains of the mhc class ii molecule and the protruding loop in the α domain of the mhc class i molecule will constrain the docking geometry of the tcr to the pmhc (figure ) . a recent report by beringer et al. ( ) had challenged the consensus idea of a highly stereotype docking of tcr atop mhc molecules ( ) . crystallographic structures of two tcr binding to proinsulin peptide presented by hla-dr (hla-dr proinsulin ) have been obtained from two clones of induced regulatory cd t cells. the ternary complexes revealed a ° polarity reversal compared to all other tcr-peptide-mhc complex structures. it remains to be address whether this singular observation could be generalizable, whether the reverse docking is a unique feature of regulatory cells and whether the potential signaling differences may influence the phenotype and the function of the t cells. cross-reactivity can be defined by the ability of a given tcr to interact with more than one pmhc complex with different presented peptides or mhc molecules. this new concept has been presented as early as by matzinger and bevan ( ) . an alloreactive t cell clone was derived by owens et al. in with three h -e reactivity (allo-e k specific, h -e k , dba/b h -e d , and self h -e d ) ( ) . since then, numerous reports have provided evidence of cross-reactivity. for instance, mouse c tcr can interact with syngeneic mhc h- k b presenting dev ( ) and siyr ( ) and with allogeneic h- k bm presenting dev /k bm ( ) and allogeneic h- l d -p ca ( ) . the study by birnbaum et al. is an elegant attempt to quantify the cross-reactivity of a given tcr ( ) . using five different cd tcr clones (three from mouse origin and two from human origin), high throughput screening of yeast libraries and deep sequencing, the authors demonstrate that a single tcr can interact with more than different peptides. jerne et al. postulated in the mid- that each cell exhibits a unique clonotype able to recognize only one antigen ( , ) . don mason has been among the first to challenge the validity of this clonal selection theory ( ) showing that the immune system will be highly incompetent to protect an individual from external insult if one and only tcr was able to recognize a single peptide presented in a given hla context. more than t cells, which would weigh more than kg, would be needed to provide efficient coverage of the potential foreign peptides. this clearly stated that the immune system could not efficiently protect individual if one tcr interacts with a single antigen. unlike the affinity maturation of b cell receptor, the protein sequence of tcr is fixed and naive t cells are required to recognize foreign antigens not encountered before. the number of potential antigens to be recognized is huge given the variability induced by the high diversity of peptide-binding groove of hla class i and ii molecules. from the proteinogenic amino acids and given that peptides from -to -mer can be presented, an incredibly high number of peptides can be potentially generated (> peptides) ( ) . the diversity can be further extending by the posttranslational modifications of amino acids. in a opinion paper, andrew sewell elegantly presents the necessity of the cross-reactivity ( ), as the number of potential foreign peptide-mhc complexes that t cells might encounter dwarfs the number of tcrs available [the number of unique tcrαβ is estimated to be in the magnitude of ( , ) ]. the mechanisms described previously to generate a diverse tcrαβ have to be envisioned at the population level. given the relatively limited number of genes encoding for tcr chain α and β and the requirement of tcr to recognize the highly diverse hla molecules, the necessity of each t cell to recognize a large array of peptides is expected ( ) . before presenting the experimental approach aiming to quantify the number of peptides recognized by a single tcr, we would like to present clear evidences of the cross-reactivity involving memory t cells without previous antigen encounter. it has been described few years ago that cd t cells with a memory phenotype can be found in mice ( ) ( ) ( ) . cd t cells specific for ovalbumin and viral antigens (hsv, vaccinia) could be detected in mice despite their germ-free environment ( ) . despite the absence of previous antigen encounter, these pre-existing memory cd t cells harbor traits of memory cells such as the ability to rapidly proliferate upon stimulation and to secrete rapidly pro-inflammatory cytokines. homeostatic proliferation, aging, and cross-recognition of alternate ligands have been postulated to drive the accumulation of these memory-like naive cd t cells ( , ) . this observation has been extended to human settings in which cd t cells specific for hiv- , cmv, and herpes simplex virus (hsv) epitopes were identified in healthy volunteers that had never been infected with these viruses ( ) . again, these cells exhibit not only memory markers but also memory-associated features (rapid proliferation and cytokine secretion). the acquisition of memory characteristics could be a consequence of homeostatic proliferation ( ) or a consequence of the cross-reactivity to other antigens in the environment. to support the latest hypothesis, su et al. have shown that hiv- specific t cells can recognize environmental peptides present in the gut and soil, bacteria and ocean algae, and plants. of interest, t cells specific for hiv- can even be purified from cord-blood ( ) , demonstrating thereby the presence of t cells able to recognize self and non-self antigens in newborns. of interest, the phenotype of cross-reactive t cells was different between newborns and adults, with a naive and a memory phenotype, respectively. the concept of clonal deletion that occurred in the thymus is challenged by the aforementioned reports and compelling evidences suggest that from an evolutionary perspective, the necessity to protect an individual against pathogens is far more important than to limit the autoreactivity. a recent study from davis team further sustained this claim ( ) . the frequency of cd t cells specific of a y chromosome specific antigen (equivalent to hy peptide) is only threefold lower in man as compared to women ( ) . of interest, whereas cd t cells purified for their specificity regarding a pool of six self peptides do not proliferate after stimulation with the same set of self peptides, cd t cells specific of a pool of six non-self peptides exhibit a potent proliferative response ( ) . the absence of response reported for self-specific cd t cells and not for foreign antigen-specific cd t cells has been linked to a different genetic programing as compared to the clones purified from woman, with a lower expression of il- r, il- r, and bcl-xl ( ) . thus, evolution has favored the absence of hole over autoimmune disease (about % of incidence). it may seem awkward that the evolution has favor the escape of antiself specific t cells from thymic selection over a more stringent deletion of all anti-self t cells. a heavier burden of maintaining tolerance is needed to prevent the development of autoimmune diseases. however, the need to defend the immune system against pathogens, especially during childhood, is far greater than the need to prevent autoimmunity as for population's survival. by limiting the deletion of self-reactive t cells and thanks to the large cross-reactivity of t cells, the holes in the t cell repertoire that pathogens might take advantage of are constrained. the analysis of the immune system in monozygotic twins is enlightened in many aspects as such studies allow the dissociation between the inborn and the acquired contributions. the team of davis has recently showed that the heritability of t and b cells parameters declines very rapidly with age ( ) . at the age of years, the heritability explained less than % of the variation in t and b cell parameters. cmv infection is a protypical example of the influence of non-heritable factor on the whole immune system. indeed, % of all parameters measured in discordant twins were influenced by cmv infection ( ) . the environment carves the immune system of each single individual, with each past immune response heavily imprinting the (present) immune system. since the initial observation that immunity against cowpox protects individual from smallpox ( ) , numerous examples of cross-reactivity had been reported in mice and in human ( ) . for instance, infections with bcg, influenza a virus (iav), lymphocytic choriomeningitis virus (lcmv), and murine cytomegalovirus (mcmv) all confer a level of protective immunity against vaccinia virus ( ) ( ) ( ) . the benefit of cross-reactivity as for pan-virus protection is more difficult to assess for obvious reasons. nevertheless, the numerous example of a single tcr able to recognize different antigens [bcg and poxviruses ( ) ; papillomavirus and coronavirus ( ) ; influenza virus and epstein-barr virus ( ) ]. the large cross-reactivity of t cells confers a more efficient protection cover using a limited number of t cells that need to screen an incredibly large array of peptides that can be presented by mhc molecules. beside the efficient use of limited t cell resource, cross-reactivity confers a spatiotemporal advantage to the immune system to scan any infected cells. cross-reactivity could also be envisioned as an evolution strategy to limit the immune recognition escape. recipient immune system can interact with foreign hla molecules under two very different circumstances: pregnancy and transplantation. thanks to evolution and adaptation of the maternal immune system to the presence of hla mismatch fetuses, allorecognition during pregnancy is not harmful and could even be beneficial as for mammalian sexual reproduction. immunological tolerance toward allogeneic fetus is obtained through a complex network of regulatory mechanisms including the lack of expression of classical mhc class i molecules by the placental trophoblast and the expression of non-classical mhc class i hla-e and hla-g. more surprisingly, hla mismatches have been proposed to be beneficial for pregnancy outcome. in the s, billington reports that the placenta is larger in h- incompatible mouse as compared to compatible fetuses ( ) . hla compatible fetuses (i.e., similar to maternal hla) have been shown to be more prone to be aborted ( ) . in contrast, recipient immune system will potently eliminate an allogeneic graft in the absence of immunosuppressive therapy. despite the absence of thymic central selection ( ) of potential graft-recipient t cells by allogeneic mhc motifs regarding their ability to recognize allogeneic potential hla, a large pool of t cells can be activated by donor hla molecules either through the direct pathway (i.e., donor hla presenting donor peptides) or the expected processing of foreign mhc molecules, coined as the "indirect pathway" (i.e., recipient hla presenting donor peptides) in transplantation immunologist jargon. the direct allorecognition pathway represents a unique example of functional and efficient cross reactivity. two main hypothesizes have been postulated to explain the basis of alloreactivity, emphasizing the role of either mhc molecule or peptide. the polymorphism between donor and mhc molecules could act as an "innate focus" that leads to the activation of unprimed recipient t cells or the allopeptide could be recognized as foreign antigen while allogeneic and self-mhc molecules exhibit a high degree of similarity (figure ) . according to the mhc centric model, the peptide plays only a minor role in the process, and alloreactive tcrs recognize structural determinants on the mhc helices of syngeneic or allogeneic mhc. the bias of tcr to interact with mhc molecules supports this theory. crystal structures of allo-pmhc complexes such as c tcr with allogeneic h- k bm presenting dev /k bm ( ) or bm . tcr with allogeneic pbm -h- k b ( ) have shown that alloreactive tcrs interact with allogeneic mhc in a similar fashion as with syngeneic mhc. to further support the role of mhc in alloreactivity, it has been reported that some hla mismatches between donor and recipient are associated with worse graft survival than others, leading to the notion of taboo mismatches based on shape rather than sequence differences ( ) . for instance, despite a single amino acid in an hla class i antigen, mismatches between hla-b* and hla-b* is associated with transplant rejection ( ) and acute graft-versushost disease ( ) . the peptide repertoire bound to hla-b* or hla-b* have been shown to be very similar ( ) . however, a recent report challenges this observation ( ) . the single amino acid mismatch induced the presentation of more unique peptides by hla-b* than hla-b* , consistent with the stronger t cell alloreactivity observed toward hla-b* compared with hla-b* ( ) . this observation supports the notion of a peptide focus tcr allorecognition, in the same line as molecular mimicry. allorecognition could also involved cross-reactivity between mhc class i and mhc class ii or even xeno mhc ( , ) . in , schilman et al. reported that cd t cell clone could be activated by both mhc class i (h- d b ) and mhc class ii (i-e k ) molecules ( ). by-directional recognition of t cells between mhc class i and mhc class ii have been reported later ( ) ( ) ( ) . these observations may have important implication in the attempt to minimize hla mismatches during the process of organ allocation. using a mixed lymphocyte reaction ( ) , it has been shown that - % of t cell in peripheral blood can be activated ( ) . as mentioned before, the number of hla mismatches between donor and recipient is a primary driving force that mobilized a larger fraction of t cells than nominal antigens. whether alloreactive t cells are activated by the high number of new antigens presented by donor hla or by the large number of different allo-phla complexes (or both) is still under debate, and the two hypotheses are not mutually exclusives. the indirect pathway further enhances the reactivity of recipient t cells toward allogeneic graft. indeed, peptides presented by mhc molecules derived predominantly from mhc-related molecules ( ) ( ) ( ) . the introduction of donor hla molecules will thus lead to the introduction of great pool of new peptides that can mobilized a large fraction of recipient t cells. it is now also well accepted that memory t cells generated prior transplantation constitute a major hurdle for long-term graft acceptance. chronic viruses such as ebv and cmv induce the generation of a large pool of memory t cells. for instance, % of both the cd and cd memory compartments in blood are reactive to hcmv ( ) . the cross-reactivity between virus-specific t cells and allogeneic hla has been extensively documented ( ) . ebv or cmv specific cd t cells exhibit frequently a crossreactivity toward allogeneic mhc class i complexes ( ) ( ) ( ) ( ) ( ) . similar observations have been reported for cd t cells specific for ebv or cmv ( ) ( ) ( ) . virus-specific t cells that cross-react with alloantigens have been shown in experimental models to proliferate in response to a transplanted allograft in vivo ( ) . for instance, lcmv-specific cd t cells generated after infection of mice with armstrong strain of lcmv are able to vigorously proliferate in vivo after skin transplantation and ultimately to mediate skin graft rejection ( ) . tracking anti-donor response by the investigation of tcr vβ repertoire: from low resolution technique to high throughput sequencing given the size of anti-donor t cell pool, great efforts have been paid to track the immune-response using the analysis of tcr vβ repertoire and to correlate specific usage of tcr vβ repertoire with graft status or graft outcome. before presenting the available reports, it is necessary to present the two major methods used to investigate tcr vβ usage; a low resolution (spectratype alone or tclandscape when combined with quantitative analysis) and, more recently, a high resolution (deep-sequencing of tcr vβ region) approach (figure ) . the low-resolution technique is based on the analysis of the length of the cdr region whereas the high-resolution technique identifies the sequence of each tcr vβ and later quantifies the abundance of the different t cell clones. each tcr vβ family is composed of t cells with various lengths of their cdr region. the distribution of the cdr length can be assessed by spectratype ( , ) . a broad spectrum of profiles can be identified ranging from a gaussian-like profile to a highly restricted profile, highlighting the absence of selection of t cell, or the expansion of t cell clones, respectively. different analytic tools have been used to characterize the cdr length distribution ( ) ( ) ( ) ( ) . the qualitative assessment of the tcr vβ repertoire can be complemented by the quantification of the different vβ families at the mrna level using qrt-pcr ( ) ( ) ( ) or at the cellular level using flow-cytometry ( ) . such techniques still offer several benefits over higher resolution techniques such as their cost, the short time frame to obtain results, and the generation of a reasonable amount of data can be also displayed as "visible" pattern as an "x-ray" of the global tcr alteration in a specific pathological context ( ) ( ) ( ) ( ) . a rapid survey of the usage of the tcr vβ repertoire can be efficiently performed, guiding further investigations focused on targeted tcr vβ families. at the other range of the resolution spectrum, deep-sequencing of tcr vβ obtains a full picture of the usage of t cell repertoire with deep or ultra-deep resolution. the availability of all tcr vβ sequences allows for the precise appraisal of the distribution of the different t cell clones especially across different biological compartments ( ) . furthermore, with a complete tcr vβ sequencing, researchers can investigate the similarity of t cell figure | characterization of the tcr vβ repertoire by low resolution and high resolution technique. immune challenge leads to the selection of t cells harboring specific tcrαβ among the highly diverse tcrαβ repertoire. antigen-specific t cells could be identified by low-resolution techniques (e.g., spectratype) or high-resolution techniques (e.g., ngs). low-resolution techniques are aiming to identify vβ families that exhibit monoclonal distribution of their cdr length distribution using vβ specific pcr and spectratyping. the clonality of the identified vβ families needs to be confirmed by the sequencing of the pcr product. vβ-specific t cell purification enables later to perform functional assay or to reconstruct the tcrαβ in order to identify the recognized antigen. deep-sequencing of tcr vβ region identify the sequence of each tcr vβ and intensive bio-informatic process is needed to quantify the abundance of the different t cell clones. given the burden of data generated, the next-generation sequencing is well-fitted to track t cell clones in time or across different anatomic sites. march | volume | article frontiers in immunology | www.frontiersin.org sequences between biological compartments or individuals and take advantage of public repository databases to assess the specificity of a sequence and potentially to reconstruct the tcr in order to search for the recognized peptides. however, the amount of data generated using this technique is extremely high and efficient bio-informatics tools specifically devoted to the analysis are needed to identify meaningful information in the ocean of data. the accessibility of deep-sequencing is likely to be broaden in the near future thanks to the advances in bio-informatics tools and the reduction of the cost. tcr repertoire in transplantation frontiers in immunology | www.frontiersin.org low-resolution techniques have been used to investigate the usage of tcr vβ repertoire in kidney transplant recipients with various clinical outcomes or at various time points posttransplantation ( ) ( ) ( ) . using the combination of spectratyping and quantitative assessment of the tcr vβ transcript, we have been able to define direct or indirect allorecognition patterns in an experiment model of allograft in congenic rats ( , , ) . using the same approach, we reported that patients with biopsy-proven chronic antibody-mediated (camr) rejection exhibits strong alterations of their tcr vβ repertoire correlating with the level of graft lesions classified with banff classification ( ) . in contrast, operationally tolerant patients [i.e., patients off-immunosuppression for more than months with a wellfunctioning graft ( ) ( ) ( ) ] exhibit a polyclonal tcr vβ repertoire ( ) . a large cohort of patients with stable graft function for more than years post-transplantation had been prospectively recruited in our center with stringent clinical and demographic inclusion criteria in order to obtain a homogeneous population. nevertheless, we could highlight that the usage of tcr vβ repertoire is highly heterogeneous ranging from the absence of clonal selection (similar to operational tolerance) to an accumulation of selected t cells (as for camr rejection) ( ) . the presence of altered tcr vβ repertoire has been previously reported in a rat model of camr ( ) in which similar cd clones could be identified in the blood and in the graft ( ) . in a large prospective study of kidney transplant recipients with a stable graft function for more than years, we show that the altered tcr vβ repertoire was due to an accumulation of temra (t cell effector memory re-expressing cd ra; cd ra + ccr − ) cd t cells with an activated profile (cd − cd − ), a high expression of cytotoxic molecules, perforin (perf) and granzym b (gzm-b) , t-bet, and cd and the ability to secrete tnf-α and ifn-γ ( ) . of interest, stable patients who have an increase in differentiated temra cd t cells have a twofold higher risk of long-term graft dysfunction ( ) . of note, using a similar strategy, kim et al. recently reported that clonal cd t cell could be evidenced in human transplanted hand, with several tcr clonal selections persisting at least days (among the days of surveillance) ( ) . collectively, these data highlight that a low-resolution technic provides key features as for the accumulation of selected t cell clones that can be used to monitor the kidney transplant recipients. a major drawback of spectratype-based method is its intrinsic low resolution as multiple t cell clones could share the same cdr length. it is necessary to sequence the tcr vβ chain with an altered cdr length distribution to assess the clonality of a given vβ family. however, we recently compared spectratype or next generation sequencing (ngs) techniques to characterize the tcr vβ repertoire in the blood, the cerebral spinal fluid (csf), and the central nervous system (cns) of patients with multiple sclerosis ( ) . both methods were as efficient to highlight the similarity of tcr vβ repertoire between csf and cns (≈ % of tcr vβ clones identified in the cns were also found in the csf) and to identify ≈ % of the tcr vβ clones using blood cd sample ( ) . as previously discussed, the size of donor-reactive t cell repertoire is large and constitutes a limitation to the use of deep-sequencing approach. it may thus be a naive approach to perform ngs on unfractioned t cells with the aim to identify t cell clones specific to a given situation, such as kidney transplantation or viral infection; a two-step approach is needed. the first step is to purify the t cell population of interest based on the expression of phenotypic (using tetramer for instance) or functional (e.g., cytokine secretion, proliferation) markers. the in-depth characterization of tcr vβ of t cell population of interest allows for the definition of a signature that can be later used as a tag when unpurified samples are analyzed. this approach has been used to track cmv-or bk-specific t cell clones ( ) or alloreactive t cells ( , ) . the first report hypothesizing such an approach in the transplant context has been published by the group of leventhal ( ) . using healthy volunteers, this study aimed to assess breadth, clonal structure, and dynamics of the alloreactive t cell repertoire. after days of mlr, the proliferating t cells were purified according to the dilution of cell division dye. by comparing the number of clones before culture and in the proliferated mlr responder, two types of alloreactive clones were identified, low-(i.e., unobserved in pre-culture sample and ≥ t cells after mlr) and high-abundance pre-culture clones (i.e., present in pre-culture sample and ≥ × enriched after mlr). more than , low-abundant clones and more than , high-abundant clones were detected in the different experiments. these data provide new evidences of the large size of the alloreactive t cell pool. this approach was used recently to track donor-reactive t cells in kidney transplant recipients ( ) . the fingerprint of donorreactive t cell repertoire was established before transplantation by deep-sequencing of proliferating cd and cd t cells after days of mlr. the fingerprint of donor-reactive t cells was monitored later after transplantation without the need to perform mlr. the team of sykes provides evidences that tolerance induction protocol based on combined kidney and non-myeloablative bone marrow transplantation results in a reduction of donor-alloreactive t cell clones. however, this decrease was neither observed in the patient that failed to respond to the tolerant inducing protocol nor in patients with standard immunosuppressive regimens. pretransplant identification of donor-reactive t cell clones before transplantation could thus be a means to track the activation of the immune system by allogeneic graft. the studies of emerson ( ) and morris ( ) showed that the anti-donor fingerprint is stable over-time in healthy volunteers. given the design of the assay, only pre-existing clones could be tracked. it would be of great value to compare the anti-donor clone repertoire before and after transplantation, starting each time from a direct mlr assay to investigate if new anti-donor t cells arise after transplantation. indeed, infections that occurred frequently after transplantation could generate virus-specific t cells with an allogeneic crossreactivity potential ( ) . moreover, not all proliferating cells after days of mlr are per se donor-specific as proliferation of t cells could also be linked to bystander stimulation ( ) . will transplant immunologists be able to track the rise and the expansion of donor-specific t cells and would this approach be widely available and useful to the clinical management are still open questions. high-through put techniques that have recently emerged are certainly an important step forward. nevertheless, the high cross-reactivity of t cells is a major hurdle to identify the trigger of the expansion of donor-reactive t cells, as donor antigen, viral peptides, and other environmental antigens can lead to the selection of donor-specific t cells. while promising, the study of tcr alteration has not overcome the double difficulties of offering an accessible technical presentation of the data and a validated correlation with clinical outcomes. therefore, longitudinal studies to test the reactivity of recipient t cells against donor antigens at different time points are needed. nd, sb, and j-p s wrote the review. this work was realized in the context of the labex igo program supported by the national research agency via the investment of the future program anr- -labx- - and the labex transplantex [anr- -labx- _transplantex], in the context of the ihu-cesti project which received french government financial support managed by the national research agency via the "investment into the future" program anr- -ibhu- and in the context of a centaure foundation. the ihu-cesti project is also supported by nantes metropole and the pays de la loire region. this work was also supported by the fp visicort project that has received funding from the european union's seventh framework program for research, technological development and demonstration under grant agreement no . we thank dr. yap and dr. haspot for their fruitful discussion. somatic generation of antibody diversity the complete -kilobase dna sequence of the human beta t cell receptor locus analysis of the . -mb human alpha/delta t-cell receptor locus with bacterial artificial chromosome clones estimating the diversity, completeness, and cross-reactivity of the t cell repertoire different tcrbv genes generate biased patterns of v-d-j diversity in human t cells exhaustive t-cell repertoire sequencing of human peripheral blood samples reveals signatures of antigen selection and a directly measured repertoire size of at least million clonotypes junctional sequences of t cell receptor gamma delta genes: implications for gamma delta t cell lineages and for a novel intermediate of v-(d)-j joining most alpha/beta t cell receptor 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cell receptor-peptide-major histocompatibility complex complexes: a buried alloreactive mutation subtly alters peptide presentation substantially increasing v(beta) interactions crystal structure of a t cell receptor bound to an allogeneic mhc molecule association between specific hla combinations and probability of kidney allograft loss: the taboo concept bone marrow-allograft rejection by t lymphocytes recognizing a single amino acid difference in hla-b hla-b -directed cytotoxic t cells associated with acute graft-versus-host disease following unrelated bone marrow transplantation characterization of natural peptide ligands for hla-b* and -b* : implications for peptide involvement in allorecognition of a single amino acid change in the hla-b heavy chain a naturally selected dimorphism within the hla-b supertype alters class i structure, peptide repertoire, and t cell recognition human peripheral blood cd t cell-engrafted non-obese diabetic-scid il rγ(null) h -ab (tm gru) tg (human leucocyte antigen d-related ) mice: a mouse model of human allogeneic graft-versus-host disease characterization of an lyt- + alloreactive cytotoxic t cell clone specific for h- db that cross-reacts with i-ek restricted mhc-peptide repertoire predisposes to autoimmunity how the t cell repertoire becomes peptide and mhc specific a single t cell receptor bound to major histocompatibility complex class i and class ii glycoproteins reveals switchable tcr conformers quantitative assay of antigenic disparity at hl-a-the major histocompatibility locus in man quantifying the frequency of alloreactive t cells in vivo: new answers to an old question predominant naturally processed peptides bound to hla-dr are derived from mhc-related molecules and are heterogeneous in size specificity and promiscuity among naturally processed peptides bound to hla-dr alleles sequence analysis of peptides bound to mhc class ii molecules broadly targeted human cytomegalovirus-specific cd + and cd + t cells dominate the memory compartments of exposed subjects advances in direct t-cell alloreactivity: function, avidity, biophysics and structure t cell receptor repertoire for a viral epitope in humans is diversified by tolerance to a background major histocompatibility complex antigen cross-reactivity of herpesvirus-specific cd t cell lines toward allogeneic class i mhc molecules an alloresponse in humans is dominated by cytotoxic t lymphocytes (ctl) cross-reactive with a single epstein-barr virus ctl epitope: implications for graft-versus-host disease cross-reactive memory t cells for epstein-barr virus augment the alloresponse to common human leukocyte antigens: degenerate recognition of major histocompatibility complex-bound peptide by t cells and its role in alloreactivity cross-recognition of hla dr alloantigen by virus-specific cd + t cells: a new paradigm for self-/nonself-recognition ebv-specific cd + t cell clones exhibit vigorous allogeneic responses cross-recognition of human alloantigen by cytomegalovirus glycoprotein-specific cd + cytotoxic t lymphocytes: implications for graft-versus-host disease allo-hla reactivity of virus-specific memory t cells is common allografts stimulate cross-reactive virus-specific memory cd t cells with private specificity circulating t cell repertoire complexity in normal individuals and bone marrow recipients analyzed by cdr size spectratyping. correlation with immune status molecular detection and in vivo analysis of the specific t cell response to a protein antigen statistical analysis of cdr length distributions for the assessment of t and b cell repertoire biases expanded cd t-cell sharing between periphery and cns in multiple sclerosis the blood of healthy individuals exhibits cd t cells with a highly altered tcr vb repertoire but with an unmodified phenotype t cell repertoire alterations of vascularized xenografts direct recognition of foreign mhc determinants by naive t cells mobilizes specific vbeta families without skewing of the complementarity-determining region length distribution highly altered v beta repertoire of t cells infiltrating long-term rejected kidney allografts immune responses elicited in tertiary lymphoid tissues display distinctive features improved assessment of t-cell receptor (tcr) vb repertoire in clinical specimens: combination of tcr-cdr spectratyping with flow cytometry-based tcr vb frequency analysis blood t-cell vbeta transcriptome in melanoma patients serial blood t cell repertoire alterations in multiple sclerosis patients; correlation with clinical and mri parameters serial evolution of tcr beta chain transcript mobilization in hiv type- -infected patients following vaccine immune stimulation and haart interruption operationally tolerant and minimally immunosuppressed kidney recipients display strongly altered blood t-cell clonal regulation analysis of the peripheral t-cell repertoire in kidney transplant patients expansion of highly differentiated cytotoxic terminally differentiated effector memory cd + t cells in a subset of clinically stable kidney transplant recipients: a potential marker for late graft dysfunction identification of a peripheral blood transcriptional biomarker panel associated with operational renal allograft tolerance the natural history of clinical operational tolerance after kidney transplantation through twenty-seven cases clinical operational tolerance after kidney transplantation indirect cd + th response, antidonor antibodies and diffuse c d graft deposits in long-term recipients conditioned by donor antigens priming functional compartmentalization following induction of long-term graft survival with pregraft donor-specific transfusion clonal cd + t cell persistence and variable gene usage bias in a human transplanted hand tcr repertoire analysis by next generation sequencing allows complex differential diagnosis of t cell-related pathology defining the alloreactive t cell repertoire using high-throughput sequencing of mixed lymphocyte reaction culture tracking donor-reactive t cells: evidence for clonal deletion in tolerant kidney transplant patients high-resolution characterization of cytokine-producing alloreactivity in naive and allograft-primed mice the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord- -kldu q x authors: oany, arafat rahman; sharmin, tahmina; chowdhury, afrin sultana; jyoti, tahmina pervin; hasan, md. anayet title: highly conserved regions in ebola virus rna dependent rna polymerase may be act as a universal novel peptide vaccine target: a computational approach date: - - journal: in silico pharmacol doi: . /s - - - sha: doc_id: cord_uid: kldu q x purpose: ebola virus (ebov) is such kind of virus which is responsible for , cases and deaths worldwide only in and with an average diseases fatality rate between % and %. although, medical technology has tried to handle the problems, there is no food and drug administration (fda)-approved therapeutics or vaccines available for the prevention, post exposure, or treatment of ebola virus disease (evd). methods: in the present study, we used the immunoinformatics approach to design a potential epitope-based vaccine against the rna-dependent rna polymerase-l of ebov. bioedit v . . sequence alignment editor, jalview v and clc sequence viewer v . . were used for the initial sequence analysis for securing the conservancy from the sequences. later the immune epitope database and analysis resource (iedb-ar) was used for the identification of t-cell and b-cellepitopes associated with type i and ii major histocompatibility complex molecules analysis. finally, the population coverage analysis was employed. results: the core epitope “fryeftapf” was found to be the most potential one, with % conservancy among all the strains of ebov. it also interacted with both type i and ii major histocompatibility complex molecules and is considered as nonallergenic in nature. finally, with impressive cumulative population coverage of . % for the both mhc-i and mhc-ii class throughout the world population was found for the proposed epitope. conclusion: to end, the projected peptide gave us a solid stand to propose for vaccine consideration and that might be experimented for its potency in eliciting immunity through humoral and cell mediated immune responses in vitro and in vivo. electronic supplementary material: the online version of this article (doi: . /s - - - ) contains supplementary material, which is available to authorized users. background evd, previously designated as ebola haemorrhagic fever, is a fatal disease in humans and other mammals (monkeys, chimpanzees and gorillas) (choi and croyle , leroy et al. , sullivan et al. . the fatality rate of edv is varied from to % with an average of about % (peters and peters ) (hoenen et al. ). the fifth one, reston virus (reston ebolavirus), is harmful to nonhuman primates, but not to humans (elisha and adegboro , geisbert et al. ). among the recognized species of ebolavirus, the notoriously deadly zaire ebolavirus is responsible for epidemics which have been taken place mainly in african countries including democratic republic of congo, uganda, sudan, the ivory coast, and gabon (baize et al. , chowell et al. , feldmann et al. , frieden et al. , hewlett and hewlett , kuhn et al. , li and chen , rouquet et al. . this virus is passed on people from wild animals and through human-to-human contact transmits in the human population. those are infected with this virus bear fearsome symptoms, including high fever, hemoptysis, impaired kidney and liver function and severe internal bleeding (gatherer , goeijenbier et al. , keiser et al. , peters and peters . in the fall of the ebola virus gained widespread attention when in west africa the largest outbreak has been reported in history. the ebov genome is a single-stranded, negative-sense, non-segmented rna approximately kb long. it codes for seven tandemly arranged viral genes which order is ′ leader-np (nucleoprotein) -vp (virion protein )-vp -gp (glycoprotein)-vp -vp -l (rna-dependent rna polymerase)-trailer − ′. transcription and translation of this viral genome result in the synthesis of seven structural proteins and a single non-structural, secreted glycoprotein (feldmann et al. ) . three of the structural proteins are membrane-associated proteins; gp is a type i transmembrane protein, while vp and vp are placed on the inner surface of the membrane. the remaining four, np, vp (transcription factor), vp (polymerase cofactor), and l (rna-dependent rna polymerase), are essential to viral genomic rna to form the ribonucleoprotein complex. these proteins have been shown to be necessary and sufficient for ebov transcription and replication (crary et al. , feldmann et al. , mühlberger et al. , takada et al. ). to date, information regarding the processing, structure and functions of ebola virus (ebov) protein l (ebol) demonstrates that it is an rna-dependent rna polymerase, with the assistance of vp . it also shows mrna (guanine-n ( )-)-methyltransferase, mrna guanylyltransferase and poly (a) synthetase activities which are essential for the replication and transcription of ebov (poch et al. ) . the viral mrna guanylyltransferase serves either as transcriptase or as replicase. the transcriptase synthesizes subgenomic rnas, assures their capping and polyadenylation. the transcriptase stutters on a specific sequence, leads to a co-transcriptional editing of the glycoprotein (gp) mrna. in replicase mode, the polymerase replicates the viral genome without recognizing the transcriptional signals. these reports suggest that ebol is an important cellular component for the transcription and replication of the ebov genome and, as such, plays a key role in the ebov life cycle. due to the emergence of ebola virus outbreak, there is an immediate need to determine novel therapeutic targets against this pathogen. the identification of specific epitopes derived from infectious pathogens has significantly advanced the development of epitope-based vaccines (evs). bettered understanding of the molecular basis of antigen recognition and hla binding motifs has resulted in the advancement of rationally designed vaccines depend on algorithms predicting the peptide's binding to human hla. in comparison to the conventional vaccines, peptide or epitope based vaccines are easy to develop, chemical stable, more specific, and free of any infectious or oncogenic potential hazard (holland and domingo , sette et al. ) . though evs have varied advantages, the wet lab based discovery of candidate epitopes is expensive and time consuming. furthermore, for the final selection of epitopes various immunological requirements are needed to be considered. as a result computational methods, an alternative in silico approaches (germain ) have recently been attracting growing interest of the researchers for predicting epitopes with reduced cost and time. the application of bioinformatics in immunology is termed as immunoinformatics. currently, numerous immunoinformatics tools are available for identifying b and t cell epitopes and human leukocyte antigen (hla) ligands (petrovsky and brusic , poland et al. , sette and fikes with high sensitivity and specificity. the 'immunoinformatics' approach has already proven its potency in the case of human immunodeficiency virus (wilson et al. ) , multiple sclerosis (bourdette et al. ) , tuberculosis (robinson and amara ) and malaria (lópez et al. ) with desired results. in the present study, we have followed immunoinformatics approaches for designing potential conserved epitope candidate for the utility of vaccine development against the deadly ebola virus, with an expectation of further wet lab validation. the protein sequences of the rna-dependent rna polymerase-l ) of the ebov were retrieved from the uniprotkb (apweiler et al. ) database in the fasta format. bioedit v . . sequence alignment editor (apweiler et al. ) was used for the identification of the conserved region among the sequences through multiple-sequence alignment (msa) with clustalw (hall ) . finally, jalview v tool (thompson et al. ) was used to retrieve the alignment and the clc sequence viewer v . . (http:// www.clcbio.com) was used for analysis of the divergence among the different strains of the ebov. vaxijen v . , a web-based server (waterhouse et al. , doytchinova and flower ) was used for the determination of the antigenicity of the conserved sequences. herein, we used the default parameters for the prediction, with a threshold value of . . for this study, two online servers were used. firstly, the netctl v . server (larsen et al. ) was used for predicting potential cytotoxic t lymphocyte (ctl) epitopes from the conserved peptides. here for predicting the epitopes, we used a combined algorithm including major histocompatibility complex class i (mhc-i) binding, transporter of antigenic peptide (tap) transport efficiency, and proteasomal c terminal cleavage prediction. depending on the score, the best candidates were picked for further investigation. the epitope prediction was confined to mhc-i supertypes. mhc-i binding and proteasomal cleavage were carried out through artificial neural networks and the weight matrix was used to estimate the tap transport efficiency. the threshold value for epitope identification was set at . for maintaining sensitivity and specificity of . and . , respectively during the analysis. this would support to assess the findings more decisively by developing more epitopes. finally, for confirming the prediction with default parameters, ctlpred (bhasin and raghava ) was employed additionally. furthermore, from the immune epitope database and analysis resource (iedb-ar), t cell epitope prediction tools was implied for the identification of mhc-i (hoof et al. , nielsen et al. ) and mhc-ii (wang et al. ; binding of the peptide. in order to calculate the half-maximal inhibitory concentration (ic ) values required for peptide binding to mhc-i molecules, stabilized matrix method (peters and sette ) was applied with a preset . -mer epitope. in case of mhc-ii binding analysis, the iedb-recommended method was used for the specific hla-dq, hla-dp, and hla-dr loci. herein, specific peptides were used to predict the mhc-ii interaction on the basis of mhc-i analysis and antigenic conservancy. population coverage for epitope was assessed by the iedb population coverage calculation tool (bui et al. ). here we used the allelic frequency of the interacting hla alleles for the prediction of the population coverage for the corresponding epitope. linear b cell epitopes are of different lengths of peptides from to in comparison to that of t cell epitopes. b-cell epitope produces immune response when it interacts with b lymphocytes. it then initiates the differentiation of b lymphocytes into plasma and memory cells (nair et al. ) . there are a number of web-based tools are available for the prediction of b-cell epitope which are hosted by iedb-ar. for the b-cell epitope prediction with high accuracy, multiple tools, including the emini surface accessibility prediction (emini et al. ) , kolaskar and tongaonkar antigenicity scale (kolaskar and tongaonkar ) , parker hydrophilicity prediction, (parker et al. ) and finally the chou and fasman beta turn prediction tool (chou and fasman ) were employed, because the antigenic parts of a protein belong to the beta turn regions (rini et al. ) . the structure of the conserved region was constructed by homology modelling using the modeller v (Šali et al. ) . modeller is a program that implements an automated approach to comparative protein structure modelling by satisfying spatial restraints (fiser et al. , sali and blundell ) . finally, the evaluation of the predicted model was verified by using two software tools, procheck (arnold et al. , laskowski et al. and qmean (benkert et al. ) . for predicting the disorder among the amino acid sequences, disopred v (ward et al. ) server was used. in order to calculate the protein variability index the protein variability server was implied where wu-kabat variability coefficient (garcia-boronat et al. ) has been used. the web-based allerhunter server (muh et al. ) was used to predict the allergenicity of our proposed epitope for vaccine development. this server predicts allergenicity through a combinational prediction, by using both integration of the food and agriculture organization (fao)/world health organization (who) allergenicity evaluation scheme and support vector machines (svm)-pairwise sequence similarity. allerhunter predicts allergens as well as nonallergens with high specificity. this makes allerhunter is a very useful program for allergen cross-reactivity prediction (liao and noble ) . epitope conservancy of the candidate epitopes was examined using a web-based epitope conservancy tool available in iedb analysis resource (bui et al. ). the conservancy level of each potential epitope was calculated by looking for identities in all rna-dependent rna polymerase-l protein sequences of different strains retrieved from database. a total of rna-dependent rna polymerase-l protein molecules from different variants of the ebov were retrieved from the uniprot database. the msa of the rnadependent polymerase-l proteins was retrieved from bioedit tool through clustalw with bootstrap replicates (additional file : figure s ). clc sequence viewer was used to construct phylograms from the msa obtained from bioedit, in order to analyze the divergence among the retrieved sequences. phylogram of rna-dependent rna polymerase-l is depicted in fig. . finally, the highly conserved region from the msa was retrieved for the further analysis. the selected conserved region is depicted in the fig. , from the msa number to . then the vaxijen v . server calculate the antigenicity of the conserved sequences with a score . . t-cell epitopes were selected firstly by using the netctl v . server where the epitope prediction was confined to mhc-i supertypes. based on the combined score, the top five epitopes (table ) were listed for further analysis. t-cell epitopes were again predicted by the ctlpred server (table ) . here a combined approach of artificial neural networks and support vector machines was applied. depending on the two analyses, the most common epitope-containing peptides, identified by both servers, was selected. the selected epitope was then used for the mhc-binding analysis. mhc-i-binding prediction, which was run through the stabilized matrix method, predicted a wide range of mhc-i allele interactions for the proposed t-cell epitopes. the mhc-i alleles for which the epitope showed higher affinity (ic < nm) are listed in table . the output of the mhc-ii interaction analysis is also shown in table . iedb population coverage tool analyzed the population coverage of the proposed epitope. the combined mhc-i and mhc-ii class were assessed against the whole world population with the selected mhc-i and mhc-ii interacted alleles (fig. ) . here, for predicting potential b-cell epitopes, we used amino acid-based methods. according to this procedure different analysis methods were applied for the identification of a continuous b cell epitope. the kolaskar and tongaonkar antigenicity scale was used for assessing the antigenic property of the peptides. the average antigenic propensity of the protein was . , with a maximum of . and a minimum of . . for the protein the antigenic determination threshold value was . , where all values equal or greater than . were potential antigenic determinants. the antigenic plot is depicted in the fig. . to be a potent b cell epitope, it must be surface accessible. hence, emini surface accessibility prediction was employed, with a maximum propensity score of . at threshold . (fig. ) . to strengthen our support for the prediction of the epitope to elicit b cell response the parker hydrophilicity and the chou and fasman beta turn prediction were employed. those are described in the figs. and . homology model of the conserved region was obtained by the modeller software, which is shown in fig. a and b. procheck server validated the stereochemical quality of the model through ramachandran plot (fig. c) , andqmean server also assessed the tertiary structure, with a qmean score of . . disopred v server predicted the disorder of the conserved peptide in order to get insight about the disorder among the conserved sequences, which is depicted in fig. . protein variability server predicted the variability of the conserved region of the rna-dependent rna polymerase-l ( fig. ) to ensure that the proposed epitope is within the invariable region. conservation analyses of the proposed epitopes were analyzed by the iedb conservancy analysis tool that is shown in table . allerhunter server predicted the our world is the habitation of more than seven billion people now. with the upgrade of medical science, new viruses along with their causing diseases are also emerging. ebola virus is such kinds of virus with a deadly outrage of their endemic nature especially in africa in recent time (evans and popova ) . till now there is no potential treatment for this virus to combat its deadly effects. recent time, the immunoinformatics approach give us some sort of hope for the design of an effective therapeutics, like vaccine, in association with the advancement of sequence based technology. similar approaches have been used successfully for identifying vaccine candidates in several pathogens viz. human corona virus (oany et al. ) , saint louis encephalitis virus (hasan et al. ) , crimean-congo hemorrhagic fever virus fig. parker hydrophilicity prediction of the epitope, with a minimum propensity score of − . and maximum score of . . notes: the x-and y-axes represent the sequence position and antigenic propensity score, respectively. the threshold value is . . the regions above the threshold are antigenic, shown in yellow fig. chou and fasman beta turn prediction of the epitope,with a minimum propensity score of . and maximum score of . . notes: the x-and y-axes represent the sequence position and antigenic propensity score, respectively. the threshold value is . . the regions above the threshold are antigenic, shown in yellow (oany et al. ) , chikungunya virus (hasan et al. ) and some others. the in vitro validation of this type of work has also been proven in recent time (khan et al. ) . though epitope-based vaccine designing has become a familiar approach, in the case of ebov no significant work yet has been done. ebov is an rna virus which has genetic blueprints made of rna instead of dna. creating vaccines is particularly difficult for rna viruses as they can quickly mutate their different exposed proteins (twiddy et al. ) . therefore the most potential way to create stable antiviral therapies against rna viruses including ebov is to target the transcription or replication machinery. scientists revealed that rnadependent rna polymerase-l (ebol) is an important cellular component for the transcription and replication of the ebov genome. when an ebov infects a cell, its rna genetic blueprint enters the cell along with rna-dependent rna polymerase-l. this polymerase normally "read" the rna genetic blueprint in order to synthesize mrna, which then leads to the formation of viral proteins as well as viral replication and more viral particles are produced. for these two vital involvements at the gateway, this protein was targeted to design most potential epitopes using in silico computational approaches. in the current study, firstly all the available sequences of rna-dependent rna polymerase-lwere retrieved from database. then antigenicity of the conserved peptides, generated by multiple sequence alignment was predicted by vaxijen, which suggested their ability to elicit potential immune response. sequence based bioinformatics approaches were applied to predict both b cell and t cell epitopes for conferring immunity in different ways. though at present, most of the vaccines are based on b cell immunity; vaccines based on t cell epitope have been encouraged recently. it is because, with time humoral response from memory b cells can be overcome easily by antigenic drift, while cell mediated immunity often provides long lasting immunity (bacchetta et al. , igietseme et al. ). cytotoxic cd + t lymphocytes (ctl) inhibit the spread of infectious agents by recognizing and killing infected cells or secreting specific antiviral cytokines (garcia et al. , shrestha and diamond ) . thus, vaccination based on t cell epitope is a unique approach to obtain strong immune response against infectious agents, such as, viruses (klein et al. ) . both netctl and ctlpred server were used to find epitopes for the activation of t-cell immunity with potential antigenicity. by examining the output it was predicted that fryeftapf would be the best epitope candidate and was further subjected for binding proficiency analysis. length is an important factor to consider for peptide antigen binding with mhc or tcr or both. t cell epitopes presented by mhc class i molecules are generally peptides between and amino acids in length. we therefore set peptide lengths at before making software based mhc class i t cell epitope identification using fig. protein variability index of the conserved peptides of all the sequences. the prediction suggests that our proposed epitope "fryeftapf" falls in the invariable region (blue line). notes: the conservancy threshold was . in this analysis. the x-axis indicates the amino acid positions in the sequences and the y-axis indicates the shannon variability score fig. disorder prediction of the conserved antigenic amino acid sequences. here, our proposed epitope lies outside ( - ) of the disordered region to secure its potentiality as an effective epitope. notes: amino acids in the input sequence are considered disordered when the blue line is above the gray dashed line, that is, when the confidence score is . . the orange line shows the confidence score of the disordered protein-binding residue predictions immune epitope database (iedb). analysis revealed that the core epitope "fryeftapf" would interact with ten different mhc class i alleles. on the other hand, the complete peptide "nlafryeftapfiey" interacts with the highest numbers of mhc class ii alleles (as many as alleles). along with the t-cell epitope, in our study, attention was also given to the b-cell epitope, which can induce both primary and secondary humoral immunity (trainor et al. ). multiple prediction methods were applied to determine the b-cell epitope considering several criteria of antigenicity, hydrophilicity, surface accessibility, and beta-turn. our proposed epitope has met all the criteria of the above b-cell prediction methods. the three-dimensional model of the conserved protein ensured the exact location of the epitope outside of the protein ( fig. a and b ) surface and the model validity was assessed by ramachandran plot (fig. c) , whereby . % amino acid residues were found within the favored region. the epitope was also treated as suitable candidate for vaccine through tenabled its position in the conserved sequence, by the discopred and protein variability server (figs. and ). conservancy is the most important criterion of an epitope to consider it for vaccine development. conservancy analysis of our proposed epitope showed % conservancy among all the available sequences. another important feature of the peptide vaccine is its allergenicity (mckeever et al. ) . in silico analysis revealed that the proposed epitope is nonallergenic in nature. wide range population coverage must be needed for a potential vaccine aspirant. at this point, our proposed epitope covers a remarkable population of . % for both types of mhc allele throughout the world population. that makes the epitope as a supreme candidate for vaccine consideration. finally, from the above in silico analysis, we are really optimistic that our proposed epitope would trigger an immune response in vitro and in vivo. a number of approaches exist for new vaccine development, such as recombinant vaccines, sub-unit protein and dna vaccines, auxotrophic organisms to deliver genes and so on. current study is an attempt to identify potential epitope targets against ebov using different computational tools. it is quite obvious that in order to minimize the deadly effects of ebov, highly potential drugs are immediately required and these in silico approaches will reduce the wet lab efforts with higher probability of success. therefore, it is concluded that the identified epitope may be exploited further for developing epitope-based vaccine against ebov. nevertheless, the initial hints we obtained will help to prioritize potential therapeutics for ebov. additional file : figure s . msa of the rna-dependent polymerase-l proteins of the different ebov. 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system for functional analysis of ebola virus glycoprotein clustal w: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice mutation analysis of the fusion domain region of st. louis encephalitis virus envelope protein inferring the rate and time-scale of dengue virus evolution characterization of the l gene and ′trailer region of ebola virus a systematic assessment of mhc class ii peptide binding predictions and evaluation of a consensus approach peptide binding predictions for hla dr, dp and dq molecules the disopred server for the prediction of protein disorder jalview version -a multiple sequence alignment editor and analysis workbench development of a dna vaccine designed to induce cytotoxic t lymphocyte responses to multiple conserved epitopes in hiv- the authors declare that they have no competing interests.authors' contributions aro has made substantial contributions to conception and design, acquisition of data, analysis and interpretation of data. ts and asc carried out the molecular genetic studies, participated in the sequence alignment and drafted the manuscript. tpj worked for computational analysis. mah conceived of the study, and participated in its design and coordination and helped to draft the manuscript. all authors read and approved the final manuscript. convenient online submission rigorous peer review immediate publication on acceptance open access: articles freely available online high visibility within the fi eld retaining the copyright to your article submit your next manuscript at springeropen.com key: cord- - t kly i authors: salmier, arielle; de thoisy, benoit; crouau-roy, brigitte; lacoste, vincent; lavergne, anne title: spatial pattern of genetic diversity and selection in the mhc class ii drb of three neotropical bat species date: - - journal: bmc evol biol doi: . /s - - - sha: doc_id: cord_uid: t kly i background: although bats are natural reservoirs of many pathogens, few studies have been conducted on the genetic variation and detection of selection in major histocompatibility complex (mhc) genes. these genes are critical for resistance and susceptibility to diseases, and host–pathogen interactions are major determinants of their extensive polymorphism. here we examined spatial patterns of diversity of the expressed mhc class ii drb gene of three sympatric neotropical bats, carollia perspicillata and desmodus rotundus (phyllostomidae), and molossus molossus (molossidae), all of which use the same environments (e.g., forests, edge habitats, urban areas). comparison with neutral marker (mtdna d-loop) diversity was performed at the same time. results: twenty-three drb alleles were identified in c. perspicillata, alleles in d. rotundus and alleles in m. molossus. the occurrence of multiple drb loci was found for the two phyllostomidae species. the drb polymorphism was high in all sampling sites and different signatures of positive selection were detected depending on the environment. the patterns of drb diversity were similar to those of neutral markers for c. perspicillata and m. molossus. in contrast, these patterns were different for d. rotundus for which a geographical structure was highlighted. a heterozygote advantage was also identified for this species. no recombination or gene conversion event was found and phylogenetic relationships showed a trans-species mode of evolution in the phyllostomids. conclusions: this study of mhc diversity demonstrated the strength of the environment and contrasting pathogen pressures in shaping drb diversity. differences between positively selected sites identified in bat species highlighted the potential role of gut microbiota in shaping immune responses. furthermore, multiple geographic origins and/or population admixtures observed in c. perspicillata and m. molossus populations acted as an additional force in shaping drb diversity. in contrast, drb diversity of d. rotundus was shaped by environment rather than demographic history. electronic supplementary material: the online version of this article (doi: . /s - - - ) contains supplementary material, which is available to authorized users. regarding adaptation to infectious diseases, genes of the major histocompatibility complex (mhc) are the most commonly studied in mammals. mhc genes play a significant role in the functionality and effectiveness of the immune response of vertebrates and are directly involved in fitness and adaptation of hosts to their pathogens [ ] . therefore, studying mhc gene diversity in non-model animals, known to be reservoirs of pathogens, is a critical tool to assess its evolutionary process and implication in ( ) the variation of individual fitness, ( ) population viability and ( ) evolutionary potential in a changing environment [ ] . studies have essentially focused on the genetic variability of exon of the mhc class ii dr beta (drb) gene given that this exon encodes the peptidebinding region (pbr) [ ] . hence, most of the variability displayed is recorded on this exon. variations within the pbr define the repertoire of antigenic determinants and subsequently the ability to recognize a variable number of circulating pathogens [ ] . optimum resistance to pathogens is, therefore, the result of all intrinsic and extrinsic factors that influence the genetic variability of individuals' immune system [ ] . most of the vertebrate populations studied so far exhibited high levels of mhc diversity both in the number of alleles and in their heterozygosities and allelic variation [ ] [ ] [ ] [ ] . pathogen-driven balancing selection and sexual selection seem to play a fundamental role in the preservation of this high level of polymorphism [ ] . pathogen-driven balancing selection acts through antagonistic host-parasite coevolution via several mechanisms: overdominant and frequency-dependent selection [ ] , as well as spatial and temporal variation in host pathogens [ ] . sexual selection pressures encompass mechanisms such as maternal-fetal interactions [ ] and mate selection [ ] . however, when assessing the genetic variability of both neutral markers and mhc genes, studies have highlighted the role of past demographic processes (e.g., fragmentation, bottlenecks, geographic isolation) in shaping the pattern of mhc variability that can sometimes surpass that of natural selection [ ] [ ] [ ] [ ] [ ] [ ] . local immunogenetic adaptation of hosts that live in different environments was associated with different parasite and pathogen pressures [ ] [ ] [ ] [ ] . indeed, differences in the diversity of pathogens (inducing different selection pressures on the hosts) are directly linked to environmental components. these latter (e.g., vegetation cover and density, landscape fragmentation, human occupation) modulate parasite and pathogen species richness, their survival and adaptability, as well as their distribution, transmission, developmental success and their ability to induce diseases [ ] . environmental components likewise impact the richness, population dynamics, immunocompetence and nutritional status of host species, all of which subsequently determine resistance or susceptibility to disease [ , ] . a strong correlation was also shown between host and parasite species richness, their life history and ecological traits [ ] [ ] [ ] . moreover, anthropogenic alterations of habitats induce changes in host-pathogenenvironment interactions and are consequently linked to the emergence of infectious zoonotic diseases [ ] [ ] [ ] . therefore, considering the role of the environment is critical for the assessment of mhc gene variability. bats (chiroptera) are the second largest species-rich mammalian order, accounting for % of all mammal species in the world. compared to other mammals, their uncommon biological features (e.g., ability to fly, long lifespan, complex social structures) and their long-term evolutionary history with pathogens make them excellent reservoirs and spreaders of viruses (e.g., rabies virus, henipaviruses, coronaviruses), which have a significant impact on both human and animal health [ ] . however, bats rarely exhibit clinical or pathological signs of diseases and appear to coexist with their pathogens in a disease-free state, characteristic of reservoir hosts [ ] . at the order level, the monophyly of chiroptera was evidenced via phylogenetic relationships inferred from intron sequences of drb [ ] . the monophyletic origin of drb genes was also evidenced at the family level via phylogenetic relationships inferred from drb sequences from saccopteryx bilineata, myotis spp., noctilio spp. and carollia perspicillata [ ] . other studies, investigating the diversity of mhc drb, showed significant differences in the polymorphism of mhc genes between species. this polymorphism was essentially influenced by ( ) a pathogen-driven selection [ ] , ( ) a social structure driven by mhc-mediated post-copulatory mechanisms [ ] , ( ) diversifying selection and recombination events [ , ] and ( ) geographical constraints resulting in spatial variation of pathogen-mediated selection and enhanced susceptibility to environmental changes [ , ] . the amazon, a major biodiversity hotspot in south america, possesses a large diversity of bat species and pathogens in a wide variety of climates and vegetation formations [ , ] . french guiana, a tropical amazonian region near the equatorial zone, harbors a great diversity of bats, with species registered [ ] . in this region, the impact of deforestation on the composition and dynamics of bat communities was assessed [ , ] as well as, the role of habitats on rabies virus circulation and maintenance [ ] . in this study, we analyzed the genetic variability of the expressed mhc class ii drb exon in three bat species: two phyllostomidae, carollia perspicillata and desmodus rotundus, and one molossidae, molossus molossus. these three widely distributed species have ecological plasticity and tolerance to disturbance. in french guiana, these species are sympatric and use the same habitats (e.g., forests, edge habitats, and urban areas). the key hypothesis of this study was that composition and distribution of mhc drb alleles were specific to the environments (forests vs. disturbed areas), rather than randomly distributed in space. consequently, we should observe local immunogenetic adaptation to the contrasting pathogen pressures or equally adapted alleles. to assess which are the best factors that predict the mhc diversity, pathogen-mediated selection, recombination, gene conversion, demographic history and population structure were investigated. there is a higher diversity of microorganisms in forest environments, compared to disturbed environnments, due to greater host species richness and better transmission-promoting parameters [ , ] . for this reason, we expect higher levels of mhc diversity in forest environments facing lower disturbance pressures, where higher parasite and pathogen diversities imply a higher selection pressure. furthermore, assuming that bats using the same roosting area and/or the same foraging areas would be subjected to similar pathogen pressures, we should observe similar trends in intra-and inter-specific mhc diversity. in contrast, once the demographic neutral genetic histories-which may also influence mhc diversity-are controlled, we should observe differences in selective histories between bats inhabiting different environments. to identify different signatures of selection in the drb exon , which would imply area-specific recognition capabilities, conformation of the hla-drb was used as a reference to detect putative sites directly involved in antigen binding (abs) and consequently subjected to selection. additionally, to reduce the misidentification bias of abs, the selection signature within the pbr was investigated without a priori identifying species-specific abs for further comparisons. mhc genes exhibit high levels of allele similarity within species as well as between related species and the occurrence of identical mhc alleles in related species is frequent. convergence and trans-species polymorphism are thought to be responsible for this trans-species evolution. to highlight which of these two mechanisms acts predominantly on the evolutionary history of the drb gene in the three species investigated, phylogenetic relationships were inferred from the drb sequences obtained here and with other available chiropteran sequences. finally, mhc spatial diversity was compared to that of neutral markers (mtdna d-loop) to highlight the impact of demographic processes and population structure on the diversity pattern in the three bat species investigated. animals were captured, handled, sampled and, whenever necessary, euthanized following asm guidelines [ ] under the supervision of researchers who had been granted the french "expérimentation animale niveau " diploma. bats are not protected by law in french guiana; however, the project was submitted to the conseil scientifique régional pour le patrimoine naturel de la guyane and approved. captures that occurred within protected areas (nature reserves) received approval by the conseil scientifique the three bat species (c. perspicillata, d. rotundus and m. molossus) were sampled at sites. three sites were located in pristine lowland forests (pf sites) with low disturbance pressure, four sites were located in edge habitats (eh sites), five in anthropized areas (aa sites), and two in urban and periurban areas (ur sites) (fig. , additional file : table s and additional file : figure s ). bats were trapped with mist nets erected near breeding sites, roosts, at forest edges, around livestock, or through putative foraging courses. bat species were identified in the field using external morphology and, prior to release, a brachial vein puncture and wing biopsies were performed. a few individuals captured were euthanized at the laboratory to collect organs. a total of c. perspicillata, d. rotundus and m. molossus were sampled. nucleic acids were extracted from blood, liver or spleen depending on sampling. extractions were performed using the nuclisens easymag bio-robot (biomérieux®). amplification and genotyping of the mhc class ii drb gene all amplifications of mhc drb genes for the three species were performed using cdna obtained from liver and spleen rnas. all cdnas were synthesized using superscript® iii reverse transcriptase (invitrogen) and random hexamers (roche) following the manufacturer's recommendations. expressed mhc drb genes of the three bat species were amplified by a minimum of two independent polymerase chain reactions (pcrs) using a standard procedure with the amplitaq gold dna polymerase pcr kit (thermo fisher scientific). to optimize the detection of different alleles, a minimum of two distinct pcrs per individual were performed. speciesspecific primers were designed for c. perspicillata and d. rotundus to avoid occurrence of non-amplifying alleles (table ) . to amplify the m. molossus drb gene, primers designed by richman et al. [ ] were modified. primers located in conserved parts of exon (jsex . , [ ] ; ex fd, modified from [ ] ), exon (js cape, [ ] ), exon (ex rd, modified from [ ] ) and exon (l , [ ] ) were used to amplify functional mhc class ii drb alleles from the cdna of three bats from each species, c. perspicillata, d. rotundus and m. molossus. the sequences obtained were used to design specific primers complementary to conserved parts of exon (batcmhiif (f) and batcmhiif. (f ), table ) and exon (batcmhiir (r) and batcmhiir. (r ), table ). the primer combinations f-r and f -r were used as the first pcr for d. rotundus and c. perspicillata (additional file : figure s ). for the second pcr, all of the newly designed primers were used together with jsex . , ex rd, ex rd and l in different combinations to screen the cdna of a total of c. perspicillata and d. rotundus (additional file : figure s ). two different primer combinations with js cape, ex rd, r and r were used to amplify the drb alleles from the cdna of m. molossus (additional file : figure s ). the pcr products were cloned using the pcr™ -topo® ta cloning® kit (invitrogen). ten clones per pcr amplification were sent for sequencing to beckman coulter genomics (takeley, uk) using t and t primers. all amplifications of mitochondrial dna (mtdna) control region (d-loop) for the three species were performed using genomic dna. the primers f(mt) and p(mt) [ ] were used to amplify the hypervariable domain hvi of mtdna d-loop. pcrs were performed using a standard procedure with biotaq™ dna polymerase pcr kit (bioline). sequencing was carried out by beckman coulter genomics (takeley, uk) using f(mt) and p(mt) primers. to assess the environmental impact on the mhc class ii drb diversity, the data were analyzed according to the location of the capture sites and the type of environment at these sites. for d. rotundus and m. molossus, each capture site corresponded to one environment type. d. rotundus sampling sites corresponded to two different roots (cave f (cf) and cave m (cm)), considered as pf sites, and one foraging area (saut athanase (sa)), an eh site ( fig. , additional file : table s ). m. molossus individuals were captured in one foraging area (cacao (cc)), an aa site, and two different roosts located at the eh site with different disturbance levels: paracou (pa) with a high level of disturbance and the sa site with a low level of disturbance ( fig. , additional file : table s ). c. perspicillata individuals were captured in the four types of environments pf, eh, aa and ur ( fig. , additional file : table s ). to explore the potential bias induced by the spatial structure of collecting sites in c. perspicillata samples, spatial structure was compared with environment structure. sequences were edited using geneious r [ ] . dna sequences were aligned using the mafft alignment tool [ ] included in the software. sequences were confirmed as mhc class ii drb and mtdna d-loop by homology analysis using the ncbi blast search [ ] . a drb clone sequence was regarded as valid if the following criteria were met: ( ) incidence in at least two independent pcrs either from the same individual or different ones, or amplification by two different primer pairs, and ( ) identified by at least three identical clones [ ] . haplotypes were reconstructed for each bat species using the dnasp [ ] . as proposed by [ ] , the nomenclature of mhc alleles for non-human species was used: to each allele a prefix (the first two letters of the genus and the species names) was given, with the serial number attached as follows: cape-drb for c. perspicillata, dero-drb for d. rotundus and momo-drb for m. molossus. cape-drb alleles identified in this study were named after the alleles previously described by [ ] . allele frequencies (f), proportion of segregating sites (s), nucleotide diversity (π), observed (h o ) and expected (h e ) heterozygosities, and gene diversity (h d ) were estimated using arlequin . [ ] . allelic richness (r), with a correction for sample size using a rarefaction method and inbreeding coefficients (f is ) were calculated using fstat . . [ ] . the mean number of differences between nucleotide and amino acid alleles were counted using mega . [ ] . analyses were performed per species for the entire dataset, and for each environment subgroup separately. evidence of genetic recombination or gene conversion events between drb sequences was assessed using gene-conv . a [ ] . this program detects lengthy patterns shared between sequences despite the existence of a pronounced polymorphism. p-values were determined from both global and pairwise permutation tests, with , replicates. no mismatches between fragments were accepted, and global p-values were corrected for multiple comparisons (gscale = ). recombination breakpoints were identified using the gard and sbp algorithms [ ] implemented in the hyphy package [ ] , available in the standard datamonkey analysis library (http://www.data monkey.org/, delport et al. [ ] ). putative antigen-binding sites (abs) were identified by comparison with the human abs of the hla-drb [ ] . the relative rates of d n and d s substitutions were calculated for putative abs, non-abs and all codons following the method of nei and gojobori with the jukes and cantor correction for multiple hits [ , ] . a z-test was performed to assess the deviation of the d n /d s (ω) ratio from . . these tests were performed using mega . . codons potentially subjected to positive selection were further identified with no a priori assumption of abs. for this purpose, two maximum-likelihood (ml) frameworks were carried out as proposed by [ ] . first, two alternative models comprised in the codeml program included in the paml package version . were executed [ ] . the first one (m ) allows codons to evolve either neutrally (ω = ) or under purifying selection (ω < ), while the second one (m ) considers a class of sites under positive selection (ω > ). these models were compared with a likelihood ratio test (ltr; Δl = -l -l ) with two degrees of freedom (α = . ) [ ] . as previously described, each analysis was run twice, with starting ω values of . and . , to ensure convergence [ ] . the f × model of codon frequencies was assumed for all analyses. the amino acids positively selected were identified using the bayes empirical bayes approach (beb), as recommended by yang [ ] , with the cutoff posterior probability set at %. secondly, five ml methods proposed in the hyphy package were used. for all analyses, the best fitting nucleotide substitution model was assessed with the automatic model selection tool available on the server. a single likelihood ancestor counting (slac) model was used to detect evidence of the non-neutral evolution of the drb gene [ ] . based on ancestral reconstruction, this model counts d n and d s changes at each codon position in a phylogeny. fixed (fel) and random effects likelihood (rel) models were performed to estimate the ω ratio on a site-by-site basis [ ] . for the fel model, ω ratios were estimated with an a priori distribution of rates across sites, while this distribution was determined by the present data for the rel model. an internal fel (ifel) model was carried out to highlight selection pressure at the population level (i.e., along internal branches). finally, a mixed effects model of evolution (meme) was performed to highlight both diversifying and importantly episodic selection at individual sites [ ] . codon positions were regarded as candidates for positive selection if the following two a see additional file: figure s , b primer modified from richman et al. [ ] to amplify the exon of mhc class ii drb gene criteria were met: ( ) p-values < . for slac, fel, ifel and meme and ( ) bayes factor > for rel. accordingly, only sites identified as being under positive selection by at least two approaches were considered [ ] . analyses were performed per species for the entire dataset and for each environment subgroup separately. for both neutral and functional markers, the genetic differentiation (f st ) among environment subgroups and collecting sites was calculated through an implementation of analysis of molecular variance (amova; , permutations) using arlequin . . isolation-by-distance (ibd) was examined with the web service idbws v. . (http://ibdws.sdsu.edu/~ibdws/; [ ] ) by assessing the statistical significance of the correlation in a mantel test with , permutations. the quantity f st / ( -f st ) was used as a genetic distance according to rousset [ ] . geographic distances between populations were determined from gps coordinates recorded at each collecting site. to add a measurement of relationships between alleles at the intraspecific level, haplotype networks were constructed for each species using the parsimony statistical algorithm tcs implemented in popart . [ ] . a bayesian skyline plot was used to investigate changes in effective population size (ne) over time with beast . . [ ] , using mtdna d-loop data only. markov chain monte carlo samples were based on × generations, logging every steps, with the first , , generations discarded as the burn-in. the best-fitted model hky + g was used with a relaxed molecular clock. the number of stepwise changes in ne was set at five. phylogenetic relationships and trans-species polymorphism between drb alleles were inferred using a bayesian inference approach implemented in mrbayes [ ] . the hla-drb sequence (accession number: ng_ ) was used as outgroup. the gtr + g + i model was selected as the best-fitted model of nucleotide substitution using jmodeltest [ , ] under corrected akaike information criteria (aicc). the program was run × generations, with a sampling frequency of and a % burn-in. validation of the inference was assessed based on the standard deviation of split frequencies, less than the expected threshold value of . . one hundred and three bats ( c. perspicillata, d. rotundus and m. molossus) were tested for the amplification of mhc drb using cdna. pcr product sizes ranged from bp to bp, for both c. perspicillata and d. rotundus and covered all of exon ( bp in length) (additional file : figure s ). for m. molossus, we successfully amplified bp, excluding primers, of exon . we successfully obtained mhc drb-like fragments from bats ( mhc drb-like fragments for c. perspicillata, for d. rotundus and for m. molossus, additional file : table s ). based on blast searches, sequences showed homologies to only drb loci. considering validation criteria, sequences were validated, corresponding to bats (additional file : table s ). one hundred and ten sequences were validated for c. perspicillata, corresponding to cape-drb alleles (previously reported cape-drb* , genbank accession number: jq , and cape-drb* - ; genbank accession numbers: ku -ku ; additional file : table s ). two hundred and thirty-three sequences were validated for d. rotundus, corresponding to dero-drb alleles (dero-drb* - ; genbank accession numbers: ku -ku ; additional file : table s ). one hundred and forty-seven sequences were validated for m. molossus, corresponding to momo-drb alleles (momo-drb* - ; genbank accession numbers: ku -ku ; additional file : table s ). all amino acid sequences were unique. a maximum of three transcribed alleles per individual was detected in one c. perspicillata and two d. rotundus individuals (additional file : tables s and s ). no more than two transcribed alleles were identified in m. molossus samples (additional file : table s ). all sequences showed blast homology with the hla-drb locus (accession number: ng_ ), with a maximum of % nucleotide identity and % amino acid identity. the percentage of nucleotide and amino acid identity with other bat species (s. bilineata, m. davidii, c. perspicillata and n. albiventris) was above % and % for all transcripts, respectively. two alleles (cape-drb* and ) presented a -bp deletion at position - of the nucleotide alignment (fig. ) . these single-codon indels did not induce a frame-shifting mutation or a stop codon. bats carrying these alleles were heterozygotes and found in eh and ur environments. two other alleles (dero-drb* and dero-drb* ) presented a -bp insertion at position - of the alignment. these two-codon indelsone serine (s) and one asparagine (d)did not alter the reading frame. dero-drb* was carried by four bats, all captured in sa, with one homozygote. dero-drb* was carried by one heterozygote bat captured in cf. no indel event was detected for m. molossus. we amplified the first hypervariable segment (hvi) of the mitochondrial dna (mtdna) control region (d-loop) for the bats tested. we obtained a -bp alignment for m. molossus, a -bp alignment for c. perspicillata and a -bp alignment for d. rotundus. thirty-nine mtdna d-loop haplotypes were identified for c. perspicillata table ) . overall, c. perspicillata presented the highest values of allelic richness (r = . ), number of segregating sites (s = ), nucleotide diversity (π = . ± . ), gene diversity (h d = . ) and mean nucleotide and amino -domain position -- alignment position -- [ ] . † indicates pss identified by [ ] , and ‡ indicates pss identified by [ ] acid differences ( three alleles (cape-drb* , , and ) were shared either between bats trapped in the same environment (eh) or between bats trapped in distinct environments (eh vs. pf). these three alleles were the most common, with an overall frequency (f ± sd) of . ± . for cape-drb* and . ± . for both cape-drb* and cape-drb* (fig. ) . cape-drb* was shared between one bat trapped in pf (n = ) and two bats trapped in eh. cape-drb* was shared between two bats trapped in eh (n = ). nine individuals in this region were homozygotes (additional file : table s ). cape-drb* was shared between two bats trapped in pf and eh. alleles found in ur were private alleles and three of the five trapped bats were heterozygotes. up to alleles were shared either between bats roosting in the same area or between bats foraging in the same area. out of these alleles, one (dero-drb* ) was shared between six bats from the three sampling sites (cf, cm, and sa sites) (additional file : table s ). five alleles (dero-drb* , , , and ) were shared between bats from sa and cm sites, and two alleles (dero-drb* and ) between the sa and cf sites. dero-drb* and had the highest overall frequency of . ± . (fig. ) . four alleles (dero-drb* , , and ) were exclusively shared between individuals trapped in sa. two alleles (dero-drb* and ) were solely shared between individuals trapped in cm. we did not find shared alleles between bats trapped in cf (additional file : table s ). sixteen of the bats captured in sa were heterozygotes, five in cm (n = ) and three in cf (n = ). up to ten shared alleles were identified but none was shared between the three sampling sites (additional file : table s ). three alleles (momo-drb* , and ) were shared between bats captured in the sa and pa sites. two alleles (momo-drb* and ) were shared between individuals from the sa and cc sites. one allele (momo-drb* ) was shared between two bats captured in the cc and pa sites. three alleles (momo-drb* , and ) were exclusively shared within sa. momo-drb* had the highest overall frequency: . ± . (fig. ) . four of the five bats trapped in cc were homozygotes, seven in pa (n = ), and in sa (n = ). table ). more specifically, in putative antigen binding sites (abs), d n /d s values were highly significant (p < - ) and ranged from . for d. rotundus to . for c. perspicillata. we found significant d n substitutions also occurring in putative non-abs for c. perspicillata (d n /d s = . , z = . , p = . ) but none in both d. rotundus and m. molossus samples (p > . ). paml analyses (performed by species and within subgroups per species) showed significant support for m and m (log likelihood estimates (ltr), Δl = [ . - . ], df = , p < . ), indicating an effect of positive selection acting on the drb gene. overall, codons appeared to be under positive selection for c. perspicillata, eight for d. rotundus, and six for m. molossus (beb posterior probability p ≥ %, slac, fel, ifel and meme p-values < . , and rel bayes factor > ; table ). three positively selected sites (pss , and ; fig. ) were common to the three species investigated. these codons were the only common between d. rotundus and m. molossus. six of the eight pss identified in d. rotundus were also found in c. perspicillata, and all of the pss identified in m. molossus were identified in c. perspicillata. regarding environment subgroups for all species, a maximum of pss was identified in ur for c. perspicillata, eight in cf for d. rotundus and in sa for m. molossus. three pss were found in all environment subgroups for d. rotundus, four in c. perspicillata and four in m. molossus. a maximum of six unique pss (sites detected as positive only within the defined subgroup) was observed for c. perspicillata, four for m. molossus and two unique pss were observed in the two roosts (cf and cm) for d. rotundus ( table ). none of the pss detected in c. perspicillata was shared between ur and pf. the same result was obtained between sa and cm for d. rotundus and between pa and cc or sa for m. molossus. thirteen of the pss identified were located within the human abs region for c. perspicillata, seven for d. rotundus and five for m. molossus (fig. ) . all the pss identified in m. molossus were described as pss for n. albiventris, for c. perspicillata and six for d. rotundus. six pss in c. perspicillata were also found in a. jamaicensis, three for m. molossus and one for d. rotundus. the sites where pss differed were located within one to six amino acid residues ( - bp) of the human abs or other bat species pss, depending on the bat species investigated. table s (b) − ). minimum spanning trees showed complex connections between the haplotypes, marked by numerous nucleotide mutations, without clustering of haplotypes either by site or by environment (figs. , , and ). we did not find relevant isolation-by-distance in our samples (additional file : table s ). the haplotype networks for c. perspicillata revealed two main groups of haplotypes that possessed all the haplotypes found in the different environments or collecting sites (figs. and ) . the same result was obtained for m. molossus, with a maximum of four mutations between haplotypes (fig. ) . the lowest number of mutations was found for d. rotundus for which the five haplotypes were linked by one connection, marked by only one or two mutations (fig. ). no population expansion or collapse was observed for the three species. we did not find relevant isolation-by-distance in our samples (additional file : table s ). the analysis included the drb sequences identified in this study ( bp; nucleotide position - ), as well as the published chiropteran drb sequences selected randomly from a. jamaicensis [ ] , from c. perspicillata [ ] , from myotis spp. (m. velifer and m. vivesi [ ] ), from noctilio spp. (n. albiventris and n. leporinus [ , ] ) and from s. bilineata [ , ] . the phylogenetic tree showed six major clades (fig. ) . we observed a clustering by species or genus supported by medium and high posterior probabilities -c. perspicillata ( . ), d. rotundus ( . ), m. molossus ( ), noctilio spp. ( . )with an intermingled clustering of n. albiventris and n. leporinus drb alleles. within the c. perspicillata clade, drb sequences clustered with the previously published sequences, with a high posterior probability ( . ). one drb sequence of c. perspicillata was related to a d. rotundus clade with a posterior probability of . . one drb sequence from s. bilineata clustered with the clade noctilio spp., with a low posterior probability value. the drb sequences of the a. jamaicensis species clustered with five drb sequences of d. rotundus and two drb sequences of c. perspicillata, with a posterior probability of . . within this phyllostomid clade, we observed a species-dependent clustering with high posterior probabilities. two drb sequences of c. perspicillata clustered with a low posterior probability with two sequences from a. jamaicensis. we did not observe a in this study, we explored the genetic variability of the expressed mhc drb genes of three sympatric neotropical bat species, looking for selection signatures within this high drb polymorphism was observed in the three species whatever the degree of habitat disturbance (heavily disturbed, slightly impacted or pristine). in addition, no loss of polymorphism induced by low host and/or pathogen diversity, expected to occur in disturbed environments [ , ] was observed. furthermore, mtdna d-loop sequences, considered as neutral, revealed high levels of genetic diversity for c. perspicillata and m. molossus. high levels of neutral diversity have been associated with either multiple geographic origin and/or population admixture [ , ] , suggesting that c. perspicillata and m. molossus populations originated from one of these two phenomena or from both. the high level of drb polymorphism can be related to population history for these two species with the effects of additional selection factors such as pathogen-mediated selection. a high number of drb homozygotes was also found in m. molossus in all habitats as well as in c. perspicillata but in edge habitats only. for these two species, the presence of a specific allele (allele-specific overdominance) ensuring an adequate immunity can be hypothesized [ ] . considering d. rotundus, a high number of heterozygotes for drb was observed in all habitats. this result suggests a heterozygote advantage related to the forest (pristine and edge habitats) environment known to encompass higher species and pathogen richness [ , ] . the most frequent mhc drb alleles encountered in each bat species (dero-drb* , dero-drb* , dero-drb* , cape-drb* and momo-drb* ) were all identified in forest environments. these alleles are likely ancient and play a significant role in the immune response, conferring a selective advantage to the bats in fig. phylogenetic relationships of the mhc class ii dr beta sequences based on part of exon ( bp) with values of posterior probabilities for nodes. species designation follows the genbank accession numbers for previously described drb sequences. arja a. jamaicensis, cape c. perspicillata, dero d. rotundus, momo m. molossus, myve m. velifer, myvi m. vivesi, noal n. albiventris, nole n. leporinus, sabi s. bilineata. hla-drb was used as the outgroup. tsp indicates trans-species polymorphism of the newly described drb alleles with previously described drb alleles sympatry in these habitats. indeed, given that forest environments are characterized by a high level of pathogen diversity, these alleles most likely possess enhanced recognition capabilities and might be able to recognize a broader range of pathogens. they thus allow a more efficient immune response in line with the ecological diversity. moreover, the geographical structure highlighted in d. rotundus, using mtdna d-loop results, did not affect the allelic distribution. indeed, most of the alleles identified in d. rotundus were shared between sites, and one allele (dero-drb* ) was present at all sites. in contrast, no geographical structure was found in c. perspicillata even if the alleles identified in urban and periurban areas were rare and not shared with other groups. these results suggest that environment rather than population structure drives the allelic distribution, corroborating a local adaptation hypothesis. although edge and forest habitats may differ in pathogenic pressures (see above), the results observed for c. perspicillata may be related to the high tolerance of this species to habitat disturbance and to its dominance in bat communities in edge habitats [ ] , likely allowing continuous exposure to the communities of pathogens of both pristine and disturbed forests, and pathogen transfer between these habitats. gene conversion and recombination events were not detected for any species. the identification of three expressed drb alleles in individuals from c. perspicillata and d. rotundus supported the existence of at least two functional drb loci. however, underestimation of the number of alleles and expressed loci can be suspected, considering the limited number of recombinant clones per primer combinations and the use of cdna only allowing the detection of the most frequently expressed alleles. the occurrence of multiple mhc drb loci varying in the number of loci between individuals and species was reported in neotropical bat species such as c. perspicillata (three drb loci), s. bilineata (ten drb loci) [ ] , a. jamaicensis (three drb loci) [ ] , as well as in other animals [ , , , ] . together with environmental and biological patterns, gene conversion and recombination processes are known to drive the extensive mhc polymorphism [ , ] . variable gene duplication between closely related taxa and among individuals from the same species is characteristic of mhc genes and plays a critical role in the adaptive evolution of the host [ ] . gene duplication can occur over different time scales and therefore predates or follows speciation events. furthermore, the absence of recombination between alleles of different species was reported. this result was regarded as evidence of recent duplication events between loci that occurred after speciation [ ] . our findings support this hypothesis since some recent radiation events occurred in neotropical bat species during the pleistocene era ( . - . mya) [ ] [ ] [ ] . despite the indels detected in some c. perspicillata and d. rotundus sequences, the reading frames remained unaltered, suggesting that these sequences encode functional proteins (fig. ) . nevertheless, the heterozygote-dominant character of individuals with these alleles suggests a turnover of these loci generated by evolutionary processes such as the silencing of duplicate genes by mutation or deletion [ ] . under the birth-and-death model, genes created by duplication can be maintained for long periods, deleted or transformed as pseudogenes and, therefore, contribute to host fitness and adaptability [ ] . the phenomenon is especially significant among heterozygote d. rotundus, for which one of the two alleles is among the most widespread in the population studied (dero-drb* , and ), and supports a shift towards an allele with better recognition capabilities. higher rates of non-synonymous (d n ) vs. synonymous (d s ) nucleotide substitutions were observed, especially in sites supposedly involved in antigen-binding. this excess of d n over d s in abs is consistent with balancing selection acting on drb loci, as a sign of historical positive selection in polymorphic mhc genes [ ] . furthermore, an unexpected excess of d n in putative non-abs for c. perspicillata suggests strong positive selection acting on these sites and highlights species-specific divergence on sites involved in antigen recognition. a high congruence was observed between speciesspecific positively selected sites (pss) in the three species and human abs demonstrating the functional homology of these sites. in contrast, pss not identified as human abs might play a role in mhc drb recognition capability or in the molecule stability for the species studied. here, nine of the pss were detected in c. perspicillata, while only one was detected in both d. rotundus and m. molossus. therefore, the observed excess of d n in c. perspicillata was attributed to pss identified as non-abs in humans. this result demonstrates the critical role of positive selection in shaping mhc diversity and corroborates that positive selection is driven by a species-specific immune response. focusing on c. perspicillata, comparisons of pss identified here with those previously described revealed high congruence, even among pss not identified as human abs [ ] . a similar result was observed between c. perspicillata and a. jamaicensis, two phyllostomidae with the same diet [ ] . taken together, these findings reveal family and species-specific selection pressures acting on mhc genes. moreover, the high similarity of pss between a. jamaicensis and c. perspicillata highlights that bat species sharing similar feeding strategies might be subjectedindependently of their habitatsto similar pathogenic pressures, mainly from their microbiota [ ] . comparisons between each environment subgroup, for each present case, revealed the existence of private pss depending on the area, suggesting a habitat-specific selection pressure process. indeed, while six private pss were counted in urban and periurban areas for c. perspicillata, only two were detected in edge habitats and one in pristine forest. phylogeny, trans-species polymorphism and demographic process two mechanisms are thought to be responsible for the trans-specific similarity of mhc genes: convergent evolution [ ] and trans-species polymorphism [ , ] . evidence of trans-species polymorphism in bats was reported in both myotis spp. and noctilio spp. [ ] and at the family level between the two phyllostomids a. jamaicensis and c. perspicillata [ ] . phylogenetic analyses in our study did not reveal any characteristic clustering of mhc alleles depending on the habitat, but rather clades at the family, genus and species levels (fig. ) . these results suggest either similar pathogenic pressures or that these alleles possess a larger antigenic recognition capability that constitutes a major asset to the immune response of these species. intermingled clusterings observed in this survey (between drb sequences from a. jamaicensis, c. perspicillata and d. rotundus) are in agreement with trans-species polymorphism reported in bats [ , ] . the observed trans-species polymorphism for myotis spp. [ ] was not detected in the present study. the results highlighted clustering between s. bilineata and m. velifer. however, this clustering was no longer observed by analyzing full-length exon . observation of trans-species polymorphism within bat families (e.g., phyllostomidae) strengthens the scenario of independent modes of evolution of mhc drb alleles, allowing balancing selection to retain substantial allelic lineages before speciation events [ , ] . extended periods of host-pathogen coevolution is thought to contribute to trans-species polymorphism given that host-sharing pathogens and exposure to similar pathogen pressures are believed to induce similar changes in different host species [ ] . although pathogen-driven selection is known to be crucial in mhc diversity [ ] , demographic processes such as genetic drift, bottlenecks, expansions, fragmentation and geographic isolation have been demonstrated to also participate in the shaping of this extensive polymorphism [ , , , ] . evolutionary analyses of mhc drb alleles for the three species did not reveal any area-dependent clustering effect. furthermore, analyses of partial sequences of the mtdna d-loop revealed no structuration for c. perspicillata and m. molossus, whereas it was observed for d. rotundus. demographic analysis performed for the three species did not detect any substantial bottleneck or expansion. while the patterns observed for the first two species corroborate the hypothesis of a similarity between the intrinsic genetic diversity of the species and that of mhc, the lack of similarity between these patterns of diversity for d. rotundus contradicts this statement. these results suggest that the signature of an area-limited pathogen-driven selection, supported by the presence of private pss rather than by demographic processes such as distance isolation, is observed in this case. abbreviations abs: antigen-binding sites; aicc: corrected akaike information criteria beb: bayes empirical bayes; drb: exon of the mhc class ii dr beta fel: fixed effects likelihood; ifel: internal fixed effects likelihood meme: mixed effects model of evolution; mhc: major histocompatibility complex; ml: maximum-likelihood; mtdna d-loop: mitochondrial dna control region; pbr: peptide-binding region pss: species-specific positively selected sites; rel: 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computing winning the invasion roulette: escapes from fish farms increase admixture and facilitate establishment of non-native rainbow trout less can be more: loss of mhc functional diversity can reflect adaptation to novel conditions during fish invasions global drivers of human pathogen richness and prevalence parasite and viral species richness of southeast asian bats: fragmentation of area distribution matters duplication and population dynamics shape historic patterns of selection and genetic variation at the major histocompatibility complex in rodents mhc class ii variation in a rare and ecological specialist mouse lemur reveals lower allelic richness and contrasting selection patterns compared to a generalist and widespread sympatric congener selection and recombination drive the evolution of mhc class ii drb diversity in ungulates natural selection and the evolutionary history of major histocompatibility complex loci phylogeography of the common vampire bat (desmodus rotundus): marked population structure, neotropical pleistocene vicariance and incongruence between nuclear and mtdna markers patterns of diversification in two species of short-tailed bats (carollia gray, ): the effects of historical fragmentation of brazilian rainforests bats (chiroptera: noctilionoidea) challenge a recent origin of extant neotropical diversity evolution by the birth-and-death process in multigene families of the vertebrate immune system balancing selection: the major histocompatibility complex phyllostomid bat microbiome composition is associated to host phylogeny and feeding strategies origin of major histocompatibility complex polymorphism: the trans-species hypothesis trans-species polymorphism in immune genes: general pattern or mhc-restricted phenomenon? evolutionary history of an mhc gene in two leporid species: characterisation of mhc-dqa in the european brown hare and comparison with the european rabbit pathogen-driven selection and worldwide hla class i diversity genetic diversity and differentiation at mhc genes in island populations of tuatara (sphenodon spp.) signatures of environmental genetic adaptation pinpoint pathogens as the main selective pressure through human evolution we are grateful to françois catzeflis and marguerite delaval for useful discussions on the biology, ecology and dynamics of bats. all field volunteers and owners and/or managers of capture sites are warmly acknowledged for their assistance in captures. nucleotide sequences for the drb alleles and for mtdna d-loop are available on genbank (accession numbers: ku -ku and ku -ku , respectively). this study was the first to investigate mhc drb polymorphism in three sympatric bat species, m. molossus, c. perspicillata and d. rotundus, from the amazonian region. our results revealed a high genetic variability in the mhc drb gene. natural selection, as well as local adaptation driven by different parasite and pathogen exposures across environments, contribute to the maintenance of an extensive mhc polymorphism. the richness of pathogens in the different habitats should be investigated to strengthen our assumptions on potential local adaptation. further studies using finer neutral markers such as microsatellites will be necessary to detect possible confounding effects, such as hidden structuring and demographic history, and thus to better understand the drivers of mhc allelic diversity. additional file : table s . supplementary methods, including the characteristics of all capture sites (table s ), the list of samples successfully amplified for the mhc class ii drb loci (table s ) , the distribution of drb alleles per species (table s - ) , the pairwise differentiation computations per species (table s - ) , as well as the correlations of genetics and geographic distance (table s ) table s . (pdf kb) additional file : figure s . positions of pcr primers used to amplify the indicated fragments of the mhc class ii loci in c. perspicillata, d. rotundus and m. molossus, based on cdna. the structure of the cdna of the mhc class ii drb gene is based on [ ] . according to each species, boxes indicate the amplified region using each couple of primers. the shaded region represents the region of interest, namely exon . sequences and references of all primers used are given in table the authors declare that they have no competing interests. submit your next manuscript to biomed central and we will help you at every step: key: cord- -ieb upi authors: papenfuss, anthony t; baker, michelle l; feng, zhi-ping; tachedjian, mary; crameri, gary; cowled, chris; ng, justin; janardhana, vijaya; field, hume e; wang, lin-fa title: the immune gene repertoire of an important viral reservoir, the australian black flying fox date: - - journal: bmc genomics doi: . / - - - sha: doc_id: cord_uid: ieb upi background: bats are the natural reservoir host for a range of emerging and re-emerging viruses, including sars-like coronaviruses, ebola viruses, henipaviruses and rabies viruses. however, the mechanisms responsible for the control of viral replication in bats are not understood and there is little information available on any aspect of antiviral immunity in bats. massively parallel sequencing of the bat transcriptome provides the opportunity for rapid gene discovery. although the genomes of one megabat and one microbat have now been sequenced to low coverage, no transcriptomic datasets have been reported from any bat species. in this study, we describe the immune transcriptome of the australian flying fox, pteropus alecto, providing an important resource for identification of genes involved in a range of activities including antiviral immunity. results: towards understanding the adaptations that have allowed bats to coexist with viruses, we have de novo assembled transcriptome sequence from immune tissues and stimulated cells from p. alecto. we identified about , genes involved in a broad range of activities with the most highly expressed genes involved in cell growth and maintenance, enzyme activity, cellular components and metabolism and energy pathways. . % of the bat transcribed genes corresponded to immune genes and a total of about immune genes were identified, providing an overview of both innate and adaptive immunity. a small proportion of transcripts found no match with annotated sequences in any of the public databases and may represent bat-specific transcripts. conclusions: this study represents the first reported bat transcriptome dataset and provides a survey of expressed bat genes that complement existing bat genomic data. in addition, these data provide insight into genes relevant to the antiviral responses of bats, and form a basis for examining the roles of these molecules in immune response to viral infection. bats make up approximately % of the extant mammalian diversity and are the second most species rich mammalian lineage after rodents [ ] . the order chiroptera is divided into two suborders: the megachiroptera and microchiroptera. these two lineages are estimated to have diverged approximately million years ago [ ] . megachiroptera consists of a single family, the old world fruit bats, while microchiroptera includes families of echolocating bats. bats have a wide geographic distribution and exploit a variety of environmental niches, being absent only from the polar regions. bats are also hosts to numerous viruses, many of which are highly pathogenic to humans and other mammals yet appear to cause no clinical consequences in bats [ ] [ ] [ ] [ ] [ ] [ ] . this group of mammals also shares a variety of unique characteristics that likely facilitate the persistence and spread of the viruses they carry. highly social species, bats live at much higher densities than other mammals. they are the only mammals capable of powered flight and have long lifespans relative to their body size [ ] . despite their diversity, unique characteristics and role as natural reservoirs for viruses, bats are also the least studied of all mammalian taxa and there is little information available on antiviral immunity in any bat species. bats are the natural reservoir hosts of more than viruses, with new viruses or viral sequences of bat origin being discovered each year [ , ] . rna viruses account for the overwhelming majority of known bat viruses, many of which are among the most deadly known to man, including ebola, hendra, nipah and sars-like coronaviruses [ ] . many of these viruses, which cause severe morbidity and mortality in humans and other mammals, appear to cause no clinical diseases in bats under natural or experimental infection. the most studied example is the henipaviruses (hendra and nipah viruses) which are members of the family paramyxoviridae. nipah virus has a mortality rate of - % in humans and close to % in experimental animal models (cats and hamsters). yet, infection of pteropus vampyrus (the natural reservoir host of nipah virus in malaysia) and p. poliocephalus (a related bat species native in australia) by a high dose of nipah virus, failed to result in clinical signs of disease [ , , ] . other examples of experimental infections of bats including ebola zaire, japanese encephalitis and st. louis encephalitis viruses have not resulted in any symptoms of disease despite the presence of viral rna in tissues [ ] [ ] [ ] [ ] . experimental infections of p. poliocephalus with nipah virus have demonstrated the presence of serum antibody and viral shedding in the absence of clinical symptoms of disease [ ] . the only viruses that have been demonstrated to cause clinical symptoms of disease in bats are rabies virus and the closely related australian bat lyssavirus [ , ] . however, results of experimental infections are inconsistent with only a small proportion of bats succumbing to infection, and rates of sero-conversion and virus recovery from tissues were reported to be very low [ ] . the long co-evolutionary history of bats with viruses has likely resulted in the adaptation of the bats immune system to cope with viral infection. one hypothesis is that the innate immune system rapidly controls viral replication to very low levels that cause no clinical consequences to bats, but still results in viral shedding and subsequent spillover to other species. however, as little information currently exists on any aspect of bat immunology and few bat-specific reagents exist, this hypothesis remains untested. recent years have seen a surge in the availability of whole genome sequence data. bats were among the organisms sequenced as part of the us national institutes of health (nih)-funded mammalian genome project. these genomic resources are an important step forward in identifying the genes that are involved in antiviral immunity in bats and in providing insights into other unique life history characteristics. there are currently two publicly available bat genome sequences: one from the megabat p. vampyrus and a second from the microbat myotis lucifugus. both bat genomes were initially sequenced to low coverage ( . x for p. vampyrus and . x for m. lucifugus, though a draft quality assembly of the m. lucifugus genome based on x coverage sequencing is now available). additionally, the annotations were predominantly based upon comparative data. despite these shortcomings, these projects have an important role to play in revealing the mechanisms that have evolved to allow bats to remain asymptomatic to so many viral diseases. in order to understand bat-virus interactions, we are developing the australian black flying fox, p. alecto, as a model bat species. p. alecto belongs to the family pteropodidae and is closely related to p. vampyrus [ ] . these two species are reservoirs for a variety of closely related viruses, the most important of which include the henipaviruses, hendra virus in p. alecto and nipah virus in p. vampyrus [ ] . a number of important resources have now been developed for p. alecto, including cell lines from a variety of tissues [ ] . we have also begun to identify some of the genes involved in immune responses in this species and carry out functional studies in bat cells [ ] [ ] [ ] [ ] [ ] [ ] . to begin to characterise the immune gene repertoire of p. alecto, we sequenced the transcriptome of bat immune tissues and mitogen-stimulated cells using the illumina platform. to our knowledge, this study represents the first analysis of the transcriptome of any species of bat. our analysis of the p. alecto transcriptome provides information on a variety of immune genes not previously identified in any bat species and represents an important starting point for examining the antiviral activity of these molecules. overview of the bat transcriptome two separate transcriptomic datasets were generated and raw sequences from each database were submitted to the sequence read archive [sra: srr . and srr . ]. the first was obtained using total rna extracted from a juvenile male flying fox thymus. due to its role in central tolerance, the thymus expresses a large proportion of the proteome and therefore allows for the identification of a broad range of genes, including those involved in the immune response. to enrich for sequences corresponding to cytokines and innate immune genes, the second dataset was derived from pooled total rna obtained from mitogen-stimulated spleen, white blood cells and lymph node and unstimulated thymus and bone marrow obtained from one pregnant female and one adult male flying fox. cells were stimulated with lipopolysaccharide (lps) and ionomycin, which stimulate the production of pro-inflammatory cytokines; polyic, a tlr ligand; pha, which triggers t cell activation and pma, which activates t and b cells. about . million bp long reads were obtained from the thymus dataset, while . million bp long reads were generated from the stimulated pooled sample. prior to assembly, the raw reads were trimmed of low quality sequence and polya/t tails, uninformative strings of 'n' and primer/adapter contaminants were cleaned. the filtered dataset consisted of , , reads from the thymus (between - bp) and , , reads from the stimulated pooled dataset (between - bp). the filtered reads were de novo assembled using the software packages velvet and oases. the resulting oases assemblies consisted of , contigs (n bp) from the thymus and , contigs (n bp) from the pooled samples. the largest contigs in the thymus and pooled samples were . kb and . kb respectively, both of which correspond to the dna-dependent protein kinase catalytic subunit (dna-pkcs) which is represented by a . kb transcript in other species, including horse. for comparative purposes, an assembly using mira was also generated. summary statistics from the velvet, oases and mira assemblies are listed in additional file : table s . all subsequent analyses were performed using the oases assemblies. to identify orthologues of known mammalian protein coding genes, the bat contigs were used to search the kegg and ncbi non-redundant (nr) protein databases with blastx (e-value < . ). of the , contigs longer than bp in the thymus sequence assembly, about % matched annotated proteins in the nr database. for the pooled dataset, about % of the , loci matched proteins in nr. similar results were obtained for both assembled libraries against the kegg database. of the assembled thymus transcripts annotated using kegg, % of all transcripts were more similar to horse sequences than to any other species, followed by dog ( %) and cow ( %) (figure ). similar results were obtained for the pooled tissue dataset (not shown). this result is consistent with the now generally accepted view that bats belong within laurasiatheria, which includes carnivora, cetartiodactyla (whales and even toed ungulates), eulipotyphla (moles and shrews), pholidota (scaly anteater) and perissodactyla (odd toed ungulates) [ ] [ ] [ ] [ ] [ ] [ ] . however, until recently, the phylogenetic relationships within laurasiatheria have been controversial. conflicting results have been reported using complete mitochondrial genome sequences to infer phylogenetic relationships with support for a sister relationship between chiroptera and fereungulata (carnivora, pholidota, perissodactyla and cetartiodactyla) or a relationship between chiroptera and eulipotyphla [ ] [ ] [ ] . analysis of the nuclear gene, protamine p , as well as large genomic datasets, has provided evidence that bats are sister to a clade containing perissodactyla, carnivora, and cetartiodactyla [ , ] . the volume of sequence data generated by transcriptome sequencing provides the opportunity for larger scale sequence comparisons than previously possible using the few full length bat genes available or by comparison with the limited whole genome sequence data. our results support the comparative analysis of retroposon loci which has also demonstrated that bats share a sister relationship with horses, forming a clade with carnivora [ ] . alignment of contigs from the thymus and pooled datasets to the kegg database identified , and , contigs respectively with homology to , and , unique human proteins. to explore gene function, gene ontology (go) terms were used. of contigs that matched proteins in the kegg database, % were assigned go terms and % could be mapped to go slim terms using go term mapper (additional file : figure s ). genes with go slim terms were further classified into twelve selected classes ( figure ). the most abundant go terms found in the thymus dataset were involved in cell growth and maintenance ( . %), enzyme activity ( . %), cellular components ( . %) and metabolism and energy pathways ( . %). similar results were obtained for the pooled tissue dataset (data not shown). the go classification demonstrates that a diverse range of genes were identified in each of our two datasets providing a broad survey of bat genes. a goal of the present study was to identify immune transcripts, particularly those that may play a role in antiviral immunity. only . % of the bat transcribed genes from each of the datasets showed homology to genes associated with immune function. this represents about different immune-related genes ( figure ). the bat immune transcripts were further categorised using go terms to annotate the transcripts into immune categories. represented in the datasets were genes involved in a broad range of immune activities with lymphocyte activation, cytokine production and t cell activation making up the largest proportions of immune transcripts ( figure ). using kegg codes to identify immune genes, our data revealed genes involved in toll-like receptor (tlr) cascades, genes involved in b cell activation, involved in t cell activation, involved in natural killer cell cytotoxicity and involved in antigen presentation. additional immune genes not identified in the kegg database were obtained by searching sequences from the nr database. the sequences of all genes described in the text are provided in the additional file . one hypothesis for the ability of bats to resist the pathological effects of viral infection is that they are able to rapidly control viral replication early in the immune response through innate antiviral mechanisms. the bat transcriptome contained representatives of a variety of immune genes including pattern recognition receptors, interferons, interferon stimulated genes and natural killer cell receptors. pattern recognition receptors (prr) including tlrs, rig-i like helicases (rlhs) and nucleotide oligomerisation domain (nod) like receptors (nlrs) recognise conserved molecular patterns associated with a broad range of pathogens. both tlrs and rlhs initiate signalling pathways that result in the induction of similar immune and inflammatory responses but are expressed in different locations within the cell and differ in the pathogens they recognise. tlrs are transmembrane proteins expressed by the plasma membrane or endosome and recognise a broad range of pathogens including viruses, bacteria and fungi. of eleven previously identified p. alecto tlr genes [ ] , only tlr was absent from the oases assemblies, however it was present in the mira assembly, which used a lower coverage cut-off and is useful for identifying genes with low expression levels. rlhs are expressed in the cytoplasm where they recognise viral rna and dna [ , ] . three bat rlh genes, retinoic-acid-inducible protein i (rig-i), melanoma-differentiation-associated gene (mda ) and laboratory of genetics and physiology (lgp ) were identified in our transcriptome datasets and have recently been described in p. alecto [ ] . these results provide further evidence that bats are able to recognise a broad range of pathogens, similar to other species. nlrs are a diverse family of cytoplasmic prrs involved in the activation of a variety of signalling pathways. nlrs are primarily involved in bacterial recognition, although more recently, evidence for recognition of viral rna and dna by some members of the nlr family has been reported [ ] [ ] [ ] . the only nlrs identified in the bat transcriptome datasets were nod-like receptor family card domain containing (nlrc ) and nlr family, pyrin domain containing (nlrp ). nlrc is a recently identified nlr proposed to function as a positive and negative regulator of antiviral immune responses [ ] . nlrp (also known as nalp ) is activated by a variety of danger signals including viral and bacterial infections and environmental irritants. activation of nlrp in turn activates caspase- in the inflammasome which proteolytically cleaves the cytokines il- β and il- into active mature peptides [ ] . the identification two nlrs with associations with antiviral immunity in the bat transcriptome is remarkable and provides a starting point for understanding the role of nlrs in antiviral immunity in bats. the interferon (ifn) response is a key component of the innate immune system and the first cytokines induced against viral infection. since the ifn response is important in the control of viral replication in other mammals, we searched the bat transcriptome for ifns and ifn stimulated genes (isgs) that may be critical to the ability of bats to remain asymptomatic to viral infections. type i (including ifnα and β) and iii (ifnλ) ifns are induced directly in response to viral infection and play a role in the earliest stages of the innate immune response. type i (α) ifn and its receptor (ifnar and ifnar ) were identified in the bat transcriptome datasets (additional file ). although type iii ifns, ifnλ and ifnλ are upregulated in stimulated bat cells [ ] , neither of these genes were identified in our datasets, likely reflecting a low expression level in our samples. the il- r chain of the type iii ifn receptor was present in the bat transcriptome, but its partner chain ifnlr was not found. both il- r and ifnlr were recently described in p. alecto and ifnlr was demonstrated to act as a functional receptor for ifnλ [ ] . the induction of type i and type iii ifns results in the transcription of hundreds of isgs including prrs that detect viral rna, transcription factors that result in the amplification of the ifn response and a small number of proteins that are directly responsible for inducing an antiviral state. the isgs, myxovirus resistance (mx) gtpases, protein kinase r (pkr), '- ' oligoadenylate synthetases (oas), ribonuclease l (rnasel) and isg are among the proteins with confirmed antiviral activity in other mammals [ ] . the bat transcriptome datasets contained genes orthologous to mammalian mx , mx , oas , oas , oas , oas-like (oasl), pkr, rnasel and isg consistent with the presence of an isg repertoire in bats that is similar to that of other species. these results provide the first evidence that the pathways activated by the ifn response are likely similar in bats to those described in other mammals. the mx gene family is among the best characterised isgs, first identified as antiviral proteins following the observation that the sensitivity of many inbred mouse strains to orthomyxovirus was solely due to mutations within the mx locus [ ] . the mx family of gtpases trap essential viral components, and in so doing prevent viral replication at early time points. although the full spectrum of mx antiviral activity is unknown, representatives of both rna and dna viruses have been shown to be sensitive to the effects of mx [ ] . a full length transcript, encoding a amino acid protein was identified in our bat transcriptome datasets and found to be orthologous to mx based on comparison with known mammalian mx and mx family members (figure a and data not shown). bat mx contained the highly conserved tripartite gtp-binding domain found in all mammalian mx proteins. in addition, a dynamin family signature and putative leucine zipper motif were found near the c terminal end, represented by a stretch of evenly spaced leucine residues. the bat protein was also conserved in the region identified as the stalk of human mxa including loop which is associated with antiviral activity. consistent with other species, loop of the mxa stalk is the least conserved region of the bat mx protein [ ] . loop has been reported to be proteinase k sensitive and may play a role in lipid binding [ , ] (figure b ). bat mx does not contain the stretch of basic amino acids (k/r) near the c terminal end associated with nuclear localisation of mouse mx , consistent with the bat protein remaining localised within the cytoplasm [ ] . the conservation of key residues important in antiviral activity is consistent with the bat mx playing a role in antiviral immunity similar to other species. the identification of the sequences of important isgs will now allow us to determine whether functional differences in the initiation and regulation of these proteins account for the differences in susceptibility of bats to viral infections compared to other mammals. natural killer (nk) cells are an important component of the innate immune response, providing a first line of defence against viruses and tumours. to our knowledge, no investigations of nk cell receptors from any species of bat have been reported previously. nk cells express cell surface receptors that recognise major histocompatibility complex (mhc) class i or class i like molecules on the surface of cells and lyse infected or abnormal cells by cytotoxicity. two families of nk receptors that bind classical mhc class i ligands have been identified: the killer immunoglobulin like receptors (kirs), which are encoded by genes in the leukocyte receptor complex (lrc), and the killer cell lectin like receptors (klrs), which are encoded by genes in the natural killer complex (nkc). different lineages of mammals make use of genes from the two different superfamilies to carry out analogous functions. kirs are used preferentially by primates, cattle, domestic cats, dogs and pigs [ , ] . similarly, the kir-like receptors, marsupial immunoglobulin-like receptors (mairs) and chicken immunoglobulin-like receptors (chirs), have expanded in marsupials and chickens respectively [ , ] . although chir-ab binds igy, the ligand for the majority of chirs is unknown and the presence of a charged transmembrane residue and a cytoplasmic immunoreceptor tyrosine-based inhibition motif (itim), are consistent with the possibility that they play a role in nk activity [ ] . rodents, horses and platypus are the only species so far described that have expanded the klrs, represented by the ly family [ ] [ ] [ ] . in the bat transcriptome dataset, no transcripts with homology to kirs or ly receptors were identified. in bony fish, novel immune type receptors (nitrs) which contain an n terminal variable domain and a c terminal ig domain have been identified as the primary activating and inhibitory receptors expressed by nk cells [ ] . nitrs were also used to search the bat transcriptome but failed to identify any orthologous transcripts. the failure to find kir or ly like receptors in the bat transcriptome may reflect low expression levels of these genes resulting in their absence from our datasets. however, blast searches of the publicly available whole genome sequence of the closely related pteropid bat, p. vampyrus revealed no evidence of kirs or ly receptors. as this is a low coverage genome ( . x), further work is required to determine whether pteropid bats have kir and/or ly receptors. overall, the absence of these important nk receptors from our datasets warrants further investigation into the nature of nk cells in bats. nk cells in a wide range of mammalian species additionally express cd /nkg (also called klrd / klrc) lectin-like receptor heterodimers. unlike the kir and ly receptors, which bind (classical) mhc class ia ligands, the cd /nkg heterodimer binds the (non-classical) mhc class ib ligands hla-e and qa- in humans and mice respectively [ ] . the cd / nkg a heterodimer generates inhibitory signals whereas the cd /nkg c heterodimer generates activating signals within nk cells. both cd and nkg a were identified in the bat transcriptome, however nkg c transcripts were not identified, possibly reflecting the low abundance of transcripts of this gene in our datasets. two and nkg a transcripts were identified in the thymus and pooled datasets respectively and six transcripts corresponding to cd were identified in the pooled dataset. two of the longest nkg a sequences were aligned with nkg a and nkg c sequences from human and mouse. as shown in figure a , the bat genes display highest conservation with other nkg a genes including the presence of conserved itim motifs in their cytoplasmic domains, designated by i/v/l/sxyxxl/v indicating that they are likely functional inhibitory receptors [ ] . the more divergent nkg d, which binds mhc class i chain-related genes, mica/b, and the ul binding proteins (ulbps) in human [ ] , was also detected. two distinct bat cd contigs were identified, one of which is missing two conserved cysteines in the stalk region, the first of which forms an interchain disulfide bond with nkg and the second which forms an intrachain disulphide bond. the second bat cd sequence is missing a conserved cysteine in the extracellular domain that forms an intrachain disulphide bond (figure b ). the absence of key cysteines in both of the bat cd sequences may have implications for the formation of heterodimers with nkg and for the unique folding of the cd chain. combined with our failure to detect kirs or ly receptors, our data may provide the first evidence for the presence of atypical nk cell responses in bats. however, confirmation of the nature of the nk response and the composition of receptors used by bat nk cells awaits further investigation. other nk receptors were also identified in our datasets including cd which acts as an activating or inhibitory receptor on human and mouse nk cells respectively [ ] and the natural cytotoxicity receptors expressed by nk cells. co-receptors including cd and cd expressed by subsets of nk cells in other species were also identified in the bat transcriptome. identification of nk cell receptors and co-receptors provides information for the development of reagents to identify bat nk cells and paves the way for further studies of nk cell function during viral infection in bats. genes involved in the adaptive immune system, including mhc class i and ii genes and t and b cell receptors and co-receptors were highly represented in both the thymus and pooled datasets providing evidence that bats have all of the components necessary to mount an adaptive immune response. mhc class i molecules play an important role in the initiation of the adaptive immune response through recognition of endogenously-derived peptides from viruses and other pathogens. in the thymus dataset, contigs had homology to mammalian mhc class i proteins, while were homologous in the pooled data. other transcripts in the mhc class i antigen-loading and presentation pathway were also identified, including beta- -microglobulin, transporter associated with antigen processing (tap ), calnexin and tapasin. class irelated genes were also present in the bat transcriptome dataset including cd a, cd b, cd d, mr , hfe, fcrn and ulbps, which have a variety of immune and nonimmune functions. the presence of ulbps is consistent with the expression of nkg d, but orthologues of mica/b or mill were not observed. the presence of nkg d suggests bats should have a mic homologue, but these may not be detected possibly due to low or tissue-specific expression. to our knowledge, these sequences provide the first class i and class i-associated transcripts from any species of bat. of the contigs with homology to mhc class i genes in the thymus dataset, contained in-frame stops. these may be expressed pseudogenes, represent assembly or sequencing errors or result from reading frame shifts due to the presence of unprocessed transcript. as the sequences were obtained from multiple individuals, it is not possible to confidently distinguish between alternative isoforms, alleles and in some cases, loci. however, clustering the remaining contigs with open reading frames (orfs), there are clearly at least distinct mhc class i genes expressed. the majority of class i contigs contained the α or α domain or partial sequence corresponding to both domains and were used for further sequence analysis. the deduced amino acid sequence of contigs with the most complete α or α domains were aligned with human hla-a ( figure ). all of the bat class i sequences contained a unique three amino acid insertion in the α domain that appears to be bat specific. as shown in figure , the bat transcripts display amino acid variation in their α and α domains, corresponding to the peptide binding region. however, they appear to be remarkably conserved from residues to of the α domain. these results may indicate that bats contain a very closely related class i gene repertoire that have coevolved with the specific viruses they carry. some of the class i transcripts represented in the thymus and pooled datasets contained an bp insertion at the end of the α domain. the longest of these transcripts corresponded to the leader peptide through to amino acids of the α domain and is shown in figure . the insertion at the end of the α domain is not present in class i sequences from other mammals and includes two in frame stop codons that would prevent translation beyond the α domain ( figure ). this sequence was figure a. alignment of deduced amino acid sequences of bat nkg with human and mouse nkg a and nkg c. sequences are divided into cytoplasmic, transmembrane, stalk, and lectin domains. the predicted itim motifs in the cytoplasmic domain are shaded. the conserved cysteine residue in the stalk predicted to be involved in interchain disulphide bond formation with cd is shaded and indicated with an asterisk. dashes indicate similarity and dots indicate gaps. b. alignment of the deduced amino acid sequences of bat cd with the human and mouse orthologues. sequences are divided into cytoplasmic, transmembrane, stalk, and lectin domains. conserved cysteines predicted to be involved in disulphide bond formation are shaded. cysteine pairs are indicated by identical numbers below the cysteine. the cysteine predicted to form a disulphide bond with nkg is indicated with an asterisk. confirmed by race pcr and transcripts were detected in a variety of tissues including lymph node, spleen, liver, lung, heart, kidney, small intestine, brain and salivary glands, thus providing evidence that they are not an artefact of the transcriptome assembly (data not shown). comparison with the closely related p. vampyrus whole genome sequence available in ensembl revealed that the bp insertion is identical to the beginning of intron of a p. vampyrus class i gene. mhc class i splice variants that retain intron sequence and result in the translation of a truncated protein have been identified in other mammals, including soluble splice variants of human hla-g that plays a role in immunoregulation at the feotal-maternal interface [ ] . further investigation will be required to determine whether the bat gene encodes a soluble protein corresponding only to the α domain or whether it represents a transcribed pseudogene. however, given the abundance of this transcript in our datasets it is possible that it plays a role in immune regulation in p. alecto. unlike class i molecules, which are ubiquitously expressed, class ii molecules are expressed only by antigen presenting and b cells and present exogenously derived peptides to t cells. the mhc class ii molecules are composed of an α and a β chain encoded by a and b genes respectively [ , ] . eutherians have three main classical class ii gene clusters: dp, dr, and dq, as well as the nonclassical dm and dn/do gene clusters [ , ] . sequences corresponding to exon of mhc class ii drb genes have been described in four species of microbats [ ] [ ] [ ] . however, prior to the present study no class ii genes have been reported from any species of megabat. sequences corresponding to genes involved in the class ii antigen processing and presentation pathway were also identified in our datasets including the class ii invariant (cd ) chain and cathepsin s (additional file ). in the p. alecto thymus and pooled datasets we identified and contigs respectively that were homologous to class ii sequences. phylogenetic analysis revealed that the alpha chain sequences were homologous to dma, doa, dqa and dra from other mammals (figure a ) and the beta chain sequences were homologous to dmb, dob, dqb and drb (figure b ). these results are consistent with orthologous relationships between the bat class ii genes and those from other mammals. t cell receptor (tcr) genes corresponding to all four chains of the t cell receptor were present in our datasets, consistent with bats having both αβ and γδ t cells. sequences corresponding to the constant and variable domains of the tcr were identified including many tcrα related contigs, tcrβ related contigs, a few tcrγ and tcrδ chain related contigs. in humans and mice approximately % of circulating t cells express the αβ t cell receptor. in contrast, γδ t cells account for up to % of circulating t cells in young ruminants, rabbits and chickens [ , ] . the low abundance of tcrγ and tcrδ related transcripts in our datasets is consistent with the possibility that αβ t cells may be the predominant tcr present in bats. in addition, a variety of t cell co-receptors, including the accessory tcrζ chain, cd , cd , cd and cd were identified in our datasets. we previously described the immunoglobulin heavy chain diversity of p. alecto, revealing the presence of a highly diverse variable region gene repertoire [ ] . sequences encoding the variable and constant domains of immunoglobulin heavy and light chains were represented in our datasets. these included heavy chain genes encoding iga, igg, igm and ige, which have previously been described in the megabat, cynopterus sphinx. no evidence for the transcription of igd was observed in the p. alecto transcriptome, a result which is consistent with c. sphinx [ ] . the two light chain subtypes, kappa and lambda and a variety of b cell co-receptors including cd , cd , cd , cd a and cd b were also identified in our datasets (additional file ). many of the bat immune transcripts showed high levels of sequence similarity compared to homologues from other mammals. among the most conserved bat innate immune genes were the prrs; the tlrs, rig-i helicases and nlrs, which displayed > % amino acid identity with homologues. this likely reflects their roles in the recognition of conserved pathogen motifs. members of the oas family were also highly conserved, in particular oas which shared % amino acid identity with the dog oas sequence. in addition, the nk co-receptor, cd shared % amino acid identity with mouse, hamster, guinea pig and human sequences. among the adaptive immune genes, mhc associated proteins, calnexin, tap and cathepsin s shared - % identity with corresponding sequences from other mammals reflecting their conserved roles in the antigen processing and presentation pathway. several members of the mhc class i and ii families were also highly conserved, including cd b and cd d which shared and % amino acid identity with horse and chimp sequences respectively. the bat mhc class ii doa and dra shared and % amino acid identity with orthologous sequences in other mammals. the t cell co-receptor, cd shared % identity with the rhinoceros cd sequence and the constant domain of igm shared % identity with camel igm. there were > , unannotated contigs in the thymus and pooled datasets. only about % of these contigs matched predicted cdnas from the p. vampyrus genome sequence, which are annotated using orthologous sequences from other species [ ] . the unannotated contigs contained a total of open reading frames (orfs) longer than bp. of these, . % (e-value < - ) aligned to the closely related p. vampyrus whole genome sequence and represent highly divergent homologues or bat specific genes. the remaining loci represent either misassembled contigs or bat-specific transcripts that are located in sequencing gaps in the low coverage p. vampyrus genome sequence. the long (> nt) orfs were searched for conserved domains using profile hidden markov models with hmmscan (hmmer v ; http://hmmer.org/) obtained from the pfam database [ ] . this identified orfs containing unique domains, including several defensins, antimicrobial peptides and dna-binding domains. searches using domain models from the pfam-b database, identified a further unique, predicted-conserved domains in orfs. a further orfs remained unannotated. a high proportion of these were rich in cysteine, tryptophan and proline, and prolines frequently appeared in low complexity regions (additional file : figure s a and b) . further characterisation of these unannotated transcripts will provide insight into whether they are functionally significant and in particular whether any unique bat specific transcripts are involved in the antiviral immune response. bats are a highly diverse, species rich group of mammals that have evolved a variety of distinctive characteristics since their divergence from other mammals [ ] . despite the central importance of bats in harbouring a variety of viruses with the potential to spillover to other species, very little is known about antiviral immunity in bats. next generation sequencing provides the opportunity to survey genes that are conserved between distantly related species as well as to provide insights into novel adaptations through the identification of previously unidentified transcripts. to identify genes involved in the immune response, we carried out a transcriptome analysis of thymus and immune cells and tissues of the australian black flying fox, p. alecto. this study represents the first survey of expressed bat immune genes and complements existing low coverage bat genome sequences. our analysis provides a broad overview of the bat transcriptome and contains representatives of all of the major classes of immune genes. the results are consistent with bats having all of the components of the immune system present in other mammals. the majority of these correspond to genes that have not previously been described in any species of bat and thus represent an important resource for future investigations into antiviral immunity in bats. animals p. alecto bats used in this study were wild caught from east brisbane, queensland, australia. bats were handled and euthanised as previously described [ ] . all experiments were approved by the australian animal health laboratories animal ethics committee (protocol aec ). the thymus was removed from a juvenile male bat and immediately stored in rnalater (ambion) for rna extraction. the spleen, lymph nodes (ln), thymus, bone marrow and peripheral blood were collected from one adult male and one pregnant female bat. single cells were extracted from the spleen, thymus and ln by tissue extrusion through a μm sterile sieve (bd biosciences) in the presence of dmem supplemented with mm l-glutamine, units/ml penicillin and units/ml streptomycin (invitrogen). splenocytes and peripheral blood lymphocytes (pbmls) were isolated by density centrifugation over lymphoprep (nycomed, oslo) as described previously [ ] . cells were resuspended in dmem with % fcs, mm l-glutamine, units/ml penicillin and units/ml streptomycin and cell numbers were determined using a haemocytometer with trypan blue exclusion. the thymus and bone marrow cells were stored in rnalater (ambion) for rna extraction and the spleen, thymus and ln were cultured with a variety of stimulants. the isolated splenocytes, ln and pbmls from each bat were pooled and were then seeded at x cells per well in well tissue culture plates (nunc) with pha ( ug/ml; sigma) and lps ( μg/ml; sigma); pma ( μg/ml; sigma) and ionomycin ( nm/ml; sigma); or polyic ( μg/ml; invivogen) and incubated in a humidified atmosphere of % co in air at °c. cells were harvested in rlt buffer (qiagen) at , , and hours and homogenised using a qiashredder (qiagen) following the manufacturer's instructions. the lysate was then stored at − °c and total rna extracted the next day ( , , and hours) or processed immediately ( hours). rna extraction was carried out as previously described using the rneasy mini kit (qiagen) with removal of genomic dna with dnase i digestion [ ] . total rna from the thymus of a juvenile male bat was used for illumina sequencing separately from all other samples. total rna obtained from the stimulated and unstimulated cells from the two adult bats was pooled as follows: % thymus total rna ( % from each bat) and % pooled total rna from the rest of the mitogen stimulated and unstimulated cells/tissues (~ . % for each sample; total of samples). sequencing mrna isolation from total rna, library preparation and single-end read sequencing was performed by geneworks pty ltd, thebarton south australia using the illumina genome analyser iix sequencing platform. library preparation was performed as per illumina's mrna sequencing sample preparation guide (part # rev. d) except μg of total rna was used for mrna selection using poly-t oligo-attached magnetic beads. the thymus library was run on a single lane of a flow cell resulting in more than . million -base sequences for a total of about . gigabases (gb) of sequence. the pooled library consisted of lanes resulting in million bp sequences for a total of about . gb of sequence. sequence pre-processing and de novo assembly the quality of the sequences were evaluated using fastqc [ ] . sequences were pre-processed in two stages. first, all bases at the ' end of the reads with quality scores of or lower were removed. second, poly a/t tails, uninformative sequences (ns) and primer/adaptor contaminants were trimmed using snowhite (version . . ) [ ], a cleaning pipeline for next-generation cdna sequences, which includes seqclean [ ] and tagdust [ ] . we ran snowhite with two runs of seqclean and one run of tag-dust and a final minimal length cutoff of bp was used. the pre-processed sequences were de novo assembled using two different approaches. ( ) the reads were assembled with velvet (version . . ) [ ] using individual kmers from bp to bp. next, the contigs produced by velvet were processed using oases (version . . ) [ ] . oases loci were then merged using cd-hit-est (version . ) [ ] with a global sequence identity threshold of . . finally, a length cutoff was set to bp and the default coverage cutoff of was used. we term the final result of this process a contig ( ) . the reads were also assembled using mira (v . . rc ) [ ] with default setting for est and illumina reads assembly, i.e. maximum front and end gap clip is bp, maximum length of the possible vector leftover allowed is bp, minimum quality score, window length and read length were all set to , allowed to clip poly a/t at ends, and minimum read coverage per contig was . the bat contigs were firstly annotated by using the best hits of blastx [ ] search against nr protein database and kegg pathway database with an e-value cutoff of . for annotating the protein coding contigs that were conserved with other species. then the unannotated contigs were further annotated by using blastn search against refseq_rna database with an e-value cutoff of - for the contigs containing conserved utrs and without significant protein coding regions. the contigs not annotated by the above two steps were further analysed by using blastn against the cdnas from megabat (p. vampyrus) and microbat (m. lucifugus). we translated the un-annotated transcripts into protein sequences from frames, extracted the orfs longer than bp. this was performed separately for the datasets. these orfs were searched against pfam-a and pfam-b databases to identify conserved domains. the two sets of long orfs were pooled and clustered based on cd-hit with sequence identity of % [ ] . the amino acid compositions were further analysed for the nonredundant longer orfs with composition profiler [ ] . all the kegg ids of the human proteins identified by the blastx searches were extracted from the annotation process and were mapped to uniprot ids. then the go analysis for the uniprot proteins (uniprotkb-goa: gene_association.goa_human) was used to assign the go terms for the transcripts. the number of genes in categories of the go slim database was counted using the go term classification counter, categoriser [ , ] and the immune category of the bat genes was annotated using the generic gene ontology term mapper [ ] . the go classifications were further grouped into twelve broad categories as follows: cell death and apoptosis go: ; extracellular matrix go: ; cell, go: and nucleoplasm, go: ). binding (binding, go: ; protein binding, go: go: ; nucleotide binding go: ; actin binding, go: ; calcium ion binding, go: ; chromatin binding, go: ; carbohydrate binding, go: and rna binding, go: ). reproduction and development (development enzyme activity (catalytic activity go: ca), based on the protein alignment to retain codon positions. based on the nucleotide and protein alignments, phylogenetic trees were constructed by the neighbour joining method [ ], maximum parsimony and minimum evolution using the mega program the genbank accession numbers for sequences used in the sequence and phylogenetic analysis are as follows: mhc class i: (cap ) hla-a; mhc class iia: human, homo sapiens (hosa) dma (nm_ ) dqa (m ), dra (u ) dra (nm_ ), dma (nm_ ) gallus gallus (gaga) b-la (ay ) mhc class iib: hosa dqb (m ), drb (nm_ ), dob (l ), dpb (m ), dmb (u ) rattus norvegicus (rano) dqb (x ) equus caballus (eqca) dqb (l ) dmb (dq ), drb (ay ) ovis aries (ovar) dqb (l ) mumu (nm_ ) hosa (af _ ) avian mx: gaga (np_ ) mammal species of the world: a taxonomic and geographic reference in the timetree of life human ebola outbreak resulting from direct exposure to fruit bats in luebo, democratic republic of congo swanepoel r: fruit bats as reservoirs of ebola virus studies of arthropod-borne virus infections in chiroptera. iv. the immune response of the big brown bat (eptesicus f. fuscus) maintained at various environmental temperatures to experimental japanese b encephalitis virus infection experimental inoculation of plants and animals with ebola virus transmission studies of hendra virus (equine morbillivirus) in fruit bats, horses and cats experimental hendra virus infection in pregnant guinea-pigs and fruit bats (pteropus poliocephalus) bats: important reservoir hosts of emerging viruses bats as a continuing source of emerging infections in humans experimental nipah virus infection in pteropid bats (pteropus poliocephalus) australian bat lyssavirus infection in a captive juvenile black flying fox pathogenesis studies with australian bat lyssavirus in grey-headed flying foxes (pteropus poliocephalus) a phylogenetic supertree of the bats (mammalia: chiroptera) establishment, immortalisation and characterisation of pteropid bat cell lines immunoglobulin heavy chain diversity in pteropid bats: evidence for a diverse and highly specific antigen binding repertoire molecular characterisation of rigi-like helicases in the black flying fox, pteropus alecto molecular characterisation of toll-like receptors in the black flying fox pteropus alecto interferon production and signaling pathways are antagonized during henipavirus infection of fruit bat cell lines type iii ifn receptor expression and functional characterisation in the pteropid bat, pteropus alecto type iii ifns in pteropid bats: differential expression patterns provide evidence for distinct roles in antiviral immunity complete mitochondrial genome of a neotropical fruit bat, artibeus jamaicensis; and a new hypothesis of the relationships of bats to other eutherian mammals parallel adaptive radiations in two major clades of placental mammals molecular phylogenetics and the origins of placental mammals resolution of the early placental mammal radiation using bayesian phylogenetics molecules consolidate the placental mammal tree pegasoferae, an unexpected mammalian clade revealed by tracking ancient retroposon insertions the phylogenetic position of the talpidae within eutheria based on analysis of complete mitochondrial sequences monophyletic origin of the order chiroptera and its phylogenetic position among mammalia, as inferred from the complete sequence of the mitochondrial dna of a japanese megabat, the ryukyu flying fox (pteropus dasymallus) maximum likelihood analysis of the complete mitochondrial genomes of eutherians and a reevaluation of the phylogeny of bats and insectivores confirming the phylogeny of mammals by use of large comparative sequence data sets characterization and phylogenetic utility of the mammalian protamine p gene differential roles of mda and rig-i helicases in the recognition of rna viruses shared and unique functions of the dexd/h-box helicases rig-i, mda , and lgp in antiviral innate immunity function of nod-like receptors in microbial recognition and host defense regulation of immune pathways by the nod-like receptor nlrc nlrp inflammasome activation: the convergence of multiple signalling pathways on ros production? interferon-inducible antiviral effectors transgenic mice with intracellular immunity to influenza virus the mx gtpase family of interferon-induced antiviral proteins. microbes and infection the interferon-induced mx protein of chickens lacks antiviral activity stalk domain of the dynamin-like mxa gtpase protein mediates membrane binding and liposome tubulation via the unstructured l loop structural basis of oligomerization in the stalk region of dynamin-like mxa transport of the murine mx protein into the nucleus is dependent on a basic carboxy-terminal sequence natural killer cell receptors in cattle: a bovine killer cell immunoglobulin-like receptor multigene family contains members with divergent signaling motifs comparative genomics of natural killer cell receptor gene clusters characterization of the opossum immune genome provides insights into the evolution of the mammalian immune system the leukocyte receptor complex in chicken is characterized by massive expansion and diversification of immunoglobulin-like loci the chicken leukocyte receptor complex encodes a family of different affinity fcy receptors the ever-expanding ly gene family: repertoire and signaling natural killer cell receptors in the horse: evidence for the existence of multiple transcribed ly genes identification of natural killer cell receptor clusters in the platypus genome reveals an expansion of c-type lectin genes the phylogenetic origins of natural killer receptors and recognition: relationships, possibilities, and realities nk gene complex dynamics and selection for nk cell receptors signaling pathways engaged by nk cell receptors: double concerto for activating receptors, inhibitory receptors and nk cells of mice and men: different functions of the murine and human b (cd ) receptor on nk cells biology and functions of human leukocyte antigen-g in health and sickness* nomenclature for factors of the hla system three-dimensional structure of the human class ii histocompatibility antigen hla-dr sequence organisation of the class ii region of the human mhc evolutionary relationships of class ii majorhistocompatibility-complex genes in mammals class ii drb polymorphism and sequence diversity in two vesper bats in the genus myotis non-neutral evolution of the major histocompatibility complex class ii gene drb in the sac-winged bat saccopteryx bilineata mhc class ii drb diversity, selection pattern and population structure in a neotropical bat species, noctilio albiventris prominence of gamma delta t cells in the ruminant immune system characterization of avian t-cell receptor γ genes the two suborders of chiropterans have the canonical heavy-chain immunoglobulin (ig) gene repertoire of eutherian mammals the pfam protein families database a molecular phylogeny for bats illuminates biogeography and the fossil record tagdust-a program to eliminate artifacts from next generation sequencing data velvet: algorithms for de novo short read assembly using de bruijn graphs oases: de novo transcriptome assembler for very short reads cd-hit: a fast program for clustering and comparing large sets of protein or nucleotide sequences using the miraest assembler for reliable and automated mrna transcript assembly and snp detection in sequenced ests gapped blast and psi-blast: a new generation of protein database search programs composition profiler: a tool for discovery and visualization of amino acid composition differences categorizer categorizer: a web-based program to batch analyze gene ontology classification categories generic gene ontology (go) term mapper multiple sequence alignment using clustalw and clustalx the neighbor-joining method: a new method for reconstructing phylogenetic trees mega : molecular evolutionary genetics analysis (mega) software version . statistics of local complexity in amino acid sequences and sequence databases accuracy of protein flexibility predictions the universal protein resource (uniprot) submit your next manuscript to biomed central and take full advantage of: • convenient online submission • thorough peer review • no space constraints or color figure charges • immediate publication on acceptance • inclusion in pubmed, cas, scopus and google scholar • research which is freely available for redistribution we thank craig smith, carol de jong, deborah middleton low complexity regions in protein sequences were detected with the seg program with default parameters [ ] . the transcription of a bat mhc class i gene was examined using quantitative pcr (qpcr) as described previously [ ] . briefly, total rna was prepared from lymph node, spleen, liver, lung, heart, kidney, small intestine, brain and salivary glands using the rneasy mini kit (qiagen) as described above. cdna was generated using a quantitect reverse transcription kit for rt-pcr (qiagen). qpcr primers were designed using primer express . (applied biosystems) with default parameter settings ( '-acgactcctattccccaggatag-f and '-gaaagc cactggtacctgtgaga-r). reactions were carried out using express sybr w greener tm qpcr supermix universal (invitrogen) and an applied biosystems fast real-time qpcr instrument. additional file : table s . summary of additive multiple-kmer velvet/ oases/mira assembly.additional file : figure s . overview of the bat transcriptome. the distribution of , and , transcriptome sequences that have mapped to human orthologues from p. alecto thymus and pooled tissue datasets based on go slim terms. sequences within the three areas of gene ontology: molecular function, biological process and cellular component are further divided into subgroups at the go slim level.additional file : sequences of all genes described in the manuscript.additional file : figure . amino acid composition of large unannotated orfs. the horizontal axis shows amino acids sorted by flexibility index [ ] .a. amino acid composition of large unannotated non-redundant orfs relative to proteins in the swissprot database [ ] . the amino acids trp, cys and pro have twice the abundance in unannotated orfs compared to swissprot proteins.b. amino acid composition of low complexity regions in unannotated orfs relative to unannotated non-redundant orfs. prolines are abundant in low complexity regions, but trp and cys are not. the authors declare that they have no competing interests. key: cord- - tfg h authors: dong, rong; chu, zhugang; yu, fuxun; zha, yan title: contriving multi-epitope subunit of vaccine for covid- : immunoinformatics approaches date: - - journal: front immunol doi: . /fimmu. . sha: doc_id: cord_uid: tfg h covid- has recently become the most serious threat to public health, and its prevalence has been increasing at an alarming rate. the incubation period for the virus is ~ – days and all age groups may be susceptible to a fatality rate of about . %. covid- is caused by a novel single-stranded, positive (+) sense rna beta coronavirus. the development of a vaccine for sars-cov- is an urgent need worldwide. immunoinformatics approaches are both cost-effective and convenient, as in silico predictions can reduce the number of experiments needed. in this study, with the aid of immunoinformatics tools, we tried to design a multi-epitope vaccine that can be used for the prevention and treatment of covid- . the epitopes were computed by using b cells, cytotoxic t lymphocytes (ctl), and helper t lymphocytes (htl) base on the proteins of sars-cov- . a vaccine was devised by fusing together the b cell, htl, and ctl epitopes with linkers. to enhance the immunogenicity, the β-defensin ( mer) amino acid sequence, and pan-hla dr binding epitopes ( aa) were adjoined to the n-terminal of the vaccine with the help of the eaaak linker. to enable the intracellular delivery of the modeled vaccine, a tat sequence ( aa) was appended to c-terminal. linkers play vital roles in producing an extended conformation (flexibility), protein folding, and separation of functional domains, and therefore, make the protein structure more stable. the secondary and three-dimensional ( d) structure of the final vaccine was then predicted. furthermore, the complex between the final vaccine and immune receptors (toll-like receptor- (tlr- ), major histocompatibility complex (mhc-i), and mhc-ii) were evaluated by molecular docking. lastly, to confirm the expression of the designed vaccine, the mrna of the vaccine was enhanced with the aid of the java codon adaptation tool, and the secondary structure was generated from mfold. then we performed in silico cloning. the final vaccine requires experimental validation to determine its safety and efficacy in controlling sars-cov- infections. in december , covid- , caused by the severe acute respiratory syndrome coronavirus (sars-cov- ) was first discovered in china and has rapidly spread across the world. as of : noon on june , a total of , , confirmed cases of covid- have been reported globally, including , deaths. the prevalence of the disease has been increasing at an alarming rate. there were , , cases in the united states, , in brazil, , in russia, , in the united kingdom, and , , in a number of other countries ( ) . the incubation period for the virus is ∼ - days, and all age groups are susceptible to a fatality rate of about . %. the most common clinical manifestations are low-grade fever, dry cough, fatigue, and gastrointestinal symptoms ( ) . about half of all patients with covid- develop shortness of breath, and severe cases may rapidly develop sars, septic shock, difficult-to-correct metabolic acidosis, and coagulation disorders ( ) . covid- may also affect other organs, most commonly the heart and kidneys ( ) ( ) ( ) . some patients may have mild symptoms, without fever, and may recover after - weeks ( ) . other patients may show signs of serious illness and some may die; however, most patients show favorable progress ( ) . male individuals with the disease and aged patients have the worst prognosis. in children, the disease is relatively mild ( ) . covid- is caused by a novel single-stranded, positive (+) sense rna beta coronavirus, which is a pathogen of the coronaviridae family, named sars-cov- ( ) . the full-length genome sequences revealed that sars-cov- has the greatest genetic similarity to bat coronavirus, ∼ - % similarity to severe acute respiratory syndromerelated coronavirus (sarsr-cov), and a smaller similarity of - % to the middle east respiratory syndrome-related coronavirus (mers-cov) ( ) . thus, a bat might be the original host of sars-cov- , but the intermediate host remains undiscovered ( ) . the genes of sars-cov- encode structural proteins and non-structural proteins. four structural proteins are absolutely vital for viral assembly and invasion of sars-cov- . spike protein homotrimers constitute the spikes on the viral surface, and these spikes are responsible for attachment to host cells by binding to their receptors ( ) . the m protein has three transmembrane domains, which determine the shape of the virion, facilitate membrane curvature, and bind to the nucleocapsid. the e protein plays an important role in virion assembly and release, as well as involved in viral pathogenesis. the n protein has two different domains, both of which bind to the viral rna genome via totally different mechanisms. in addition, some reports have shown that non-structural proteins are essential for the replication of coronaviruses ( ) . vaccination is a vital tool for the control and elimination of the virus, and the development of a vaccine for sars-cov- remains an urgent need ( ) . traditional methods of vaccine development are time-consuming and very labor-intensive ( ). the realm of immunoinformatics tools considers the mechanism of the host immune response to yield additional methodologies in the design of vaccine against diseases are cost-effective and convenient, as in silico predictions can reduce the number of experiments needed ( , ) . dozens of studies have generated epitope-based peptide vaccine of sars-cov- . baruah and bose ( ) used immunoinformatics tools to discover cytotoxic t lymphocyte (ctl) and b cell epitopes for the spike protein of sars-cov- . then, abraham et al. developed a multi-epitope vaccine that was designed using immunoinformatics tools that potentially trigger both cd + and cd + t-cell immune responses ( ) . although there are many vaccines generated by immunoinformatics tools, most of these are based on spike protein. the spike protein is responsible for attachment to host cells by binding to angiotensin-converting enzyme (ace ) ( ) . a vaccine based on the spike protein could induce antibodies to block sars-cov binding and fusion or neutralize virus infection ( ) . but there are still many obstacles, spike protein-based sars vaccine may induce harmful immune responses that cause liver damage of the vaccinated animals ( ) . other virus proteins are considered as the candidates for designing vaccine with protective and less harmful immune responses ( ) . vaccine-based on structural and non-structural proteins of the virus is revealed potential vaccine inducing protective immune responses ( , ) . pandey et al. reported the more scientifically rigorous strategy of multi-epitope subunits based on multiple proteins against parasitic and viral diseases, such as malaria, visceral leishmaniasis, and hiv ( ) ( ) ( ) . in this present, we employed immunoinformatics to predict multiple immunogenic proteins from the sars-cov- proteome and thereby design a multi-epitope vaccine. these proteins included non-structural and structural sequences of sars-cov- , their reference sequences were retrieved from the national center for biotechnology information (ncbi) database. the proteins of the sars-cov- have been reported and reference could get from ncbi ( , ) . the reference sequences of sars-cov- proteins were retrieved from ncbi protein database (https://www.ncbi.nlm.nih.gov/protein) and accession numbers in table , then we stored the reference sequences as a fasta data type. the proteins with < amino acid sequences which are too short to predict epitopes were excluded, the remaining proteins were used for further analysis. vaxijen is the first server for alignment-independent prediction of protective antigens, which overcome the limitations of alignment-dependent methods ( ) . to identify the potential antigenicity of sars-cov- proteins, an online prediction server, vaxijen v . (http://www.ddg-pharmfac.net/vaxijen/ vaxijen/vaxijen.html) was used to predict the antigenic values of each protein ( ) . this identification was applied according to the default parameters of the server. proteins having antigenicity were sorted according to an antigenic score of ≥ . (threshold for this model is . ) and were selected for further structural modeling ( ) . there are no available experimental structures of sars-cov- proteins, phyre provide model regions trough a new ab initio folding simulation with no detectable homology ( ) . the sars-cov- proteins were modeled by phyre server (http://www.sbg. bio.ic.ac.uk/phyre /). because the sars-cov- proteins with no detectable homology protein to finish the modeling, we chose the intensive search and output the accurate alignment by the alignment of hidden markov models. modrefiner was used by the galaxyrefine server (http:// galaxy.seoklab.org /cgi-bin/submit.cgi?type=refine) ( ) . the structure assessment was performed by the swiss-model workspace (https://swissmodel.expasy.org/assess) ( ) . the three dimensional ( d) models were used for the conformational (discontinuous) b-cell epitope predictions while the sequences were utilized in linear b-cell and t-cell epitope predictions. netctl- . is demonstrated to have a high predictive performance ( ) . the netctl . server (http://www.cbs. dtu.dk/services/netctl/) was applied to predict ctl epitopes for the sars-cov- at the threshold value of . with high sensitivity and specificity ( ) . to cover ∼ % of the world's population, three supertypes (a , a , and b ) were selected based on artificial neural networks, to predict mhc class i binding epitopes ( ) . the best candidates for the sars-cov- vaccine construction were sorted for further prediction, based on a half-maximal inhibitory concentration (ic ) < nm and an integrated score. the ic < nm represents epitope has a high affinity to receptor. the integrated score indicated the transporter of antigenic peptides (tap) transport efficiency, class i binding, and proteasomal cleavage prediction ( ) ( ) ( ) . then the specific treg epitopes were screened and excluded by the epitoolkit (https://epivax.com/). for mhc class ii t cell epitope predictions, the immune epitope database server predicted binders based on the percentile rank or mhc binding affinity ( ) . the immune epitope database server (iedb; http://tools.iedb.org/mhcii/) was used to predict helper t lymphocyte (htl) epitopes ( ) . we chose the combinatorial approach which recommended by iedb to predict htl epitopes. the combinatorial approach combined nn-align, smm-align, comblib, sturniolo, and netmhciipan methods ( ) ( ) ( ) ( ) ( ) . the alleles of the human leukocyte antigen (hla) were selected for the prediction at α and β chains, separately ( ) . for final construction, epitopes were selected based on their scores (low scores indicated favorable binding), the release of interferongamma (ifn-γ), induction of emergent properties, and the ic < nm. the ifn-γ cytokine makes a major contribution to antiviral mechanisms. it excites both native and specific immune responses by activating macrophages and natural killer cells ( ) . further, ifn-γ augments the response of mhc to antigens. the ifn-γ epitope server (http://crdd.osdd.net/raghava/ifnepitope/ scan.php) was used to recognize ifn-γ epitopes ( ) . we entered the htl epitopes with low scores into the ifn-γ epitope server. positive ifn-γ induction was predicted based on the support vector machine (svm) hybrid approach. the final htl epitopes were determined based on ifn-γ induction and mhc class ii binding, both of which facilitate the stimulation of t-helper cells ( ) . the abcpred (http://crdd.osdd.net/raghava/abcpred/) and bepipred linear epitope prediction (http://tools.iedb.org/bcell/ result/) servers were utilized to predict linear b cell epitopes. the abcpred server is based on an artificial neural network (ann) ( , ) . the linear b cell epitopes of the sars-cov- protein were predicted at a threshold of . . the bepipred linear epitope prediction server is based on seven methods: ( ) ( ) ( ) ( ) ( ) ( ) . we used these seven methods separately to predict the average threshold. the overlap between abcpred and bepipred severs was selected to determine the candidate epitopes for the sars-cov- vaccine construction ( ) . unlike t-cell epitopes that are linear continuous stretches of residues, b-cell epitopes are generally conformational (discontinuous) ( ) . in this study, the ellipro servers (http://tools.iedb.org/ellipro/) were applied to predict the conformational b-cells epitopes ( ) . the server predicts epitopes based on pi (protrusion index) value. the epitope with pi = . would include % of residues with % being outside the ellipsoid, discontinues b-cells epitopes with the top pi value was selected for vaccine designing ( ) . to develop the final vaccine, epitopes determined by various immunoinformatics software were linked together with the aid of separate linkers. the ctl epitopes were linked by the aay linker, htl epitopes by the gpgpg linker, and b cells were linked by the kk linker ( , , ) . to increase the vaccine immunogenicity, the β-defensin ( mer) amino acid sequence was adjoined to the n-terminal of the vaccine with the help of the eaaak linker ( ). the β-defensin peptides provoke innate immunity cells and recruit naive t cells through the chemokine receptor- (ccr- ) ( ). the pan-hla dr binding epitopes ( aa) as well as added to the n-terminal of the vaccine with the aid of the same linker ( ) . the pan-hla dr binding epitopes in vaccine construct facilitating binding to many different types of mouse and human mhc-ii alleles to induce cd -helper cell responses ( ) . to enable the intracellular delivery of the modeled vaccine, a tat sequence ( aa) was appended to c-terminal ( ) . linkers (ayy, kk, and gpgpg) play vital roles in producing an extended conformation (flexibility), protein folding, and separation of functional domains, and therefore, make the protein structure more stable ( ). the allergenic proteins induce a harmful immune response, allergenicity of the vaccine should be non-allergic ( ) . the non-allergic character of the vaccine sequence was evaluated by the algpred server (http://www.imtech.res.in/raghava/algpred/) ( ) . we predicted allergenicity of vaccine sequences choosing a hybrid approach (svmc+ige epitope+arps blast+mast) with the highest accuracy and sensitivity ( ) . the vaxijen v . server (http://www.ddgpharmfac.net/ vaxijen/vaxijen/vaxijen.html) was applied to evaluate the antigenicity of the vaccine ( ) . the antigenicity prediction method was solely based on the physicochemical properties of proteins without recourse to sequence alignment. the precision rate of the server ranged from to %. to determine immune response profile of this multi-epitope vaccine, computational immune simulations were performed by the c-immsim online server at (http://kraken.iac.rm.cnr.it/ c-immsim/) ( ) . the c-immsim utilizes the celada-seiden model for describing both humoral and cellular profiles of a mammalian immune system against designed vaccine. as per the literature, three injections were administrated at different intervals of month. the simulation was performed with default parameters. the vaccine sequence was administered weeks apart. the simulation volume was , , simulation steps was , , random seed was , , and the vaccine injection with no lps ( ). the protparam tool (http://web.expasy.org/protparam/) was used to evaluate the physicochemical properties of the final vaccine protein ( ) . the physicochemical properties included the number of amino acids, molecular weight, theoretical isoelectric point (pi), amino acid composition, atomic composition, formula, extinction coefficients, estimated half-life, instability index, aliphatic index, and grand average of hydropathicity (gravy) ( ) . the molecular weight and theoretical pi were computed by user-entered sequences. the amino acid and atomic compositions were self-explanatory. the extinction coefficient of a protein was based on information about its amino acid composition. the instability index of a protein indirectly indicated the stability of the protein. if the computed instability index of protein was < , it was regarded as a stable protein, while values > were regarded as unstable. in vivo half-life evaluation of proteins was based on the principle of the "n-end rule." furthermore, gravy is a measurement of the hydrophobic nature of the protein, which is calculated by determining the total hydropathy of all amino acids divided by the number of amino acid residues in the protein. to avoid inducing pathogenic priming and autoimmunity, the sequence homology of the final vaccine to human protein was screened by blastp online server (https://blast.ncbi.nlm.nih. gov/blast.cgi) ( ) . an ideal vaccine should have non-sequence to human proteins. the designed vaccine was a reconstructed protein with no detectable homology ( ) . phyre incorporates an ab initio folding simulation to model regions of proteins with no detectable homology. the phyre server (http://www.sbg.bio. ic.ac. uk/phyre /) was used to predict the three-dimensional structure of the designed vaccine. the server generates a fulllength d model of a protein sequence by employing both multiple template modeling and simplified ab initio folding simulation ( ) . to enhance the overall and partial structural quality of the protein, the output d structure of the final vaccine from the phyre server was further refined by the galaxyrefine server (http://galaxy.seoklab.org/cgi-bin/submit.cgi?type=refine) ( ) . the galaxyrefine server predicted five refined models of our developed vaccine construct, in which model was made by the structural perturbation based simply on the clusters of the side chains; whereas, models - were generated by deeper perturbations of loops and secondary structural elements ( ) . for the assessment of the tertiary structure of the final vaccine protein, a ramachandran plot was performed by the swiss-model workspace (https://swissmodel.expasy.org/ assess) ( ) . the ramachandran plot illuminates favored regions for backbone dihedral angles against amino acid residues in protein structure ( ) . the structure assessment page shows the most relevant scores provided by molprobity and help we easily identify where residues of low quality lie in their model or structure ( ) . then, prosa-web (https://prosa.services.came. sbg.ac.at/prosa.php) was employed in the final vaccine protein structure validation. a positive z-score commonly means an erroneous or erratic section found in the generated d protein model ( ) . to revealing the binding affinity between the vaccine construct and antigenic recognition receptors of toll-like receptor- (tlr , a z) and major histocompatibility complex (mhc-i, wuu, and mhc-ii, c j) present on the surface of immune cells ( ) . docking analysis was performed using the cluspro server (https://cluspro.bu.edu/login.php?redir/queue. php). tlr act as receptors for antigenic recognition. the cluspro server computed the models based on electrostatic interactions and desolvation energy ( ) . to reconfirm the binding affinity of the designed vaccine construct between these receptors, the patchdock server (https://bioinfo d.cs.tau.ac.il/ patchdock/) was used for docking ( ) . the server predicted the potential complex with the help of three algorithm-molecular shape representations, surface patch matching, filtering, and scoring ( ) . after the acquisition of the output from the patchdock server, the complexes were refined by the firedock algorithm, which predicted the optimal complex with the aid of energy functions ( ) . the pdb file of vaccine protein and receptor complex (tlr , mhc-i, and mhc-ii) were used to start the molecular dynamic (md) simulations. the complexes were placed in a octahedron box of water molecules represented by the threepoint charge spc model, whose boundary is at least Å from any protein atoms. the solvated protein was subsequently neutralized by chloridions. covalent bonds involving hydrogen atoms were constrained using the lincs algorithm, and longrange electrostatic interactions were treated with particle-mesh ewald employing a real-space cutoff of Å. the system was first briefly minimized with backbone atoms restrained to the initial coordinates to remove close contacts, and the restrained system was gradually heated to k under constant volume conditions in ps. each system was equilibrated for ns using the constant isothermal-isobaric ensemble at atm and k without any restraints. the parrinello-rahman barostat and a v-rescale thermostat were used with an integration time step of fs. production run md simulations were performed for ns with coordinates recorded every ps. all simulations were performed using gromacs . along with the gromos a force field ( , ) . for the purpose of cloning, codon adaptation of the designed vaccine was performed for analyzing the codon usage by the prokaryotic organism (escherichia coli, e. coli). the java codon adaptation tool (http://www.jcat.de/) was used to optimize codon ( ) . then the secondary structure of mrna was predicted by mfold (http://unafold.rna.albany.edu/? q=mfold) ( ) . for raising the expression efficiency of the final vaccine protein, the e. coli k strain was chosen. for the valid translation of the vaccine gene, we proofread and avoided rho-independent transcription termination, prokaryote ribosome binding site, and cleavage site of restriction enzymes. restriction endonuclease sites xhoi and bamhi were appended to n and c terminals of vaccine, respectively. then, it was inserted into the pet a (+) vector between the xhoi and bamhi. the flow chart of the designed work is shown in figure . the strategy of vaccine construction is presented in figure . the proteome of sars-cov- was retrieved, which comprised proteins. the reference sequences of those proteins were retrieved in the fasta format and their details are presented in table . five proteins with < amino acid sequences are too short to predict epitopes (orf protein, orf protein, orf b protein, nsp , and envelope protein) were excluded. in order to develop a subunit vaccine, it is critical to identify candidate proteins that are important for inducing a protective immune response ( ) . the remaining proteins sequence were relayed to the vaxijen server to determine their antigenicity based on the antigenic scores ( table ) . proteins with antigenic scores > . were selected for further analysis ( ) . nine proteins, namely orf a protein, orf protein, nsp , nsp , nsp , endornase, orf a protein, membrane glycoprotein, and nucleocapsid phosphoprotein were finally selected for further epitope prediction. there is no available experimental structures of these nine proteins, we predicted homology models for the nine proteins applying the normal mode of phyre online server. the most suitable templates for the nine proteins were identified to be the pbd entries ( table s ). all of the modeled structures were showed over % residues in the ramachandran favored region figure s and table s . the prediction of ctl epitopes ( mer) was performed by the netctl server. the binder sites were determined based on three supertypes (a , a , and b ), with a % coverage rate of the world's population. nine proteins were selected based on antigenicity. one epitope of each supertype was selected based on the highest score and an ic value < nm. then the specific treg-inducing epitopes were excluded by epitoolkit. a total of epitopes were selected from nine proteins as the candidates for the construction of the vaccine ( table ) . the htl epitopes ( mer) were evaluated for three hla supertypes: hla-dr (drb * : , drb * : , drb * : , drb * : , drb * : ); hla-dq (dqa * : /dqb * : , dqa * : /dqb * : , dqa * : /dqb * : , dqa * : /dqb * : , dqa * : /dqb * : , dqa * : /dqb * : ); and hla-dp (dpa * /dpb * : , dpa * : /dpb * : , dpa * : /dpb * : , dpa * : /dpb * : , dpa * : /dpb * : ). we sorted the top epitopes with the lowest scores (low scores indicated the highest binding capability) from three supertypes. the best candidate was then selected based on positive ifn-γ induction and an ic < nm. then the specific treg-inducing epitopes were excluded by epitoolkit. thus, a total of epitopes were selected for vaccine design ( table ) . we used the abcpred and bepipred servers to identify the line b cell candidate epitopes. all predicted epitopes from both servers were compared, and only the overlapping epitopes were selected for the development of the vaccine. the line epitopes identified by abcpred had prediction scores ranging from . to . , and line epitopes identified by bepipred had prediction scores ranging from . to . among these line epitopes, only ( mer) were found to be common or partly common in both servers ( table ) . these line epitopes were selected for vaccine construction ( table ) . the non-continuous b cell epitopes were predicted by the ellipro severs, a total number of non-continuous b cell epitopes were generated from ellipro. amino acid residues, sequence location, the number of residues, and the pi scores of the predicted conformational epitopes are shown in table and the graphical depiction of these epitopes can be seen in figure s . twenty-four epitopes were excluded because it added the allergenicity of vaccine, three epitopes were marked red and selected for vaccine construction. the best candidate epitopes were used for the construction of the vaccine. a total of ctl epitopes, htl epitopes, the half-maximal inhibitory concentration (ic ) value was > nm, which ensured a higher binding capability of the selected epitopes to mhc molecules. linear, and three non-continuous b cell epitopes were fused together with the aid of linker sequences. the ctl epitopes were linked by ayy (the aay liner helps the epitopes produce suitable sites for binding to tap transporter and enhances epitope presentation), the htl epitopes were combined with the aid of gpgpg (the gpgpg linker stimulate htl responses and conserve conformational dependent immunogenicity of helpers as well as antibody epitopes), and b cell epitopes were merged with the aid of kk. the final to enhance vaccine immunogenicity, the human β-defensin- sequence ( aa) and pan-hla dr binding epitopes (the pan-hla dr binding epitopes in vaccine construct facilitating binding to many different types of mouse and human mhc-ii alleles to induce cd -helper cell responses.) was added to the nterminal of the vaccine with the aid of the eaak linker. to enable the intracellular delivery of the modeled vaccine, a tat sequence ( aa) was appended to c-terminal. the vaccine was developed to be amino acids in length ( figure s ) . the sequence homology of final vaccine protein to human protein sequence shown that there were no significant alignments ( figure s ). the allergenic character of the vaccine was determined by the algpred server and was based on the hybrid approach (svmc + ige epitope + arps blast + mast) with a . % coverage. the vaccine was non-allergen with % accuracy and . % sensitivity at threshold value was − . . similarly, the antigenic the half-maximal inhibitory concentration (ic ) value was < nm, which ensured a higher binding capability of selected epitopes to mhc molecules. nature of the vaccine construct was evaluated and showed that the protein was a favorable antigen with a global prediction score of a protective antigen of . (probable antigen). the default threshold value for antigenicity was . in the virus model. moreover, the vaccine constructs contained amino acids, and its molecular weight was . kda. the theoretical pi was predicted to be . . the vaccine contained negatively charged residues and positively charged residues. the vaccine construct was composed of , atoms, and its chemical the immune response profile in silico immune simulation the immune stimulation of the final vaccine was performed using c-immsim online server, which gives the immune profiles of the designed vaccine. the proliferation in the secondary and tertian immune response were identified by igg + igg and igm, as well as, the decreasing of the antigen count igg + igm showed the proliferated (figure a) . the stimulation result revealed the development of immune response after immunization. b cell population was highly stimulated upon immunization ( figure b) . similarly, the cytotoxic and helper t cell levels were proliferated that suggested the development of secondary and tertian immune response (figures c,d) . during the exposure time, it was also observed that the production of ifn-γafter immunization ( figure e) . these results were significant for the immune response against sars-cov- . hence, the tertiary structure of the full-length vaccine sequence was predicted by phyre , and it was applied for refinement and further analysis. twenty-five templates were employing modeling as figure s shown. there were three templates from human defensin which were we added in to enhance the immunogenicity, others from virus ( figure s ) . the immune epitopes were not structural homology to human proteins that could avoid inducing autoimmune. the secondary structure of the predicted model contained % alpha-helix, %tm helices % beta-sheets, and % disordered figure s . to optimize the d structure of the modeled protein, the initial model was refined in the galaxyrefine server. the galaxyrefine server-generated five models based on the rootmean-square deviation (rmsd) and molprobity algorithm. the details of the five models are shown in table s . model with the top ramachandran favored, therefore selected for docking purposes (figure ) . a model with more residues in the ramachandran favored region, less in outliers region and rotamer region was considered as a more ideal one. the initial model generated from phyre server and refine model from galaxyrefine were evaluated with the aid of the swiss-model workspace. the initial model was . % of residues in the ramachandran favored region, . % in the ramachandran outliers region, and only . % in the rotamer region (figure ). the refine model was . % of residues in the ramachandran favored region, . % in the ramachandran outliers region, and only . % in the rotamer region (figure ) . other favorable parameters of the refined model were as follows: gdt score of . , rmsd value of . , molprobability of . , clash score of . , and poor rotamers totaling . ( table s ). the quality and potential errors in the final vaccine d model were verified by prosa-web. the z-score indicates overall model quality, the model with a lower z-score was considered as the higher quality one. the z-score of the initial model was − . , refine model is − . (figure ). to further evaluate the binding affinity between the developed vaccine construct and the relative antigenic receptors (tlr , mhc-i, and mhc-ii), molecular docking was performed. the server yielded candidate models with different binding energies. twenty-nine model complexes of tlr and covid- vaccine were determined, from which just one complex with the lower binding energy score of − . was selected to show ( table and figure ) . a total of model complexes of mhc-i and the covid- vaccine were discovered, and the lowest binding energy score was − . ( table and figure ) . a total of complex models of mhc-ii and the covid- vaccine were predicted, among which, one model complex with the lowest binding energy score of − . was chosen to show ( table and figure ) . further, the vaccine construct was evaluated using the patchdock server, which identified different models and produced a score table. the top complexes identified were refined by the firedock algorithm. among those top models, the model with the lowest binding energy was further selected to show in this paper. the refinement outcomes of tlr and the vaccine complex was solution number with global energy of − . , attractive van der waals energy (vdw) of − . , repulsive (vdw) of . , and atomic contact energy of − . ( table and figure ). the complex of mhc-i and the vaccine was ranked number nine, with global energy of − . , attractive vdw of − . , repulsive vdw of . , and atomic , g , t , t , l , k , e , p , c , s , s , g , p , h , p , l , a , d , n , k , c , c , p , d , g , v , r , s , v , s , p , k , l , f , i , r , e , e , e , t , c , g , q , q , q , t , t , l , k , g , k , k , g , v , q , i , p , c ,t , c , g , k , q , a , t , k , y , l , v , q , q , e , s , p , f . k , p , q , v , n , g , l , t , w , a , d , n ,n , c , l , s , a , p , p , a , q , y , e , l , k , h , g , t , f , t , e , y , t , g , n , y , q , c , g , h , k , t , s , k , e , t , l , y , c , i , d , g , a , l , l , t , k , s , s , e , y , k , g , p , i . d , d , t , l , v , e , f . k , e , n . endornase s , l , e , n , v , a , f , n , v , v , n , k , g , h , f , d , g , q , q , g , e , v , p , v , s , i , i , n , n , t , v , y , t , k , v , d , g , v , d , v , e , l , e , n , k , t , t , l , p , v , n . e , g , s , v , k , g , l , g , e , a , v , k . l , p , s , m , i , d , l , e , l , a , m , d , e , f , i , e , r , y , l , e , g , y , a , f , e , h , i , y , g , d , f , s , h , s , q , l , g , k , r , f , k , e , s , p , e , f , t , d , a , q , t , g , s , s , k , c , k , s , q , d , l , s , v , v , s , k , v , m , l , w , c , k , d , g , h , v , e . t , i , g , c , s , m , t , d , i , a , k , k , p , t , e , t , i , c , a , p , l , t , g , r , v , d , g , v , d , l , f , r , n , a , r , n , k , v , d , g (table and figure ). to accomplish the estimate of the stability of the vaccinereceptor complex, we performed the simulation of the docked complexes (vaccine and tlr- , mhc-i, and mhc-ii) with the help of gromacs. then, various analysis like energy minimization, pressure assessment, temperature, and potential energy calculations were performed. the temperature and pressure of the simulation system during the production run was around k and atmosphere, respectively, indicating a stable system and successful md run. the temperature and pressure of the three simulation systems (vaccine and tlr- , mhc-i, and mhc-ii complexes) during the production run were around k and atmosphere, respectively, indicating the stable systems and successful md run (figures a-f) . the complex root mean square deviation (rmsd) plot represents the structural fluctuation of the overall structure of the complex of vaccine and immune receptor. the rmsd of vaccine-tlr complex has large fluctuation during - ns simulation. after ns, the rmsd value was kept around . nm, indicating that the conformation of this complex was stable ( figure g) . otherwise, the rmsd of vaccine-mhc-i and -mhc-ii complexes has large fluctuation during - ns simulation. after ns, the rmsd value were kept around nm, indicating that the conformation of the two complexes were stable (figures h,i) has low rmsf value, indicating these residues has low structural flexibility. by contrast, residue - and - has relatively higher rmsf value, indicating the larger flexibility during those regions (figures j-l) . to fuse the final vaccine to an expression vector, codon conversion of the vaccine protein was performed by the java codon adaptation tool. restriction site xhoi and bam hi were added to n and c terminals of the codon sequence, then was inserted into the pet a (+) vector between the xhoi and bamhi (figure ) . the rna secondary structure using the mfold program was generated foldings contain , base pairs out of . % in the energy dot plot. mfold predicted an identical secondary structure of , bp formed by nucleotide fragments (figure s ). sars-cov- is characterized by high infectivity and high transmission speed; thus, a prophylactic vaccine is needed ( ) . the availability and advantages of the multi-peptide vaccine developed by immunoinformatics methods have been confirmed by previous studies ( , ) . ojha et al. used the immunoinformatics methods to develop a multiepitope subunit vaccine to epstein-barr virus-associated malignancy ( ) . in recent studies, genomics and proteomics information of sars-cov- have been retrieved, stored, and utilized ( , ) . in the present research, we tried to develop a multi-epitope subunit prophylactic vaccine of sars-cov- , with the help of immunoinformatics tools. a line of research have tried to develop the vaccine of sars-cov- by immunoinformatics tools. baruah and bose ( ) used immunoinformatics tools to discover cytotoxic t lymphocyte (ctl) and b cell epitopes for the spike protein of sars-cov- . then, abraham et al. developed a multi-epitope vaccine that was designed using immunoinformatics tools that potentially trigger both cd + and cd + t-cell immune responses ( ) . most of those research just focus on the spike protein-based vaccine. a vaccine based on the spike protein could induce antibodies to block sars-cov- binding and fusion or neutralize virus infection ( ) , as well as induce harmful immune responses that cause liver damage ( ) . other proteins should be ideal candidates for designing vaccines. in the present report, we selected nine proteins with positive antigenicity for further epitope prediction. all proteins from sars-cov- with < amino acid sequences were excluded, and the antigenic nature of the remaining proteins was evaluated. this method can facilitate the discovery of potential antigens of sars-cov- when the precise immunity mechanisms are unknown. to design an effective vaccine, we selected the sars-cov- protein through the above-mentioned methods for epitope prediction. in recently, asaf et al. reported that identify multiple epitopes for cd + and cd + t cells based on muti-protein ( ) . their protein list was the same as this in our research. in asaf 's report, they just predicted the t cell epitopes, non-b cell, b cell peptide was not predicted ( ) . the b cell epitopes are antigenic determinants from the antigen that are recognized by the b cell surface membrane receptor and evoke the production of specific antibodies. the persistent challenge in immunological prediction tools is the prediction of epitopes to a higher level of accuracy ( ) . to determine accurate linear b cell epitopes from the antigenic proteins, we used two bioinformatics tools based on different algorithms of prediction. we identified nine overlapping linear b cell epitope candidates from two different bioinformatics tools. this method was superior to the prediction of epitopes from a single tool ( ) . moreover, we also have predicted the noncontinue b-cell epitopes. the b cell immune response is preferred in the design of a vaccine. however, t cells may also elicit a strong immunoreaction. the vaccine that activates both ctls and htls should be more effective than a vaccine that only targets ctl responses ( ) . to generate a more effective vaccine, we predicted both ctl epitopes and htl epitopes. the t cell epitopes were decomposed fragments from the antigen presented by the mhc molecules of t cells and stimulated the production of effector t cells, immunological memory t cells, and ifn-γ. the cellmediated immune response induced by ctls plays a vital role in the defense against viral infections through the recognition of intracellular viral pathogens by mhc class i molecules. in the present report, mhc-i binding epitopes were predicted by choosing a , a , and b alleles, which cover ∼ % of world's population. we selected ctl epitopes. the htls play a vital role in the antiviral immune response by producing ifn-γ. moreover, htls are able to induce and maintain ctl responses. furthermore, htls epitopes were chosen based on both the binding capability and ifn-γ induction. bhattacharya et al. also used the spike protein sequence predicted for mhc-i and mhc-ii epitopes of sars-cov- , but not predicted capability of producing ifn-γ ( ). the t cell epitopes enhanced ifn-γ inducing capability, which evokes both the native and specific immune responses by activating macrophages and natural killer cells, and augmenting the response of the mhc to the antigen ( , ) . in this study, the immunogenic epitopes from b cells, ctls, and htls were chosen to develop a more valid, reliable, and effective vaccine against sars-cov- . a multiepitope approach was used by splicing together epitopes with the aid of their respective linkers. to improve the immunogenicity of this multiepitope vaccine, an adjuvant β-defensin and pan-hla dr binding epitopes ( aa) were fused to the n-terminal with the aid of an eaaak linker, then a tat sequence ( aa) was appended to c-terminal with the added of kk. the final vaccine constituted amino acids. the allergenicity, antigenicity, and stability of the designed vaccine constructs were then evaluated. the tertiary structure of the generated vaccine was predicted by using the phyre server and then refined by the galaxyrefine server. the binding affinity of complexes of the developed vaccine and receptors, in which tlr- , mhc-i, and mhc-ii (present on the surface of the immune cell) were confirmed by the cluspro server was based on molecular docking. furthermore, to ensure the translation efficiency of the designed vaccine in a specific expression system, the mrna of the vaccine was enhanced with the aid of the java codon adaptation tool. the restriction enzyme cutting sites of xho? and bamh? were then appended to the n and c terminals, respectively. the vaccine sequence was subsequently cloned in pet a (+), the expression vector. further experimental validation of the safety and efficacy of the designed vaccine for sars-cov- is warranted. all datasets presented in this study are included in the article. rd and zc performed the experiments. rd and yz wrote the paper. yz and fy edited the final version. all authors participated in the experimental design, data analysis, and agreed with the final version of the paper. available online at clinical characteristics of patients infected with sars-cov- in wuhan severe acute respiratory 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coronavirus (sars-cov- ): immunoinformatics approach the use of databases, data mining, and immunoinformatics in vaccinology: where are we? role of allograft inflammatory factor- in the pathogenesis of diseases we extend the sincerest appreciation to nhc key laboratory of pulmonary immunological diseases, and guizhou provincial people's hospital, for their technical assistance. the supplementary material for this article can be found online at: https://www.frontiersin.org/articles/ . /fimmu. . /full#supplementary-material conflict of interest: the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.copyright © dong, chu, yu and zha. this is an open-access article distributed under the terms of the creative commons attribution license (cc by). the use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord- -dnsdg n authors: nan title: poster sessions date: - - journal: eur j immunol doi: . /eji. sha: doc_id: cord_uid: dnsdg n no abtract the humoral pattern recognition receptors of innate immunity include collectins, ficolins and pentraxins. ptx , the prototype of long pentraxins, plays a nonredundant role in resistance against a. fumigatus lung infection. the model proposed suggests that upon binding, ptx facilitates recognition, phagocitosis and killing of a. fumigatus conidia by alveolar macrophages, dendritic cells and neutrophils and the subsequent development of a properly th -oriented adaptive response. actually, ptx -deficient mice are highly susceptible to aspergillosis and develop th skewed responses; moreover, ptx -deficient resident macrophages and neutrophils show defective conidia phagocytosis. both in vitro and in vivo defects can be rescued by the administration of recombinant ptx , which does not show direct activity on fungal cells. finally, ptx alone or in combination with antifungal agents, induces a curative response in mice with aspergillosis, even when given prophylactically. in the present study, we investigated the mechanisms underlying the ptx -mediated opsonic activity and the involvement of complement, complement receptors and fcg receptors, by in vitro studies and genetic approaches in vivo. in vitro ptx amplified the complement-dependent effects on a. fumigatus conidia phagocytosis by human neutrophils, activated through the alternative pathway. accordingly, in the presence of ptx -opsonised conidia, cd b activation, internalization, recruitment to the phagocytic cup and cd b-dependent phagocytosis were increased. as pentraxins interact with fcgreceptors, which in turn can control cd b activation, the phagocytic assay was performed in the presence of fcgr blocking abs. data obtained strongly suggest that upon conidia opsonisation with ptx , fcgriia/cd mediates inside-out activation of cd b and consequently phagocytosis of c b-opsonised conidia. in vivo phagocytosis experiments performed with c q-and fc common gamma chain-deficient mice and complement inhibitors support in vitro data. these data confirm and extend the paradigm of cooperation among innate receptors, in particular among the humoral arm of innate immunity (complement, ptx ) and the cellular arm (fcgrs, cr ). moreover, they confirm previous studies on the interaction between pentraxins and fcgrs and support the idea that pentraxins behave as predecessors of antibodies. innate immunity is the first line of defence against pathogens and plays a key role in the initiation, activation and orientation of adaptive immunity. the humoral arm of the innate immunity includes soluble pattern-recognition receptors (prrs) such as collectins, ficolins, complement components and pentraxins. the prototypic long pentraxin ptx is rapidly produced and released by diverse cell types in response to proinflammatory signals. ptx binds selected microorganisms such as aspergillus fumigatus and restores protective immunity against this pathogen in ptx -/-mice. neonates have an immature innate immune system and are more susceptible to bacterial infection than older children or adult. a beneficial effect of breast feeding on newborn health is highly demonstrated. this protective effect is mediated by nutrients, immunomodulatory mediators (ifn-g, tnfa, or tgf-b), innate immunity factors (soluble cd , immunoglobulins, lactoferrin), and leukocytes contained in milk that can penetrate the newborn circulation. we thus hypothesized that milk may contain ptx . we found high concentration of ptx in human colostrum ( . ± . ng/ml at day post-delivery) compare to the one found in human serum ( x ng/ml). the presence of ptx in human colostrum seems to be due to the secretion of ptx by human mammary gland since we report the production of ptx by these cells. this prr is also found in human milk cells (hmc), mainly in leukocytes, and penetrate into newborn tissus after suckling. furthermore, human colostrum upregulated the ptx production by adult and neonate immunocompetent cells and we demonstrate that neonate mice present a deficit in their ptx production after lps injection. collectively, these data demonstrate that newborn have three distinct ways of ptx supplying by breast feeding: (i) soluble ptx in colostrum (ii) hmc that can secrete ptx upon stimulation in the specific tissue, (iii) an increase of ptx production by immune cells in the presence of colostrum. thus, soluble or cell-derived ptx may participate to the beneficial role of breast feeding on the newborn health. a. m. baru , j. stephani , h. wagner , t. sparwasser twincore, institute for infection immunology, hannover, germany, technical university of munich, institute for medical microbiology, immunology and hygiene, munich, germany toll-like receptors (tlrs) represent the best characterized pattern recognition receptor family in mammalian species. the family currently comprise of receptors in humans (tlr - ) and in mice (tlr - , - ). as transmembrane receptors, tlrs are expressed on the cell surface (tlr , , , , , ( ) ( ) ( ) ( ) and at endosomal membranes (tlr , , and ) . toll-like receptors recognize specific patterns of microbial components and regulate the activation of both innate and adaptive immunity. bacterial dna has been shown to possess immunomodulatory activity about a decade prior to the identification of cpg motifs. about years later to this, toll-like receptor (tlr ) was identified and shown to be the receptor for unmethylated cpg dna which is present mainly in non-vertebrate genome. studies have defined potential role of tlr as adjuvant enhancing protective immune responses against tumours and infectious diseases in murine models. although promising results are obtained from a few human clinical trials, overall efficacy and safety could not yet be translated entirely from murine studies to human trials. one explanation for these discrepancies could be the fact that expression of human-tlr (hutlr ) is restricted to b-cells and plasmacytoid dendritic cells (pdcs) whereas murine-tlr (mutlr ) is also expressed on conventional dendritic cells (cdcs). consequently, tlr ligands induce distinct cytokine profiles in mice and human thereby probably regulating immune responses in a different manner. by employing bacterial artificial chromosome (bac) technology, we generated transgenic mice with hutlr (henceforth called as hut mouse) integration in their genome under human epigenetic control. to avoid effects seen due to overlapping ligand specificities, we crossed this mouse onto mutlr knock-out background. we expect that hut -mutlr -/mice mimic the human specific expression pattern of tlr , i. e. exclusively in b-cells and pdcs, allowing us to investigate detailed in vivo functions of hutlr . by studying infection and tumour models as well as models for autoimmunity, allergy and transplantation we could then define appropriate and safe implications for employment of tlr ligands in human immunotherapy. the fractal analysis provides unique physical insights into the interactions between c q and the prp protein. if one may take the liberty to extend this to cellular surfaces, where presumably these reactions are taking place, then one has access to a possible avenue by which one may control these reactions in desired directions. if this is true, then surely, this is worth exploring further. any effort, no matter how small that assists in help providing better insights into these debilitating and neurodegenrative disorders such as alzheimers is defintely worth the effort. interleukin- is a heterodimeric cytokine consisting of the two subunits p and p . the main inducers of il- p are microbial components activating toll-like receptors with the magnitude of il- p induction depending on the specific tlr engaged. differential induction of il- p upon tlr stimulation correlated with striking differences in the kinetics of nfkb activation. cpg-dna strongly induces il- p due to its outstanding capacity (i) to induce nucleosomal remodelling in proximal il- p promoter region and (ii) to stimulate prolonged rela activity. here we were interested in further changes in chromatin structure of the il- p promoter upon tlr triggering. we did not observe a change in dna methylation, but using chormatin immunoprecipitation (chip) we were able to detect a strong increase in histone and acetylation in specific regions of the proximal promoter region. acetylation of h showed a specific distribution pattern and occured mainly in regulatory elements within the il- p promoter, whereas acetylation of h took place over all regions analyzed. tlr tolerance has been reported to be associated with specific chromatin alterations. methylation status of lysine residue on h turned out to be important for the inhibition of gene expression upon repeated stimulation. modifying the chromatin structure of gene promoter regions therefore seems to be a sensitive mechanism to modify cytokine expression to exogeneous stimuli in innate immune cells thereby allowing adaption of innate immune responses. a. d. koepruelue , w. ellmeier medical university of vienna, institute of immunology/division of immunobiology, vienna, austria macrophages are important in innate and acquired immunity. failures are associated with inflammatory and autoimmune diseases. understanding their stimulation is the basis for therapeutic targeting. members of the tec kinase family (bmx, btk, itk, rlk and tec), expressed in the haematopoietic system, constitute the second largest family of non-receptor tyrosine kinases. mutations in btk represent the source of human x-linked agammaglobulinemia (xla). a mutation in the murine btk gene accounts for a similar syndrome, x-linked immunodeficiency (xid). although the tec family members tec, btk and bmx are expressed in monocytes/macrophages, little is known about their function there. tec kinases become activated upon signaling via divers receptors including antigen receptors, receptor tyrosine kinases or tlrs. several studies in xla or xid macrophages and in monocyte/macrophage cell lines implicated roles for tec kinases in tlr signaling and as well as other macrophage effector functions like phagocytosis. inspired by these findings, we aim to determine the role of tec kinases in bone marrowderived macrophages (bmm), during macrophage activation and in other macrophage functions such as recruitment or phagocytosis. in a comprehensive functional analysis of tlr-mediated bmm activation from mice deficient for one or more of the tec family members in vitro, we reveal which of the kinases play a role in which tlr pathway. based on the results of this analysis, we set the goal to further study how tec kinases regulate the respective signaling cascades. our study will contribute insights into the role of tec kinases in this important cell population of the innate immune system. g. lunazzi , m. buxadé , j. minguillón , r. berga , j. aramburu , c. lópez-rodríguez universitat pompeu fabra, department of experimental and health sciences (dcexs), barcelona, spain nfat is a transcription factor that regulates the expression of cytokines such as tnfa and lymphotoxin b in response to osmotic stress. in addition, nfat participates in multiple processes not linked to the response to hypertonicity. in this regards, it has been recently reported that nfat is required as a novel host factor that supports hiv replication in macrophages. given the established connections between nfat , the expression of certain inflammatory cytokines, and its role in the response to specific pathogens in macrophages, we aimed at studying whether nfat could be activated by receptors for pathogens expressed in macrophages. the activation of toll-like receptors (tlrs) is central to innate immunity. upon stimulation of tlrs, cells of the immune system induce signalling pathways that lead to the activation of different transcription factors. as a result of that, cells such as macrophages and dendritic cells induce the expression of genes that participate in the response to pathogens such as those encoding proinflammatory cytokines, antimicrobial products, survival factors or mediators of cellular migration. we have analyzed whether nfat is expressed in primary macrophages through the activation of different toll-like receptors. likewise, we have explored whether the activity of nfat is induced during the response to tlrs. in addition, we have studied whether the specific inhibition of different signalling pathways positioned downstream of tlrs could interfere with the expression of nfat . our work indicates that nfat is a novel transcriptional regulator acting in response to the activation of tlrs. our work extends the knowledge about mechanisms that participate during the innate immune response to pathogens and offers a new regulatory pathway as a possible target to modulate this response. objectives: compelling evidence support a link between inflammation, cell survival, and cancer, with a central role played by nf-xb, a master switch of inflammation. recent studies implicate some tlrs in tumor development or regression, and immune escape. however, mechanisms leading to tumor growth or apoptosis induced by tlr stimulation are not fully understood. several studies strongly suggest that chronic inflammation in lungs induced by chronic bronchitis, chronic obstructive diseases, emphysema, asbestos or tobacco smoke, increases the risk of carcinogenesis. we hypothesized that some tlrs can contribute to lung inflammation and tumor development in vitro and in vivo. methods: tlr expression in lung cancer was assayed by immunohistochemistry or flow cytometry. nfxb activation was determined by western blot and nuclear translocation assay after tlr stimulation. clonogenicity of stimulated cells was analyzed by colony assay. transcriptomic analysis were performed by taqman lda technology. tumor growth in vivo was analyzed in nod/scid mice after subcutaneously engraftment of human lung tumor cell lines. we have observed that primary human lung tumors express tlr , tlr , tlr and tlr and that stimulation of these receptors in lung tumor cell lines by poly i:c, lps, loxoribine or poly u induces nfxb activation through atypical signaling pathway, with phosphorylation of ixba without its degradation and nuclear translocation of p and p nfxb subunits. interestingly, we observed that tlr stimulation induces apoptosis depending of the histological type of the tumor. on the contrary tlr , tlr and tlr stimulation induces cell survival and increases clonogenicity. this is correlated with an up-regulation of bcl- expression. moreover, despite a common atypical activation of nfxb, our transcriptomic analysis revealed major differences in gene modulation after triggering of tlr , tlr , tlr and tlr . finally, in vivo tlr stimulation of human lung tumor cells dramatically increases tumor size and metastasis. conclusions: altogether, these data emphasize that tlr , tlr or tlr triggering can directly favor tumor development whereas tlr signaling can induce tumor cell death. these data suggest that anticancer immunotherapy using tlr adjuvants should take into account the expression of these tlrs in lung tumor cells. objective: dasatinib (bms- ) is a small molecule src/abl tyrosine kinase inhibitor approved for the treatment of chronic myeloid leukaemia and philadelphia chromosome-positive acute lymphoblastic leukaemia. members of the src family of kinases are involved in normal physiological processes, and play a significant role in the induction and regulation of innate and adaptive immunity. the purpose of this study was to evaluate the inhibitory action of dasatinib on toll like receptor (tlr) signalling, natural killer (nk) cell cytotoxicity as well as antigen-specific cd + and cd + t cell function. methods: to analyse tlr signalling in vitro murine bone marrow derived (bmd) macrophages were stimulated with the tlr ligand lipopolysaccaride (lps) in the presence of dasatinib and tumour necrosis factor a (tnf-a) in the culture medium was measured. the response to tlr stimulation was also tested in vivo, dasatinib-treated mice were challenged with lps and tnf-a in the serum was quantified. in addition, the clearance of the rma-s cells, a mhc class i deficient thymoma sensitive to nk cell lysis, was analysed in mice undergoing dasatinib treatment. to investigate the inhibitory effects of dasatinib on adaptive immune responses, transgenic cd + and cd + t cells specific for ovalbumin were utilised to measure antigen specific t cell proliferation. endogenenous cd + and cd + t cell responses were determined following immunisation of dasatinib-treated mice with a nonreplicating recombinant virus. results: we show that dasatinib impairs: . innate immune response; dasatinib treatment reduced the (a) production of tnf-a following tlr stimulation of bmd macrophages in vitro, (b) production of tnf-a in vivo in response to lps and (c) ability of nk cells to eliminate mhc class i deficient cells in vivo . . adaptive immune response; dasatinib treatment inhibited (a) proliferation of antigen-specific murine transgenic t cells, (b) endogenous antigen-specific helper t cell recall-responses and (c) t cell-mediated cytotoxic effector function. conclusions: these findings suggest that dasatinib has the potential to modulate the host immune response and highlights scope for off target applications, for example therapeutic immunosuppression in the context of autoimmune pathogenesis, or in combination with other interventions for the treatment of endotoxic shock. i. zanoni , r. oatuni , m. collini , m. caccia , p. castagnoli , g. chirico , f. granucci university of milano-bicocca, biotechnology and bioscience, milan, italy, university of milano-bicocca, physics, milan, italy, singapore immunology network (sign), biomedical sciences institutes, immunos, singapore, singapore the recognition of mamps by tlrs expressed on dendritic cells (dcs) plays an essential role for the regulation of the immune responses. by recruiting different combinations of adapter proteins, individual tlrs turn on signal transduction pathways leading to the activation of different transcription factors. interleukin- (il- ) is one of the molecules produced by dcs shortly after stimulation with different tlr agonists. based on this observation and by analogy with the events following t-cell receptor (tcr) engagement leading to il- production, we hypothesized that the stimulation of tlrs on dcs might lead to activation of the ca +/ calcineurin and nfat pathway. we found that dc stimulation with lps induces extracellular ca + influxes, leading to calcineurin-dependent nfat activation. the activation of this pathway was independent of tlr engagement, depending instead exclusively on cd . we also found that lps-induced nfat activation in dcs was necessary for the efficient synthesis of cyclooxygenase- (cox- ) that, by generating prostaglandins (pgs), such as pge , regulates different dc functions including migration and polarization of t cell responses. our findings reveal novel aspects of the molecular signaling triggered by lps in dcs and define a new role for cd . given the essential involvement of cd in many diseases, including sepsis and chronic heart failure, the discovery of signal transduction pathways activated exclusively via cd represents a major step towards the development of potential treatments with modes of action involving interference with cd functions. we have examined the interaction of cd , a -kda glycosyl-phosphatidylinositol (gpi)-anchored membrane protein, with the monocyte signalling receptor, cd . human monocytes were isolated from healthy adult donor's peripheral blood. this involved labelling molecules at saturation with different coloured fluorophores and determining their positions separately by dual wavelength imaging. the cells were labelled at saturation with anti-cd antibody coupled to biotin visualised by qd- -streptavidin and anti-cd antibody coupled to allophycocyanin. the images are analysed to quantify the overlap of the particle images and hence determine the extent of co-localization of the labelled molecules. single particle fluorescence imaging (spfi) uses the high sensitivity of fluorescence to visualize individual molecules that have been selectively labelled with small fluorescent particles. the images of particles are diffraction-limited spots that are analysed by fitting with a two-dimensional gaussian function providing the basis for determining the dynamic and associated behaviour of receptors on living cells. changes in the numbers of receptors, and in the proportion of receptors showing colocalisation, indicated that lps promotes the interaction of cd and cd , suggesting a new functional role of cd as a member of a multimeric lps receptor complex. l. lundvall , r.r. schumann charité -universitätsmedizin berlin campus mitte, institute for microbiology and hygiene, berlin, germany meningitis is a life-threatening disease mainly caused by bacteria and viruses. bacterial components such as lipopolysaccharide (lps), lipoproteins or peptidoglycan breakdown products (i. e. mdp, mesodap) stimulate pattern recognition receptors (prrs), such as toll-like receptors (tlrs) and the intracellular nod-like receptors (nlrs) for an inflammatory response. we hypothesize that a synergistic effect of tlr-induced nf-xb activation and nlr-mediated caspase- induction leads to an increased release of mature il- b during bacterial meningitis in brain-derived cells. a mouse meningitis model with s. pneumoniae (d ) was established for assessing il- b induction during this disease. the murine raw . cell line, the human astroglial u- mg and the murine microglial cell-line bv- were stimulated with the tlr ligand lps, the tlr ligand pam cys, the nod ligand mdp, or the nod ligand c -ie-dap, and, as control, atp alone or in combination. we assessed il- b by elisa and caspase- and pro-il- b expression by western blot. furthermore, primary mouse astrocytes isolated from the cortices of mouse puppies were used for stimulation followed by sirna suppression of elements of the il- b induction pathway. s. pneumoniae (d ) infected mice showed a significant increase in il- b release after hours. in vitro, an increase in il- b levels after costimulation with lps or pam cys, and mdp or c -ie-dap was observed in a dose-dependent manner. a synergistic enhancement of il- b by tlr-and nlr-ligands was observed in raw cells, bv- cells, u- cells and primary astrocytes. active caspase- (p ) was induced by mdp or c -ie-dap, corresponding with high il- b responses when lps or pam cys was added. sirna experiments show that a knock-down of nod leads to a diminished il- b release after lps-and mdp-stimulation. the precursor forms of il- b and caspase- seem to be constitutively expressed in astrocytes and microglia. a synergistic enhancement between tlrs and nlrs in il- b release in brain-derived cells was observed. so a two-step stimulation seems necessary for the release of high levels of mature il- b by astrocytes and microglia. bacteria containing both, tlr-and nlr-ligands thus have the potential to induce high levels of il- b which may contribute to disease pathology and may point to novel intervention strategies. j. rosenberg toll-like receptors (tlrs), nod-like receptors (nlrs), and rig-i-like (rlrs ) are more well-characterized in their identity and expression as signaling markers which effect the ealry innate immune response and elicit adaptive immunity , . in the case of tlrs most sutides to date have delineated tlr expression and function on antigen presenting cells like dendritic cellof this research. extension of the profiling and presence of tlrs on cell characterized as adaptive immune cells such as t cells is the subject of this line of research. using a cd and cd activation model system -tlr presence on cd + cells is found in mouse t cells, human t cells and jurkat cell lines. following cd /cd activation for hours we have identified a small but distinct populationof tlr + cells. further characterization indicates these cells to be cd +cd + cells. further characterization of the expression and functional acitvity of the tlr + t cells indicates co-expression of tlr with md- indicating a functional tlr receptor. in addition lps activiation did not lead to upregulation of tlr expression in t-cells. the data indicate that tcr activation leads to tlr expression. the expression appears to be associated with cd +cd + cells and refelecting an activated t cell phenotype which will be further characterized as perhaps related to tregs or other tcell subsets. s. m. lehmann , d. kaul , c. krüger , f. zipp , r. nitsch , s. lehnardt charité-universitätsmedizin berlin, cecilie-vogt-clinic for neurology, berlin, germany, charité-universitätsmedizin berlin, institute for cell biology and neurobiology, berlin, germany the innate immune system is the first line of defense against various pathogens and requires the expression of toll-like receptors (tlrs). in macrophages, tlr plays a crucial role in immune responses elicited by gu-rich ssrna (i. e. ssrna ) as well as synthetic antiviral chemicals, including imidazoquinoline components (i. e. imiquimod) and some guanine nucleotide analogs (i. e. loxoribine). these compounds were initially described to activate mouse tlr (and human tlr ) and are potent immune response modifiers leading to important antiviral and antitumor activities. microglia serve as the major innate immune cells in the central nervous system (cns). employing various techniques including pcr, in situ hybridization, and immunocytochemistry, we demonstrate that tlr is expressed in these cells. incubation of microglia with all three of the above mentioned tlr ligands leads to activation of these cells displaying an ameboid shape and releasing inflammatory cytokines such as tnf-a and il -b in a dose-and time-dependent fashion. analysis of wild type (wt) and tlr knock out (ko) microglia by real- because neutrophil apoptosis plays a key role in resolving inflammation, identification of proteins regulating neutrophil survival should provide new strategies to modulate inflammation. using a proteomic approach, coronin- was identified as a cytosolic protein cleaved during neutrophil apoptosis. coronin- is an actinbinding protein that can associate with phagosomes and nadph oxidase but its involvement in apoptosis was currently unknown. in coronin- -transfected plb cells, coronin- overexpression did not modify the kinetics of granulocyte differentiation as assessed by cd b labeling. concerning apoptosis, increased coronin- expression in dmf-differentiated plb significantly decreased gliotoxin-induced mitochondrial depolarization as compared with controls. likewise, coronin- significantly decreased trail-induced apoptosis with less mitochondrial depolarization, caspase- and caspase- activities, but not caspase- or bid truncation suggesting that coronin- interfered with mitochondria-related events. to validate the prosurvival role of coronin- in a pathophysiological condition involving neutrophil-dominated inflammation, neutrophils from cystic fibrosis (cf) patients were studied. circulating neutrophils from cf patients had more coronin- expression assessed by immunoblotting or proteomic analysis of cytosolic proteins. this was associated with a lower apoptosis rate than those from controls evidenced by delayed phosphatidylserine externalization and mitochondria depolarization. in addition, inflammatory neutrophils from cf patients lungs showed an intense coronin- immunolabeling. we concluded that coronin- could constitute a potential target in resolving inflammation. p.-n. hsu national taiwan university, graduate institute of immunology, taipei, taiwan, republic of china human osteoclast formation from mononuclear phagocyte precursors involves interactions between tumor necrosis factor (tnf) ligand superfamily members and their receptors. many of the proinflammatory cytokines and growth factors implicated in inflammatory processes have also been demonstrated to impact osteoclast differentiation and function. recent evidence indicates that the tnf-related apoptosis-inducing ligand (trail) of the tnf ligand superfamily, which was initially thought to induce apoptosis in many transformed cell lines, can serve as an effector molecule in activated t cells. we show in this work that trail can induce osteoclast formation from human monocytes and murine raw . macrophages. we demonstrated that both cell models differentiate into osteoclast-like cells in the presence of trail in a dose-dependent manner, as evaluated in terms of tartrate-resistant acid phosphatase (trap)-positive multinucleated cells and bone resorption activity. the trail-induced osteoclast differentiation is independent of caspase activation and apoptosis induction activity. however, trail-induced osteoclastogenesis is dependent on activation of nf-xb, erk, and p map kinase. the trail-induced osteoclastogenesis was significantly inhibited by treatment with traf- sirna and traf- decoy peptide, indicating this pathway is traf- dependent. thus, our data demonstrate that trail induces osteoclast differentiation via direct engagement with the trail death receptor through a signaling pathway distinct from apoptosis. our results indicate that in addition to triggering apoptosis, trail induces osteoclast differentiation. it provides a novel role for trail in regulating osteoclast differentiation and in osteoimmunology. microglia are considered to be the local antigen presenting cells (apcs) of the central nervous system (cns) which are thought to play a crucial role in local reactivation of autoreactive t cells during cns autoimmunity e. g. in multiple sclerosis (ms) and its animal model experimental autoimmune encephalomyelitis (eae) . in this study we investigated if the anti-inflammatory nuclear transcription factor peroxisome proliferator-activated receptor gamma (pparg) that has been described to negatively regulate macrophage activation has an influence on microglia immunogenicity. sustained activation of pparg both reduced microglial signalling via mhc molecules and costimulatory molecules and concomitantly increased signalling via the coinhibitory molecules b -h and b -dc. moreover, also production of pro-inflammatory cytokines like tnf-a and il- was profoundly reduced if microglia were pre-treated with the pparg-agonist pioglitazone (pio). in contrast to this, the lack of pparg in microglia resulted in increased expression of pro-inflammatory cytokines not only following an inflammatory stimulus but also in the steady-state indicating that pparg might play a cell-intrinsic role in controlling microglia immunogenicity. importantly, if pparg was activated in microglia, the capacity to prime ovalbumin-specific t cells was impaired. t cells primed by pio-treated microglia produced reduced amounts of il- and ifn-g which could not be overcome by restimulation with acd . this indicates that t cells primed by pio-treated microglia did not undergo functional differentiation but were impaired in exhibiting effector functions. furthermore, microglia were able to induce antigen-specific differentiation of naive cd t cells into t helper (th ) cells, which have been associated with autoimmune pathogenicity during eae. however, if pparg was activated, microglia were no longer able to induce th differentiation. in conclusion, activation of pparg impairs microglial apc function leading to reduced activation of antigen-specific t cells and, in addition, inhibits the induction of th cells. therefore, activation of pparg in microglia is a promising approach to limit local activation of autoreactive t cells in the cns in cns-autoimmune deseases. bacterial lipopolysaccharide (lps) triggers monocytes and macrophages to produce several inflammatory cytokines and mediators. however, once exposed to lps, they become hyporesponsive to a subsequent endotoxin challenge. this phenomenon is defined as lps desensitization or tolerance. previous studies have identified some components of the biochemical pathways involved in negative modulation of lps responses. in particular, it has been shown that the il- receptor-related protein st could be implicated in lps tolerance. the natural ligand of st was recently identified as interleukin- (il- ), a new member of the il- family. in this study, we investigated whether il- triggering of st was able to induce lps desensitization of mouse macrophages. we found that il- actually enhances the lps response of macrophages and does not induce lps desensitization. we demonstrate that this il- enhancing effect of lps response is mediated by the st receptor since it is not found in st ko mice. the biochemical consequences of il- pretreatment of mouse macrophages were investigated. our results show that il- increases the expression of the lps receptor components myeloid differentiation protein- (md ), cd and tlr- and the myeloid differentiation factor (myd ) adaptor molecule. in addition, il- pretreatment of macrophages enhances the cytokine response to tlr- but not to tlr- ligands. thus, il- treatment preferentially affects the myd -dependent pathway activated by the tlr. c. klotz , b. lenz , r. lucius , s. hartmann humboldt-university berlin, molecular parasitology, berlin, germany chronic helminth infections are shown to be negatively associated with allergic disorders in humans and animal models and parasite cysteine protease inhibitors (cystatins) have been identified as a major class of modulators from filarial parasites. recently we showed that recombinant parasite cystatin (avcystatin), derived from the model parasite acanthocheilanema viteae, effectively abolished ova-induced allergic airway responsiveness in a mouse model of asthma (schnoeller et al., ) . the cystatin effect was blocked by the application of anti-il- receptor antibodies and by depletion of macrophages using clodronate liposomes. we hypothesize that parasite cystatin induced regulatory macrophages characterized by secretion of immune suppressive interleukin- (il- ). the aim of the present study was to elucidate the molecular mechanisms by which avcystatin induces il- in primary macrophages. in vitro experiments with peritoneal macrophages from balb/c mice confirmed specific and concentration dependent il- production after avcystatin stimulation. application of specific inhibitors revealed that the il- induction was p and erk dependent, and inhibitor titration indicated a higher sensitivity towards p . western blotting experiments confirmed the phosphorylation of p and erk in macrophages after avcystatin stimulation. in addition, by using specific inhibitor and western blotting, we showed that avcystatin induced il- is also regulated by the phosphatidylinositol- -kinase (pi k) -proteine kinase b (akt) pathway. further analysis indicated a hierarchical signalling pattern and cross regulation of the identified pathways. hence, we conclude that avcystatin renders macrophages into a regulatory state by addressing a broad range of signalling cascades that ultimately lead to the expression of il- and possibly other regulatory markers. in general, revealing fundamental knowledge about induction of regulatory macrophages by helminth immunomodulators will help to design new strategies for the treatment of inflammatory disorders. we screened approximately half the (putative) human kinome to identify novel candidates interfering with macrophage activation in response to endotoxin. this screen revealed the impact of several novel kinases as well as kinases with previously established function. one of the top candidates identified to block endotoxininduced tnf-a secretion was carkl, a gene with no previously described function. subsequent biochemical analyses unequivocally revealed that carkl is a phosphotransferase protein using sedoheptulose as a phosphate acceptor and atp as a donor. sedoheptulose is a monosaccharide consisting of seven carbon atoms and a functional ketone group. the product sedoheptulose- -phosphate (s- p) is also an intermediate metabolite of the pentose phosphate pathway (ppp) and so far was only known to be produced by condensation of ribose- p and xylulose- p via a transketolase reaction. to identify the molecular mechanism by which carkl modulates the immune response, we investigated its endogenous regulation and function in the course of macrophage activation. so far, our data favor a model where post-stimulatory downregulation, i. e. loss of carkl is essential for the activation of macrophages by various pro-inflammatory stimuli. disentangling the signaling pathways responsible for the rapid regulation of carkl unearthed nf-kb and p /jnk but not erk as driving forces. counterbalancing endotoxin induced loss of carkl by over-expression led to an impaired cytokine response and a concomitant block of free radical production. comparison of wild type and catalyticinactive forms of carkl unveiled that most of the effects of carkl on the inflammatory response were due to its phosphotransferase activity. expression profiling using gene chip analysis further supported the concept that carkl may represent a new key modulator of inflammatory processes. taken together, detailed analyses to study the molecular function of carkl should ultimately lead to a more profound understanding of cellular metabolism and especially clarify new mechanisms involved in the regulation of inflammation. in addition, connecting the ppp and its impact on the cellular redox state with inflammatory disease models might reveal new therapeutic targets. in this context, the sedoheptulose kinase carkl and its product s- p may provide a novel basis for interfering with adverse immune responses. t. bosschaerts , y. morias , p. de baetselier , a. beschin vib, cmim, vub brussels, brussels, belgium the development of classically activated monocytic cells (m ) is a prerequisite for effective elimination of parasites, including african trypanosomes. however, persistent m activation causes pathogenic damage including liver injury during infection, resulting in death of the host. we aim to identify mechanisms involved in regulation of m activity in order to dampen their pathogenicity and increase the resistance of the host to parasitic diseases. methods: we have scrutinized the phenotype and cellular origin of liver m in trypanosoma brucei infected by facs analysis and bone marrow transfer experiments. the contribution of different signaling pathways, including myd , ifng, il- , ccr and nf-kb to the development and/or recruitment of pathogenic m in the liver was investigated using knock-out mice or by delivering il- in infected mice. results: we established that cd b+ly c+cd c+ tnf and inos producing dcs (tip-dcs) represent the major m liver subpopulation. tip-dcs differentiated in an ifng/myd -dependent manner from cd b+ly c+ inflammatory monocytes in the liver of infected mice. ccr promoted the egression of inflammatory monocytes from bone marrow to blood but not their entry, differentiation and maturation to tip-dcs in the inflamed liver. as a consequence, ccr ko mice experienced reduced pathogenic symptoms. on the other hand, the absence of il- enhanced the recruitment of inflammatory monocytes as well as their differentiation and maturation to tip-dcs, resulting in exacerbated pathogenicity and early death of the host. in addition, the therapeutic liver-specific delivery of il- in t.brucei infected mice efficiently limited the differentiation and maturation to tip-dcs, hereby limiting disease-associated pathogenicity. finally, the absence of the nf-kb member p was associated with increased tissue injury associated with increased production of pathogenic tnf and no by inflammatory monocytes, but not by tip-dcs. conclusion: our data demonstrate that nf-kb p and il- play a role in preventing infection-associated pathogenicity in hosts confronted with a chronic inflammatory situation by limiting the activity of pathogenic m , in particular tip-dcs. the inflammatory activity of liver m is controlled by il- and/or p nf-kb at different levels, including recruitment of inflammatory monocytes to the liver, their differentiation to pathogenic tip-dcs, or their production of tnf and no. a. popov , j. driesen , z. abdullah , a. niño castro , t. chakraborty , m. krönke , o. utermöhlen , c. wickenhauser , j.l. schultze limes institute, laboratory for genomics and immunoregulation, university of bonn, bonn, germany, institute of molecular medicine and experimental immunology, bonn, germany, institute of medical microbiology, university of giessen, giessen, germany, institute for medical microbiology, immunology and hygiene, university of cologne, cologne, germany, institute for pathology, university clinic leipzig, leipzig, germany dendritic cells (dc) and macrophages play an important role in pathogen sensing and antimicrobial defense. here we report on a new role for the myeloid antigen presenting cells (apc) in granulomatous infections. infection of myeloid dc and macrophages with listeria monocytogenes results in a distinct regulatory phenotype characterized by expression of multiple inhibitory molecules, including indoleamine , -dioxygenase, cyclooxygenase- and cd and production of prostaglandin e (pge ) and interleukin . all these molecules are strictly dependent on autocrine tnf, released during infection, and are in concert suppressing t-cell responses; cd , expressed by regulatory myeloid cells, acts as an il- scavenger. importantly, myeloid cells with regulatory phenotype are characterized by increased resistance to infection and demonstrate significantly improved bactericidal activity against intracellular bacteria. furthermore, infected cells can transfer the regulatory phenotype to the uninfected ones in a cell-cell contact independent manner, thereby extending the pool of infection-resistant myeloid cells. induction of regulatory and protective phenotype in macrophages and dc require at least two signals provided by tnf and either pge or tlr ligands. transcriptional changes in human macrophages, infected by mycobacterium tuberculosis, resemble the ones induced in dc during infection with l.monocytogenes. in fact, granuloma in patients with tuberculosis and listeriosis are enriched for cd + ido + cox- + regulatory myeloid cells, whereas most effector cell populations, such as t cells, b cells and nk cells, are expelled from the granuloma. of note, in tuberculosis granuloma consist mostly of macrophages, whereas in listeriosis dendritic cells predominate. altogether, our studies provide strong evidence that intracellular pathogens such as m.tuberculosis and l.monocytogenes induce a specific polarization of myeloid dc and macrophages characterized by a functional preponderance of inhibitory mechanisms. we postulate that these regulatory myeloid cells play a dual role during life-threatening granulomatous infections. on one hand, they promote pathogen containment by efficiently killing intracellular bacteria; on the other hand, these myeloid cells inhibit granuloma-associated t cells and thereby might be involved in the retention of granuloma integrity protecting the host from granuloma break-down and pathogen dissemination. the interferon-gamma (ifn-g) component of the immune response plays an important and essential role in infectious and non-infectious diseases. induction of ifn-g secretion by human t and nk cells through synergistic co-stimulation with interleukin (il- ) and il- in the adaptive immune responses against pathogens is well known, whereas a similar activity by macrophages is still controversial, largely due to criticisms based on the contamination of macrophages with nk or t cells in the relevant experiments. the possible contribution of macrophages to the interferon response is, however, an important factor relevant to the pathogenesis of many diseases. to resolve this issue, we have determined the production of ifn-g at a single cell level by inmunohistochemistry and by enzyme-linked immunosorbent spot (elispot) analysis and have unequivocally demonstrated that human macrophages derived from monocytes in vitro through the combined stimulation of il- and il- or with macrophage-colony stimulating factor (m-csf) were able to produce ifn-g when further stimulated with a combination of il- and il- . in addition, naturally activated alveolar macrophages immediately secreted ifn-g upon treatment with il- and il- . therefore, human macrophages in addition to lymphoid cells contribute to the ifn-g response, providing another link between the innate and acquired immune response. a. j. denzel , m. rodriguez gomez , m. niedermeier , y. talke , n. göbel , k. schmidbauer , m. mack unversity hospital regensburg, internal medicine ii, regensburg, germany, university hospital regensburg, regensburg, germany we have shown previously that basophils recognize and react to free antigen during a memory immune response in vivo and release large amounts of il- and il- . activation of basophils is dependent on the presence of free antigen, antigen specific immunoglobulins and expression of immunoglobulin fc-receptors. we now have analysed in more detail the binding of antigen to basophils, the recruitment of basophils to lymphoid organs and the basophil dependent migration of other leukocytes during the first days of a memory immune response. following restimulation with soluble antigen only antigen specific basophils but not basophils from naïve mice migrate from bone marrow and spleen to the site of restimulation (e.g. the peritoneum) and the draining lymph nodes. peripheral blood basophils are markedly reduced during the first hours after restimulation. in the blood, spleen lymph nodes and bone marrow basophils can bind intact antigen on their surface for up to h, with basophils in the bone marrow binding the lowest amount of antigen. depletion of basophils also affects the recruitment of various other leukocyte subsets in immunized mice. our datas show that basophils are recruited to draining lymph nodes during a memory response. tnf-a is a pro-inflammatory cytokine that mediates inflammation in response to various pathogens, including mycobacterium tuberculosis. it is also a key factor in the pathogenesis of autoimmune diseases like rheumatoid arthritis. three tnf-a-blocking drugs have been approved to treat selected autoimmune diseases; two are monoclonal antibodies against tnf-a (adalimumab and infliximab); the other is a soluble tnf receptor/fc fusion protein (etanercept) . tnf-a-blockers have been shown to increase the risk of reactivation of latent tuberculosis and this risk appears to be higher in patients treated with the monoclonal antibodies. we studied the effects of tnf-a blockers on the maturation of mycobacteria-containing phagosomes in human macrophages. all three drugs had an inhibitory effect on ifn-g-induced phagosome maturation in pma-differentiated human thp- cells infected with m. bovis bcg, the avirulent m. tuberculosis h ra strain and the virulent m. tuberculosis h rv strain. adalimumab and infliximab, but not etanercept, suppressed phagosome maturation in primary human peripheral blood monocyte-derived macrophages (mdm) in the presence or absence of ifn-g. macrophages secreted tnf-a in response to infection with mycobacteria and this response was enhanced by activation of the cells with ifn-g. treatment of infected macrophages with tnf-a increased maturation of mycobacteria-containing phagosomes. these results suggest a role for tnf-a in activating phagosome maturation and highlight a novel mechanism through which tnf-a blockade can affect the host innate immune response to mycobacteria. z. g. dobreva , l.d. miteva , s.a. stanilova trakia university, faculty of medicine, molecular biology, immunology and genetics, stara zagora, bulgaria il- is a heterodimeric cytokine composed of a p subunit associated with the il- / p subunit. like il- , il- is expressed by the activated antigenpresenting cells and both cytokines induce ifn-gamma secretion by different t cell subsets. the proper balance between il- p -related cytokines controls the appearance of normal th and pathological th mediated immune responses. in this study, we examined the dynamics of inducible il- p and il- p mrna expression and protein production in purified human monocytes and how jnk and p mapks inhibitors influenced il- p and il- production. the cytokines' quantity determination was performed by elisa. quantitative real-time polymerase chain reaction (qrt-pcr) was performed for mrna transcripts detection. results were calculated in fold increase compared with gene expression in nonstimulated monocytes. il- p gene expression was higher than those observed for il- p gene at all time-points. the level of il- p mrna increased after th h and reaching a maximum level at th h ( . fold for c bgp and . fold for lps). c bgp and lps triggered il- p gene transcription were almost equal at the rd h ( . and . fold) and at th h ( . and . fold, respectively) after stimulation. the higher level of il- p gene expression was detected at th h in lps compared to c bgp stimulated monocytes ( . vs. . fold). however, il- p and il- protein production was increased in the highest level after c bgp stimulation. the inhibition of p led to the statistical significant augmentation of c bgp stimulated il- p production. the inhibition of the same map kinase enhanced lps stimulated il- p production without significant difference. the inhibition of jnk and p mapks significantly decreased c bgp stimulated il- production from human monocytic cells.in summary, the present study demonstrates the different time-course and ability of c bgp and lps to induce the expression of il- p and il- p mrnas in purified human monocytes. we showed that inhibition of p mapk down regulated il- and upregulated il- p production in stimulated monocytes. we concluded that in human monocytes p map kinase activation has an opposite effect on the il- p and il- p expression. neutrophils represent key components of the innate immune system with the ability not only to phagocytose and killing invading pathogens, but also to produce a variety of proteins, including cytokines and chemokines, with important consequences on the recruitment and activation of other immune cells, such as monocytes, dendritic cells, t and b cells. for instance, it has been shown that neutrophils can directly interact with, and induce functional maturation of, immature monocyte-derived dendritic cells (modc). indeed, upon interaction with neutrophils, modc up-regulate the expression of costimulatory molecules, such as cd , cd and cd , and secrete il- , thus acquiring the ability to induce proliferation and th polarization of naï ve t cells. in order to extend these findings, the present study was designed to address whether human neutrophils interact with peripheral blood-derived dendritic cells and the pathological consequences that such interaction could eventually produce. in human peripheral blood, dendritic cells can be divided in plasmacytoid dendritic cells (pdc) and myeloid dendritic cells (mdc), the latter further divided in three different subsets based on the expression of cd c, bdca- , and cd . by analyzing different chronic inflammatory pathologies, such as crohn's disease, psoriasis and sweet's syndrome, we found that neutrophils co-localize with a subtype of myeloid dendritic cells (mdc) with characteristics resembling the cd + subset of mdc. in order to characterize the interaction between the two cell types, autologous neutrophils, highly purified by an in-house built immunonegative selection protocol, and cd + dc were isolated from healthy donors and analyzed in a co-culture system under different stimulatory conditions. here we show that neutrophils modulate different effector functions of cd + dc, including their survival and their ability to produce il- p . besides providing the basis for a better understanding of the cellular interactions that occur in pathological conditions, our results further emphasize the importance of neutrophils in the modulation of the inflammatory response. chitin is a linear polymer of n-acetyl-d-glucosamine (glcnac) residues present in human pathogenic fungi or nematodes. chitotriosidases (cht) and acetic mammalian chitinase (amcase) have been identified as the only functional chitinases in mammalians. the expression of both chitinases appears to be strongly species dependent, indicating distinct physiological functions. amcase is considered as predominant chitinases in mice while cht is regarded as major chitinases in humans. interestingly, cht is constitutively expressed by human phagocytes at high levels while it is absent in mice phagocytes. although, amcase received increased attention as modulator of the innate immune response against chitin in mice, the physiological function of cht in humans is virtually unknown. to evaluate the physiological function of cht we have characterised the substrate specificity of human cht and the mode of substrate cleavage by analysing chtproduced fragments of chitosan, a close but water soluble derivate of chitin. degradation products of chitosan have been investigated by gel electrophoresis and maldi-tof mass spectroscopy. moreover, the application of a computer-based model of cht activity revealed the mode of substrate cleavage. we found that cht is a processive endo-cleaving chitinase resulting in the production of only small diffusible chitin/chitosan fragments. in further studies we could show that those cht-produced small chitin/chitosan fragments exhibit a strong ability to induce a pro-inflammatory response in human blood derived monocytes/macrophages as indicated by an increased release of the pro-inflammatory cytokines tnf-a, il- , il- and mcp- involving the transcription factor nfxb. moreover, these stimulated monocytes/macrophages revealed an increase of cht expression indicating an autocrine positive feed-back regulation. our data suggest that human cht is involved in the early recognition of chitin/chitosan containing human pathogens due to the generation of immuno-stimulatory chitin/chitosan fragments. m. hasenberg , s. wolke , a. brakhage , m. gunzer otto-von-guericke universität, institut für molekulare und klinische immunologie, magdeburg, germany, hans-knöll-institut, abteilung für molekulare und angewandte mikrobiologie, jena, germany since their discovery in nucleic extracellular traps (nets) released by certain cell types including neutrophil and eosinophil granulocytes were shown to play a crucial role in mediating innate immune responses towards different bacterial und fungal pathogens. recently it was found by us and others that neutrophil granulocytes release nets also upon contact to the filamentous fungus aspergillus fumigatus. in the present study we aimed to characterize this process in more detail focusing on the kinetics of net-formation as well as clarifying the responsible cell-biological mechanisms. by the use of several microscopic techniques (scanning electron microscopy, fluorescence widefield microscopy, confocal-and -photon microscopy) we initially demonstrated the generation of net like structures after coincubation of a. fumigatus germlings and freshly isolated murine or human pmn. the analysis of our time lapse video microscopy data allowed us to examine the exact time course from initial contact to the fungal surface to explosive release of nets up to hours later. moreover, we investigated the dependency of this phenomenon on the induction of an oxidative burst. therefore we added the nadph-oxidase inhibitor dpi to the cell coincubation and found clearly reduced net formation. by fluorescence staining of reactive oxygen species we could demonstrate that ros are released prior to net detection. interestingly, our data currently suggest that in contrast to other pathogens investigated so far, nets are not directly toxic to fungal elements. whether and how nets control growth of a. fumigatus currently remains open. to summarize our data, we found rapid net formation as a commonly observed immune response of neutrophil granulocytes contacting a. fumigatus. consistent with studies on different pathogens this mechanism seems to be ros-dependent, however not toxic for the fungus. thus, in the future we will have to clarify whether net-formation really occurs in vivo and how nets can control the outgrowth of a. fumigatus at sites of infection. production of type i interferons (ifn-i, mainly ifn-a and ifn-b) is a hallmark of innate immune responses to all classes of pathogens. when viral infection spreads to lymphoid organs, the majority of systemic ifn-i is produced by a specialized 'interferon-producing cell' (ipc) that has been shown to belong to the lineage of plasmacytoid dendritic cells (pdc). it is unclear whether production of systemic ifn-i is generally attributable to pdc irrespective of the nature of the infecting pathogen. we have addressed this question by studying infections of mice with the intracellular bacterium listeria monocytogenes. protective innate immunity against this pathogen is weakened by ifn-i activity. in mice infected with l. monocytogenes systemic ifn-i was amplified via ifn-b, the ifn-i receptor (ifnar) and transcription factor interferon regulatory factor (irf ), a molecular circuitry usually characterisitic of non-pdc producers. synthesis of serum ifn-i did not require tlr . in contrast, in vitro differentiated pdc infected with l. monocytogenes needed tlr to transcribe ifn-i mrna. consistent with the assumption that pdc are not the producers of systemic ifn-i, conditional ablation of the ifn-i receptor in mice showed that most systemic ifn-i is produced by myeloid cells. furthermore, results obtained with facs-purified splenic cell populations from infected mice confirmed the assumption that a cell type with surface antigens characteristic of macrophages and not of pdc is responsible for bulk ifn-i synthesis. the amount of ifn-i produced in the investigated mouse lines was inversely correlated to the resistance to lethal infection. based on these data we propose that the engagement of pdc, the mode of ifn-i mobilization, as well as the shaping of the antimicrobial innate immune response by ifn-i differ between intracellular pathogens. t. naessens , s. vander beken , p. bogaert , j. grooten ghent university, biomedical molecular biology, zwijnaarde (ghent), belgium introduction: although the effector and modulator functions of activated macrophages in innate and adaptive immunity are well documented, their exact role in the initiation and propagation of immune pathologies is still not fully understood. recent insights in monocyte and macrophage heterogeneity render the picture even more complex. in addition, it is unclear to what extend resident and elicited macrophages differ functionally and hereby differentially contribute to immune pathologies. in this study we focused on the dynamics and function of resident alveolar macrophages (ram) during and after allergic bronchial inflammation. strategy: we used an ovalbumin (ova)-alum based mouse model of allergic asthma and an ova-complete freund's adjuvant (cfa) based mouse model of hypersensitivity pneumonitis, constituting a th -driven immunological counterpart of the th -driven experimental asthma. ram were distinguished by prior in situ labelling with fluorescent polystyrene microspheres. as complementary approach, ram and elicited alveolar macrophages (eam) were distinguished using cd bone marrow chimeric mice. combined with flow cytometry and fluorescence activated cell sorting, both approaches allowed us to trace resident and elicited am populations in the course of th -and th -driven allergic airway inflammation. results: during the acute phase of the allergic response, isolated ram and eam showed distinct gene expression signatures, reflecting a possible functional heterogeneity between these two macrophage subsets. in both types of allergic inflammation, microsphere-tagged cd . + ram remained constant in cell number for the first days of chronic ova-exposure and then dropped sharply, having nearly completely disappeared from the alveoli by day of ova-exposure. as a consequence, following the clearance of inflammation, inflammation-experienced ram replaced the initial ram population. strikingly, in both types of allergic inflammation, this secondary ram population showed a markedly altered responsiveness to lps stimulation. this involved macrophage activation markers and nf-kb inducible inflammatory genes. however, especially genes induced by ifn-beta showed strongly increased expression in secondary ram as opposed to their near lack of induction in primary ram. this switch from an ifn-beta deficient to an ifn-beta adequate phenotype may increase the inflammatory sensitivity of allergic inflammationexperienced lungs as also observed in asthmatic patients, showing an increased sensitivity to bacterial infection. e. schlecker , a. stojanovic , a. cerwenka german cancer research center, innate immunity, heidelberg, germany myeloid-derived suppressor cells (mdsc) are a heterogeneous population of cells that expand during cancer, inflammation and infection. these cells play a critical role in suppressing t cell responses. the exact nature and function of mdsc remain unclear. here we show that a subpopulation of mdsc (gr- + cd b + f / + ) isolated from rma-s tumor-bearing mice did not suppress but rather activated nk cells to produce ifn-g. additionally, nk cells eliminated this subpopulation both in vitro and in vivo. in order to identify molecules and pathways that might be involved in mdsc accumulation in tumor bearing mice and their suppressive/activatory function, gene expression profiling of mdsc subpopulations was performed using whole genome microarrays. understanding the reciprocal interaction of mdsc with nk cell could improve the efficiency of cancer immunotherapy. g. solinas , f. marchesi , m. fabbri , s. schiarea , c. chiabrando , a. mantovani , , p. allavena istituto clinico humanitas, rozzano, italy, istituto mario negri, milano, italy, università di milano, milano, italy experimental and clinical evidence has highlighted that tumor-associated macrophages (tam) represent the principal component of the leukocyte infiltrate and are usually associated with tumour growth, progression and metastasis. macrophage population is generally divided into two distinct subsets: m and m . m macrophages act as a first line of defence against pathogens whereas m cells participate in wound repair and maintenance of tissue integrity. in the tumour micro-environment tam interactions with the extracellular matrix, neighboring cells, and soluble stimuli largely influence their gene expression and behavior. to investigate the role of the tumor micro-environment on macrophage differentiation, we cultured freshly isolated human monocytes with pancreatic cancer cell line supernatants, in the absence of exogenous cytokine addition.. in selected cultures, about % of the monocytes differentiated after days into macrophages. the phenotype analysis of tumor-conditioned macrophages (tc-macro) demonstrated high expression of the mannose receptor, cd , cd and low levels of mhc class ii. tc-macro produced il- , il- , tnf but not il- , even after lps stimulation. moreover, tc-macro produced a panel of chemokines including ccl , cxcl , ccl and cxcl . the transcriptional profile of tc-macro revealed that several genes in line with an m polarization are highly expressed. the nature of the tumor-derived factors inducing macrophage differentiation is currently under investigation; biochemical analysis indicated that the biological activity is excluded from exosomes and have a high molecular weight ( g . kda). il- and il- were not detectable in tumor supernatants whereas m-csf was present at low levels. by mass spectrometric techniques, we surprisingly found that the tumor-derived m-csf had peculiar migration patterns which were different from those expected for the common human homodimeric glycosilated protein, suggesting an interesting structural differences for the tumor-secreted isoforms of this primary regulator of mononuclear phagocyte. the characterization of tumor-derived factors inducing macrophage differentiation could better clarify the intricate cross-talk between tumor cells and macrophages and thus might aid in the process of devising novel anti-tumor treatments. genomic effects of glucocorticoid hormone (gc) are exerted by glucocorticoid receptor (gr)-mediated changes of gene expression. this is relatively timeconsuming process, needing hours to develop. in contrast, non-genomic effects may occur within minutes. gcs are used for a long time for the therapy of anaphylactic reactions, where mast cells play crucial role. moreover, many cells and cell lines of haemopoetic origin are sensitive to gc-induced apoptosis. recent findings indicate, that non-genomic gc effects mediated by mitochondrial gr may have important function in generating pro-apoptotic signals. we aimed the investigation of non-genomic gc effects on in vitro cultured rbl- h rat mast cell line. we demonstrate that gr nuclear translocation begins within minutes and completes after minutes in dx treated rbl- h cells. since genomic effects occur in the nucleus through gene expression changes, we considered gc effects within minutes as non-genomic. studying gc-caused apoptosis, rbl- h cells proved to be gc-resistant and no mitochondrial gr translocation neither impaired mitochondrial function could be observed upon gc treatment. in further experiments we used rbl- h cells sensitized with anti-dnp (dinitrophenyl) ige and dnp-conjugated bovine serum albumin was used for stimulation. minutes of dx treatment inhibited ca + -signaling in antigen stimulated rbl- h cells in the concentration range of nm - mm. moreover, minutes of dx treatment altered the tyrosine phosphorylation pattern of rbl- h cells. dx treatment alone caused slight increase in tyrosine phosphorylation, while dx treatment of activated cells caused also an increase in tyrosine phosphorylation compared to the solvent-treated controls. the tyrosine kinase syk plays indispensable role in regulating mast cell activation through the fc[epsilon] receptor i. our immunoprecipitation studies show, that dx treatment results in decreased syk phosphorylation in both resting and activated cells. this finding raises the possibility, that syk phosphorylation thus kinase activity may be directly or indirectly regulated by gcs via non-genomic pathway. taken together, our experiments along with the clinical experiences suggest that gcs rapidly influence mast cell activation via a non-genomic pathway, too. the elucidation of the exact signal transduction mechanisms behind rapid gc effects need further experiments. high mobility group box (hmgb ) is a non-histone nuclear protein that binds chromatin and has transcriptional and architectural functions. notably, hmgb is highly mobile in the nucleus and is passively released by necrotic cells, while it is bound firmly to apoptotic chromatin ( ) . extracellular hmgb can act as a cytokine and a chemoattractant, mediating inflammatory responses. interestingly, hmgb exerts antibacterial functions in human adenoid and testis ( ) . recent investigations have revealed that neutrophils eliminate microbes not only by intracellular phagocytosis but also by trapping them in three-dimensional structures called neutrophil extracelluar traps (nets), made of dna fibers, nuclear proteins (histones) and granule proteins. it has been shown that histones on nets have an anti-microbial activity ( ). we asked whether hmgb from neutrophils is a component of nets and whether it has a function in nets. we purified human primary neutrophils from peripheral blood of healthy volunteers on ficoll gradients. to induce net formation, we stimulated cells for or minutes with nm phorbol ester (pma), ng/ml interleukin (il- ), or ng/ml lps. the presence of nets was assessed by immunofluorescence using antibodies directed against the granule protein myeloperoxidase (mpo) and against a dna-histone h a-histone h b complex. dna was stained with hoechst. using a polyclonal antibody we found hmgb in the euchromatin of polylobulated nuclei of resting neutrophils and on the filamentous structure of nets induced by all stimuli. elisa assays revealed that hmgb is not present in the supernatants of activated neutrophils, confirming its binding to nets. in conclusion, we found that hmgb localizes on nets. we hypothesize that net-bound hmgb might exert a direct antimicrobial function, or that nets might concentrate hmgb locally to recruit macrophages to the site of infection. these receptors were present on the mast cell surface. incubation ( °c, h) of hlmc with vegf-a, vegf-b, vegf-c, vegf-d and placental growth factor- induced concentration-dependent chemotaxis that was blocked by a combination of anti-vegfr- and anti-vegfr- antibodies. these data indicate that human mast cells represent both a source and a target of vegfs and therefore may play a role in inflammatory and neoplastic angiogenesis through the expression of proangiogenic factors and their receptors. macrophages are important effector cells in immunity to intracellular pathogens and at the same time are exploited as host cells by a number of microorganisms such as mycobacterium tuberculosis. a very important mechanism of intracellular killing is delivery of invading microbes to phagolysosomes. whilst mycobacteria can block phagosome maturation in resting macrophages, and hence survive and replicate inside the host cell, the ifn-g activated macrophage utilizes a diversity of defense mechanisms to eliminate the invader. these include putative killing by antibacterial peptides/proteins and overcoming phagosome maturation block, possibly by induction of autophagy, production of reactive nitrogen or oxygen intermediates and deprivation from nutrients such as iron. mycobacteria are not eliminated even upon onset of protective immunity rather leading to persistence. we hypothesize that the very early steps of pulmonary infection directs the outcome of disease. therefore, we investigate initially infected lung cells and their role in infection in the lung with respect to their anti-microbial mechanisms against mycobacteria in vitro as well as in vivo. preliminary data show that m. tuberculosis is able to persist in the alveolar space for several weeks and bacterial numbers do barely drop even after very low dose infection, indication that bacterial killing is inefficient from the very beginning. cells harboring mycobacteria are found during early and late stages of infection. both, autophagy and nitric oxide production seems to contribute to growth restriction of mycobacteria by macrophages. neutrophils, although recruited in vast numbers to infected lungs, are not able to reduce bacterial numbers in the absence of il- . altogether, the initial response in the barrier organ lung executed by resident and immigrating cells restricted by the local environment can determine the outcome of infection. human cd molecules are dedicated to lipid presentation to t cells and are implicated in inflammatory and auto-immune responses. the cd a protein is almost exclusively expressed at the cell surface of dendritic cells and is dedicated in surveying extracellular environment. our previous studies have demonstrated that ii associated with cd a and cholesterol-dependent lipid rafts impact on cd a surface expression and cd a-restricted t cell response. bacterial infections can induce an increase in self glycolipid synthesis in dendritic cells and such activated dcs acquire the ability to stimulate cd -restricted autoreactive t cells. this mechanism of self recognition induced by bacterial infection is believed to be involved in the development of auto-immune disorders. sulfatide, which is a major component of the myelin sheath, is also the only known self-antigen presented by cd group i molecules. the functional role of these molecules has not been investigated in auto-immune diseases and we propose that regulation of glycolipid presentation by cd a molecules could impact in such pathologies. we have thus conducted a preliminary study to understand the implication of cd molecules in multiple sclerosis. we first analyzed cd expression on monocytes from ms patients and the influence of sera and plasma from these patients on dendritic cell differentiation from healthy donors. results obtained in this preliminary study demonstrate that cd a was not expressed on ms patient monocytes, while the other members of the cd family were expressed. moreover ms sera and plasma induced an earlier and more rapid dendritic cell differentiation than ab sera. these preliminary results confirm our hypothesis that cd molecule expression is modified in ms and also reveal that serum from patients with ms modifies lipid-antigen presenting cells. further studies should contribute to define precise mechanisms involved in lipid presentation by cd molecules in this context. c. ohnmacht , d. voehringer ludwig-maximilians-universität munich, institute for immunology, munich, germany basophils are effector cells of the innate immune system which are associated with allergic inflammation and infections with helminth parasites. however, their development and in vivo functions are largely unknown. here, we characterize basophil turnover, tissue localization and effector functions during infection with the gastrointestinal helminth nippostrongylus brasiliensis. for this purpose, brdu incorporation experiments and in situ fluorescence microscopy of il- reporter ( get) mice as well as in vivo depletion of basophils are used to uncover their role during type immune responses. our results demonstrate that under homeostatic conditions basophils have a lifespan of about h. n. brasiliensis induced basophilia is caused by increased de novo production of basophils in the bone marrow. basophils are found near the marginal zone in the red pulp of the spleen, in the lamina propria of the small intestine and in the lung parenchyma. activated basophils promote systemic eosinophilia, were associated with differentiation of alternatively activated macrophages in the lung and contributed to efficient worm expulsion of n. brasiliensis in the absence of th cells. these results demonstrate that basophils play a crucial role as effector cells in type immune responses which might hold great potential for the treatment of helminth infections and allergic diseases. during acute bacterial infections such as meningitis, neutrophils enter the tissue where they combat the infection before they undergo apoptosis and are taken up by macrophages. neutrophils show pro-inflammatory activity and may contribute to tissue damage. in pneumococcal meningitis, neuronal damage despite adequate chemotherapy is a frequent clinical finding. this damage may be due to excessive neutrophil activity. we here show that transgenic expression of bcl- in haematopoietic cells blocks the resolution of inflammation following antibiotic therapy in a mouse model of pneumococcal meningitis. the persistence of neutrophil brain infiltrates was accompanied by high levels of il- beta and g-csf as well as reduced levels of anti-inflammatory tgf-beta. significantly, bcl- -transgenic mice developed more severe disease that was dependent on neutrophils, characterized by pronounced vasogenic edema, vasculitis, brain haemorrhages and higher clinical scores. in vitro analysis of neutrophils demonstrated that apoptosis inhibition completely preserves neutrophil effector function and prevents internalization by macrophages. the inhibitor of cyclin-dependent kinases, roscovitine induced apoptosis in neutrophils in vitro and in vivo. in wild type mice treated with antibiotics, roscovitine significantly improved the resolution of the inflammation after pneumococcal infection and accelerated recovery. these results indicate that apoptosis is essential to turn off activated neutrophils and show that inflammatory activity and disease severity in a pyogenic infection can be modulated by targeting the apoptotic pathway in neutrophils. objectives: to investigate the existence of systemic inflammatory response to subchronic oral warfarin (wf) consumation in rats. methods: dark agouti (da) rats were treated with warfarin in drinking water ( mg and mg daily) for days. oxidative activity (cytochemical nbt reduction) and myeloperoxidase (mpo) intracellular content of peripheral blood neutrophils, plasma levels of il- and tnf-a (elisa) and superoxide dismutase (sod) activity (red blood cell lysates) were analyzed as inflammatory parameters in rats following warfarin consumation. changes in prothrombin time (pt), as basic biological warfarin activity was determined as well. results: significantly increased pt was noted at the lower wf dose, with tremendous rise after the higher dose. increase of pma-stimulated neutrophil nbt reduction capacity (neutrophil priming) was noted at both wf doses, while increase in mpo intracellular content was noted at the higher wf dose solely. warfarin consumation resulted in no changes in plasma levels of il- and tnf-a. significant decrease in the sod activity was detected in red blood cell lysates at both wf doses, suggesting systemic oxidative activity. conclusion: increased neutrophil priming as well as prooxidant activity in peripheral blood of rats following subchronic warfarin consumation imply proinflammatory effects of oral warfarin administration. absence of the rise in inflammatory cytokines in circulation, suggest low-grade inflammation in these rats. this work is funded by serbian ministry of science and technological development (grant ). objectives: although many different macrophage receptors and serum proteins have been shown to play a role in phagocytosis of apoptotic cells, the unique microenvironment of an inflammatory site will have considerable influence upon the molecular pathways which are utilized in apoptotic cell removal. we have recently reported that immune complexes (ic) are able to specifically bind to the surface of human apoptotic neutrophils which may have profound implications for their physiological clearance. in disease situations where immune complexes are present neutrophils undergoing apoptosis would be predicted to become coated with ic. here we address the consequences of ic opsonisation of apoptotic cells upon phagocytosis and cytokine response by macrophages that would be expected to be present at the earliest stages of inflammatory responses (type- macrophages, mph ), and during resolution of inflammation (type- macrophages, mph ). methods: mph / were generated by culturing cd + human monocytes for days in the presence of gm-csf or m-csf, respectively. phagocytosis by mph / of ic opsonised and unopsonised neutrophils was assessed by flow cytometry. after phagocytosis mph / were stimulated with lps and secreted il- , il- , il- , il- p and tnf were quantified by elisa. results: mph are relatively efficient phagocytes for apoptotic neutrophils whereas mph are only poorly phagocytic. opsonisation with ic leads to enhanced neutrophil uptake by both mph and mph which is specifically inhibited in the presence of a blocking mab for macrophage fcyrii. uptake of ic opsonised neutrophils causes a shift towards an anti-inflammatory cytokine profile. in both macrophage subsets il- , il- and tnf production is suppressed while il- secretion is increased. in contrast, engagement of macrophage fcyr with ic alone induces the release of pro-inflammatory cytokines. conclusion: our data demonstrate that ic opsonisation of apoptotic neutrophils increases the proportion of macrophages capable of phagocytosis and that apoptotic cell recognition interactions provide a dominant anti-inflammatory signal, suppressing macrophage responses, even in the presence of ic opsonisation. we suggest that ic present in the inflammatory milieu would opsonise apoptotic neutrophils, enhance macrophage phagocytosis and thereby facilitate the process of resolution of inflammation. excessive production of reactive oxygen species (ros) produced by neutrophils is known to be a factor accelerating ageing because of damaging effect on cells. on the other hand, intracellular heat shock proteins (hsp) are involved in protecting cells from the damaging effects, and provide cell resistance to stress. in this work, correlation analysis was applied to analyze relationship between ros production and intracellular hsp in neutrophils of elderly people. neutrophils were isolated from peripheral blood of donors of years old and older (long-livers). intracellular ros and hsp levels were registered by flow cytofluorimetry upon labeling with ', '-dichlorofluorescin diacetate (invitrogen) and anti-hsp antibody (brm- , sigma), respectively. intracellular level of hsp was also estimated in neutrophils after heat shock (hs) performed at °c for min. extracellular ros production from zymosan-activated neutrophils was detected by luminol-dependent chemiluminescence. a positive correlation was determined for intracellular ros level and zymosan-mediated extracellular ros release although the dynamics of ros release at - min time range varied within the group. the correlation was unaffected by hs of neutrophils performed for min at °c, although this short heat treatment decreased significantly ros release. there was no correlation between basal intracellular hsp (hsp basal ) and ros level, both intracellular and extracellular. at the same time increased hsp level immediately after hs (hsp ( min)) correlated negatively with intracellular ros (initial and after hs). the hsp increase value (hsp ( min) -hsp basal ) correlated negatively also with intracellular ros and extracellular ros release in response to zymosan; and the correlation with ros level became lower when hsp increase was registered in min after hs (hsp ( min) -hsp basal ). thus it was found that within this age group the alteration in hsp induced by hs in neutrophils but not basal hsp itself is the parameter associated negatively with both spontaneous ros level and ros production in response to activating action of zymosan. this work is supported by istc grant # . d. goyeneche-patiño , z. orinska , f. mirghomizadeh , s. bulfone-paus forschungszentrum borstel, borstel, germany several studies have shown different roles of mast cells (mc) in innate and adaptative immune responses. in fact, crosstalk between cd + t cells and mc has shown to induce multiple genes implicated in the signaling of specific programs such as type ifn. two novel genes, receptor transporter protein (rtp ) and virus inhibitory protein, endoplasmic reticulum-associated, interferon-inducible (viperin) are ifn inducible and were found to be over-expressed in chip analysis. the aim of this study is to characterize the expression and protein production of rtp and viperin in mast cells after tlr ligand stimulation in mice lacking of ifnra and the adapter proteins myd and trif. bone marrow derived mast cells (bmmc) from wt, ifnra -/-, mydd -/and trif -/mice, were exposed to tlr ligands (lps, pic, cpg, p(da-dt) and new castle disease virus (ndv)) during and h. mrna and protein extraction were performed for further qrt-pcr and sds page analysis for rtp and viperin. intracellular stimulation of tlr was performed transfecting cells with nucleic acids using lipofectamine . stimulation of wt cells with pic, pda-dt and ndv showed an increased expression of viperin and rtp in comparison to control cells (untreated). the same trend was observed for mc from the trif and myd knockout mice. in contrast, in the ifnra deficient mice, expression of genes and protein production was abrogated to the same levels of wt untreated cells. lipofectamine stimulation does not increase the expression/production of the genes. direct stimulation of the well recognized viral sensors tlr and , as well as, infection of mast cells with ndv (rna virus) induce the expression of rtp and viperin. the findings suggest that activation of mc with the ulterior expression of genes is type i ifn dependent. in contrast, the adaptor proteins myd and trif and the pathways that they represent are not relevant in the expression of rtp and viperin. these findings provide bases for performing further studies focused to elucidate the functions of these proteins and show an alternative role of mc in innate immune responses. in recent years, it has been suggested that the phenomenon of "myc-dependent cell competition" described in drosophila melanogaster, could be a critical step when a cell initiates nascent tumour field. we have taken a step forward and applied the phenomenon of cellular competition to the human macrophage system: inflammatory macrophages theoretically have the ability to eradicate cancer due to their tumoricidal capability and, at the same time, acting as antigen-presenting cells (apcs) to activate lymphocytes; but inflammatory macrophages do not express c-myc and, within the tumour, they encounter two powerful rivals: tumoral cells and alternative tumour-associated macrophages which express c-myc. we studied some phenomenons suggested to be myc-dependent such as the ability to feed, the ability to survive in a competitive medium, the ability to proliferate and the ability to eliminate competitors and we observed that alternative macrophages have more resources to survive in a tumoral microenvironment and could be involved in tumour growth by collaborating with tumour cells in transforming inflammatory macrophages into anergic cells which enter into apoptosis and are then phagocyted. finally, using lentiviral vectors, we over-expressed exogenous c-myc in inflammatory macrophages in an attempt to increase their chances of survival in the tumour microenvironment, in vitro and in vivo, and to determine whether it can be utilized as a potential anti-tumoral cell therapy. g. germano , e. erba , r. frapolli , m. d'incalci , a. anselmo , s. pesce , p. allavena , a. mantovani , humanitas clinical institute, rozzano, italy, mario negri institute, milan, italy, institute of general pathology, university of milan, milan, italy several lines of evidence suggest a strong association between chronic inflammation and tumor progression; therefore the use of anti-inflammatory drugs may be beneficial in anti-tumor therapies. inflammatory mediators (e. g. cytokines, chemokines) are produced at the tumor site both by tumor-associated macrophages (tam) as well as tumor cells, and are attractive target of novel anti-tumor therapies. trabectedin (et- ) is a natural product derived from the marine tunicate ectenascidia turbinate, it binds the minor groove of dna, affects transcriptional factor activity and blocks cell cycle. this novel anti-tumor agent is currently used in phase ii studies in patients with sarcoma, ovarian and breast cancer, with clinical regressions. we previously demonstrated that trabectedin is selectively cytotoxic, in vitro, to monocytes/macrophages, being active at concentrations that spared lymphocytes suggesting a possible alternative target for the anti-tumoral role of this drug. we tested the effect of trabectedin on primary cultures and liposarcoma cell lines showing that at sub-cytotoxic concentrations the production of some inflammatory mediators were down-modulated. trabectedin significantly reduces ccl , cxcl and the inflammatory protein pentraxin (ptx ) either at transcriptional and protein level, especially after tnfa/il b stimulation. down-regulation of ccl , cxcl , ptx and also of il and vegf were confirmed in primary cultures of liposarcoma. according to the previous in vitro data we now show in a mouse model , using the fibrosarcoma mnmcai , that trabectedin treatment selectively affects monocytes in the blood and bone marrow. moreover trabectedin treatment strongly reduces the number of macrophages and of cd + vessels in the tumor microenvironment, in line with its selective activity on monocytes/macrophages. overall these results suggest a possible triple role of trabectedin. besides its direct cytotoxic effect on tumor cells, trabectedin also affects tumor associated macrophages and at low dose the transcriptional activity of inflammatory genes involved in the tumor-microenvironment cross-talk. as the local expression of inflammatory mediators may play a role in tumor progression, this newly recognized effect of trabectedin makes it an attractive candidate in inflammation-associated tumors. interleukin- (il- ) is a key cytokine of the t helper cell response. il- has been found to be a major regulator of immunoglobulin class switching to ige and has important functions in the regulation of allergic diseases. here, the onset of the il- production after birth was investigated in equine neonates. the form of equine placentation does not support the transfer of cytokines or immunoglobulins in utero and maternal immunity is exclusively transferred to the neonate with the colostrum after birth. il- producing cells were measured in peripheral blood mononuclear cells (pbmc) of neonates and foals by flow cytometric analysis. at day - after birth, a small population of il- producing cells was observed in the absence of any stimuli. the il- + population was not detectable at or weeks of age. other cytokine producing cells (ifn-g, il- ) were not detected using these conditions. the stimulation of neonatal pbmc with pma and ionomycin did not alter the il- + cell population. phenotyping of the neonatal il- + cells showed that they were ige + /mhcii -/cd cells. the occurrence of cd + il- producing cells after pma stimulation increased slowly with age and did not reach adult levels by weeks after birth. magnetic cell sorting of the ige + /mhciicells identified them as basophils. previous work has shown that foals do not produce endogenous ige for at least six months of life. ige bound to the surface of neonatal basophils was found to be of maternal origin and transferred with the colostrum after birth. here, the stimulation of neonatal pbmc with anti-ige induced the secretion of il- at day after birth. neonatal pbmc collected before colostrum uptake did not produce il- in response to anti-ige. in summary, equine neonates provide a model to investigate ige mediated il- responses after birth. the transfer of maternal ige from allergic individuals could potentially provide a direct mechanism for the early induction of an allergen-specific neonatal il- response mediated by the mare's accumulated acquired immunity to allergens. s. schmechel , d. voehringer ludwig-maximilians-university munich, institute for immunology, munich, germany macrophages display broad phenotypic heterogeneity depending on their microenvironment. the initial inflammatory response to th cytokines is predominantly mediated by classically activated macrophages whereas macrophages undergo alternative activation in a stat -dependent manner when stimulated with the th cytokines il- or il- . alternatively activated macrophages (aam) are implicated in diverse disease pathologies such as host response to parasitic infection and asthma. furthermore, it has been shown that aam suppress the proliferation of t cells by a yet to be determined mechanism. currently there is still very limited information about the phenotype, migration and function of aam. we began to elucidate whether macrophage turnover and recruitment to inflammatory sites is regulated in a stat -dependent manner. to this end we generated mixed bone marrow chimeras with bone marrow from congenic wild-type and stat -deficient mice and infected these chimeras with the helminth nippostrongylus brasiliensis to determine whether lack of stat in macrophages affects their turnover and recruitment to the lung side-by-side in the same animal. the highest turnover of macrophages was found in the peritoneum, irrespective of stat expression. no major differences in tissue distribution and turnover were observed between both populations suggesting that macrophage proliferation and recruitment during parasite infection is not dependent on stat expression in macrophages. we could further confirm that in vitro generated aam from wild-type but not from stat -deficient mice have a strong inhibitory effect on t cell proliferation. we are now trying to identify the mechanism(s) by which t cell proliferation is inhibited. furthermore, we work on the cellular cross-talk between eosinophils and macrophages and try to determine the plasticity of macrophage differentiation. we have previously shown that aam can recruit eosinophils to inflammatory sites and we now try to clarify which chemotactic factor are involved in this process. to identify potential aam-derived eosinophil chemotactic factors we currently compare the gene expression profile of il- exposed macrophages from wild-type and stat -deficient mice. candidate genes will be expressed using retroviral transfections of stat -deficient macrophages and supernatants from these cells will then be used to induce eosinophil recruitment in transwell assays. macrophages are an essential component of leukocytes infiltration in the tumor. they are identified as tumor associated macrophages (tams). these cells are also present in pleural effusions which appear as a consequence of spreading of neoplasm in the pleural cavity. the aim of the study was to assess the influence of the pleural macrophages on cells from human malignant cell lines. we tested the dynamics of growth of the malignant cells, their apoptosis and expression of proteins regulating this process under the influence of conditioned media from cultures of macrophages isolated from pleural effusion. we have also attempted to interpret our results by assessing the expression of a variety of immune modulating factors, their receptors on the malignant cells surface as well as the transcription factors. in the study we used macrophages isolated from a total of pleural effusions, including malignant and nonmalignant tumors. the following human malignant cell lines were tested: a , ht , hct , sw , mcf , mda-mb , jurkat and hl . results: our results suggest that the conditioned media isolated from the cultures of pleural macrophages can up-regulate the proliferative activity of the human malignant cell lines. macrophages from pleural effusions can act as a factor promoting or inhibiting apoptosis of malignant cells. down-regulation of apoptosis may depend on modulation of expression and activity of proteins regulating this process. macrophages can affect the apoptosis regulatory proteins and their activity through the immune-modulatory molecules, e. g. cytokines, chemokines, and growth factors. the up-or down-regulation of transcription factors expression may control the expression of pro-and anti-apoptotic proteins. the results indicate that macrophages from malignant and non-malignant pleural effusions differ from each other insignificantly; however the macrophages isolated from the non-malignant tumors show a pattern comparable to m , and the tams isolated from the malignant effusions similar to m . among the alternative stimuli, glucocorticoids are the most effective stimulus up-regulating ms a a and ms a a: highest trascriptional level after h of stimulation with - m dexamethasone. ms a murine genes are differently expressed respect to the human counterpart and only the homologs of ms a a (ms a b, c and d) have a similar regulation. finally, egfp-tagged ms a a, ms a a, and ms a expressed in cho cells showed that all molecules traffic to the cell membrane. though the biological functions of these ms a proteins has not jet been defined, their membrane localization and the structural relationship with other better characterized ms a members suggest a potential involvement in signal transduction, either as components of multimeric receptor complexes or as components of ligand-gated ion channels. during inflammatory reactions endogenously produced cytokines and chemokines act in a network and interact with hormones and neurotransmittors to regulate host immune responses. these signaling circuitries are even more interfaced during infections in which microbial agonists activate toll-like (tlr), rig-like (rlr) and nod-like (nlr) receptors. on the basis of the discovery of synergy between chemokines for neutrophil attraction, we here extended this phenomenon between the chemokine monocyte chemotactic protein- (mcp- )/ccl and the gpcr ligand fmlp or the tlr agonist lipopolysaccharide (lps) on monocytes. in fact, the bacterial tripeptide fmlp, but not the cytokines il- b or ifn-g, significantly and dose-dependently synergized with ccl in monocyte chemotaxis. furthermore, lps rapidly induced the expression of interleukin- /cxcl , but not of the ccl receptor ccr in monocytic cells. in turn, the induced cxcl synergized with ccl for mononuclear cell chemotaxis and the chemotactic effect was mediated by cxcr /cxcr , because cxcl receptor antagonists or antibodies were capable of blocking the synergy, while keeping the responsiveness to ccl intact. these data recapitulate in vitro the complexity of innate immune regulation, provide a novel mechanism of enhancing monocyte chemotaxis during bacterial infections with gram-negative bacteria and demonstrate the importance of local contexts in inflammatory and infectious insults. objectives: in recent years the existence and effects of cell-derived vesicles (e. g. exosomes, microparticles) have been revealed in several physiological functions, such as antigen presentation, hemostasis or receptor transfer to innocent cells. most data were collected on endothelial cells and on thrombocytes. however, there are only few data on vesicles derived of neutrophilic granulocytes (pmn), and most of these investigations applied only pharmacological agents. our aim is to investigate pmn-derived cell-free particles and their possible role in bacterial killing methods: preparation of pmn and investigation of bacterial killing by our semi-automatic method was described by rada et al. (blood, ) . cell-free vesicles were prepared after co-incubation of human pmns with different activating agents for min at °c with gentle shaking, followed by spinning down of pmns for min, at °c and g. the supernatant was sedimented at g for min, °c, and we used the sedimented fraction for our investigations. formation of particles was followed by fluorescent and electron microscopic assays. the amount of particles was estimated with flow cytometer and by their protein content. we observed that upon co-incubation of pmns with s. aureus, opsonized by mixed human serum, pmns produce a well detectable amount of vesicles. omitting opsonization or opsonizing with heat inactivated serum caused a minimal amount of particles. production of particles could be inhibited with diphenyl-iodonium (dpi), cytochalasin b (cb) or with azide treatment. treating pmns with dnase or withdrawing glucose during co-incubation had no effect on vesicle formation. in killing assays we detected remarkable antibacterial effect, which correlated well with the protein content of the used fraction. this antibacterial activity could be inhibited by dpi, cb, azide treatment or by withdrawing glucose from the medium during the killing assay. however, treatment of the microvesicles with dnase had no effect on their antibacterial capacity. for long, cd has been used for the detection and identification of natural killer (nk) cells. recently, the presence of a minor subset of cd low cd + blood monocytes (mo) in healthy individuals and the increase in cd +cd + blood mo in patients with inflammation has been reported. the functional activity of human cd + blood mo has been studied in vitro but not tested ex vivo so far. healthy people living permanently in malaria endemic areas are exposed to plasmodium infection, and we hypothesized that blood mo of these individuals could be activated and display increased cd expression. we tested if this phenotypic expression was associated with detectable changes in the mo anti-parasitic activity. the mo phenotype of healthy malaria naï ve and malaria exposed individuals was determined by three-color flow cytometry. myeloid cell markers included cd and activation markers such as hla-dr and trem- . percentages of blood mo involved in phagocytosis activity either with or without immune sera were then identified by flowcytometry, and the potential association between a given mo phenotype and phagocytosis activity was then looked for, using spss ® and statview ® softwares. our results showed that, compared with malaria naï ve individuals, there was a . fold increase (p x . ) in the total number of circulating cd low mo present in the blood samples of healthy malaria exposed asian individuals living in thailand. according to the density of surface antigen determined by fluorescence intensity (fi), the decrease in cd and the concomitant increase in hla-dr expressions indicated that in this malaria endemic area, blood mo were mature and highly activated by comparison with surface markers of mo from malaria naï ve donors. the relative levels of cd + blood mo were associated with the percentages of membrane-bound ifn-g present at the mo surface. in conclusion, (i)-a subset of cd + blood mo expressed increased levels of cd on mo of healthy malaria exposed individuals; (ii)-blood mo with activated (hla-dr+) and mature (trem- +) phenotypes were present in these healthy individuals; (iii)-increased expression of hla-dr and cd on cd high mo was associated with a high phagocytosis activity. introduction: adipokines, initially described for their function within metabolism, have been characterized to exert a regulatory role on the immune response. for instance the appetite-regulating hormone leptin has been identified to modulate the response of the innate as well as the acquired immune system. the present work focuses on the effects of leptin on the reactivity of m -and m -polarized human macrophages. methods: monocytes were isolated from the peripheral blood by magnetic cell sorting. polarization to m and m macrophages was induced by culture in the presence of mcsf or gmcsf respectively. polarized cells were characterized by flow cytometry, stimulated with lps and response assessed by characterization of cytokine profiles via cytometric bead array (cba). results: culture of monocytes in the presence of mcsf or gmcsf induced two different phenotypes. cells cultured in the presence of gmcsf represented the m type and were cd negative but cd and mhcii positive and produced high levels of il- , tnfalpha and il- following lps stimulation. culture in the presence of mcsf resulted in induction of the m phenotype. these cells were cd positive with intermediate expression of cd and mhcii expression and produced high levels of il- , il- and il- following lps stimulation. interestingly, already baseline il- production was high in these cells. stimulation with leptin alone increased cytokine production in both cell types as compared to cells cultured in medium alone. however, if leptin was present in cultures stimulated with lps, the induction of cytokine production was significantly reduced in both, m -and m -polarized cells as compared to cells stimulated with lps alone. summary: whereas presence of leptin enhances baseline cytokine production in polarized macrophages, it reduces the cytokine production in response to stimulation with the tlr ligand lps. thus, abundant leptin levels like present in obesity or in the hypertrophied fat as present in crohn's disease patients might exert modulating effects on macrophage response to bacterial antigens. methods: hl cell line was used as a model of leukemic myeloid cell differentiation cultured in suspension or on fibronectin matrix prior to pma ( ng/ml) treatment for h. morphological evaluation was performed with conventional microscopy and electron microscopy. immunephenotype and phagocytic activity of the cells were determined by flow cytometry and immunocytochemistry. a colorimetric nitro-blue-tetrazolium reduction assay was performed to assess the production of reactive oxygen species (ros). results : besides their distinctive macrophage morphology and ultrastructure with spindle cell-like features and high granularity, the pma-treated fibronectinadherent hl cells expressed antigen receptors cd , tlr , tlr and cd , and displayed enhanced phagocytic activity and production of ros. expression of cd , cd and cd was also maintained however the cells were hla-dr and cd a negative. conclusion: we describe the enhanced ability of fibronectin-adherent hl cells to differentiate into macrophages in response to pma. hl may provide a functional model for macrophage differentiation. above all, this finding may stimulate further research on myeloid leukemia biology and potential adjuvant therapies. a. aporta , n. ferrer , a. gómez , j. gonzalo , a. arbués , a. anel , c. martín , j. pardo , apoptosis, immunity and cancer university of zaragoza, molecular and cellular biochemistry and biology, zaragoza, spain, university of zaragoza, mycobacterium genetics, zaragoza, spain mycobacterium tuberculosis is an intracellular pathogen that uses alveolar macrophages as its preferred habitat, being capable of produce both a progressive disease and an asymptomatic latent infection. it has been postulated that infected macrophage apoptosis may contribute to host defence against this intracellular infection by, firstly, eliminating supportive environment for bacterial growth and, secondly, by leading to the formation and release of apoptotic vesicles containing mycobacterial antigens. it has been proposed that m. tuberculosis inhibits host cell apoptosis thus interfering with the immune system response. however the biological relevance of this process is not clear. our group has generated so , a m. tuberculosis phop mutant strain that was shown (perez et al ) to be more attenuated than the present attenuated vaccine strain bcg and conferred protective immunity against m. tuberculosis infection in mice and guinea pigs (martin et al ) . in the present study, we compare the time course and phenotype of cell death induced by so , bcg and wild type m. tuberculosis on the murine macrophage cell line j and on bone marrow derived mouse macrophages. our results indicate that wild type m. tuberculosis induces macrophage cell death analysed by a clonogeneic assay much faster than the attenuated bacteria. of note cell death presented apoptotic features like caspase- activity and nuclear condensation. in order to analyse the consequences of this apoptotis-like cell death, it has been invetigated whether dead cells translocate phosphatydilserine to the outer part of the plasma membrane and if this traslocation is enough to promote phagocytosis by fresh macrophages. experiments are ongoing with macrophages derived from trl x deficient mice and wt animals in order to study the role and implication of those receptor on the susceptibility to infection and death induced by the virulent and attenuated phop m. tuberculosis strain. objectives: vip is a potent anti-inflammatory peptide, mainly acting as endogenous macrophage deactivating factor. type receptor for vip (vipr ) gene is highly conserved through species and, in humans, is highly polymorphic. vipr has been reported to be down-modulated in cells of the immune system after activation. an association of some snps with some autoimmune diseases has also been reported. in this study we have investigated the correlation between these snps and gene expression in monocytes exposed to lps. methods: monocytes from blood donors were separated from pbmc and stimulated with lps. total rna was reverse transcribed and the level of vipr in untreated or lps-stimulated monocytes was measured by real-time rt-pcr and protein expression. protein level was measured by western blot and densitometric analysis. the kinetic of expression of vipr after , , and h of exposure to lps was firstly analysed in monocytes from five individuals. there were two kinetics: one in which a reasonable high levels ( g %) of mrna was maintained trough time and a second one in which the decrease of mrna was pronounced. the experiments were repeated using monocytes from donors that had been typed for the relevant vipr snps. the down-regulation of vipr correlates with the presence of a t at rs mapping in the '-end of the gene (p= . ). the vipr protein level was decreased about % in monocytes of subjects typed as t/t at rs whereas subjects typed as c/c at rs maintained a high level of expression after h of lps treatment. the data show that different haplotypes of the vipr gene correlate with a different kinetics of gene expression in activated monocytes. a possible consequence of these data is that the anti-inflammatory properties of vip governed by the vipr vary in different individuals and can eventually contribute to the genetic predisposition to some autoimmune diseases. j. oujezdska , t. vavrochova , d. filipp , immunobiology institute of molecular genetics as cr, immunobiology, prague, czech republic phagocytes which appear in early mouse development (e . - . ) represent a unique embryonic macrophage lineage that differs from adult macrophages phenotypically, biochemically and by their origin. recent studies suggested that there are at least three waves of macrophages populating an early embryo: a maternallyderived one and two waves of extraembryonal, ys-derived phagocytes. in addition, the occurence of early embryonic phagocytes of undetermined origin in the anterior head mesoderm in several invertebrate and vertebrate species is well documented. this origin-related heterogeneity among early embryonic phagocyte subpopulations coupled with the lack of their specific surface markers makes it difficult to distinguish them phenotypically and study their potentially distinct physiological roles in early development. the aim of this study is to identify a set of surface markers expressed on embryonal phagocytes suitable for phenotypic distinction among embryonic phagocyte subpopulations. here we report the temporal and spatial expression of toll-like receptors (tlrs) and cd in the early mouse embryo (me). facs analysis of cell suspension prepared from . day me showed that about . - % of cells were positive for cd b. these cells exclusively were also positive for cd , tlr , and cd antigens. using qpcr and flow cytometry we show that tlrs and other tir domain-containing signaling molecules are expressed in the embryo through embryonic days , - , . reciprocal matings between wild type and mhcii-egfp knock-in mice revealed that while maternallyderived mhcii + macrophages are present in the embryo from early developmental stages (e , ), embryo-derived mhcii + macrophages start to appear in the embryo around day . multicolor facs analysis of cd b, cd , cd , f / , tlr , tlr , c-kit and mhcii surface markers revealed differential expression of tlr and c-kit on embryonal phagocyte subpopulations. moreover, the microarray analysis of cd b + tlr + cells isolated from the e , embryos has revealed significantly upregulated expression of several novel genes in comparison to their expression in murine peritoneal macrophages. these molecules are currently being tested for their use as embryonic phagocyte specific-lineage markers. these results are first to characterize the regulated expression of tlrs on early embryonal phagocytes and demonstate their potential to serve as novel markers for their detection and isolation. humans may be exposed to a variety of mycobacteria ranging from environmental or bcg vaccine to more pathogenic mycobacteria. only a minority of individuals exposed develop disease, this susceptibility may result in part from variability of host immune responses genes through simple (mendelien disease) and complex (polymorphisms with milder effect) inheritance mechanisms. interestingly, key elements of inflammatory pathways are particularly involved in this susceptibility to mycobacteria. il /il -dependent ifng pathway of macrophage activation plays a central role in inflammation and cell-mediated immune responses to mycobacteria. due to the high rate of consanguineous marriages in the north african countries, recessive genetic disorders including primary immunodeficiencies occur with a relatively high prevalence. in tunisia, among patients affected with primary immunodeficiencies presented with disseminated bcg infection (bcg-osis). among them, five have an underlying well-defined primary immunodeficiency either a severe combined immunodeficiency or a chronic granulomatous disease and have a mendelien susceptibility to mycobacterial disease. using a candidate gene strategy, we have identified in out of these patients mutations in several ifng pathway genes, other candidate genes are being investigated for the other patients. in the general population, common polymorphisms with milder effect on the risk of tuberculosis have been identified including mhc and nramp . we did focus on the study of genes which are considered as important pathogen recognition receptors of the innate immune system: tlr is the principal mediator of macrophage activation in response to mycobacteria through nfkb pro-inflammatory signaling pathway and dc-sign is the major receptor of m. tuberculosis on human dendritic cells and in contrast induces anti-inflammatory il- cytokine. using a case/household-contact cohort we did investigate polymorphisms of these genes in tunisian patients affected with active pulmonary tuberculosis and have shown specific patterns of snp and microsatellite polymorphisms associated with susceptibility/resistance to tuberculosis. host inflammatory responses play a major role in granuloma formation and control of the infection. unraveling these pathways might be crucial in order to identify new therapeutic targets and strategies including immunotherapy e. g. ifng therapy for tuberculosis, particularly in this era of emergence of multi-drug and extensively-drug resistant m. tuberculosis strains. francisella tularensis is a gram negative bacterium that is the causative agent of tularemia. research into francisella has expanded over recent years due to its designation as a potential biological warfare agent. several species of francisella exist and have varying degrees of pathogenicity. f. tularensis live vaccine strain (lvs) is an attenuated strain of the holarctica subspecies and has been shown to be an effective vaccine in humans. however, it is pathogenic in mice which can, therefore, act as a useful model of human tularemia. f. tularensis is an intracellular pathogen and is able to invade several different cell types, in particular macrophages, most commonly through phagocytosis. therefore, if phagocytosis could be disrupted via the addition of inhibitors, uptake of f. tularensis would decrease and antibiotic treatment may be more effective. a flow cytometric assay was developed to measure bacterial uptake. this method used a fitc labelled anti-f. tularensis antibody in conjunction with antibodies to cell surface markers to determine specific cell phenotypes that were positive for bacteria. a series of phagocytic inhibitors have been tested in vitro on an alveolar macrophage derived cell line (mhs) and on ex-vivo mouse lung tissue to determine whether uptake of f. tularensis lvs could be altered. the presented data shows that several inhibitors work efficiently to reduce lvs uptake by up to - % in both the in vitro and ex vivo assays. however, cytotoxicity of some of the inhibitors was high and, therefore, it was essential to concentrate on inhibitors with low cytotoxicity for further assessment. in addition, bacteriological data suggests that the combination of inhibitors with antibiotics may be a useful therapeutic against f. tularensis. it may also work against other intracellular pathogens that use phagocytic mechanisms to enter their optimal niche.ã crown copyright. dstl, . hsp are intracellular proteins but it is known that these proteins can be expressed on cell surface and contained in extracellular medium, in particular in peripheral blood serum. it is also known that extracellular hsp have pronounced immunomodulatory properties. to study the pathways of the protein modulating action on immune system we investigated effect of exogenous and cell surface hsp on reactive oxygen species (ros) release from phagocytes, namely human neutrophils, during process of phagocytosis (respiratory burst). neutrophils were isolated from human peripheral blood by using a standard protocol. respiratory burst induced by opsonized zymosan was measured by method of luminol dependent chemiluminescence. for the experiments human recombinant hsp (low endotoxin) and paraformaldehyde fixed mouse thymocytes exposed surface hsp were used. exogenous hsp was used in concentration - ug/ml, fixed thymocytes were added to neutrophil samples in quantitative ratio : and : directly before the measuring. as the control we registered amplitude of oxidative burst in samples supplemented with pbs or live mouse thymocytes having no hsp on their surface. results demonstrating effect of exogenous hsp on phagocytosis-induced ros release from human peripheral blood neutrophils have been obtained. it was demonstrated marked dose-dependent inhibiting action of exogenous hsp on amplitude of respiratory burst. the cells expressing surface hsp impacted on ros production in this model similarly. the results of chemiluminescence analysis demonstrated that zymosan induced ros production was essentially decreased under action of fixed thymocytes, and was decreased slightly in presence of live thymocytes in the neutrophil samples. the effect was more pronounced for increased amount of thymocytes added to the samples. thus, immunomodulatory effects of exogenous hsp might be caused by influence of the protein on ros release from phagocytes. we suppose that the registered effects are connected with ability of hsp to inhibit activity of nadp-oxidase -the key enzyme for ros production during respiratory burst. results: we recruited pts, with so far five complete pathological remission, five partial responses and five no responses. no substantial changes were detectable in the number of circulating monocytes. in contrast we observed a clear expansion of cd /cd and cd /cd double positive subsets. this event was transient; it abated at the later time point suggesting a causal relationship to the treatment. it correlated with sensitivity to the treatment. in fact we observed that in the responder patients the expansion of the cd / subset was clear in the first weeks of treatment and decreased there after. in contrast in non-responder patients it was already expanded before the neo-adjuvant therapy. all the patients had an initial expansion of the cd / subset. in the responder patients this population was still present at the time of surgery. the immunohistochemical study revealed a massive tumoral infiltration by macrophages that displayed clear features of alternative m polarization. conclusion: these data suggest that neo-adjuvant therapy modulates the cellular components of innate immune responses that could represent valuable predictive factors. m. dimitrijević , i. pilipović , s. stanojević , k. mitić , k. radojević , v. pešić , g. leposavić , institute of virology, vaccines and sera "torlak", immunology research centre "branislav janković", belgrade, serbia, faculty of pharmacy, university of belgrade, department of physiology, belgrade, serbia the primary aim of our current study was to ascertain whether rat resident peritoneal macrophages synthesized catecholamines and to unmask putative effects of catecholamines on nitric oxide (no) and hydrogen peroxide (h o ) production and phagocytic activity of these cells. in addition, given that chronic administration of b-adrenoceptor antagonist increases the density of b-adrenoceptors on both non-immune and immune cells and thereby affects their sensitivity to catecholamine action, we hypothesized that such treatment could also affect macrophage responsiveness. to address our proposition, we determined adrenoceptor expression on peritoneal macrophages from rats subjected to -day-long propranolol treatment and measured both no and h o production and phagocytic activity of these cells. using both immunocytochemical and flow cytometric analyses of rat peritoneal exudate cells constitutive expression of tyrosine hydroxylase and both b -and a -adrenoceptors on macrophages was revealed. furthermore, according to the characteristic assemblage of tyrosine hydroxylase and adrenoceptor subtype expression different macrophage subsets were identified. in vitro treatment of macrophages with the non-selective a,b-adrenoceptor agonist arterenol and/or the b-adrenoceptor antagonist propranolol indicated that b-adrenoceptors potentiated no production and suggested a-adrenoceptor-mediated suppression of hydrogen peroxide h o production. an increase in h o production in the presence of the a -adrenoceptor antagonist ebrantil provided support for this. chronic propranolol treatment in vivo led to increased no and h o production by peritoneal macrophages. furthermore, this treatment resulted in opposing effects on the expression of b -and a -adrenoceptors on peritoneal macrophages (a stimulatory effect on b -adrenoceptors and a suppressive effect on a -adrenoceptors). in conclusion, a subset of resident peritoneal macrophages synthesizes catecholamines, which may exert differential effects on h objectives: monocytes display great phenotypical and functional heterogeneity and are divided into two major subsets: cd ++ cd -('classical') and cd + cd + ('pro-inflammatory') monocytes. a central monocyte function is cytokine production in response to toll-like receptor (tlr) ligation. the cd + cd + monocytes display higher tlr and - expression, produce higher levels of pro-inflammatory cytokines and have increased potency for antigen presentation than the cd ++ cd monocytes, suggesting that the two subsets could play different roles in antimicrobial responses. newborns are vulnerable to infections and an immaturity of both adaptive and innate immunity has been described. studies of neonatal monocyte antimicrobial responses show contrasting results and much remains to be learned, especially regarding monocyte subpopulations. thus we aimed to compare monocytes from newborns and adults, focusing on monocyte subpopulations and responses following tlr stimulation. methods: cord blood (n= ) and peripheral-blood (n= ) mononuclear cells were stimulated in vitro for hrs with peptidoglycan and subsequently analysed for cd and cd and intracellular il- p and tnf expression. the mann-whitney u-test was used to evaluate differences between groups. results: a significantly higher percentage of neonatal monocytes were positive for il- p , both unstimulated and after peptidoglycan stimulation, as compared to adults. geomfi of il- p was low and similar between groups, although significantly higher in newborns after stimulation. in both newborns and adults, il- p (% positive cells and geomfi) was significantly higher for cd + cd + cells than for cd ++ cd cells, unstimulated and stimulated. regarding tnf, neonatal and adult monocytes did not differ in unstimulated cultures, however geomfi of tnf was significantly higher in neonatal monocytes after stimulation. whereas the tnf response following stimulation was similar between the adult monocyte subsets, in newborns the cd ++ cd cells were positive for tnf to a significantly higher extent than the cd + cd + cells. in particular the tnf response to tlr stimulation differed between newborns and adults, with neonatal monocytes having a higher per cell production of the cytokine. notably, in newborns the cd ++ cd monocytes were positive to a higher extent for tnf following stimulation pointing towards a functional immaturity of neonatal monocyte subset responses. objective: chronic granulomatous disease (cgd) is an uncommon congenital phagocyte disorder characterized by recurrent life-threatening infections. cgd generally present with recurrent suppurative infections; however, intracranial fungal abscess complicating cgd may cause a diagnostic problem to anyone who is unfamiliar with its clinical and radiological features. we report a -year-old boy who admitted with complaints of seizures during the previous months. there was a history of axillary and perianal suppurative skin infections and cavitary pneumonia. the family history was unremarkable, and the parents were unconsanguineous. physical examination was only remarkable for oral moniliasis and skin scars at axillary and perianal region. a large frontol mass with diffuse peripheral vasogenic edema was discovered on mri. subfalcine herniation was noted secondary to mass effect. cgd was suspected and the analysis with flow cytometric dihydrorhodamine assay (dhr assay), for functional analysis of neutrophils was compatible with the diagnosis of cgd and no bimodal histogram pattern spesific for x-cgd was found in the mother and sister. after the diagnosis of cgd, neurosurgical removal of the abscess cavity was performed due to peri-lesional edema and herniation risk. aspergillus fumigates grew from the culture; liposomal amphotericin b and voriconazole were started; which were found to be sensitive to the cultured species. in addition, interferon-g ( mgr/m /day, subcutaneously every other day) was started. after months, control mri showed regression of the lesion, and the anti-fungal treatment was continued for months. the screening of the other family members with dhr assay demonstrated that one of his sisters had also cgd and phenotype was autosomal recessive. mutaton analysis in "hot spot" in ncf gene concerns the well-known gt deletion in the second exon of ncf gene both at the patient and his sister. results: this was an atypical clinical presentation of cgd in an adolescent boy with cerebral aspergillosis, mimicking intra-cranial tumor. we documented a good response to the combination of ifn-g, liposomal amphotericin b and voriconazole after surgery. conclusion: cgd should be considered in the differential diagnosis for all children presenting with invasive fungal infections particularly, those involving the central nervous system. recent data suggest that ubiquitin has anti-inflammatory properties and therapeutic potential after severe trauma and brain injuries. therefore, we hypothesized that ubiquitin treatment can modulate the local inflammatory response triggered after brain injury. to test this hypothesis, a focal cortical contusion was induced using a controlled cortical impact (cci) model in sprague-dawley rats. animals (n = ) subjected to moderate brain injury were randomized, and received either . mg/kg ubiquitin or vehicle (placebo) intravenously within min after cci. levels of tnf-a, il- b, il- , il- and il- receptor antagonist were analyzed in brain tissue using real time rt-pcr at and hours after treatment. immune cell infiltration was studied by immunostaining for neutrophils and macrophages/ microglia at h and days. data were analyzed with the mann-whitney u test and a two-tailed p x . was considered significant. all cytokines were highly up-regulated hours after cci but no differences between the groups were observed at this time point. three days after trauma the levels of il- were significantly lower in the ubiquitin treated animals, whereas the levels of il- and tnf-a were higher when compared to the placebo group. interestingly, macrophages/ activated microglia were significantly increased in the pericontusional cortex after ubiquitin treatment at day . the infiltration of neutropils was not affected by ubiquitin treatment. here, we could demonstrate for the first time that a single injection of ubiquitin immediately after brain trauma is able to modulate the inflammatory response triggered after brain injury at the cellular as well as at the cytokine level. macrophage activation and oxidative metabolic changes are commonly implicated in pulmonary tuberculosis (ptb) patients. efficient plasma antioxidant activities are needed to neutralize high free radical load in pulmonary tuberculosis (ptb) patients. there is limited information about the plasma levels of neopterin (a marker of macrophage activation) and oxidative stress indices such as total plasma peroxide (tpp), total antioxidant activity (taa), malondialdehyde (mda), and oxidative stress index (osi) in ptb patients during chemotherapy with or without micronutrient supplementation. the present study was designed to assess the levels of neopterin, tpp, taa, mda, and osi during chemotherapy with (c+m) or without (c-m) micronutrient supplementation using elisa and spectrophotometric methods. thirty-eight ( ) newly diagnosed ptb patients and forty non-ptb apparently healthy subjects volunteered to participate in this study. twenty of the ptb patients were on anti-tuberculosis drugs supplemented with micronutrients (c+m) while were treated with anti-tuberculosis drug alone (c-m) for a period of four weeks. the levels of neopterin (p= . ), tpp (p= . ), osi (p = . ), mda (p = . ) were significantly raised but taa (p = . ) was significantly reduced in ptb patients compared with controls. the levels of mda (p = . ), neopterin (p= . ) and tpp (p= . ) were significantly reduced in c+m after two weeks of treatment compared with baseline values before commencement of treatment. the levels of tpp (p= . ), mda (p= . ), neopterin (p= . ), osi (p= . ) were significantly reduced while taa (p= . ) was significantly raised in c+m after weeks of treatment compared with the baseline concentrations. in c-m, only mda showed significant decreased after weeks of treatment when compared with the baseline values. plasma level of neopterin, tpp, osi and mda declined faster in c+m than c-m. therefore, micronutrient supplementation of ptb drugs with synthetic antioxidants or naturally occurring ones (fruits and vegetables) should be attempted. this will improve deranged macrophage activation and reduce oxidative stress indices in ptb patients. a. p. aguas , e.m. cunha , m.j. oliveira icbas, university of porto, anatomy, porto, portugal the acute in vivo intake of mercury (hg) microparticles ( nm in diameter) by neutrophils and macrophages was studied with the use of in situ detection of hg by scanning electron microscopy coupled with x-ray elemental microanalysis (sem-xem). the intracellular distribution of hg particles was compared, at high resolution, between macrophages and neutrophils, and between activated and non-activated phagocytes. balb/c mice were injected intraperitoneally (ip) or in a subcutaneous air-pouch with mercury chloride, and the animals were sacrificed up to minutes after the injection. in some mice, before the hg injection, peritoneal phagocytes were activacted by ip injection of bsa. pre-injections with a selenium (se) salt were also performed in order to study the putative modulatory role of se on hg intake by phagocytes. peritoneal cells were collected by washing of the peritoneal or subcutaneous cavities with pbs, they were cytospinned, fixed with formaldehyde, and processed for observation by sem-xem. five min after the hg injection more than half of the mouse phagocytes were positive for hg. a higher percentage ( %) of macrophages contained the metal particles than neutrophils ( %). phagocyte activation enhanced the number of hg particles seen inside the phagocytes. pre-injection of the peritoneal cavity of mice with se resulted in finding that more than half of the hg intracellular particles were coupled with se. subcellular topography of hg particles showed that they were presented in individual small cytoplasmic vesicles. we conclude that hg microparticles are rapidly ingested by macrophages and neutrophils, a processed that is enhanced by cell activation. hg particles are ingested by pinocytosis and sorted in the cytoplasm of macrophages and neutrophils inside individual small vesicles. this study was supported by a grant from fct, portugal. mast cells play central roles in allergic inflammatory reactions and innate immunity. swap- is a rac-interacting protein expressed in several cells types of the hematopoietic system including mast cells. in b cells and mast cells swap- regulates f-actin cytoskeletal rearrangements, cell polarisation and cell migration. (pearce et al., ; sivalenka and jessberger, ) . swap- -/-bone marrow derived mast cells (bmmc) are specifically impaired in fceri-mediated activation and degranulation and in c-kit-induced activation, migration and cell adhesion (gross et al., ; sivalenka and jessberger, ; sivalenka et al., ) . crucial regulators of these processes are members of the rho family of small gtpases such as rac and rhoa. swap- interacts with rac in vitro and preferentially binds the active gtp-bound rac . swap- supports the increase of active rac in vitro by a yet to be defined mechanism (shinohara et al., ) . in this study, in vitro pull-down assays with purified recombinant proteins were employed to characterize the interaction between swap- and rac . it was found that fulllength swap- preferentially binds to constitutively active rac (rac q l) but not to its dominant negative form (rac t n). binding assays with swap- truncated mutants showed interaction of swap- 's n-terminus with gtpgs rac or rac depleted of guanine nucleotide, whereas swap- central or c-terminal regions do not bind to any form of rac . preliminary competitive-binding assays with overlapping mer peptides, spanning the entire swap- sequence, mapped the rac binding site near the n-terminus of swap- . full-length swap- site-specific mutants will be generated to test the relevance of these interactions in mast cells in terms of adhesion, migration and activation of rho gtpases. elucidating the molecular interactions of swap- with rho gtpases and the relevance of these will shed light on the biology and biochemistry of mast cells and possibly other hematopoietic cells, which express swap- . v. c. barbosa , c. d. polli , m.c. roque-barreira , m.c. jamur , c. oliver , g. pereira-da-silva mast cells are essential cells in ige-associated immune responses. fceri crosslinking induces mast cell degranulation and release of proinflammatory mediators. we have previously shown that the lectin artinm induces mast cell activation but the mechanisms involved in this activity remain unknown. objective: the present study was undertaken to further characterize the ability of artinm to activate mast cells. methods: rbl- h cells were sensitized with ige anti-tnp and stimulated with dnp -hsa or artinm. artinm binding to rbl- h cells was assessed by flow cytometry. mast cell degranulation was determined by measurements of released b-hexosaminidase activity. microplate binding assays were utilized to assess artinm binding to ige. to investigate fceri recognition by the lectin, western blots of cell lysates were stained with biotinylated artinm and be's antibodies specific for fceri b-subunit. intracellular protein phosphorylation was detected by specific antibodies and analyzed by confocal microscopy. mcp- and tgf-b levels released by mast cells were measured by elisa. results: artinm binding to the cell surface was dependent on sugar recognition and resulted in mast cell degranulation in the presence or absence of ige. the release of b-hexosaminidase doubled when cells were sensitized by the immunoglobulin and was abrogated in the presence of d-mannose, suggesting that mast cell degranulation induced by artinm might be the result of interactions between the lectin crds and glycosylated components on the cell surface, like fceri or ige. indeed, it was observed that the lectin bound to ige in a dose-dependent manner and recognized the fceri b subunit in western blot analysis. exposure to artinm resulted also in phosphorylation of intracellular proteins, mcp- release and tgf-b production. significant increases in these activities were observed upon sensitization with ige. conclusions: these results suggest that artinm may bind to glycans of the high affinity ige receptor and/or of the ige (bound to fceri) and that such interactions would be implicated in its ability to activate and degranulate mast cells. in view of the well-established significance of mast cells in allergic inflammation, the participation of sugars as binding receptors on mast cell surface opens new ways of controlling allergic disorders. the adhesion receptor l-selectin is a key player of the innate immune response in the process of leukocyte migration from the blood stream to inflamed tissue. it is expressed on leukocytes and promotes the initial contact to the endothelium resulting in steady rolling and eventually diapedesis. a distinct feature is the exclusive presentation of l-selectin on the tip of finger-like cell membrane protrusions called microvilli which cover the entire leukocyte surface. this topography was shown to facilitate the first transient interactions of the free flowing cell to the static counterreceptor particularly in the context of high dynamic shear. other adhesion molecules such as p-selectin glycoprotein ligand (psgl- ), b and b -integrins also share this special phenotype. taken together, prominent adhesion receptor positioning reflects a widespread biological principle contributing to inflammation as well as hematogenic tumor metastasis. despite the functional relevance and frequent occurrence, however, molecular mechanisms of cell surface receptor compartmentalization remain largely unknown. in this study we identified the highly conserved transmembrane domain of l-selectin to regulate microvillus receptor positioning and adhesion under flow. taking advantage of the inverse surface expression pattern of cd (cell body) compared to l-selectin (microvilli) in a myeloid cell line, we investigated domain swapped chimeric receptors regarding their substructural surface localization and their ability to initiate rolling under flow. transmission electron microscopy showed a crucial impact of the transmembrane domain to target the chimeric receptors to a certain cell surface compartment independent of the intracellular anchorage. in turn, the receptor shift from microvilli to the cell body goes along with a substantial decrease of rolling cells in an in vitro parallel flow chamber assay. thus, contrary to the common view of single membrane spanning domains to simply act as a mechanical anchor, our results attach an important functional component as well and might point out a new general principle for targeting receptors to specific membrane compartments. objectives: macrophages are one of the principal effector cells involved in the innate immunity response. they kill microbes through phagocytosis and upon activation, secrete pro-inflammatory cytokines such as il- b, il- and tnf-a. herpes simplex virus (hsv- ) is an enveloped dna virus that infects mostly oral mucosa and sensory neurons. innate immunity responses activated by hsv infection consist of: activation of macrophages; activation of the complement cascade, and production and secretion of a variety of cytokines and chemokines. il- and tnf-a are cytokines produced by macrophages that contain known anti-hsv properties. the objective of this study was to characterise the secretome of human primary macrophages infected with hsv . methods: human monocytes were purified from the peripheral blood mononuclear cells of healthy blood donors and differentiated in vitro into macrophages. macrophages were left untreated or primed with poly(i:c) ( ug/ml), a mimetic of double-stranded rna, after which cells were left uninfected or infected with hsv- for h. after this, cell culture supernatants were collected, concentrated and proteins purified. the secreted proteins were digested into peptides, identified and quantified using itraq (isotope tagged relative and absolute quantitation) -labelling of the peptides followed by peptide fractionation by cation exchange chromatography and analysis by nanolc-ms/ms. the raw ms/ms data was analysed using proteinpilot . software. results: in the first itraq experiment over human proteins were identified in the hsv infected cell supernatants. from these proteins had at least fold increase after poly(i:c) + hsv infection compared to the uninfected cells. hsv infected cells had clearly more proteins in their cell supernatants after infection compared to the uninfected cells: itraq labelling showed a total of . fold increase in the protein amount in the poly(i:c) + hsv infected cell supernatant and a . fold increase in the hsv infected cell supernatant when compared to the uninfected cell supernatant. amongst the upregulated proteins there were known inflammatory proteins: chemokine (c-x-c motif) ligand , il- , tnf-a induced protein , complement factor b, galectin- and mxa. at present, further experiments are on-going for more detailed analysis of the hsv infected macrophage secretome. h. p. prakash , german cancer research centre, translational immunology, heidelberg, germany, max planck institute for infection biology, molecular biology, berlin, germany chlamydophila pneumoniae are the major etiological factors for worldwide pneumonia, chd and copd. chlamydia lives and multiplies inside their host epithelial cells where they confer resistance for apoptosis by inducing expression and stability of anti-apoptotic proteins called inhibitor of apoptosis proteins (iaps). the significance of cellular inhibitor of apoptosis protein- (ciap- ) and x-linked inhibitor of apoptosis proteins ( xiap) in chlamydia pneumoniae pulmonary infection and innate immune response of macrophages was investigated in ciap- and xiap knockout (ko) mice using a novel non-invasive intra-tracheal infection method. in contrast to wildtype, iap knockout mice failed to clear the infection from their lung. wildtype mice responded to infection with a strong inflammatory response in the lung. in contrast, the recruitment of monocytes and macrophages was reduced in iap ko mice compared to wildtype mice. the concentration of interferon gamma (ifn-g) was increased whereastumor necrosis factor (tnfa) was dysregulated in the lungs of infected iap ko mice compared to infected wildtype mice. ex vivo experiments on mouse peritoneal macrophages and splenocytes revealed that iaps are required for innate immune responses of these cells. our findings thus suggest a new immunoregulatory role of iaps in c.pneumonaie pulmaonry infections. methods: human monocytes were purified from venous blood of normal volunteers by ficoll density gradient centrifugation. hrgal- ( mg/ml) binding to monocytes, in the presence or absence of mm lactose or sacarose, was assessed by flow cytometry and confocal microscopy. in transwell systems, assays were performed using hrgal , laminin or fibronectin immobilized or not on the filters. these were added to wells containing soluble hrgal or rpmi and monocytes ( x ) were added into each insert. when necessary, hrgal was pre-incubated with mm lactose or sacarose. mcp- ( ng/ml) was used as positive control. we observed that hrgal- binds to the surface of human monocytes through its crd, since this interaction can be inhibited by lactose. we corroborated some data of literature that hrgal- is able to induce monocyte migration in a dose-dependent manner, resulting in a bell-shaped curve as seem with other known attractants. when we evaluated the participation of the ecm laminin and fibronectin in monocyte migration induced by hrgal- , we observed that the association between these glycoproteins and hrgal- resulted in a % increase in the number of migrating cells. both n-and c-terminal domains of hrgal- are involved in the association between laminin or fibronectin and hrgal- , since the presence of lactose resulted in % and % inhibition of monocyte migration induced by the lectin, respectively conclusions: our results showed that hrgal- induces monocyte migration by haptotaxis, through the interactions established between both n-and c-terminal domains of the lectin and ecm glycoproteins, laminin and fibronectin. in a vertebrate embryo, macrophages develop in two sites (yolk sac and liver) and constitute the primary mechanism of host defense. their phagocytic function may be required during the earliest stages of development both for survival and for organogenesis. recent studies have shown that monocyte heterogeneity is conserved in humans and mice. the different monocyte subsets seem to reflect developmental stages with distinct physiological roles but nothing is known whether the macrophage diversity arises in early ontogeny. in order to study the ontogeny of the monocyte-macrophage lineage, we developed a new culture technique using human embryonic stem cells (hesc).culturing of embryoid bodies for weeks in the presence of bmp ,vegf and a mixture of hematopoietic cytokines resulted in a generation of a significant cell population of cd +cd + cells. the sorted cd +cd + cells were further cultured for - days in the presence of m-csf and gave rise to a homogenous population of adherent mature macrophages. embryonic stem cells derived macrophages were identified by several criteria including morphology and ultrastructural features observed by microscopy and by expression of nonspecific esterase and myeloperoxidase by histochemical staining. while virtually all embryonic-derived macrophages expressed the lps-receptor cd , m-csf receptor cd and the scavenger-receptor cd , we characterized two distinct subpopulations of macrophage based on their difference in size and density and the expression of the cd and cd (fcgammariii) : the cd lowcd -and cd + cd +. trancscriptional, phenotypic and functional assays suggest the alternative (m ) polarization of cd +cd + embryonic stem cell-derived macrophages.(anti-inflammatory cytokines secretion, active phagocytosis, m -related gene expression).the exact chemokine receptor expression pattern, phenotype and transcriptional activity of their foetal counterparts are currently under investigation. collectively, our data provide insight into alternative macrophage polarization in humans and and adds further data to the growing body of evidence that establishment of macrophage heterogeneity is related to early ontogeny. b.-s. choi , p. kropf imperial college london, immunology department, london, united kingdom the balance between t helper (th) and th cell responses is a major determinant of the outcome of experimental leishmaniasis, but polarized th or th responses are not sufficient to account for healing or nonhealing. we have recently shown that arginase-induced l-arginine depletion results in local suppression of antigen-specific t cell responses in nonhealing leishmaniasis. healing, induced by chemotherapy, resulted in control of arginase activity and reversal of local immunosuppression. moreover, supplementation with l-arginine restored t cell effector functions and resulted in reduced lesions size and parasite load. however, despite the efficient production of ifn-g by cd + t cells at the site of infection and despite the reduced pathology, the mice did not heal. we hypothesised that arginase-expressing macrophages contribute to persistent disease and become refractory to ifn-g mediated signals. to test this hypothesis, we used a well-defined model of bone marrow derived macrophages and determined whether the differentiation state of parasitized arginase-expressing macrophages could be altered. in addition, we also tested whether alternatively activated macrophages can be induced to switch off arginase and upregulate inducible nitric oxide synthase (inos) to kill the intracellular parasites. vg vd t lymphocyte are activated following recognition of non-peptidic phosphorylated metabolites. the phosphoantigen isopentenyl pyrophosphate (ipp) is overproduced by tumors following hyperactivation of the mevalonate pathway of isoprenoid synthesis. previous work has shown that a molecular complex homologous to mitochondrial atp synthase (ecto-f -atpase) is expressed on many cell types and is a possible specific ligand for the vg vd tcr. the present study aims at understanding the role of f -atpase in antigen regognition. using video microscopy calcium imaging in single vg vd t lymphocytes, we can now show that the t cell response to ipp requires contact with bystander cells of variable tissue origin but that this requirement is not fulfilled by a cell line deprived of surface f -atpase. purified f -atpase immobilized on polystyrene beads can partly replace the need for cell-cell contact. ipp in soluble form is highly sensitive to terminal phosphatases and addition of these enzymes in t cell activation assays clearly shows that it is not recognized as such on tumors. however, we could detect nucleotide derivatives of phosphoantigens which are resistant to terminal phosphatases in the cell lysates of stimulatory tumors. one of these, a derivative of ipp, is barely able to stimulate vg vd cells in the absence of apcs, as opposed to the non-nucleotidic antigen ipp. however it can bind stably to f -atpase. thus the f -atpase complex acts as a presenting structure for nucleotide phosphoantigens. altogether, our data suggest that vg vd t cells are dedicated to the recognition of phosphoantigens in the form of nucleotide derivatives, on the surface of tissue cells and that antigen recognition involves multiple antigen modification steps, in including final cleavage by a nucleotide pyrophosphatase activity. surface plasmon resonance was used to analyse the molecular interaction between tcr and f -atpase. by using purified f -atpase and peptides derived from vg vd tcr sequences, interaction sites between f -atpase and tcr were identified on both ligands. based on these findings a generalized model for vg vd t cell activation is proposed. ligands for the cytotoxic lymphocyte activating receptor nkg d are highly expressed on cells stressed by numerous agents including genotoxic damage, thereby contributing to the elimination of transformed cells by nkg d(+) lymphocytes. a key question is whether this represents a primary inductive means of immune surveillance, or merely enhances responses initiated by dendritic cells and antigen-specific t cells. a second key issue is the scope and scale of events that follow nkg d activation in vivo. by transiently overexpressing the nkg d ligand rae- -beta in the skin of transgenic mice, we showed that this alone provoked rapid, coincident and reversible changes in the organization, morphology and activation state of tissue-resident vgamma vdelta gamma-delta t cells and langerhans cells (lc), that were swiftly followed by epithelial infiltration of unconventional alpha-beta t cells. these data indicate a novel primary immune surveillance pathway whereby epithelial upregulation of nkg d ligands is sufficient to provoke a series of multicomponent immunological changes. the effects on lc, which lack nkg d and presumably respond to changes initiated by local gamma-delta t cells, are particularly interesting. ongoing microarray and co-culture experiments are now providing a molecular definition of the immume surveillance response to nkg d ligands in vivo. to assess the scope of this response, ovalbumin was applied to the skin concomitant with rae induction. the primary systemic th response is increased by concomitant responses to a stress antigen. we will now resolve whether this increased response contributes to the adaptive memory pool, or whether it is a primary, regulatory response that may limit adaptive responses to auto-antigens exposed during stress. in addition, the many ligands available to the nkg d receptor suggest that different ones may play unique roles. a novel nkg d-ligand, h c, is uniquely expressed in mouse skin. when the expression of this was further increased in a novel transgenic system, there was again an overt alteration in the local immune compartment, but with features that are seemingly distinct from the action of rae- induction. such studies may help resolve a long-standing puzzle over the pleiotropy of nkg d ligands, and dissect immune surveillance of changes in gene expression levels rather than absolute levels. a.-s. invariant natural killer t (inkt) cells are a distinct lineage of t lymphocytes that co-express a highly conserved ab t cell receptor (tcr) along with typical surface receptors for natural killer (nk) cells. these lymphocytes recognize glycolipid antigens presented by the non-classical class i molecule cd d. inkt cells are characterized by their capacity to produce rapidly large amounts of both th (ifn-g, tnf) and th (il- , il- ) cytokines, which enables them to play a role in the regulation of many different types of immune responses, ranging from self-tolerance to responses against pathogens and tumors. converging studies in mouse models suggest that inkt cells can prevent the development of type diabetes. the frequency of inkt cells is lower in non-obese diabetic mice (nod mice). manipulation of inkt cells, either by increasing their frequency or by stimulating them with agonists such as a-galcer, inhibits diabetes onset in nod mice. recently, a new population of cd -nk . -inkt cells producing high levels of the pro-inflammatory cytokine il- has been identified (inkt cells). given that this cytokine has been implicated in several pathologies including autoimmune diseases, we investigated the role of inkt cells in type diabetes. interestingly, nod mice exhibit a higher frequency of inkt cells producing il- as compared to c bl/ mice. this increased frequency was observed in the thymus as well as in peripheral lymphoid tissues. as previously described in normal mice, inkt cells present in nod mice were mainly cd -nk . -, express the ror-g transcription factor and il- receptor, both molecules being usually associated with th commitment. we are currently analyzing, using co-transfer experiments, whether these inkt cells play a beneficial, a deleterious, or any role in the development of type diabetes in nod mice. j. s. dodd , r. muir , s.s. affendi , p.j. openshaw imperial college london, respiratory medicine, london, united kingdom natural killer t (nkt) cells are a heterogeneous population of innate t cells that have attracted interest because of their potential to regulate immune responses to a variety of pathogens. upon activation with their cognate glycolipid antigen presented by cd d molecules, activated nkt cells produce copious and numerous cytokines which endow these cells with potent immunoregulatory properties. consequently, nkt cells have become the focus for the development of vaccine adjuvants, cancer immunotherapeutics and modulators for autoimmune and inflammatory conditions. respiratory syncytial virus (rsv) is a common cold virus of the family paramyxoviridae. it is the most frequent viral cause of serious lower respiratory tract infection in infants and children worldwide and a significant contributor to winter deaths in the elderly. despite its global impact, there is still no safe and effective vaccine and our understanding of the immunological mechanisms that regulate protection and pathology is incomplete. it is known that cd d-deficient mice with poor nkt cell responses have inefficient induction of cd t cells and reduced clearance of rsv, perhaps because of ifn-g release by activated nkt cells. we now show that activation of lung nkt cells with intranasal agalcer during rsv infection of mice boosts th immunity (increasing il- and il- ), promoting pulmonary eosinophilia and ablating cd t cell recruitment. by contrast, intraperitonal injection of agalcer enhances nk cell recruitment and boosts pulmonary cd t cell activity (as measured by cd expression), increasing ifn-g production in the airway and lung and inhibiting viral replication. effects on illness (as measured by weight loss) were similarly distinct: intranasal agalcer induced early (d ) weight loss independent of conventional t cells, whereas intraperitonal agalcer enhanced late (d ) weight loss by a cd t cell dependent mechanism. therefore, nkt cells stimulated by agalcer administered via different routes induce distinct types of immune response to viral infection in the lung with the intraperitonal route leading to optimal viral clearance. in general, neonatal conventional t cells, especially cd + ab t cells, are regarded as immature or t h biased. vg + vd + t cells are unconventional lymphocytes: they are mhc-unrestricted and can react rapidly upon activation with pyrophosphates (e. g. (e)- -hydroxy- -methyl-but- -enyl pyrophosphate (hmb-pp)) or aminobisphosphonates (e. g. zoledronate) in adults. until now, little is known on the functional reactivity of neonatal vg + vd + t cells towards these activators. because il- is preferentially secreted by neonatal dendritic cells (dc) upon tlr stimulation, we investigated the potential costimulatory effect of this cytokine on hmb-pp and zoledronate-treated neonatal vg + vd + t cells. herein, we observed that zoledronate induced neonatal vg + vd + t cell proliferation and ifn-g production in cord blood mononuclear cells (cbmc) cultures. other t h -like cytokines like tnf-a and gm-csf were also produced upon this stimulation, but less than ifn-g, while t h -like cytokines such as il- and il- were not induced. addition of il- to zoledronate selectively costimulated ifn-g production from neonatal vg + vd + t cells. furthermore, zoledronate/il- treatment resulted in neonatal vg + vd + t cells expressing high levels of the cytotoxic mediators perforin and granzyme a. zoledronate induced the expression of the receptor for il- (il- r) and the transcription factor t-bet, which is known to be important for the production of ifn-g in gd t cells. in addition, costimulation with il- resulted in a further increase of t-bet expression in neonatal vg + vd + t cells. these changes in the expression of il- r and t-bet likely contribute to the observed selective ifn-g response towards zoledronate/il- treatment. of note, in contrast to adult peripheral blood vg + vd + t cells, hmb-pp had no or only a minor effect on the functional reactivity of neonatal vg + vd + t cells. altogether, these observations show that neonatal vg + vd + t cells are functionally active and that this t cell population might play a role in protective immune responses to infections with intracellular pathogens in early life, in particular when dc-derived il- is produced in response to microbial stimuli. the evasion of antigen presentation is a feature common to herpesviruses. one of the strategies employed to inhibit antigen presenting molecules is ubiquitination, internalisation and lysosomal breakdown by viral e ligases such as hhv encoded k , k or mhv encoded mk . these viral genes represent homologues of the march family of cellular genes whose function is the regulation of cell-surface antigen presentation and reduction of the lifetime of loaded antigen complexes. ubiquitination targets surface molecules to the lysosome via the multivesicular body (mvb), a structure which also has an important role in the budding of many viruses. we investigated the existence of alternative fates for antigen presenting molecules post-ubiquitination, and how viral e ligases manipulate them. we discovered that both the cellular march and viral e ligases ubiquitinate cd molecules. however, whereas viral molecules inhibit cd -antigen presentation, the march molecules are essential for the recirculation and function of the long-lived and lysosome-resistant cd molecules. in contrast mhc class ii was only targeted by cellular and not by viral e ligases. furthermore cd molecules could be found in viral particles as a result of ubiquitination, presumably via the mvb. thus, virally expressed and cellular e ligases have opposite effects, despite their homology. how this is achieved is a matter of active investigation. gamma delta (gd) t cells recognize stress-induced auto-antigens and contribute to immunity against infections and cancer. our previous study revealed that vd negative ( neg ) gd t lymphocytes isolated from transplant recipients infected by cytomegalovirus (cmv) killed both cmv-infected cells and ht colon cancer cells in vitro. in order to investigate the anti-tumor effects of vd neg clones in vivo, we generated hypodermal ht tumors in immunodeficient mice. concomitant injections of vd neg clones, in contrast to vd + cells, prevented the development of ht tumors. vd neg clones expressed chemokine c-c motif receptor (ccr ) and migrated in vitro in response to chemokines secreted by ht cells, among which were the ccr ligands macrophage inflammatory protein (mip)- d and monocyte chemoattractant protein (mcp)- . more importantly, a systemic intraperitoneal (i. p.) treatment with vd neg clones delayed the growth of ht subcutaneous (s. c.) tumors. the effect of in vivo gd t cell passive immunotherapy on tumor growth could be reverted by addition of a blocking anti-ccr antibody. gd t cell passive immunotherapy was dependent upon the cytotoxic activity of the gd effectors towards their targets since vd neg clones were not able to inhibit the growth of a hypodermal tumors. our findings suggest that cmv-specific vd neg cells could target in vivo cancer cells, making them an attractive candidate for anti-tumor immunotherapy. more recently, we generated ht cells expressing the luciferase and realized orthotopic injection of ht -luc cells. progressive tumor development and regression following « gd treatment » will be observed in vivo using bioluminescent imaging. intraepithelial lymphocytes (iel) compose large, oligoclonal, tissue-associated repertoires of non-mhc-restricted t cells that play key roles in immunosurveillance. it is commonly considered that the characteristic iel repertoires are positively selected by thymic epithelial molecules that are also stress-induced in specific tissues, thereby activating iel function. however, no such molecules have been identified. here we characterise skint , currently the only known determinant of a canonical iel compartment, that is selectively required for vg vd + dendritic epidermal t cell (detc) development. we show that both peripheral and thymic skint expression is essential for full detc development. its effects are highly specific since even substantial and ubiquitous over-expression neither negatively selects detc, nor affects any other t cells. unexpectedly, however, skint genes are not expressed by cell lines and are downregulated rather than activated by carcinogenesis. mouse genetic models allow powerful insight into skint function; for example, we demonstrate that the constitutive expression of wild-type skint fully restores detc development in a skint mutant mouse, but does not rescue normal detc function. thus, skint provides a novel perspective into how epithelia regulate the development and function of specific tissue-associated t cell compartments, and how normal versus dysregulated tissues may be demarcated. marginal zone (mz) b cells are strategically localized in the mz of the spleen. since most of the blood reaching the spleen is passing through this region such localization favors contact with blood born antigens and pathogens. besides being able to rapidly secrete antibodies, mz b cells may also act as professional antigen presenting cells (apcs). they are known to express high levels of cd d which is the presenting molecule for nkt cells which are also located in the mz. therefore we hypothesised that mz b cells may be efficient activators of nkt cells. to test this hypothesis, we used freshly sorted splenic mz b cells (cd + cd hi cd lo cd c -) and splenic conventional dendritic cells (cdcs) (cd c hi cd a +/-cd b +/-b -) from wt and cd d -/mice as apcs for nkt cells from va -ja transgenic or wt mice. the apcs were treated with agalactosylceramide (agalcer) or heat killed (hk) listeria monocytogenes or salmonella typhimurium. both mz b cells and cdcs proved to be highly efficient apcs for priming of nkt cells and induced robust proliferation. in contrast, other populations of b cells failed to activate nkt cells. we showed, using cd d -/mice as well as blocking antibodies to icosl, that proliferation of nkt cells depends on tcr/cd d and in case of mz b cells, also on icos/icosl interactions. importantly, apcs primed with hk bacteria were not able to induce nkt cell proliferation. interestingly, mz b cells exclusively induced production of il- by nkt cells. in contrast, cdcs mostly induced production of ifn-g and il- producing cells were scarce under these conditions. cytokine production by nkt cells proved to be independent of tcr signalling, but dependent on icos/icosl interactions when mz b cells were used as apcs, and gitr-dependent when cdcs were used. taken together, our data suggest that both mz b cells as well as cdc act as professional apcs for nkt cells. notably, the nature of apcs appears to be critical for polarization of the immune response: mz b-cell-primed nkt cells induce cytokine milieu fostering a t h response, whereas cdc-primed nkt cells rather favor a t h response. objectives: il- is an innate cytokine present in elevated levels in sera from patients suffering from autoimmunity (eg. sle and ra) and the allergic disease atopic eczema. in mice, injections of il- give rise to an early polyclonal isotype switched antibody response which is absent in inkt cell deficient (cd d -/-) mice. we set out to investigate the activated b cells in il- injected mice and how these are regulated by inkt cells. methods: mice received daily i. p. injections of il- ( mg) for days and the antibody response in serum was monitored using elisa. the b cell activation in the spleen at day was evaluated by flow cytometry and immunohistology. results: mice injected with il- developed self reactive (anti-pc and anti-dna) antibodies in the serum, in line with the autoreactive antibodies in patients with e. g. sle and atopic eczema. the antibody producing cells formed cd + cell clusters in the red pulp of the spleen, a typical feature of extrafollicular activation frequently associated with autoreactive responses. surprisingly, the antibody response induced by il- was increased in inkt cell deficient (cd d -/-) mice, in contrast to published data. an increased response to il- was also observed in ja -/mice, which lack the a-chain of the tcr used by inkt cells, and thus our data suggest that inkt cells inhibit antibody producing cells in il- induced antibody responses. further characterization of the recruitment of b cells in il- injected mice revealed a marked expansion of the marginal zone b cell (mzb) population in the spleen, suggesting an important role for mzbs in the il- induced autoreactive antibody response. mzbs are innate-type b cells that express high levels of cd d, are prone to autoantibody production and often involved in early immune responses. the il- induced antibody response in mzb deficient (cd -/-) mice was either decreased (igg) or delayed (ige), supporting the importance of mzbs in il- induced antibody responses. we conclude that the role for inkt cells in il- induced antibody responses is to inhibit the production of autoreactive antibodes from mzbs in extrafollicular foci. objectives: amoebiasis is a widespread human parasitic disease caused by the intestinal protozoan entamoeba histolytica. there are two major clinical manifestations of the disease, amoebic colitis and amoebic liver abscess (ala). interestingly, only a small proportion of e. histolytica-infected individuals develop invasive disease, whereas the majority harbors the parasite within the gut without clinical symptoms. so far, cells of the innate immune system have been described to constitute the main host defense mechanism for the control of amoebiasis, relying largely on the early production of interferon-g (ifn-g). however, information is lacking about the sources of early ifn-g production as well as the amoeba antigens involved in this activation process. methods: using a recently developed c bl/ mouse model for ala, the contribution of natural killer t (nkt) cells for protection against amoebic disease was investigated. applying nkt cells and dendritic cells as antigen-presenting cells from various ko-mice, the signaling pathways implicated in recognition of amoebic antigens and activation of cytokine-secretion by nkt cells was analysed. results: nkt cells were found to play a key role in the defense against ala. specific activation of nkt cells by a-galactosylceramide (a-galcer) induced significant protection, whereas jalpha -/-and cd d-/-mice lacking inkt as well as dnkt cells suffered from more severe abscess formation. a lipopeptidophosphoglycan, which is present in large quantities on the surfcae of e. histolytica trophozoites (ehlppg), was identified as a major amoeba antigen that activates nkt cells resulting in the production of ifn-g, but not of il- . moreover, ifn-g production required the presentation of ehlppg by cd d and signaling through the tlr receptor cascade in combination with a simultaneous secretion of il- . similar to a-galcer application, treatment of mice with purified ehlppg significantly reduced the severity of ala in amoeba-infected mice. our study provides a mechanism for the innate control of amoeba invasion that might explain why the majority of e. histolytica-infected individuals do not develop amoebic disease. a few years ago, we have observed a significant expansion of circulating effector gamma delta t cells following cytomegalovirus (cmv) infection in kidney transplant recipients (ktr). these unconventional t cells display tcr dependent cytotoxicity against both cmv-infected cells and carcinoma cells. in the present study, an extensive phenotyping of gamma-delta t cells allowed us to demonstrate an over-expression of cd in cmv-infected individuals. cd is the fcgammariiia, a natural killer cell marker usually absent on conventional t cells. we found that . ± . % of gamma-delta t cells from cmv-infected ktr expressed cd , when compared with only . ± . % in non cmv-infected ktr (p x . ). similarly, . ± . % of gamma-delta t cells from cmv-seropositive blood donors expressed cd compared to . ± . % in cmv-seronegative donors (p x . ). cd + gamma-delta t cell lines generated from cmv-infected individuals were able to produce ifn-g (a potent anti-viral cytokine) in a cd -dependent manner when activated by cmv/igg immune complexes. this production greatly increased in the presence of il- and ifn-alpha, two cytokines highly produced during cmv-infection. the supernatants of gamma-delta t cells activated with agonist anti-cd mab inhibited cmv replication in vitro and this effect was abrogated in the presence of a blocking anti-ifn-g antibody. cmv/igg immune complexes were also able to induce the expression of the cytotoxicity marker cd a on cd + gamma-delta t cell lines. cd is well-known to mediate antibody-dependant cellular cytotoxicity (adcc), especially in natural killer cells. accordingly, we demonstrated that cd + gamma delta t cell lines could make adcc against the daudi lymphoma cell line and the a skin carcinoma cell line pre-incubated either with rituximab (anti-cd ) or cetuximab (anti-egfr), respectively. in contrast, no addc could be observed against cmv-infected fibroblasts pre-incubated with polyclonal anti-cmv igg (cytogam), probably because cytogam weakly stained infected cells. these data reveal a new cd -dependent anti-cmv function of gamma-delta t cells through recognition of immune complexes and secretion of ifng. moreover, they demonstrate that these cells are able to kill through adcc lymphoma and skin carcinoma cells, two tumour types frequently encountered in ktr. dendritic epidermal t cells are a prototypic population of intraepithelial gd t cells in the mouse skin. found in the basal layer of epidermis and in close contact with langerhan's cells and keratinocytes detc facilitate vital immunological and physiological processes e. g. wound healing, homeostasis, tumor surveillance and regulation of inflammation. gd t cells respond rapidly to non-peptidic microbial and stress induced self antigens in a non-mhc restricted manner and are therefore proposed to bridge the gap between innate and adaptive immunity. by using gd t cell knock-out mice tcrd-/-, ovalbumin transgenic k mova mice and a skin grafting model we aimed to elucidate the role of gd-detc in adaptive immune responses associated with elimination of foreign antigen presented in the skin.we show that in the absence of gd t cells in the skin there is a decrease in rejection of ovalbumin expressing skin grafts compared to wildtype mice. we show that optimal regimens of antigen delivered subcutaneously in conjunction with adjuvant elicits comparable responses in wildtype and knockout mice. however frequency of primed host animals is reduced in tcrd-/-mice when antigen is delivered epidermally via skin grafting; suggesting detc enhance cross presentation of classical mhc bound antigens in the skin. considering the incapability of gd t cells to recognize peptide antigens in the context of mhc we plan to dissect the relationship between detc and professional antigen presenting cells in the skin. understanding the underlying mechanisms of this relationship will expand our knowledge of enhancing professional apc function in skin by detc and potentially other epithelia by intraepithelial gd t cells and can be useful in designing therapies to epithelial infections and malignancies. we demonstrate a rapid and hmb-pp-dependent crosstalk between gd t cells and autologous monocytes that resulted in the production of inflammatory mediators including il- , ifn-g, tnf-a, osm, ccl , cxcl , cxcl , and trail. moreover, under these co-culture conditions monocytes showed enhanced survival and differentiated overnight into inflammatory dcs with antigen-presenting functions. these cells expressed cd , cd , hla-dr, and dc-sign, and lost cd , ccr , ccr , and cxcr . addition of further microbial stimuli (lps, peptidoglycan) induced ccr and enabled these inflammatory dcs to trigger antigenspecific cd + effector ab t cells expressing ifn-g and/or il- . importantly, our in vitro model replicated the responsiveness to microbes of effluent cells from pd patients and translated directly to episodes of acute pd-associated bacterial peritonitis, where vg /vd t cell numbers and soluble inflammatory mediators were elevated in patients infected with hmb-pp-producing pathogens. conclusion: our findings suggest a direct link between invading pathogens, microbe-responsive gd t cells, and monocytes in the inflammatory infiltrate, which plays a crucial role in the early response and the generation of microbe-specific immunity. the mechanism(s) responsible for their dichotomous behaviour are poorly understood, and the outcome of nkt cell manipulation remains unpredictable. there is growing evidence that the nkt cell pool is composed of functionally distinct subsets, but such a possibility has not yet been investigated in a model of nkt cellmediated immunosuppression. we examined the differential ability of nkt cell subsets from the thymus and liver to prevent type i diabetes when transferred into prediabetic nod mice. the transfer of abtcr+dn thymocytes (a population enriched for nkt cells) has previously provided robust protection against tid development; however it has not been formally shown that nkt cells are solely responsible for the protection. our study found that while the transfer of thymic dn nkt cells can prevent tid and severe insulitis in nod mice, not all nkt cell subsets show the same tolerogenic capabilities. these findings both formally demonstrate the disease-preventing effects of nkt cell transfer in nod mice and provide further evidence that nkt cells are a functionally heterogeneous population. objective: vg /vd t cells constitute a minor t cell population in human blood that expands specifically and rapidly in response to the microbial metabolite hmb-pp. our previous microarray studies showed that vg /vd t cells stimulated with hmb-pp in the presence of il- express markers associated with a possible follicular b cell helper function. we therefore investigated in more detail whether and how hmb-pp and il- regulate expression of the b cell attracting chemokine cxcl /bca- , its receptor cxcr , and co-stimulatory molecules involved in b cell help. purified peripheral vg /vd t cells were co-cultured with autologous monocytes or b cells (as feeder cells) for up to days with and without hmb-pp, in the absence or presence of il- or il- , or in medium alone. cells were analysed by flow cytometry and immunofluorescence microscopy. results: high levels of cxcl protein were detected in co-culture supernatants only when both il- and hmb-pp were provided, implying an il- -dependent and tcr-dependent expression. vg /vd t cells were confirmed as producers of cxcl by flow cytometry and immunofluorescence. under the same conditions, activated vg /vd t cells expressed cd , cd , cd l, cd , icos and ox . in contrast, neither cxcr nor ccr changed markedly by il- stimulation of peripheral vg /vd t cells. conclusion: our findings confirm on the protein level that stimulation of vg /vd t cells with hmb-pp and il- induces markers typically associated with follicular b helper t (t fh ) cells. these data suggest that gd t cells contribute to humoral immune responses and play a role in germinal centre formation and production of high-affinity antibodies in microbial infection. ongoing analyses of gd t cells in inflamed and non-inflamed lymphoid tissues (tonsils, appendices) aim at demonstrating the physiological relevance of our findings. y. emoto , m. emoto gunma university school of health sciences, department of laboratory sciences, maebashi, japan invariant (i) natural killer (nk)t cells become undetectable after stimulation with a-galactosylceramide (a-galcer) or interleukin (il)- . although downmodulation of surface t cell receptor (tcr)/nkr-p c (nk . ) expression has been shown convincingly after a-galcer stimulation, it is unclear whether this holds true for il- stimulation. to determine whether failure to detect inkt cells after il- stimulation is caused by dissociation/internalization of tcr and/or nkr-p c or by block of de-novo synthesis of these molecules, and to examine the role of il- in disappearance of inkt cells after a-galcer stimulation, surface (s)/ cytoplasmic (c) protein expression as well as mrna expression of tcr/nkr-p c by inkt cells after stimulation with a-galcer or il- , and influence of il- neutralization on down-modulation of stcr/snkr-p c expression by inkt cells after a-galcer stimulation were examined. the s/ctcr + s/cnkr-p c + inkt cells became undetectable after in-vivo administration of a-galcer, which was partially prevented by il- neutralization. whereas s/cnkr-p c + inkt cells became undetectable after in-vivo administration of il- , s/ctcr + inkt cells were only marginally affected. mrna expression of tcr/nkr-p c remained unaffected by a-galcer or il- treatment, despite the down-modulation of ctcr and/or cnkr-p c protein expression. in contrast, ctcr + cnkr-p c + stcr -snkr-p c -inkt cells and cnkr-p c + snkr-p c -inkt cells were detectable after in-vitro stimulation with a-galcer and il- , respectively. our results indicate that tcr and nkr-p c expression by inkt cells is differentially regulated by signaling through tcr and il- r. they also suggest that il- participates, in part, in the disappearance of inkt cells after a-galcer stimulation by down-modulating not only snkr-p c but also stcr. the fetus and infant are highly susceptible to viral infections. a number of viruses, including human cytomegalovirus (cmv), cause more severe disease in early life compared to later life. it is generally accepted that this higher susceptibility to viral infections is due to the immaturity of the immune system. gd t cells are unconventional t cells that can react rapidly upon activation and show mhc-unrestricted activity. herein, we show that upon cmv infection in utero, fetal gd t cells expand and become differentiated. the response was restricted to vg -gd t cells, irrespective of their vd chain expression. differentiated gd t cells expressed high levels of ifn-g, transcription factors t-bet and eomes, natural killer receptors and cytotoxic mediators including perforin and granzymes. in addition, congenital cmv-infection induced a highly restricted complementary-determining region d (cdr d ) and cdr d repertoire, with a striking enrichment in a specific germline-encoded cdr d sequence. differentiated gd t cells and the enriched cdr d sequence were detected as early as after weeks of gestation. our results indicate that functional fetal gd t cell responses can be generated during development in utero and suggest that this t cell subset could participate in anti-viral defense in early life. results: spectratyping showed only in-frame selection for vd -jd and vgi-jg . / . rearrangements in tcrgd thymocytes and to a lesser extent in tcrgd cb cells. in contrast, clear in-frame vd -jd and vg -jg . selection was seen in pb tcrgd cells. detailed analysis of the cdr motifs revealed selection determinants in both vg -jg . (canonical length and cdr motif) and vd -jd (minimal cdr length in combination with an invariant t nucleotide) rearrangements. upon evaluation of the replication history we found a clear increase in the number of cell divisions from naïve tcrgd thymocytes (˚ ) and tcrgd cb cells ( - ) to tcrgd pb cells (˚ or more). no increase was seen between cb and pb tcrgd t cells within the first year of life, suggesting that peripheral proliferation occurs later in life. our results indicate that the human peripheral tcrgd repertoire is shaped by (antigenic) selection and proliferation processes. moreover, the ontogenetic changes in the gd repertoire between the central and peripheral immune systems are clearly influenced by proliferation. background: natural killer (nk) t cells have been implied in the regulation of disease in the non obese diabetic (nod) mouse model of type diabetes (t d). we have previously shown that transgenic expression of a cd d-restricted, va . -vb tcr in nod mice lead to an increase in cd d-restricted type ii nkt cells ( abnkt cells), and prevention of the development of t d in the transgenic mice. in this study we have investigated the requirements and underlying mechanism of disease protection by type ii nkt cells in a disease transfer model. to investigate the mode of regulation by abnkt cells, we explored a disease transfer model into nod.scid mice using transgenic diabetogenic bdc . cd + t cells, in the presence or absence of selected cells from abnkt cell transgenic mice. results: in ab transgenic mice a high frequency of activated transgenic nkt cells was found in the pancreas of the protected mice. in this organ, abnkt cells expressed a high level of cxcr and a low level of ccr and cd l, a pattern similar to that observed in t cells homing to inflammatory tissues. adoptive transfer of cd + bdc . t cells into nod.scid recipients rapidly induced onset of diabetes. using this model, we found that co-transfer of spleen cells from ab transgenic mice with bdc . cd + cells resulted in the prevention of diabetes development. the protection from disease required a minor cd + subset of ab+ nkt cells, but was independent of cd + t regulatory cells. analogs of alpha galactosylceramide (a-galcer) that may modulate the strong activation of inkt and at the same time prolong their effect upon in vivo administration are a long standing goal of research in this area due to their putative immunotherapeutical applications. a new class of non glycosidic analogues bearing an aminocyclitol ring as galactose surrogate have been synthesized and assayed in their capacity to be presented by cd d and recognized by inkt. the structural novelty of these compounds resides in the presence of a cyclohexane that substitutes the sugar moeity and the substitution of the o glycosidic linkeage with the ceramide by a n. in this basic structure, substitutions in the cyclohexane ring with oh in different conformations mimicking different sugars, differences in the length of the sphingosine lipid and differences in the orientation of the n linkeage conform a series of analogs that have been analyzed in their capacity to stimulate inkt cells. proliferation assays in bulk splenocyte cultures and cytokine secretion determinations show that inkt cells are specifically stimulated by some of the analogs tested. in particular, the active compound hs , induces in vitro inkt cell expansion and ifng and il- secretion in a similar fashion but less potently than a-galcer. dose response assays show a bias towards a th profile response after recognition by nkt cells, more similar to the response induced by och. the degree of structural similarity of the cyclitol ceramides with a-galcer parallels their cellular activities. these data open the way towards the development of a new class of a-galcer lipid analogues having charged amino substituted polar heads resistant to glycosidase degradation, thus enhancing their in vivo biodisponibility, and expands the range of potential inkt cell sphingolipid agonists that can modulate the immune response due nkt cell activation. objectives: invariant natural killer (ink) t cells represent an innate lymphocyte subset with important modulatory functions. in the presence of pathogens or tumors, inkt cells play an adjuvant function that boosts t cell immunity through cytokine secretion and dc maturation. in steady-state conditions, i.e. in the absence of pathogens, inkt cells acquire a regulatory function that promotes t cell tolerance and prevents autoimmune disease. our aim was to assess the mechanism of action of inkt cells in the steady state and, specifically, to test the hypothesis that inkt cells promote immune tolerance through modulation of dcs. methods: to assess the direct influence of regulatory inkt cells on dc maturation in resting conditions, we derived murine inkt cell lines in vitro and, after staining with agalcer-loaded cd d tetramers and magnetic purification, we tested their capacity to modulate bone marrow-derived myeloid dcs in the absence of any other maturation signals. we analyze the transcriptional profile (microarray analysis) as well as maturation, cytokine expression profile and pro-tolerogenic antigen-presenting function of inkt cell-modulated dcs (inkt-dcs). the cell-cell interaction with inkt cells provoked dramatic phenotypical changes on immature dcs that acquired the cardinal features of tolerogenic dcs such as intermediate levels of mhc class ii and co-stimulatory molecules expression and high secretion of il- with no release of pro-inflammatory cytokines. most importantly, inkt-dcs acquired tolerogenic antigen-presenting function inducing the differentiation of regulatory tr cells and immune tolerance in vivo. dcs, simultaneously stimulated with inkt cells and through toll-like receptor (lps) completely lost the pro-tolerogenic phenotype and acquired a proinflammatory cytokine profile. conclusion: it is still mysterious how inkt cells can play a dual role and either boost t cell immunity or promote immune tolerance. our results suggest that the same mechanism could underlie both inkt cell functions. in the presence of pathogen-driven maturation signals, the inkt cell-modulation of dcs favors their acquisition of a pro-inflammatory phenotype and function. on the contrary, if inkt cells are activated in the absence of pathogens, e. g. during autoimmune conditions, their interaction with immature dcs promotes their tolerogenic maturation to maintain peripheral tolerance and counter-regulate autoimmune diseases. th -type immune responses have been reported to fight extracellular bacterial infection, but as well to cause autoimmune diseases and allergy. the th immune response is characterized by the secretion of il- a and il- f. the il- locus encodes the highly conserved il- a and il- f cytokines that are syntenic in kb distance to each other. besides cd + th and nkt cells, approximately % of the il a producers are gd t-cells. like cd + th cells, il- producing gd tcells have recently been implicated to play a major role in the immune response to infections with extra-and intracellular bacteria. our findings show a difference between the il- production of gd t cells in the peripheral system and mucosal epithelia. mucosal gd t-cells generally do not produce th cytokines. in the periphery, we define novel subsets of gd t-cells that can produce either il- or ifn-g. combined with the well known classification of il- producing gd t-cells along the markers cd and cd , our data point at specialized functions of the different gd t cell subsets depending on their location and origin. functional studies are currently carried out in order to address the role of the different gd t-cell subsets for th -type immune responses in vivo. in this context, the potential redundancy of il- a and il- f may complicate the analysis. so far, most studies were carried out with il- a single-deficient or il- f single-deficient mice. to further clarify these issues, we will have to address the above mentioned findings in il- a and il- f double-deficient mice. several subsets of gd tregs have been described and intensively studied, but the potential regulatory role of innate t cells in controlling immune responses remains unclear. lymphocytes expressing gd tcr are involved in both innate and adaptive immune responses. vg vgd t cells, which represent a major peripheral blood gd t-lymphocyte subpopulation in humans, display a broad reactivity against microbial agents and tumors.here we report that tgf-b and il- differentiate in vitro a subset of gd t lymphocytes with regulatory functions (vd tregs) in the presence of specific antigen stimulation. these cells express the forkhead/winged helix transcription factor (foxp ) and, similarly to ab tregs, suppress the proliferation of anti-cd /anti-cd stimulated-pbmc. detailed knowledge about the phenotype and functionality of vd tregs will improve our understanding of the role of gd t cells in the pathogenesis and regulation of autoimmune, infectious and cancer diseases. a-galactosylceramide (a-galcer) has the potential to activate invariant (i) nkt cells, which in turn release a wide variety of cytokines that stimulate immunocompetent cells. although this rapid and vigorous cytokine release appears critical for regulation of various immune responses, it remains elusive whether protection against intracellular bacteria can be induced by a-galcer. here we show that treatment with a-galcer ameliorates murine listeriosis, and inhibits inflammation in the liver and spleen following listeria monocytogenes infection. liver infiltration of granulocytes and g/d t cells was accelerated by a-galcer treatment. granulocyte and g/d t cell depletion exacerbated listeriosis in a-galcer-treated mice, and this effect was more pronounced in granulocyte than in g/d t cell depletion. although secretion of gm-csf and il- was detected among the nkt cell population in the liver and bone marrow immediately after a-galcer treatment, infiltration of granulocytes into the liver was not prevented by neutralizing mab. yet, in parallel to the numerical increase of granulocytes expressing cd b in the liver following a-galcer treatment, numbers of cells lacking cd b diminished in the bone marrow. in addition, respiratory burst in granulocytes was enhanced by a-galcer treatment. our results indicate that a-galcer-induced antibacterial immunity is caused, in part, by accelerated infiltration of inflammatory cells, in particular granulocytes and to a lesser degree g/d t cells, into the liver. we also suggest that the infiltration of granulocytes is caused by an accelerated supply of granulocytes from the bone marrow, rather than by accelerated granulopoiesis. objectives: the aim of this work is to evaluate whether phenotypic and functional features of vgamma /vdelta t cells are influenced by the activity of mevalonate pathway in tumor cells and contribute to determine disease aggressiveness in cll. methods: eighty seven previously untreated cll patients were evaluated for in vitro vgamma /vdelta t cells expansion upon stimulation with zoledronic acid (za) and interleukin- (il- ). gammadelta t cells subset distribution and natural killer receptors profile were evaluated by multicolor flowcytometry. the mutational status of the tumor immunoglobulin heavy chain variable region (igvh) was analyzed by dna sequencing. the activity of the mev pathway was determined by ) the bioinformatic analysis of gene expression profiling data ) the quantification of mev pathway metabolites. results: proliferation of gammadelta t cells was observed in patients ( %) (responders, r), whereas patients ( %) were non-responders (nr). vgamma /vdelta t-cell subset distribution was well balanced in r patients, whereas effectors subsets [i. e., effector memory (tem), and terminally differentiated effector memory (temra)] were largely predominant in nr patients. temra of nr patients mainly expressed the inhibitory receptor ilt , whereas temra of r patients had an higher expression of the costimulatory molecule nkg d. the proliferative response of vgamma /vdelta t cells was significantly associated with igvh mutational status, which is a well known prognostic factor in cll. indeed, % of r patients were m, whereas % of um patients were nr (p x . ). given this association, we evaluated the activity of the mev pathway in tumor cells of m and um patients. the pathway was more active in tumor cells of um than m patients, suggesting that the former can more easily engage gammadelta t cells and drive their differentiation into functionally exhausted t emra . given the association between the r/nr status and the igvh mutational status we also analyzed the independent prognostic impact of r/nr status in multivariate cox analysis. nr patients had a significantly shorter time to first treatment thus pointing to r/nr status as an independent prognostic factor. conclusion: these data define a novel mechanism of immune escape which can contribute to determine disease aggressiveness in cll patients. the studies reported here were undertaken to ascertain and delineate the ability of kupffer cells to regulate the response of inkt cells to biliary obstruction. methods: c bl/ mice were not treated or rendered kupffer cell-depleted by intravenous inoculation of liposome-encapsulated dichloromethylene diphosphonate. to clarify the factors that elicit inkt cell activity, additional mice were administered anti-il- p (clone r - f ; atcc) or anti-cd- d (clone b ) monoclonal antibody (mab) prior to surgery. midline laparotomies were performed; the common bile duct was ligated twice and divided. sham-operated animals served as controls. blood and liver samples were collected at periodic intervals post-surgery. the hepatic lymphoid population was purified and characterized by flow cytometry. the nkt cell population was increased significantly in the livers of control, but not kupffer cell-depleted, mice at hours post-bdl. the response of inkt cells was diminished in mice pretreated with mab specific for il- p , a component of both il- and il- ; pretreatment with anti-cd d mab had no effect. il- rb-deficient mice also exhibited a marked increase in hepatic inkt cells following bdl suggesting that il- was not a critical factor. this suggestion is supported by the increased expression of il- p and il- p (but not il- p ) mrnas by kupffer cells purified from the livers of bdl animals. these findings imply that il- production by kupffer cells promotes the response of hepatic inkt cells to biliary obstruction. objectives: p-glycoprotein (pgp or abcb ) is a member of the abc family of transporter proteins which are characterized by their ability to pump molecules across membranes in an atp-dependent manner. although pgp was first identified for its ability to confer resistance to chemotherapeutic agents in tumor cells, it has now also been described in cells of the immune system. our work primarily focuses on gd t cells that complement and regulate the activities of ab t cells, particularly in tissues. we have recently described functional subsets of gd cells based on cd expression. gd + cells secrete interferon-g, while gd cells are capable of producing il- . this study investigates the role of pgp in gd cells with specific reference to these recently-identified cd -defined subsets. methods: pgp activity was measured based on the expulsion of rhodamine . cells were incubated with rho followed by a period in the presence or absence of the pgp inhibitor cyclosporine-a. cell populations were identified using monoclonal antibodies and flow cytometry. percentages of subpopulations were compared by anova, statistical results are shown as p values that were calculated using a newman-keuls multiple comparison post-hoc test. results: up to % of intraepithelial lymphocytes (iels) from the small intestine are tcrgd + . of these, virtually all displayed pgp activity. indeed, pgp activity was generally higher in tcrgd + than tcrab + iels. in the thymus, pgp activity was observed in only˚ % of gd + cells but not at all in gd cells. by contrast, in peripheral lymph nodes, mesenteric lymph nodes and peyer's patches, - % of gd + cells were positive for pgp activity, although their gd counterparts remained largely negative (p x . ). conclusion: this study demonstrates that subsets of gd cells display different levels of pgp activity depending on their location in the body and their expression of the newly identified functional marker cd . as pgp activity may play a role in cytokine release, cytotoxicity and protection from harmful toxins, it confirms our hypothesis that gd + and gd cells have very different roles in immune responses and provides insight into the mechanism by which gd cells cope with diverse body locations. objectives: an effective immune response orchestrates different cellular activities of both innate and adaptive immune compartments. in this context, the vgamma vdelta t cell biology presents some critical features for their ability to display a broad antimicrobial activity by directly killing infected cells and by inducing an effective adaptive immune response. the activation of vgamma vdelta t cells by aminobisphosphonate drugs such as zoledronic acid (zol) results in a massive release of cytokines and chemokines that may induce a bystander activation of other immune cells such as dendritic cells (dcs) and b lymphocytes. the aim of this work was to evaluate the ability of activated vgamma vdelta t lymphocytes to orchestrate granulocytes functions in terms of migration capability, phagocytic activity and alpha defensin release. methods: peripheral mononuclear cells (pbmc) and purified vgamma vdelta t cells from healthy donors were stimulated with different compounds (zol, ipp) for hours and supernatants from these cultures were tested for their ability to induce granulocytes activation. briefly, we analysed the migration activity, the phagocytic activity and the degranulation process by perforimg migration assays, flow cytometry and elisa tests. we showed that soluble factors released by zol-stimulated vgamma vdelta t cells activate granulocytes by inducing their chemotaxis, phagocytosis, and alpha-defensins release. proteomic analysis allowed us to identify a number of cytokines and chemokines specifically released by activated vgamma vdelta t cells. moreover, mcp- depletion by neutralizing ab revealed a critical role of this chemokine in induction of granulocyte alpha-defensins release. altogether, these data show a vgamma vdelta -mediated activation of granulocytes through a bystander mechanism, and confirm the wide ability of vgamma vdelta tlymphocytes in orchestrating the immune response. conclusion: an immune modulating strategy targeting vgamma vdelta t cells may represent a key switch to induce an effective and well-coordinated immune response, and can be proposed as a way to strengthen the immune competence during infectious diseases. objectives: the aim of this study was to analyse the activity of vg vd t lymphocytes against glioma cells and to verify the possibility to target these innate cells in new immunotherapeutic approaches. human vg vd t cells recognize and kill several cancer cells presenting a disregulation in mevalonate pathway. interestingly, drugs already in clinical use, such as zoledronic acid, are able to promptly activate vg vd t cells through an indirect mechanism involving the block of farnesyl pyrophosphate synthase of the mevalonate cycle. the vg vd t cell activation by zoledronic acid results cytokines and chemokines synthesis and cytotoxic activity. glioma are tumors arising from glia in the central nervous system. unfortunately, the majority of glioma patients die in less then of a year from diagnosis and new treatment strategies are therefore hardly needed. methods: in order to analyse the activation of vg vd t cells and their effects on the viability of glioma cells, we expanded in vitro vg vd t cells from pbmcs of healthy donors by using phosphoantigen stimulation and tested the ability of vg vd t cell lines to kill three different glioma cell lines (t , u , u ) by cytokinic/cytotoxic mechanism by flow cytometry. results: our results demonstrated that vg vd t cells lines are able to recognize glioma cells, to differentiate in effector memory cells, and to kill glioma cells by releasing perforin. moreover, we analysed whether zoledronic acid treatment could improve the susceptibility of glioma cells to vg vd t lines. we showed that zoledronic acid is able to directly induce cell death on glioma cells and to strongly enhance the cytotoxic activity of vg vd t lines. conclusions: altogether, our results suggest that the induction of a strong antitumor response in vitro of vg vd t cells by using aminobisphosphonates could represent a new interesting immunotherapeutic approach for glioma treatment. viral-induced cancers, such as cervical cancer and liver cancer, contribute to approximately % of all cancers and represent a failure of host immunity to control chronic viral infection. natural killer t (nkt) cells are a population of regulatory t lymphocytes that are pivotal to the outcome of host protection to a range of viral infections and cancers, but their role in controlling host defenses to oncogenic viruses in epithelial and cutaneous tissue is virtually unexplored. using a mouse model of chronic viral infection in the skin, in which human papillomavirus (hpv) oncoproteins are expressed as a transgene in epithelial cells, we investigated the role for nkt cells in abrogating protective immunity to viral antigens in cutaneous tissue. we show that local hpv-e protein expression in the skin attracts a large lymphocytic infiltrate, including a population of cd d-restricted nkt cells. this nkt infiltrate is required to maintain local hpv-e -induced immune suppression and results in graft survival when transplanted onto a naive, immunocompetent host. the local suppressive environment evident in e -expressing transplanted skin is dependent on interactions between populations of cd d-expressing cd c+/f + myeloid cells and nkt cells. removal of either donor-resident or host-infiltrating nkt cells is sufficient to break immune suppression and allow e graft rejection. dissecting the suppressive properties of nkt cells in this novel model of chronic viral antigen presentation in the skin will provide valuable new insight into the potential for clinical manipulation of nkt cell populations to restore chronic anti-viral and anti-tumour immunity in epithelial tissues. nkt cells were expanded from total pbmcs from healthy donors by treatment with il- and a-galcer. expression of cd a, cd d and the costimulatory molecules cd and hla-dr, was established by flow cytometry. rna was quantified by real time-pcr. functional assays were performed by analysis of nkts cytokine production (ifn-g, il- ) and cytotoxicity against treated-idcs. results: idcs stimulated with olive pollen lipids up-regulated cd d expression on the cell surface in comparison with control cells. in contrast cd a expression was decreased. cd and hla-dr slightly increased, indicating certain grade of maturation. the amount of cd d mrna was higher in treated cells than in control cells. by contrast, there was less transcription of cd a, cd b and cd c genes than in control cells. nkt cells efficiently killed treated idcs as "in vitro" cytotoxic killing assays showed. ifn-g producing cells increased slightly in response to treated idcs compared to unstimulated cells, but the number of il- producing cells was not modified. similar results were obtained using monocytes as antigen-presenting cells. conclusions: idcs treated with lipidic extracts from olive pollen up-regulate the expression of cd d on the cell surface. in addition, nkt cells are able to recognize idcs and monocytes treated with lipids from pollen, producing ifn-g and cytotoxicity. all these data suggest that nkt cells may play a role in the control of the immune response to allergens, such as the lipids present in pollen grains. outline: in humans, . - % of circulating lymphocytes express a vg vd t cell receptor, yet strikingly little is known about the function and properties of such unconventional t cells. we performed cdna microarrays to find vg -enriched genes compared to conventional mhc-restricted cd + ab t cells, and found reciprocal enrichment of nectin-like adhesion molecules igsf & crtam in gd and ab t cells respectively. because igsf binds to crtam, the data fuel a hypothesis that this may be a novel axis of communication between the two cell types. interestingly, previous studies show that activated nk, nkt and cd + ab t cells express crtam, and that engagement of igsf on epithelial cells renders the latter targets for enhanced cytolytic and cytokine responses. our data extends this to the prospect of cytolytic immunoregulatory interactions between t cells mediated by igsf /crtam. we therefore sought to answer: . what is the function of igsf /crtam on gd t cells? . how is the igsf -crtam axis regulated in t cells? results and conclusions: flow cytometry showed igsf enrichment on resting gd t cells, with expression also detected on˚ % of ab t cells. the properties of those cells are being examined. however, igsf generally correlates with markers of activation/antigen experience such as cd ro. thus, igsf cells may comprise activated-yet-resting/pseudo-memory unconventional t cells and memory-effector conventional t cells. stimulating vg + t cells in vitro led to rapid crtam induction, resulting in the majority of cells co-expressing both igsf and crtam within hours. however, engagement of igsf by crtam or vice versa is not sufficient to induce cytotoxicity, as stable cho cell transfectants expressing either molecules were not specifically lysed by pbmc in vitro, compared to efficient and parallel targeting of mica + cells. instead, our current experiments address the possibility that crtam-igsf may regulate cytotoxic interactions promoted by other receptor-ligand interactions, such as mica-nkg d. this may explain why cells can tolerate co-expression of both molecules, and would refute the hypothesis that crtam-igsf interactions are sufficient for cd t cells to kill gd t cells and/or vice versa. instead, crtam-igsf interactions may set the threshold for cytotoxic immune-surveillance responses. t cell receptor (tcr) is a multisubunit complex in which the invariant subunit cd z is a kda transmembrane protein indispensable for coupling antigen recognition by tcr to diverse signal transduction pathways. approximately - % of human peripheral blood lymphocytes express the gd tcr and the majority of these cells express the vd tcr variable segment associated with the vg segment, and recognize phosphorylated non-peptidic metabolites from microbial or self origin. these compounds trigger vg vd t cells without antigen presentation. in vitro stimulated vg vd t cells with antigens are able to produce ifn-g and tnf-a and exert a powerful cytotoxic activity against infected cells as hiv-infected cells. however, during hiv infection a marked decrease of vg vd t cells was observed and the remaining cells are unable to respond to their non-peptidic ligands. aim of the present work was to study the mechanisms of vg vd t cell anergy observed in hiv+ patients. to this aim, cd z expression and ifn-g production by vg vd t cells from hiv+ and hiv-subjects were analyzed. we show that vg vd t cells from hiv-infected patients expressed lower level of cd z compared with healthy donors. a direct correlation between cd z expression and ifn-g production capability by vg vd t cell was found. however, pkc activation by pma is able to restore cd z expression and ifn-g production. our findings may contribute to clarify the molecular mechanisms of vg vd t cell anergy found in hiv+ patients and have implication in the design of effective immune-based therapies. l. abeler-dörner , m. swamy , s.l. clarke , a. hayday king's college london, immunobiology, london, united kingdom gut intraepithelial lymphocytes (iel) constitute one of the largest t cell compartments in mice and in man. their functions and their interactions with surrounding epithelium are likely to be crucial to the fine-tuned balance between tolerance to harmless food antigens, immunity to gut-associated pathogens, and overall intestinal immune surveillance. intestinal iel comprise many unconventional t cells including tcrgd cells and tcrab cd aa or cd -cd cells, which have been assigned innate-like immune functions and key roles in surveillance of stressed tissue. unlike conventional t cells, iel might initiate an immune response rather than simply being late effector cells. it is therefore important to elucidate the "immunological information flow" in the gut. to this end, this project characterizes different subsets of iel and their interactions with epithelium in steady state and under immunostimulatory conditions in vitro and in vivo. in the past, it has been notoriously difficult to study iel ex vivo. to solve this problem, we developed a novel culture system that allows us to expand the cells ex vivo and study their responses for up to days. the cells are initially activated by plate-coated acd antibody and a cytokine cocktail and maintained further in medium containing low levels of il- . after a resting period, the cells can be restimulated in vitro. in this new system, we studied responses of different iel subsets to stimulation via tcr, nkg d and cytokine receptors, either alone or in coculture with epithelial cells. as readouts we monitored proliferation, cytokine secretion (ifng, il- ) and expression of activating and costimulatory molecules. reactivation in response to various stimuli could already be observed after hours. the in vitro data set forms the basis for analysing iel responses in vivo to stimulatory molecules ectopically expressed as transgenes in the gut. the characterization of iel responses opens new insights into the nature of gut immune responses and should provide a better understanding of the immunology of inflammatory bowel diseases which still remain a major problem in the clinic today. objective: behcet's disease (bd) is a multisystemic disorder with a possible underlying pathology of immune-mediated vasculitis. increased expression of cd in bd patients suggested that nk receptors may play a pathogenic or regulatory role in the pathogenesis. considering the regulatory functions of nkg molecules in heterodimer with cd , we screened the presence of these receptors on t cell subsets in bd. the expression of nkg a/c/d molecules on gd and cd + t cells were analyzed in active and inactive patients with bd and healthy controls. expression of nkg molecules was evaluated on cd +, gd t and cd + nk cells by using flow-cytometry. results: gd t cells were increased in patients with bd compared to controls ( . vs. . %, p= . ). in addition to the increase of gd t cells, increased expression of activating nkg c molecules was also observed on gd t cells ( % vs. %, p= . ). nkg a expression on gd t cells was found to be higher than nkg c expression in patients and controls; but nkg a expression on the t cells was not statistically different in both groups ( . vs. %). nkg d receptors were present on most of the gd t cells in both groups. however these activating molecules on cd + cells were decreased in patients with bd compared to controls ( revlimid is a therapeutic agent used to treat myelodysplastic syndrome (mds), a group of haematological disorders characterised by ineffective haematopoiesis. the mechanism of action for revlimid is poorly understood, but there has been increasing interest in the strong association reported between mds and defects within the immunoregulatory nkt cell compartment. indeed, some studies now suggest an important outcome of revlimid treatment is the restoration of normal cytokine production by nkt cell levels and an increase in their overall numbers. we have conducted the most thorough study to date of the nkt cell compartment of mds patients treated with revlimid/ancestim and can report that mds patients had normal nkt cell levels prior to treatment, and no significant increase as a result of revlimid/ancestim treatment. furthermore, nkt cells from mds patients produced high levels of th and th cytokines when stimulated with pma/ ionomycin and the proportion of nkt cells capable of cytokine production did not increase significantly after revlimid/ancestim treatment. these are highly significant findings given the recent emphasis on nkt cells as a potential therapeutic target for mds. our study provides an extensive analysis of the impact of revlimid/ ancestim treatment on the nkt cell compartment and sheds new light on the role of nkt cells in mds and the mechanism of revlimid immunomodulation. objectives: human gd t cells are potent killers of a variety of tumour cell lines, and mice lacking gd t cells suffer from high incidence of experimentally-induced tumours. however, the molecular mechanisms mediating tumour cell recognition by gd t lymphocytes remain largely unknown. we aim at identifying potential tumour antigens and co-stimulation molecules expressed in ex vivo tumours and in tumour cell lines that activate human gd t cells for tumour cytolysis. as immune evasion mechanisms that down-regulate tumour antigens may operate in vivo, we have identified candidates from human tumour cell lines of hematopoietic origin that constitute in vitro cytolysis targets for vg /vd + lymphocytes. we have screened a panel of lymphoma and leukaemia cell lines using a conventional in vitro killing assay using vg /vd + cells, and selected two susceptible ("target") cell lines (over % death in the assay) and two vg /vd + resistant ("non-target") cell lines (under % death) for cdna microarray analysis. we compared the differential expression in pairs of tumour cell lines of identical origin: the burkitt's lymphoma cell lines daudi (target) vs raji (non-target), and the pre-b cell leukemia cell lines rch-acv (target) vs (non-target), and validated the results by rt-qpcr quantification. results: we identified commonly up-regulated and commonly down-regulated genes that encode cell membrane-associated proteins in susceptible tumours. ulbp , ifitm and prame, for example, are up-regulated, whereas cd and clec d are down-regulated in target cell lines. as these encode membrane-bound proteins with relevant functions in tumour immunity, they constitute potential ligands for gd lymphocyte recognition of tumour cells. the expression of these candidate genes was studied by rt-qpcr in a broader panel of cell lines and primary biopsies. we are currently testing, in functional assays based on rna interference and overexpression, these and other candidate genes in order to determine whether they provide activating or inhibitory signals to gd t cells. the comparison between the transcriptomes of vg /vd + target versus non-target cell lines allowed the identification of candidate genes, whose individual function we are currently dissecting, that may be involved in tumour cell recognition by human gd t cells. mice and humans are the only species in which phenotype and function of inkt cells have been properly described. our aims are to directly identify this cell population and to investigate cd d, in the rat. mice and rats have very similar cd d and inkt tcr genes, with the exception of the va gene segment, which is a multimember gene family in the rat. novel monoclonal antibodies with nearly identical binding capacities to mouse and rat cd d revealed a very similar pattern of cd d distribution, and could inhibit cytokine production after agalcer stimulation of primary cells in both species. response to agalcer was studied in five different rat strains, showing big inter strain differences. notably, ifn-g and il- production was - fold lower in the best responder rat strain (f ) compared to mouse (c /bl ). since nkrp a (rat homologue of mouse nk . ) and tcr are not appropriate markers for rat inkt, cd d oligomers where tested for binding to inkt-tcr transduced cells. newly generated agalcer loaded rat cd d dimers, recognized rat inkt tcr and, although less efficiently, bound to mouse inkt tcr. however, mouse cd d agalcer dimers did not bind to rat inkt tcr. agalcer loaded rat cd d dimers were then used to stain primary intrahepatic lymphocytes. but, although mouse inkt cells were stained to some extent, the identification of a discrete population in the rat was not possible. the reasons behind could be: that the avidity of the dimers for the tcr is not high enough to stain primary cells and/or that the frequencies are so low that the detection by facs analysis is difficult. in order to clarify these issues we currently produce and test rat cd d tetramers. burkholderia pseudomallei is a highly virulent bacterium which causes the potentially fatal disease melioidosis in humans. this disease is endemic in tropical regions, especially thailand and northern australia, and has a serious outcome for many infected individuals. b. pseudomallei is an intracellular bacterium and many b. pseudomallei strains are resistant to antibiotics so antibiotic treatment is aggressive and relapse of the disease is frequent. in addition to this, no vaccine is currently available to prevent the disease. human g d t cells are involved in the immune response to infection with a number of intracellular pathogens including brucella suis and mycobacterium tuberculosis. g d t cells respond to non-peptidic phosphorylated molecules known as 'phosphoantigens' which are byproducts of essential metabolic pathways in both bacteria and mammals. phosphoantigens cause expansion and activation of g d t cells during infection with intracellular pathogens including fransicella tularensis and m. tuberculosis. analogues of natural phosphoantigens have been developed to manipulate g d t cell responses as a cancer therapeutic and are currently in clinical trials for the treatment of hepatitis c virus. we aimed to determine in vitro whether enhancing gd t cell responses in human blood using the synthetic phosphoantigen picostim could reduce growth of intracellular b. pseudomallei in the human monocytic cell line thp- . a significant (p x . ) reduction in intracellular bacterial numbers was observed (n= ) in the presence of pbmcs cultured with picostim+il- in comparison with pbmcs cultured with il- or media alone. picostim+il- caused significant expansion and activation of gd t cells following culture of pbmcs for - days. purified gd t cells stimulated with picostim were able to reduce intracellular b. pseudomallei numbers -fold. this data demonstrates that pbmcs, stimulated with the synthetic phosphoantigen picostim+il- , reduced growth of intracellular b. pseudomallei in a gd t cell-dependent manner. objectives: vgamma /vdelta (gd) t cells play a major role in innate immunity against microbes, stressed and tumor cells. they represent less than % of peripheral blood lymphocytes (pbl), but can be expanded in vitro by zoledronic acid (za)-treated monocytes or dendritic cells (dc).the purposes of this study are: ) to determine whether dc generated from multiple myeloma (mm) patients are as effective as their normal counterparts in the ability to activate gd t cells; ) to evaluate whether gd t cells can exert immunoadjuvant activity on dc generated from mm patients and primed with tumor-specific antigens (survivin-sv); ) to establish whether the same issues could be solved using a simplified protocol of dc generation. ) dc were generated from cd + cells of healthy donors/mm patients; immaturedc on day were induced to fully mature by incubation for hours with tnfa + il- b + pge in the presence or absence of mm za. after days of co-culture dc:pbl, percentages and total counts of gd t cells were determined by flow cytometry; ) idc generated from cd + cells of hla-a* + healthy donors/patients were pulsed with sv-peptide and stimulated for hours with tnfa + il- b + pge in the presence or absence of mm za; after rounds of autologous t cells stimulation by dc, the frequency of sv-specific cd + t cells was determined by svpentamers staining; ) the same experiments were performed both with dc generated following a standard protocol and a h protocol (dc fast objective: depletion of or deficiency in gd t cells aggravate colitis in different animal models. additionally, reconstitution of mice with syngeneic gd t cells ameliorated chemically-induced colitis indicating a suppressive or regulatory role for murine gd t cells in intestinal inflammation. therefore, we asked whether human gd t cells possess also suppressive or regulatory potential, which could be of therapeutical use in chronical inflammatory diseases such as ulcerative colitis or crohn's disease. hence, the proliferation, suppressive activity, and cytokine profile of human peripheral gd t cells were determined in vitro. methods: human gd t cells were isolated from whole blood of healthy donors by macs technology. the proliferation was determined by [ - h]-thymidine incorporation, while suppression of responder cell proliferation was measured by flow cytometry via cfse fluorescence intensity. the cytokine profile was determined by elisa from culture supernatants as well as by flow cytometry intracellularly. finally, the in vitro characteristics of gd t cells were compared to those of cd + cd + regulatory t cells (treg). human peripheral gd t cells show suppressive activity against responder cell proliferation, though being themselve anergic, that is, they produce negligible amounts of interleukin- on stimulation and proliferate poorly. while the proliferation of gd t cells and treg cells is comparable, the suppression of gd t cells on responder cell proliferation is even stronger than the suppression by treg cells though gd t cells being foxp negative. additionally, gd t cells are strong producers for tgf-b, particularly by the vd subset. conclusion: human peripheral gd t cells possess regulatory potential and could be of therapeutical use in treatment of chronical inflammatory diseases as they are anergic and act suppressive. their suppressive activity is even superior to treg cells and might be due to strong tgf-b secretion. for application of human gd t cells in therapy their expansion under maintenance of their regulatory properties should be elucidated. there are previous descriptions of gamma-delta t lymphocytes (gd) from behçet's disease patients (bd) but, in most of cases, they are incomplete or contradictory. it has been suggested that nkg d on gd is involved in bd lesions through interaction with mica molecules. furthermore gdcd + have been recently proposed as a new regulatory t subset (treg). objectives: to study gd phenotype in bd active (bda) (n= ) and inactive (bdna) (n= ), versus healthy controls (hc) (n= ) and patients with recurrent oral ulcerations (ru) (n= ). to determine gd cytokine profile and surface markers treg-related in bd (n= ) and hc (n= ). methods: we obtained mononuclear cells from peripheral blood (pbmc). we determined by flow cytometry: -surface expression of: gd tcr, vdelta , vdelta , cd alpha, cd beta, nkg d, nkg a and cd . -intracellular expression of ctla- , and foxp . -intracellular expression of il- , il- , ifngamma, il- and tgfbeta after pbmc polyclonal stimulation. we used two tailed test for means comparison (mann-whitney u or student's t test). -vdelta + cells were significantly increased in ru. vdelta + and gdcd + lymphocytes were significantly increased in bd versus ru and hc. -the mean fluorescence intensity of nkg d was slightly increased in gd from bda. -nkg a expression by gdcd + was not different in bd versus hc. -most of gdcd + presented cd alpha-alpha homodimers in bd and hc and were negative for cd , foxp and ctla- . gdcd + and gdcd -subsets were (in bd and hc): -high ifngamma-producers without differences. -low il- -producers: il + cells were lower in gdcd + than in gdcd -. -low il- -producers: il + cells were lower in gdcd + than in gdcd -. -low tgfbeta-producers: tgfbeta+ cells were lower in gdcd + than in gdcd --very low producers of il in most of cases. the hallmark in bd was the increase of gdcd /vdelta +. this subpopulation has recently been described as immunosuppressive in infiltrates of human tumours and its function related to nkg a in intraepithelial intestinal lymphocytes from celiac patients. we did not find a cytokine profile or a phenotype t-reg-related for gdcd +, except a lower percentage of il- + cells than in the gdcd -subset. gdcd + from bd did not show significant differences versus hc. natural killer t (nkt) cells comprise a highly heterogeneous subset of t lymphocytes that co-express a t cell receptor (tcr) and nk cells markers such as cd in humans. a subgroup, the invariant nkt cells (inkt), expresses the va vb tcr rearrangement representing a minority subset in peripheral blood and virtually absent in the newborn. objectives: to establish a method to growth cord blood-derived nkt cells (cd + cd + ), in order to evaluate their phenotypic characteristics and the tcrvb repertoire. methods: mononuclear cells were isolated from healthy umbilical cord blood samples and stimulated with ifn-g ( ng/ml), anti-cd ( ng/ml) and il- ( ui/ml). these cells were cultured for days and the expanded cd + cd + cells were isolated by immunomagnetic methods. surface markers were determined by flow cytometry. total rna was extracted from the purified cd + c + cell suspension using trizol ® reagent and mrna expression of twenty tcrvb gene families was measured by semiquantitative rt-pcr. statistical analyses were performed using mann-whitney u test and one-way anova, a p value of x , was considered significant. results: we could significantly expand cord blood cd + cd + nkt cells from , ± , % to achieve an enrichment of , ± , % (p= , ). table shows the percentage (mean±sd,n= ) of phenotypic markers in cd + cd + cells at baseline (day ) and after days of culture. expression of mrna for the vb families studied was confirmed in each individual cell culture with a significant high expression of vb and vb families (p x , ). conclusion: our results show that cord blood-derived nkt cells are mainly cd + and cd + subsets, similar to peripheral blood nkt cell with a low percent of inkt cells. additionally, we confirm a diverse tcr vb repertoire with a significant expression of the vb and vb families in these cells. l. marischen , d. wesch , p. rosenstiel , a. till , d. kabelitz institute of immunology, kiel, germany, institute of clinical molecular biology, kiel, germany gd t cells account for a minority of t cells in human blood, but represent the majority of intraepithelial t cells in the intestinal tract. due to their ability to respond rapidly and in an mhc-independent fashion to particular antigens by cytokine production, gd t cells are considered as a link between innate and adaptive immunity. in addition, the expression of distinct pattern recognition receptors such as toll-like (tlr) and nod-like receptors (nlr) are characteristic for cells of the innate immune response. recent reports have demonstrated the tlr expression in human and murine gd t cells. here we provide evidence also for a gd t cell responsiveness to muramyl dipeptide (mdp), the putative ligand of the nlr family member nod . peripheral blood mononuclear cells (pbmcs) containing gd t cells as well as freshly isolated gd t cells were stimulated via the gd t cell receptor in the absence or presence of mdp and analyzed for proliferation and ifng-production. while the proliferation of gd t cells within pbmcs was decreased, ifng-production was increased after costimulation with mdp compared to the stimulation with a non-activating dd-stereoisomer of the ligand (mdpi). the enhanced ifng production of pbmcs after costimulation was mediated mainly by gd t cells as shown by intracellular flow cytometric staining. with regard to the ifng-production after co-stimulation with mdp vs. mdpi, freshly isolated gd t cells from different healthy blood donors can be divided into responder and non-responder. responder gd t cells showed a significant increase of the ifng-production due to mdp-stimulation, whereas ifng-production was not influenced in non-responder gd t cells. in further experiments, as first approach to explain the different reactivity patterns of gd t cells, it is planned to analyze the polymorphisms of the nod gene in various donors. taken together, our preliminary data indicate that gd t cells are a major source of ifng-producing cells among pbmcs when challenged with specific antigens plus mdp, and support the role of gd t-cells as an important team player in the early immune response against bacteria. objectives & methods: an increasing of gamma-delta t cells during acute p. vivax infection and convalescent period has been reported. moreover, the activation of gamma-delta t cells leads to the inhibition of blood stage p. falciparum parasites in vitro. to determine the killing mechanisms of p. vivax parasites by gammadelta t cells comparing with what has been found in p. falciparum, the gamma-delta t cells were enriched by isopentenylpyrophosphate (ipp) from naïve pbmc. different number of gamma-delta t cells and normal pbmc were incubated with intact of p. vivax parasites and protein extract of p. vivax parasites, recombinant pvmsp and pvama proteins. gamma-delta t cells was daily determined the cytokine and granzyme intracellular releasing by flow cytometry until day culturing. results: among the enriched gamma-delta t cells, the percentage of cells expressing cd + and cd + was elevated after co-culturing with intact and the proteins of p. vivax parasites. the overall gamma-delta t cells showed proliferation at day after the co-cultivation. moreover, the gamma-delta t cells expressing ifngamma + and cd a + (lysosomal associated membrane proteins: lamp- ) elevated from the first day of pbmc collection after co-culturing with the intact and p. vivax antigens. this level was correlated with the significantly decreasing number of parasites and the increasing percentage of parasite growth inhibition. our results showed the activation of gamma-delta t cells during p. vivax infection in vitro. this suggests that gamma-delta t cells could be stimulated by p. vivax parasites and these actively activated gamma-delta t cells could kill the parasites via mechanism of granzyme and cytokines at the early stage of cell activation. this study provides more understanding in activation of the innate immunity during acute malaria infection which may lead to the selection of appropriate malaria proteins as vaccine candidates in the future. objectives: several evidence suggest that invariant nkt cells (inkt) connect innate and acquired immune system. they are able to produce both th and th cytokines after stimulation. atopic dermatitis (ad) is a chronic inflammatory skin disease. th -like and th -like cytokines have been implicated in the pathogenesis of ad, but there are controversial data on their role in ad. the frequency and absolute number of inkt cells in mononuclear cells (pbmcs) of peripheral blood of patients with atopic dermatitis (ad) (n= ) and healthy controls (n= ) were determined by flow cytometry using anti-cd and monoclonal antibody specific for the cdr loop of the invariant tcr a chain of inkt cells (clone: b ). furthermore, after pma/ionomycin stimulation for hours, intracellular ifng and il- cytokines were detected in cd +cd -, cd -cd -(dn), cd -cd + and cd +cd + subsets of inkt cells by five colour flow cytometry in patients with ad (n= ) and healthy controls (n= ). results: both frequency and absolute number of inkt cells were significantly lower in patients with ad (p x . ) compared to healthy controls. the frequency of dn subpopulation was significantly lower in ad patient (p x . ). there was a positive correlation between the frequency of dn cells and inkt cells both in ad patients (r= . and p x . ) and healthy controls (r= . and p x . ). in the intracellular ifng level there were no significant difference in any of the inkt subsets of ad patients, however the intracellular il- level was significantly higher in dn subpopulation of inkt cells of ad patients compared to healthy controls (p x . ). the frequency, the number of inkt cells and the cytokine producing capacity of the cd /cd inkt subsets are different in peripheral blood obtained from ad patients compared to healthy controls. our result suggest that the dn inkt cell subset can serve as a source of il- that promotes the th differentiation in ad patients and might play a role in the pathogenesis of this disease. introduction: intrahepatic immune cells (ihic) are known to play central roles in immunological responses mediated by the liver, and isolation and phenotypic characterization of these cells is therefore of considerable importance. aims: in the present investigation, we developed a simple procedure for the mechanical disruption of mouse liver that allows efficient isolation and phenotypic characterization of ihic. these cells are compared with the corresponding cells purified from the liver after enzymatic digestion with different concentrations of collagenase and dnase. results: the mechanical disruption yielded viable ihic in considerably greater numbers than those obtained following enzymatic digestion. the ihic isolated employing the mechanical disruption were heterogeneous in composition, consisting of both innate and adaptive immune cells, of which b, t, natural killer (nk), nk t cells, granulocytes and macrophages were the major populations (constituting . %, . %, . %, . %, . % and . % of the total number of cells recovered respectively). the ihic obtained following enzymatic digestion contained markedly lower numbers of nk t cells ( . %) . the b, t and nk t cells among ihic isolated employing mechanical disruption were found to be immunocompetent, i. e. they proliferated in vitro in response to their specific stimuli (lipopolysaccharide, concanavalin a and alpha-galactosylceramide respectively) and produced immunoglobulin m and interferon-gamma. conclusions: thus, the simple procedure for the mechanical disruption of mouse liver described here results in more efficient isolation of functionally competent ihic for various types of investigation. nature killer t cells (nkt) are a special t cell population with co-expresses nk and t cell surface markers. murine nkt cells include cd + nkt and cd -cd -nkt cells. nk . + nkt cells may release large amounts of il- , il- , ifn-g and il- after they are activated. it has been reported that a-galactorsykeramide (a-galcer), a glycolipid, may induce proliferation of nkt cells with the role of immune regulation by stimulating mouse spleen cells. this study demonstrated that superantigen staphylococcal enterotoxin b (seb) , a kind of peptide, can activate the nkt cells with the function of immune tolerance. the response ability of seb-activating effect cells to cona, lps and il- had significantly decreased compared with that of normal lymphocytes. the effect cells exerted an inhibitory effect for the response of normal lymphocytes to cona and il- . there was a significantly increase in the percent of cd + nk . + and tcrvb + nk . + nkt cells identified from the seb-activated cells. based on the cell distribution detected in the upper part of the facs picture, expression of cd molecule existed in . % of the cells from large-scale selection. the percent of cd + nk . + and tcrvb + nk . + nkt cell subsets in the giant lymphocytes were enhanced to . and . folds, respectively. under a light microscope at x magnification, the seb-activating lymphocytes in size were larger than not only the cona-activated cells but also the adherent macrophages with an increase of fold observed under a microscope. there were a few granules seen in cytoplasm. the value of cytoplasm vs nuclei was less than . and they are non-adherent cells. the differentiation pathway of the seb-activating cd + and tcrvb + nkt cells was not relative to a nk source. they were produced directly from t cell population and were considered as a subsets of t lymphocytes. our results suggest that the superantigen seb can act on the cd + nkt cell and tcrvb + nkt cells. and the two nkt cell subsets may play a critical role in seb mediated tolerance. gd t cells in the intestinal intraepithelial compartment (gd iiel) show an intrinsic activated phenotype. we hypothesised that their t cell receptor gd (tcrgd) is implicated in the activation of gd iiel. because the tcr gd ligands in mice are not well described, monoclonal antibodies (mab) directed against the gd tcr, like the clone gl which binds the d subunit of tcr gd, are important tools to specifically activate gd t cells. using cytometric indo- am measurement, we could detect calcium flux of intestinal and peripheral gd t cells from tcrd-h begfp reporter mice. stimulation with anti-gd clone gl or anti-cd clone c elicited activation of gd t cells suggesting that tcr gd and cd molecules in gd t cells are functional and signalling competent. next, using elisa and cytometric bead array, we found that iiel stimulated with plate bound gl in vitro produced ccl , ifng and tnfa. therefore, we were interested whether the ccl production of gd iiel influenced the homing of ccr cells such as lamina propria (lp) cd + foxp + cells (tregs). to test this, wt mice were i. p. treated with gl mab and lp tregs were analysed by cytometry at various time points post inoculation. we found similar frequencies of lp tregs population but a slight decrease in ccr + tregs. however, when we compared wt and tcrd -/mice, we found both lower percentages of total lp tregs and of lp ccr + tregs in tcrd -/mice compared to wt mice. in conclusion, our data suggest that intraepithelial activation of gd t cell may directly or indirectly induce changes in the iiel and lamina propria (lp) lymphocyte compartment and influence the ccr expression and the homeostasis of lp treg. the ability of nkt cells to serve a variety of different immunoregulatory functions in vivo may reflect a diversity in function of different nkt cell subsets. diversity in cytokine production by nkt cell subsets has been observed in murine and human studies, although this analysis has largely been following in vitro restimulation. here, we investigated cytokine production by murine nkt cell subsets in vivo under conditions where minimal manipulation of the cells was required. to this end, we examined il- production in g reporter strains in which dna encoding green fluorescent protein (gfp) was inserted into the first exon of the il- gene. in the absence of any manipulation gfp was expressed from the il- locus in populations of immature thymic nkt cells (predominantly cd +cd lotcrhi cells on a balb/c background, and cd +cd lonk . -on a c bl/ background) and some splenic nkt cells, with overall numbers of gfp+ cells in both tissues decreasing with age. after i. v. administration of the nkt cell ligand a-galactosylceramide, il- production was induced predominantly in cd + nkt cell subsets of the liver and spleen, and after i. n. administration, in cd + nkt cells of the airways. spontaneous and a-galcer-induced expression from the il- locus occurred in the absence of stat signalling, and did not require initial exposure to il- protein from other sources in the host. diversification in cytokine expression by nkt cells subsets therefore occurs early in ontogeny, and is also a significant feature of responses to exogenous activating stimuli. interleukin- (il- ) plays an important role in neutrophil recruitment. herein, we investigated the role of il- receptor signaling in polymicrobial sepsis induced by cecal ligation and puncture (clp). methods: adult c bl/ (wt) and il- receptor gene-deficient (il- r ko) mice were subjected to non severe (ns-clp) sepsis. intraperitoneal neutrophil migration, bacteremia, cytokine, chemokines and liver injury were evaluated hours after surgery. the ability of il- mediate the neutrophil microbiocidal activity in vitro, as well the neutrophil migration in vivo and in vitro were also evaluated. the means of different treatments were compared by analysis of variance (anova), followed by bonferroni's t test and the survival rate by the mantel-cox log rank test. results: it was observed that il- r ko mice, subjected to ns-clp sepsis, show reduced neutrophil recruitment into peritoneal cavity, spread of infection, and increased systemic inflammatory response as compared to wt. as a consequence, the mice showed an increased mortality rate. moreover, il- induced neutrophil migration in vivo and in vitro. besides, we demonstrated that neutrophils harvested from il- r ko mice already show reduced microbiocidal activity, compared with wt, suggesting a physiological role of il- receptor signaling in the microbiocidal activity of neutrophils. furthermore, wt neutrophils treated with il- showed strongly enhancement of microbiocidal activity by a mechanism dependent of nitric oxide. conclusion: during ns-clp besides the importance in recruit neutrophils to focus of infection, il- also enhances the microbiocidal activity of neutrophils. therefore, our results demonstrated that il- receptor signalization plays a critical role on host protection during polymicrobial sepsis. objectives: members of the toll-interleukin- receptor (tir) family are important for host defense, inflammation, and immune regulation. their canonical signaling pathway involves adaptor proteins and il- r associated kinases to activate nfxb and p mitogen-activated protein kinase. the il- -induced signal transduction in mast cells is poorly understood. in this work we studied the signal transduction of il- in different mast cell subsets. methods: different mast cells subsets (hmc- , human cbmcs and murine bmmcs) were stimulated with il- . the resulting signal transduction was investigated by immunoblot for activated signaling molecules (pc-kit, perk / , pakt, pnfxb, p and pjnk). additionally, we studied the signal transduction of il- in il- r transfected hek t cells. results: we found, that a tir family member, il- r, transactivates the receptor tyrosine kinase c-kit in mast cells and that il- -induced cytokine production depends on c-kit transactivation. il- r and il- r accessory protein (il- racp) form a physical complex with c-kit. thereby the complexation is dependent on the activity of c-kit. conclusion: these results show for the first time that the biological function of an il- r family member is dependent on the presence of an activated receptor tyrosine kinase. furthermore, these results reveal that certain il- -induced signaling pathways and effector functions are dependent on activated c-kit and could therefore explain the effects of il- in mast cells in absence of iger activation. ( ) . we now provide a molecular mechanism underlying this pathogenic effect by which free heme sensitizes hepatocytes to undergo tnfmediated programmed cell death. independently of newly gene transcription and/or protein synthesis, free heme cytotoxicity is mediated by the unfettered generation of free radicals in response to tnf, presumably due to the participation in the fenton reaction of the fe atom present in the protoporphyrin ix ring. once exposed in vitro to free heme, a sustained c-jun n-terminal kinase (jnk) activation was observed in hepatocytes in response to tnf, an effect that promotes further free radicals production. pharmacologic or genetic (shrna) inhibition of jnk in hepatocytes avoids free radicals accumulation and caspase- activation, also mimicked by the anti-oxidants n-acetylcystein (nac) or butylated hydroxyanisole (bha). expression of the heme catabolyzing enzyme heme oxygnease- (ho- ) in hepatocytes affords protection against heme sensitization to tnf cytotoxicity. recombinant adenovirus mediated ho- expression in the liver suppresses tnfmediated hepatocyte apoptosis and prevents the lethal outcome of plasmodium infection in mice. in conclusion our data reveals a novel signal transduction pathway via which heme sensitizes hepatocytes to undergo tnf-mediated cytotoxic effect, critically involved in the outcome plasmodium infection. the multi-step leukocyte extravasation process is governed by adhesion molecules and chemotactic factors dynamically interplaying in the presence of shear forces. responsiveness to chemotactic ligands is mediated by g protein-coupled receptors (gpcrs) which are finely regulated by a family of cytosolic proteins, betaarrestin and . recent evidence indicates that, in addition to playing a regulatory role in gpcr desensitization and internalization, beta-arrestins may contribute to gpcr signaling by functioning as scaffolds for the recruitment of signaling proteins into complexes with agonist-occupied receptors. on this basis, we investigated the physiological role of beta-arrestin in chemokine-driven dynamics associated with leukocyte extravasation, with special interest to the activation of the rap small gtpase, recently emerged as pivotal regulator of integrin function. the analysis of kc the (keratinocyte-derived chemokine) rap activation profile in rbl (rat basophilic leukemia) cells expressing mcxcr shows a bimodal kinetic, with the first peak at ''/ ' and the second at ' after stimulation. rna interference-mediated depletion of beta-arrestin specifically inhibits the occurence of the second wave of rap activation, whilst it has no effect on the early pick, thereby suggesting that beta-arrrestin is involved in rap activation and that the oscillations in the formation of rap -gtp are regulated by different molecular mechanisms. in order to elucidate the gefs and gaps involved in the gtpase regulation we are at present down-regulating the expression of c g (rap gef) and spa (rap gap): preliminary results suggest that spa- has probably a role in the early activation peak. since this oscillatory chemokine-induced rap activation is present on other myeloid cell lines (hl , d) and fresh pmn's we are also translating our research to these more appropriated cells. interestingly betaarrestins amino acid sequence and three-dimensional structure reveal a unique and evolutionary conserved proline-rich sequence in beta-arrestin , localized in a solvent exposed loop which may serve as a docking site for migration-associated transducers/adaptors. in order to find sh containing proteins that interact with beta-arrestins, we have performed an overlay screening assay of different sh domains that revealed over putative beta-arrestins putative interactors, some of which isoform specific. granulocyte-macrophage colony-stimulating factor (gm-csf), interleukin (il)- and il- stimulate proliferation, differentiation, survival and functional activation of myeloid cells. the cell surface receptors for these cytokines consist of cytokine-specific a subunits and a common b-receptor (bc), required for the activation of intracellular signaling following cytokine engagement. aberrant signalling, stimulated by these cytokines, has been implicated in the pathogenesis of many diseases, including arthritis, asthma and leukemia. as a result, we have sought to define key molecular determinants of these receptor-cytokine interactions in order to gain a greater understanding of receptor activation. here we present novel insights into the role of the ig-like domain of the gm-csfra in gm-csf binding. deletion of the ig-like domain abolished direct gm-csf binding and we identified specific residues directly involved in ligand binding by site directed mutagenesis and binding studies. the results indicate a previously unrecognized role for the ig-like domain of gm-csfra. furthermore, we address a longstanding controversy in the field of gm-csf, il- and il- receptor biology, by performing a systematic study of the role of n-glycosylation upon on the bc, and related murine b il- , in ligand-binding and receptor activation. these data demonstrate definitively that n-glycosylation does not play a role in mediating ligand-binding or receptor activation. these findings clearly establish that the determined human bc structures lacking glycosylation at asn are biologically relevant conformers of the human bc ectodomain. our results appear to suggest that the potency of receptor signalling can be influenced by the biophysical and structural properties of the extracellular receptorligand interactions and it also addresses important, poorly-understood aspects of mechanisms underlying ligand recognition and activation of the gm-csf: gm-csfra: hbc receptor complex. reference: ( ) micrornas (mirnas) are endogenous small non-coding rna molecules acting as key regulators of immune cell differentiation and innate immune responses. mirna- expression is induced by activation of the toll-like/interleukin- receptor pathway (tirpathway), where it targets essential adaptor and signaling molecules, thus serving as a regulator preventing the cells from an exacerbated pro-inflammatory response. since tnfa also up-regulates the expression of mirna- a, we decided to explore whether this mirna is involved in the regulation of apoptosis. to this end, we used the hela human epithelial cell line as a model system for tnfa signaling. following tnfa and cycloheximide (chx) treatment mirna- a transfected cells showed significantly reduced levels of the active proapoptotic caspases and (casp / ). in line with this, mirna- a conferred enhanced protection against tnfa-induced dna fragmentation and mitochondrial potential drop-down. our results demonstrate that mirna- a is a regulator of receptor-mediated apoptosis. similar to the tir-pathway, mirna- a seems to be part of a negative feedback mechanism of the tnfa signaling cascade. ongoing research focuses on the identification of the specific pro-apoptotic molecules targeted by mirna- a. furthermore, we are exploring the relevance of our observations for the mycobacterial infection of human macrophages, where the regulation of apoptosis is critical. objectives: the aim of this study was to evaluate the role of single nucleotide polymorphisms (snps) located in il- , il- r a-chain and il- r a -chain genes in hiv disease progression. methods: we studied antiretroviral treated patients (progressors) and long term non progressors (ltnp). we analyzed snps in the il- gene, snps in the il- r gene and snps in the il- r gene. in univariate analysis, we found an association between the presence of at least one mutated a allele in il- r aa and a higher possibility of being ltnp ( our study suggests that genetic polymorphisms located in il- r and il- r genes can influence the rate of disease progression in hiv+ patients, especially when a combination of aplotypes is present. mutations in the coding regions might compromise the binding of the cytokines or the intracellular signal transduction pathways, therefore leading to the alteration of cd and cd t cells homeostasis. aims: mono-adp-ribosyltransferases (arts) are gpi-anchored ectoenzymes that covalently modify cell-surface or soluble target proteins by transferring an adpribose moiety from extracellular nad+ to specific arginine residues of target proteins. in this study, we report that human tumor necrosis factor (tnf) is adpribosylated by art , and that adp-ribosylation affects both the release of tnf from cells and its cytolytic action. methods: transcription of art in human leukocytes was analyzed by rt-pcr. adp-ribosylation of tnf was detected by monitoring the incorporation of adpribose from labeled nad. release of tnf from transfected hek cells was monitored by elisa. binding of tnf to tnf receptors was analyzed by biacore. tnf cytotoxicity was monitored by flow cytometry. the adp-ribosylation site on tnf was analyzed by lc/ms mass spectrometry. results: we identified art transcripts by rt-pcr analysis in human blood leukocytes. soluble art , released from the surface of transfected cells by phosphatidylinositol-specific phospholipase c (pi-plc), adp-ribosylated recombinant human tnf in vitro. co-transfection of hek cells with art and tnf resulted in modification of tnf at at least distinct sites, i. e. one within the tnf ectodomain, and one on the stalk that remains connected with the cell membrane after cleavage by tnfa converting enzyme (tace). analysis of modified recombinant tnf by mass spectrometry provided evidence that the tnf ectodomain is adp-ribosylated at r , a site that has previously been implicated in binding to tnfr . binding assays indicated that adp-ribosylation inhibited binding of tnf to its receptors. importantly, modified tnf was less potent at inducing cell death in the human t cell lymphoma line kit than wildtype tnf. furthermore, cell surface adp-ribosylation of hek cells co-transfected with tnf and art resulted in reduced release of tnf into the supernatant. conclusions: adp-ribosylation of tnf or other cell surface proteins interferes with the biology of tnf signals by at least two distinct mechanisms. adpribosylation of tnf blocks binding to its receptors, thereby inhibiting tnf-mediated cytotoxicity. additionally, adp-ribosylation of tnf or another protein on the surface of tnf-producing cells inhibits the proteolytic release of tnf. noninflammatory chronic pelvic pain syndrome : immunological study in ejaculate g. n. drannik , t.v.poroshina institut of urology amsci of ukraine, laboratory immunology, kyiv, ukraine chronic prostatitis (cp) is a disease which likely is associated with abnormalities in local immune responses. secretions of the urinary and reproductive tract mucosa contain various protective effector molecules, produced by mucosal cells, lymphocytes, macrophages and neutrophiles. the aim of this prospective study was to observe local immunophenotypic patterns in patients with noninflammatory chronic prostatitis/chronic pelvic pain syndrome for further description and as possible surrogate markers for diagnosis and treatment. methods: patients with noninflammatory chronic prostatitis/chronic pelvic pain syndrome (cp/cpps) and control men were assessed for slpi, tnf-alpha, il- and free tgf-b in ejaculate by elisas using stat-fax plus. a to day sexual abstinence period was required from the subjects before semen collection. after liquefaction and centrifugation, seminal plasma samples were kept at - degrees c°until assayed. the materials were processed after the standard programmes for statistical analysis.the study was approved by the local ethics committee. the slpi concentration was elevated in all patients ( . ± . pg/ml, p x . ) in seminal fluid, in comparison with the healthy control subjects. the tnf-alpha concentration was elevated in all patients in seminal fluid ( . ± . pg/ml; p x . ). the il- concentration was elevated in all patients ( . ± . pg/ml; p x . ) in seminal fluid. free tgf-b was present in normal seminal plasma in high concentrations ( . ± . pg/ml), while in ejaculates of patients with noninflammatory cp/cpps tgf-b concentrations were . ± . pg/ml. conclusion: ejaculate's slpi, tnf-alpha, il- and free tgf-b are possible surrogate markers for the diagnosis and treatment of patients with noninflammatory chronic prostatitis/chronic pelvic pain syndrome. m. r. marrakchi , e. a. elgaaeid faculté des sciences de tunis, biology, tunisie, tunisia ulcerative colitis (uc) and crohn's disease (cd), collectively referred to the inflammatory bowel disease (ibd), represent a group of multifactorial autoimmune disorders of the gastrointestinal tract sharing many clinical and pathological characteristics, however, differing in histological features and cytokine profiles. the excessive production of either th or th cytokines due to perturbed regulation of immune system activation results in chronic inflammatory processes and loss of immune homeostasis that may be implicated in the genesis of ibd. studies have identified a gene that encodes the nod /card protein, which is involved in the immune system's response to bacterial infection and confirmed to influence susceptibility to cd. indead, it has been suggested that high rates of asca (saccharomyces cerevisiae ) in absence of panca (perinuclear anca: anti-neutrophil cytoplasmic) antibodies were associated with aggressive forms of cd and that the important rise of panca was more frequent at uc . in a sample of tunisian patients, we examined the contribution of nod /card gene in cd. we performed a cases /controls study upon cd patients and healthy controls. this study suggests that in northen tunisian population, insc mutation in nod /card gene is a prevalent mutation leading to the typical crohn's disease including ileal location, stricturing and penetrating clinical types and asca expression. since conflicting results were obtained on il- polymorphisms as risk factor for ibd, the aim of our study was also to explore anti-inflammatory il- cytokine genetic profile in patients with ibd. we examined the contribution of il- gene promoter polymorphisms (- and - ) to crohn's disease (cd) phenotype, and the possible genetic epistasis between these polymorphisms and card /nod gene mutations in cd presentation and location. in tunisian population, the insc insertion in nod /card gene is a marker of susceptibility to cd, while the a allele at position - in the il- promoter increases the risk of cd ileal location and severe disease presentation. a genetic epistasis between il- gene polymorphisms and card /nod gene mutation was suggested. in conclusion, genetic and serologic markers might be useful in defining patien gangliosides were shown to inhibit the il- -dependent proliferation of t-cells, implying that gangliosides interfere with one or more of the il- -driven events. it is known that the major mechanism of inhibition is the direct interaction between ganglioside and the cytokine and, as a result, the capture of il- molecule by ganglioside. but gangliosides apparently can also form complexes with il- r; such complexes influence on the signal transduction through il- r. this effect of gangliosides may lead to the failure of this pathway. unfortunately, the biological and structural aspects of this problem are poorly understood. in this study we propose possible modes of interactions between exogenous gangliosides and il- r subunits. in our work we use il- -dependent cytotoxic t-cell murine line ctll- . two different approaches for study of possible interaction types between exogenous gangliosides and il- r subunits were applied: antibody staining of il- r subunits followed by flow cytometry analysis, and photoaffinity labeling of living cells with i-dcp-gm followed by immunoprecipitation of il- r subunits. the fluorescence intensity of the antibody-labeled il- r a-subunit substantially decreases after the treatment of cells with gangliosides. it has been shown that the fluorescent labeled cell fraction decreases by . % after cells incubation with ganglioside gm , and by . % after incubation with gm . labeling of the cells with antibodies to the il- r b-subunit results in a less significant fluorescence decrease after cells incubation either with gm ( . %) or with gm ( . %). to determine the mode of this impressive masking influence of ganglioside gm , photoaffinity cross-linking has been used. in our modification this method could identify is interaction of gm with subunits of il- r occur with or without incorporation exogenous ganglioside into plasma membrane. electrophoresis followed by immunoprecipitation with appropriate antibodies resulted in appearance of the radioactive band only for b-subunit of il- r, but not for il- r a-subunit. these results demonstrate that exogenous ganglioside gm can interact with a-and bsubunits of il- r in different modes. interaction of il- r b-subunit with ganglioside gm requires incorporation of the ganglioside into plasma membrane, but it is not the case for interaction with a-subunit. a. respa , j. bukur , s. purpose: loss of interferon (ifn)-g inducibility of hla class i antigens has been found in some tumor cell lines, but the underlying molecular mechanisms of such deficiencies have not yet been elucidated in detail. this kind of tumor escape mechanism might lead to an inefficient recognition of tumor cells by the immune system. methods: phospho-specific western blot analyses were performed to verify the functionality of the different ifn-g pathway components, intra-and extracellular flow cytometry experiments were employed to determine the expression of antigen processing components and hla class i cell surface antigens, quantitative real time-pcr experiments to confirm the absence of jak and presence of pathway relevant molecules as well as, genomic pcr and chromosome typing technique to prove the deletion of jak . results: different ifn-responsive phenotypes were defined in human melanoma cell lines varying from loss to low, delayed as well as strong ifn-g inducibility. resistance to ifn-g treatment was associated in one melanoma cell line with the lack of jak expression due to a gene deletion on chromosome , whereas the expression and functionality of other ifn-g signal transduction components like stat and jak were not affected. jak blockade by two jak -specific inhibitors resulted in reduced levels of hla class i surface antigens. conclusion: structural abnormalities of jak leading to a lack of jak expression are associated with loss of ifn-g inducibility as well as reduced constitutive hla class i surface expression. in addition the jak inhibition modulates the expression of the hla class i antigen processing components. of renal cell carcinomas (rccs) are associated with the size, grade, t, n, m, stage and death of rccs patients. the genotypes were compared with those of a random sample of controls of the spanish population. methods: two il polymorphisms located on the il- promoter region, snps - a/c (rs ) and - g/c (rs ) were genotyped using taqman snp genotyping assays. the functional il- gene polymorphisms studied do not influence the susceptibility to rccs in the spanish populations (il- - p= , . il - p= , ) but may contribute to disease onset and aggressiveness: the genotype il- - cc genotype, which is associated with higher il- production, was significantly associated with higher tumor size (p= , ), grade (p= , ), t (p= , ), m (p= , ) and stage (p= , ). the influence of the il- - gg genotype was less relevant, however was correlated with higher tumor size (p= , ), grade (p= , ), t (p= , ) and stage (p= , ). the multivariate analysis with cox proportional hazard model revealed that, in this serie, nuclear grade and stage grouping were independent prognostic factors, whereas il- polimorphism can not be considered as independent prognosis factors. our results suggest that the polymorphism variants from the il- gene (il- - and - ) may be associated with an worse prognosis of rcc. high levels of il- production can play an important role in grow, invasion and metastasis of renal cancer. interleukin- (il- ) is a pleiotropic cytokine that is involved in regulation of both the innate and acquired immune response. the most prominent biologic property of il- is its ability to induce the production of ifn-gamma in presence of il- . moreover, it stimulates the expression of tnf-a and il-l, enhances the differentiation of t cells to the thl and impairs the synthesis of the anti-inflammatory cytokine il-l . then it seems that il- has a crucial role in immunity against brucella infection. since the expression of il- can be affected by polymorphisms in its gene, we decided to investigate any probable relation between six different il- gene polymorph isms and brucellosis. methods: a total of patients with brucellosis and healthy farmer who consumed contaminated row milk and dairy products from animals with brucellosis, were included in this study. all individuals were genotyped for six il- polymorphisms at positions - , - , , + , + and codon / using polymerase chain reaction-restriction fragment length polymorphism (pcr-rflp). the distribution of alleles for il- polymorphisms at position - g/+ t/+ c (correlated with high production of il- ), codon / c and - g/- c (correlated with higher production of il- ) were significantly higher in the healthy controls than the patients (p= . , p= . and p= . , respectively). discussion: as data revealed genotypes that have correlation with higher production of il- are more frequent in the controls than the patients. then it might be deduced that individuals who inherited these genotypes/alleles are able to produce higher level of il- at the onset of infection and it leads to more ifngamma production and control brucella infection before appearing brucellosis. abstract withdrawn by author objectives: a dsyregulated cytokine response has been shown to play a role in inflammatory bowel disease (ibd) pathogenesis. to dissect the influence of these cytokines, specifically interferon gamma (ifng), on the inflammatory response and colitis we have created a cell specific ifng receptor (ifngr ) mouse mutant. we have generated a mouse line carrying a conditional ifngr allele using the loxp -flp recognition target (frt) approach. a targeting vector with loxp sites flanking exons - and frt sites flanking the neomycin resistance cassette was generated. after confirmation of homologous recombination the neomycin resistance cassette was deleted by crossing with flp deleter mice. cell specific deletion is being performed by crossing conditional 'flox/flox' mice with specific cre expressing mouse lines. functional inactivation of the receptor has been demonstrated by performing western blots to detect phosphorylated and total stat following ifng stimulation. results: successful deletion of the gene and conditional mutants without the presence of the neomycin resistant cassette have been generated. functional inactivation of the receptor with no stat activation following ifng stimulation has been demonstrated in the complete knock out mice. furthermore, flox/flox mice retain full responsiveness to ifng. breeding with lysm cre and cd cre mice will been completed to create a cell specific deletion of the ifng receptor in macrophages/granulocytes and t cells respectively. the specificity of this deletion will be confirmed through cell sorting and functional assays. in order to determine the influence of ifng on a specific cell type a conditional gene targeting approach has been utilised. this has allowed the generation of conditional mice mutants with deletion of ifngr on macrophages and t cells. this has generated a very powerful tool for dissecting the role of this cytokine in numerous disease models. moreover, the ability to cross the conditional mice with additional cre lines will enable the analysis of more cell types in the future. a. gonzalez , r. carretero , p. saenz-lopez , j. cantón , j. carretero , f. ruiz-cabello , l.m. torres , cts- hospital universitario virgen de las nieves, departamento de ginecología, granada, spain, hospital universitario virgen de las nieves, departamento de análisis clí nicos, granada, spain objetives: cervical cancer is almost associated with infection by human papillomavirus (hpv). however, only a subset of infected women will ever develop the malignancy. ifng dinucleotide (ca) repeat polymorphism is responsible for genetic differences in interferon-gamma production. our objective was to investigated the relationship between ifgn polymorphism and cervical intraepithelial neoplasia (cin) methods: we have studied nineteen women with cin and normal women control. dna was extracted from blood samples, and was genotyped for functional microsatellite (ca) repeats in the first intron of ifn-gamma gene. results: heterozygosis in (ca) allele of ifgn is significant more frequent in healthy control than in cin patient (p= , ) in contrast homozygosis for (ca) allele did not show significant differences between both population. analysis of relation between this polymorphism and the cin stage showed that both heterozygosis and homozygosis is correlated with advanced stage (p= , and p= , respectively). conclusions: our study suggest that ifng (ca) allele may influence in cin risk and progression. ifng is associated with hpv clearance, but it also plays a role as inflammatory cytokine, promoting cervical malignant progression. further studies of the role of ifng and other cytokines may contribute to the understanding of cin promotions and progression. introduction: macrophages play a fundamental role in controlling of brucella infection, mainly through the secretion of cytokines such ifn-y and tnf-a. interleukin- (il- ), a th -related cytokine, triggers inflammatory cell recruitment and increases the expression of ifn-y. as the cytokine production is under the genetic control, we decided to investigate the association between il- single-nucleotide polymorphisms (snps) and susceptibility to brucellosis in iranian patients. methods: patients with brucellosis and healthy animal husband men who kept animals with brucellosis, were included in this study. all individuals were genotyped for il- (c t, g a, c a and a t) polymorph isms using pcr-rflp. results: at position , the distribution of c allele and cc genotype were significantly lower in the patients than the controls (p= . and p= . , respectively). at position , the distribution of c allele and aa genotype were significantly higher and lower in the patients than the controls, respectively (p= . , p= . ). discussion: as shown the frequency of cc genotype and c allele at position were higher in healthy controls than the patients. hence, our study provides evidence that presences of cc genotype and/or c allele are significantly associated with susceptibility to brucellosis. the frequency of aa genotype at position is lower in patients than the healthy controls and the frequency of c allele at position is higher in patients than the healthy controls. hence, our study provides evidence that presences of c allele and lack of aa genotype are significantly associated with susceptibility to brucellosis. objectives: increasing evidence is emerging regarding the ability of mammary epithelial cells to respond to various cytokines. our aim was to investigate the cytokine receptor phenotypes of two distinctive human mammary cell lines, mcf- and skbr- , as well as their ability to respond to several cytokines and to the g- gpr agonist. the mcf- and skbr- human adenocarcinoma cell lines were investigated by immunocytochemistry and flow cytometry for the expression of the following cytokine receptors: il- ra, il- rg, il- /il- /gm-csfr, il- /il- r, il- ra, il- ra, il- r (gp ), il- ra, il- r, tnfr i, tnfr ii, ifngra, cxcr , cxcr , cxcr , cxcr , cxcr . cells were incubated with il- , ifn-g, tnf-a and tnf-b (for which both cell lines displayed receptors) alone and with the gpr agonist. cytosolic ca + concentrations [ca + ] i were measured by the microfluorimetric technique. results: different cytokine receptors phenotypes emerged for the two cell lines. the less well differentiated skbr- cells were found positive for a larger number of receptors than the mcf- cells. both cell lines displayed an important heterogeneity for each of the investigated molecules, in terms of number of positive cells and expression intensity, with chemokine receptors percentages constantly higher than for the other receptors. pretreatment of mcf- cells with il- reduced the calcium response to g- , while pretreatment with ifn-g and tnf-a potentiated the calcium response to g- . tnf-b had no effect on mcf- g- stimulation. no direct effect on basal [ca + ] i stimulation could be noticed when administering the cytokines alone. in skbr- cells, pretreatment with il- or tnf-b had no effect on basal [ca + ] i and did not significantly alter the calcium response to g- , while pretreatment with ifn-g induced calcium oscillations and potentiated the calcium response to g- . pretreatment with tnf-a produced calcium oscillations and reduced the response to g- . conclusions: mcf- and skbr- cell lines express distinctive cytokine receptor phenotypes. furthermore, their ability to respond to cytokines in terms of modulating the gpr stimulation proved to be different. the susceptibility towards various soluble factors of these cell lines can offer us some insights on the complexity and individuality of each primary tumor and thus the distinctive evolution of each particular patient. results: in initial analyses of the early infection phase we identified splenic pdcs expressing ifnb/yfp. furthermore, we show for the first time in vivo that these ifnb producing pdcs are not directly infected with mcmv and that not only cdcs but also cd a -pdcs expressed gfp as a marker for mcmv infection. we observed that at early time points equal frequencies of cd a -pdcs and cdcs were infected with mcmv, whereas after h p. i. the frequency of infected cdcs wins over that of the pdcs. conclusions: with this experimental system we are able to visualize the ifnb response vs. the infection status of mcmv in vivo on a single-cell level. from our initial results we can conclude that infected cells in the spleen induce ifnb production in noninfected pdcs which initiate the antiviral immune response in this organ. recently, we have developed live-attenuated arenavirus vaccine candidates based on lymphocytic choriomeningitis viruses (lcmv) carrying the vesicular stomatitis virus (vsv) envelope gene (rlcmv/vsv-g). since interferon (ifn) signaling is known to be crucial for adaptive immune responses against wildtype (wt) lcmv and control thereof, we wanted to assess the innate and adaptive immune requirements for containing rlcmv/vsv-g infection. mice lacking both, ifn type i and ii receptors generated potent virus-specific cd t cells and neutralizing antibody responses against rlcmv/vsv-g, even exceeding the respective responses in wild type animals. in further contrast to wt lcmv infection, ifn type i and ii signaling was dispensable for rlcmv/vsv-g control. rlcmv/vsv-g infection of rag -deficient mice (lacking mature t and b cells) resulted in persistent levels of circulating viral rna that was solely detectable by qrt-pcr but not by classical measures of infectivity. overt viremia was only found in mice lacking both rag and ifn type i receptor (ar). thus, viral attenuation for vaccine use was found to considerably relax the ifn-dependence of adaptive immune responses and virus control. this redundancy of ifn and adaptive immune responses in rlcmv/vsv-g control provides a better understanding of the attenuation of this vector. it furthermore suggests that the virulence of a particular virus may influence the interferon-dependence of cd t cell and antibody responses, which may have implications for vaccine development. objectives: the class of type i interferons (ifns) consist of multiple ifnas and a single ifnb subtype. although being important for anti-viral defence they have been shown to be detrimental for the host during bacterial infections. while in general the first ifn to be produced is ifnb, the cell types responsible for its initial production remain unclear. to assess the cellular sources of ifnb and its role for the outcome in bacterial infections we use an ifnb reporter-knockin mouse model, in which yellow fluorescent protein (yfp) is expressed from a bicistronic mrna linked by an internal ribosomal entry site (ires) to the endogenous ifnb mrna. methods: to induce a type i interferon response we intravenously injected the tlr agonists poly(i:c) and cpg , respectively, or infected reporter mice or bone marrow derived macrophages (bmdms) or dendritic cells (bmdcs) with the facultative intracellular pathogen listeria monocytogenes. the spatiotemporal tracking of the ifnb response was done using facs analysis and immunofluorescent microscopy. results: after poly(i:c) injection in vivo a small subpopulation of ifnb + cd a + dendritic cells relocalize from the red pulp via the marginal zone to the t cell areas of the spleen whereas ifnb + activated pdcs were localized in the splenic white pulp at the t/b-cell interface after cpg administration. in vitro infection of bmdms, bmdcs and flt -l derived dcs with listeria monocytogenes resulted in a low frequency of ifnb + cells depending on the listeria strain used and multiplicity of infection with bmdms being the most potent producers of ifnb. in vivo mostly activated f / + macrophages were accountable for the ifnb expression. simultanous visualization of the cellular state of infection shows that ifnb + bmdms carry a higher bacterial load then ifnbcells. the cellular detection of ifnb expression reveals a remarkably low frequency of ifnb-producing cells in response to distinct pamps or the infection with intracellular bacteria. this hints at a superior role of few specialised cells for mounting a significant response against distinct stimuli or bacterial disease. additional data will be presented further resolving the timecourse of the ifnb response vs. the state of infection after bacterial challenge. the spleen is a secondary lymphoid organ that is characterized by highly specialized structures and plays a crucial role in the defense of blood-borne pathogens. we investigated the role of the spleen in the generation of antigen-specific cytotoxic cd t cell (ctl) responses in a systemic infection with recombinant adenovirus expressing ovalbumin (adova). although adenovirus mainly infects the liver, the ctl response requires the spleen, as splenectomized mice were incapable to mount an antigen-specific ctl response upon systemic challenge with adova. additionally, dendritic cells (dc), macrophages and an intact splenic structure were mandatory for the induction of an efficient ctl response. we detected adova specific ctl responses only in splenectomized mice that received splenic autotransplants but not after adoptive transfer of single cell splenocytes. furthermore, we asked how toll-like receptor (tlr) ligands influence adova-specific ctl responses. tlr ligands as "danger signals" are generally known to exert immune stimulatory effects. importantly, we observed that systemic administration of single-stranded rna (ssrna) prior to adova infection inhibited the generation of antigen-specific ctl responses in a tlr and type i interferon (ifn) dependent manner. ssrna injection induced the production of type i ifns as detected in sera and supernatants of splenocytes isolated from wild-type mice. experiments performed in ifnbeta reporter mice revealed that splenic plasmacytoid dcs represented the cellular source of type i ifns. additionally, splenic cd c+ dcs purified from ssrna pretreated mice showed a reduced capacity to cross-prime ova-specific cd t cells upon adova challenge. in vivo pre-activation of endogenous ova-specific cd t cells as well as an adoptive transfer of in vitro activated transgenic ova-specific cd t cells prevented the ssrna mediated suppression of the ctl activity. we assume that dcs preactivated by systemic ssrna were impaired in their ability to activate naive cd t cells in response to an adova infection due to an impaired cd t cell help. taken together, we show that type i ifns cannot only stimulate, but also inhibit the induction of ctl responses in the spleen. within hours after infection many viruses induce early type i interferon (ifn) responses that can confer protection against lethal infection. modified vaccinia virus ankara (mva) is a highly attenuated vaccinia virus (vacv) strain generated by more than passages in chicken embryo fibroblasts. mva stimulates systemic ifn responses, whereas vacv-induced cytokine milieus are dominated by il- . to study the impact of virus-triggered ifn on the induction of t cell responses, we used recombinant mva-gp and vacv-gp expressing the gp epitope of lymphocytic choriomeningitis virus. for adoptive transfer experiments transgenic mice expressing the p t cell receptor recognizing the gp epitope were intercrossed with mice devoid of the ifn receptor (ifnar -/-) to obtain p ifnar -/mice. upon adoptive co-transfer of p ifnar -/and p ifnar wt/wt t cells and subsequent challenge with mva-gp , a massive expansion of p ifnar wt/wt t cells was observed, whereas p ifnar -/-t cells expanded less efficiently. in contrast, challenge with vacv-gp induced a rather similar expansion of ifnar competent and ifnar deficient p t cells. in the absence of ifnar-triggering t cell expansion was associated with reduced proliferation capacity and increased apoptosis. to study the impact of ifnar-signaling on the expansion of endogenous t cells, conditional mice with a t cell-specific ifnar-ablation were infected with mva. interestingly, in those mice the virus-specific t cells showed a reduced expansion compared to t cells of wt mice. additionally, we found that ifnar-triggering of dendritic cells but not of macrophages further supported specific t cell expansion. thus, upon virus infection virus-associated properties affected the overall cytokine milieu that influenced the quality and the quantity of expansion of virus-specific t cells. to delineate h n -specific signaling patterns we used a genome-wide comparative systems biology approach analyzing gene expression in endothelial cells infected with three different human and avian influenza strains of high and low pathogenicity. blocking of specific signaling pathways revealed that h n induced an exceptionally nf-kb dependent antiviral response. irf is essential part of this interferon-response of human endothelia. furthermore, we identified hmga as a novel transcription factor specifically responsible for the overwhelming proinflammatory but not anti-viral response induced by h n . finally, nfatc was found to be a transcriptional regulator for specifically h n -induced genes. we therefore describe for the first time defined signaling patterns specifically activated by h n which, in contrast to low pathogenic influenza viruses, are responsible for an imbalance of overwhelming proinflammatory and impaired anti-viral gene programs. objectives: to study the early events of immune antagonism by influenza virus in vivo and how this process impacts the timing at which adaptive immunity is generated. methods: to dissect the contribution of the unique viral antagonist ns , delta-ns influenza virus (a recombinant influenza virus lacking the ns gene) was compared side by side with wild type influenza virus in vivo. mice infected with influenza virus were sacrificed at different time points after infection. to study the onset of inflammation during infection lung and blood samples were isolated with a selected panel of inflammatory and antiviral proteins that were measured by multiplex elisa and quantitative pcr (qpcr). to determine the bridging between innate lung inflammation and adaptive immunity, the kinetics of lung antigenbearing dendritic cell (dc) migration to the draining lymph nodes was quantitated in infected mice. further, functional in vitro and in vivo assays in infected animals with influenza-ova viruses were used to determine whether antigen-bearing migratory dcs were capable of priming transgenic t cells. to investigate the effect of ns during the onset of immunity in vivo, we systematically studied the early events occurring in the lungs and draining lymph nodes upon infection with influenza virus. strikingly, no sign of innate immunity was detected in the lungs for almost two days after infection when a sudden inflammatory burst including ifns, cytokines, and chemokines occurred. this burst preceded the robust dc migration and t cell activation in the lymph nodes. virus-loaded dcs appear in the lymph node starting days post-infection, reached its maximum at day , and triggered t cell priming in vivo. a direct comparison of delta-ns virus with wild type virus infection demonstrates that virus can only trigger rapid inflammation in vivo when it lacks the ns protein. we demonstrate that the delay in the generation of immediate lung inflammation is mediated by the influenza ns protein. thus, we propose that the virally encoded ns protein establishes a time limited "stealth phase" where replicating influenza virus remains undetected thus preventing the immediate initiation of innate and adaptive immunity. keratinocytes represent the major cell population of human epidermis which provides a first line of defense barrier for the host. in addition, keratinocytes actively participate in immune response. viral double-stranded rna (dsrna) is the most important viral structure involved in activation of innate immune response. intracellular detection of dsrna triggers secretion of soluble signaling molecules, interferons, and activates pro-inflammatory response and programmed cell death, apoptosis. here we have used subcellular proteomics to characterise dsrna-induced human keratinocytes. cells were transfected with a mimetic of dsrna, poly(i:c), after which the cells were fractionated into cytoplasmic and mitochondrial fractions. these proteomes were analysed using two-dimensional electrophoresis in combination with mass spectrometry, immunoblotting and confocal microscopy. we show that several proteins involved in apoptosis, cytoskeleton reorganization and intracellular transport are up-regulated upon dsrna stimulation. in mitochondrial proteomes the expression of structural proteins, especially fragments of cytokeratins, is highly up-regulated. we show that cytokeratin is cleaved during poly(i:c) stimulation and fragments are solely localized in mitochondria. similar degradation of cytokeratin is seen in emcv-and vsv-infected keratinocytes. cytokeratin fragmentation after dsrna stimulation is dependent on caspase activation, which indicates a role for cytokeratins in the regulation of apoptosis during viral infection. in addition, we show that - - proteins are upregulated in both mitochondrial cell fraction and cytoplasm after dsrna-stimulation and during viral infection. viral dsrna also induced transient phosphorylation of - - target proteins. thus, these results suggest that - - proteins have a regulatory role in host defence against viral infections. i. wessels , d. fleischer , l. rink , p. uciechowski institute of immunology, rwth aachen university hospital, aachen, germany objectives: the proinflammatory cytokine interleukin (il)- b mediates the expression of a variety of proteins responsible for acute inflammation and chronic inflammatory diseases. however, the molecular regulation of il- b expression is not elucidated, yet. it is known that the il- b promoter is packaged into a nontranscribed but poised architecture in monocytes, rapidly producing il- b when stimulated. b-cells which are il- b non-producers reveal an inaccessible promoter structure. in this study the chromatin structure of the il- b promoter and the impact of methylation on il- b expression were examined in a cellular monocytic differentiation model. methods: promyeloid hl- cells were differentiated into monocytic cells after dihydroxyvitamine d treatment. the monocytic phenotype was confirmed by flow cytometry. the il- b promoter was analyzed using the chromatin accessibility by real time pcr (chart) assay. to test the influence of methylation, cells were treated with -aza- -deoxycytodine (aza) and changes in il- b expression were measured by pcr, elisa and western blot analyses. results: in contrast to undifferentiated hl- cells, differentiated cells displayed upregulation of cd antigen and acquired the ability to express il- b. by comparing the accessibilities of il- b promoter we detected that the il- b promoter was not accessible in undifferentiated hl- cells but highly accessible in differentiated monocytic cells. the accessibilities of differentiated cells were comparable to that observed in primary monocytes. lps stimulation did not affect promoter accessibility in promyeloic and monocytic hl- cells, demonstrating that the chromatin remodeling of the il- b promoter depends on differentiation but is independent of the transcriptional status of the cell. demethylation via aza led to the induction of il- b expression in both undifferentiated and differentiated cells which could be increased after lps stimulation. conclusion: two independent mechanisms involved in the regulation of il- b expression were found. our data indicate that the il- b promoter is reorganized into an open conformation during monopoiesis and that the established poised structure is a privilege of mature monocytes but not of the entire myeloid lineage. as a second mechanism, il- b expression is regulated by methylation acting independent of the developmental stage of myeloid cells. a. holweg , g. wetzel , h. arnold , a. gessner microbiological institute-institute for clinical microbiology, immunology and hygiene, university hospital erlangen, erlangen, germany the p family members of interferon (ifn) inducible gtpases also known as guanylate binding proteins (gbps) are among the most abundantly induced transcripts after stimulation with ifn-g. although the stimulatory capacities of the toll like receptor ligands lps and cpg, cytokines like ifn-b and il- b and the intracellular pathogens listeria monocytogenes and toxoplasma gondii have been described to induce their expression, the function of gbps during bacterial infections is still ill defined. here we report for the first time the massive induction of murine gbps in two independent in vivo models of pneumonia (infection with streptococcus pneumoniae and pseudomonas aeruginosa). a strong and rapid induction of mgbp , , , and mrna after intratracheal infection was detected by realtime pcr analysis of bronchoalveolar lavage (bal) cells. using newly generated antibodies in western blots and fluorescence microscopy, we identified macrophages as the main producers of mgbps in bal samples. although the signaling cascade involved in the upregulation of mgbps after ifn-g stimulation has been extensively studied, the mechanisms responsible for mgbp induction upon bacterial stimuli are unclear. in this study we could show that the induction of mgbps upon infection is abrogated in ifn type i/ type ii receptor double-deficient mice and thus absolutely dependent on endogenous interferon production. in contrast, the tlr adaptor molecule myd was found to be dispensable arguing for a trif-mediated interferon production subsequentially resulting in enhanced gbp expression. based on these findings our future experiments will address the functional role of gbps in innate and adaptive immune responses against extracellular bacteria. ( )). activation of resident microglia cells and infiltrating brain macrophages appeared to play a role in modulating virus replication shortly after infection but also appeared to be responsible for the inflammation in brains of infected mice. clearance of replicating virus from the cns required mcmv specific cd + t lymphocytes whose effectors functions still remain incompletely defined (j. immunol. aug ; ( )). humoral immunity appeared to also play a role in the control of mcmv infection in developing brain. infected newborn mice treated with mcmv-specific antibodies had lower viral titers in the cns, significantly less cns inflammation and improved neuronal migration and increased cerebellar area as compared to control mice (j. virol. dec; ( ) ). conclusion: peripheral inoculation of the virus induces focal infection and inflammation in the developing mouse cns followed by strong innate and specific immune response that could also alter developmental programs required for normal development of mouse brain. passive immunization of infected newborn mice reduces mcmv-related pathology in infected brain suggesting that antiviral antibodies are an important component of immunological responses during cmv infection of developing cns. chronic inflammation is associated with the promotion and enhancement of malignancy and tumor growth. tumors enhance the accumulation of myeloid derived suppressor cells (mdsc), which contribute to tumor escape, immune tolerance and immune suppression. previously, we have shown that tumor-derived inflammatory cytokines, such as il- b in the tumor microenvironment can induce a massive accumulation of mdsc in the spleen of tumor bearing mice and induce t cell suppression. in this work, we describe a novel polymorphnuclear mdsc subpopulation -inflammatory mdsc (infmdsc) which accumulates in the bm and spleen of mice bearing inflammatory t breast cancer cells over expressing il- b ( t /il- b) tumor cells, but rarely in untransfected t cells. secretion of il- b from tumor cells is crucial for infmdsc generation and accumulation. infmdsc have the ability to suppress nk cell activity via reduction of the nk activating receptor nkg d in vivo, and in a cell-cell contact dependent manner in vitro. inflammation up-regulates il- ra expression on the cell surface, which correlates with tumor growth and induction of suppression on nk cells. our data suggest that tumor derived inflammation enhances a specific mdsc subset which has the ability induce suppression of nk cells, and perhaps can serve as a new chemotherapy target. objectives: lps constitutes a main target of innate immune recognition of gram-negative bacteria and other lps carrying pathogens. cytokine production after in vitro stimulation of whole blood with lps is used as an expression of individual lps reactivity. we assess genome-wide data to analysed the association to lps induced cytokine response, and replicated the top findings in two independent cohorts. materials and methods: we used healthy caucasian blood donors as discovery samples, and replicated snps from affymetrix -genome-wide human snp array . having p x - in two independent cohorts each consisting of blood donors using a customized chip from illumina and real-time pcr respectively. in all the three cohorts whole blood samples was stimulated for h and we measured the levels of il- , il- , il- , tnf-alfa and il -ra with r&d ® assays on luminex platform.. the association analysis was performed with wald statistical test assuming an additive model. the discovery sample statistical analysis revealed snps with p x , * - . to identify/replicate the association of cytokine production for these we reanalysed these on a cohort. a combined analysis revealed snps with p x , * - .these results are not genome wide significant. the snps showing nominal association to lps cytokine response are being analysed in a replication cohort conclusion: we find nominal associated snps between lps stimulated cytokines in blood samples of healthy caucasian blood donors. the importance of these snps are to be determined in a replication cohort. adipocytes, so far known for their lipid-storage capacity came into the focus of interest because of their immunoregulatory properties resembling those of innate immune cells. adipocyte-derived pro-inflammatory mediators contribute to the development of chronic inflammation, thereby promoting the progression of insulin-resistance/metabolic-syndrome and diabetes. the physiological signals inducing the secretion of inflammatory mediators by adipocytes are unknown. heat shock proteins, such as hsp have been identified as potent regulators of inflammatory innate immune cell-activities, whereas their influence on adipocyteactivities remained elusive. here, we investigated the regulatory effects of hsp on adipocytes. for the first time we could show a hsp -stimulated release of the proinflammatory cytokines il- , cxcl and mcp- in a time-and concentration-dependent manner from murine t -l adipocytes. analyses of hsp -signalling in these adipocytes revealed that members of the mapk-family (erk / , p ) and the transcription factor nfkb are involved in hsp -mediated induction of the mediators il- , cxcl and mcp- . binding-studies with fluorescence-labelled hsp demonstrated that the interaction of hsp with adipocytes exhibits basic features of a receptor-mediated binding. hsp -binding to adipocytes was saturable and reached its maximum at . mm. binding was inhibitable only by the unlabelled ligand ( %), but not by unrelated proteins, thereby proving the specificity of hsp -binding. further analyses to characterize hsp -receptor structures on adipocytes revealed the presence of toll-like receptor (tlr) on adipocytes. tlr has been found to be expressed on macrophages and to interact with hsp , therefore suggesting tlr as a potential receptor candidate for hsp on adipocytes. in order to identify the responsible binding-epitope of hsp we investigated the effect of specific antibodies directed against different epitopes of the hsp -molecule. incubation with antibodies directed against the n-terminus of hsp (aa - ; - mg/ml) were capable of inhibiting the hsp -binding to adipocytes ( - %) indicating that the n-terminal region of hsp is involved in receptor binding. our experiments demonstrate that hsp stimulates the release of proinflammatory adipocyte mediators. the findings implicate that the hsp -mediated induction of adipocyte mediators contributes to inflammatory processes observed in obesity-associated disorders and could serve as a target for the development of therapeutic strategies for patients suffering from diabetes or diabetes-related disorders. legionella pneumophila, a gram-negative facultative intracellular bacterium, is the causative agent of a severe pneumonia known as legionnaires' disease. classically, type i ifns (ifna/b) have been associated with antiviral immunity. ifna/b signal through the ifna/b receptor (ifnar) leading to the induction of hundreds of ifn-stimulated genes (isgs), many of which have anti-microbial activities. recently, it was demonstrated that ifna/b are also produced in host cells infected with (intracellular) bacteria or stimulated with cytosolic dna. here we show by rnai that l. pneumophila infected host cells produced ifnb dependent on irf . we observed enhanced l. pneumophila replication in mouse macrophages lacking ifnar and human cells after irf knock-down, suggesting that endogenously produced ifnb activates a cell-autonomous defence against legionella. moreover, ifnb treatment restricts legionella replication in human and murine host cells. ifna/b impacts formation of large replication vacuoles, but appears not to influence the recruitment of er markers nor fusion of the legionella-containing vacuole with the lysosome. moreover, ifna/b-mediated cellautonomous defence was independent of autophagy and pyroptosis. we thus hypothesize the crucial involvement of antibacterially acting isgs. ongoing studies focus on the role of ifn-induced immunity-related gtpases (irgs). mesenchymal stem cells (mscs) are identified by their capacity to differentiate into connective tissue cell types. mesenchymal stem cells also show a high plasticity and account for a potential therapeutic efficacy. several authors have reported the expression of alfa-smooth muscle actin (a-sm actin) by msc. this protein has been considered a marker for the myofibroblast, has the capacity to polymerize into the cytoplasm and contribute to the cell contractility. this activity may be crucial for the changes of the cell shape, for cell-cell interactions and may therefore be relevant for the physiology of msc. in this work, we study the presence of alfa-sm actin in human msc by flow cytometry and immunoflurescence. we also study the contractility of these cells under the effect of different cytokines. human bone marrow samples were obtained from bone marrow aspirates. bone marrow mononuclear cells were isolated by density gradient centrifugation and cultured in opti-mem culture medium with % of fetal calf serum at °c and % co . bone marrow nonadherent cells were removed after h, and culture medium was refreshed twice per week thereafter. cells grew adherent, with a fibroblastic morphology and expressed cd , cd , cd , cd and stro- and were negative for cd . intracellular staining was performed for alfa-sm actin. cell contractility was measured with the collagen gel contraction assay. alfa-smooth muscle actin was detected in almost % of msc. interleukin- , ifn-gamma and tnf (th cytokines) increased msc contractility, whereas il- (a th cytokine) decreased msc contractility. by immunofluorescence, we observed that il- , ifn-gamma and tnf increased the incorporation of alfa-sm actin into the stress fibers of msc, whereas il- decreased that incorporation. our results suggest that th and th cytokines regulate msc physiology by acting on their contractility. aims: thapsigargin (tg) is a sesquiterpene lactone (sl) of guaianolide type isolated from the mediterranean plant thapsia garganica l. it is widely recognized as an inhibitor of sarco-endoplasmic reticulum ca + -atpase leading to elevation of intracellular calcium. this activity is shared by trilobolide (tb), a sl from laser trilobum (l.) borkh. tg has been shown to possess prospective immunotherapeutic properties. it kills slowly proliferating and non-proliferating cells, and inhibits replication of viruses. the aim of our work was to investigate possible immunostimulatory potential of tg and tb. methods: the effects of the agents were analyzed under conditions in vitro using rat and mouse resident peritoneal cells (pecs), and human peripheral blood mononuclear cells (hpbmcs). they were cultured in density of × /ml in complete rpmi- medium. supernatant levels of ifn-g were determined by elisa. production of no by animal pecs was assayed using griess reagent. possible contamination of the samples with lps was excluded using the chromogenic limulus amoebocyte assay. results: we have found that both tg and tb at as low doses as nm and nm induce ifn-g secretion in rat pecs and hpbmcs, respectively. the concentration of ifn-g produced by the highest dose of the agents ( mm) at the -h culture interval reached the values of approximately ng/ml and ng/ml in rat pecs and hpbmcs, respectively. the increase was apparent within the interval of - h in rat pecs. the -h interval was optimal for accumulation of ifn-g in cultures of hpbmcs. only modestly enhanced secretion of ifn-g was observed in mouse pecs. production of ifn-g was found to depend on activation of nf-xb and map kinases p and erk / . it was not suppressed by the calcium chelating agents bapta-am and tmb- . the tg-and tb-induced ifn-g production was associated with activation of the high-output production of no. conclusions: sesquiterpene lactones tg and tb are potent inducers of ifn-g in animal and human cells. the effect is independent of their serca inhibitory potential. underlying mechanism(s) of the immunostimulatory effects remain to be elucidated. acknowledgements: the work was supported by the grant gacr / / . pin is a peptidil-prolil-cis-trans isomerase that specifically binds phosphorylated ser/thr-pro protein motifs and catalyzes the cis/trans isomerization of peptides. mitotic proteins, cytoskeleton, transcription factors and apoptotic proteins are pin substrates and targeting sites. recent data show that pin interacts with apo-bec g (a g). the pin /a g interaction results in a reduced a g expression and a diminished a g-mediated restriction of hiv. two single nucleotide polymorphisms (snps) in the promoter region of the pin gene (- g/c and - t/c) modulate pin expression; in particular, the - gc genotype or cc haplotype are associated with reduced protein levels (neurobiol. aging, ; - , ) . the - c/g and - t/c polymorphisms in the promoter of pin gene as well as pin protein levels were analyzed in exposed seronegative individuals (esn), heterosexual partners of hiv-infected patients; hiv-infected patients (hiv) and healthy controls (hc). the genotype and allele distributions of the - snp was skewed in esn (genotype: p= . ; allele: p= . ). in particular esn showed a significantly lower frequency of the - gg genotype compared to hiv and hc (p= . and p= . , respectively) and consequently a lower g allele frequency (p= . and p= . , respectively). no significant differences were found for the - snp. these snps are in linkage disequilibrium and combine to form haplotypes. conclusions: our findings support the role of hiv viral replication as the most important promoter of immune activation and prove the importance of art in reducing immune activation and viral replication even in a sub-saharan african environment, where patients are exposed to an abundance of other infectious agents. our data further indicates that hiv replication rather than host genetics is the key determinator of circulating cytokine levels among the studied hiv infected participants. aims: acyclic nucleoside phosphonates (anps) are synthetic analogues of natural nucleoside monophosphates. they have proved to be outstanding antivirals inhibiting replication of both dna-viruses and retroviruses. tenofovir, -(r)- [ -(phosphonomethoxy) propyl]adenine [(r)-pmpa] is broadly used for therapy of aids, adefovir, -[ -(phosphonomethoxy)ethyl]adenine (pmea) has been approved for the treatment of hepatitis b. the aim of our work was to investigate possible interactions of anps with production of cytokines and nitric oxide (no) implicated in antiviral defence mechanisms. the immunobiological effects of anps were screened in vitro using mouse resident peritoneal cells. they were cultured in density of × /ml in complete rpmi- medium. secretion of cytokines was determined after the -h culture by elisa. production of no was assayed at the interval of h using griess reagent. approximately compounds belonging to several distinct anp groups were included in the study: a) pmea derivatives, b) pmedap i. e. -[ -(phosphonomethoxy)ethlyl]- , -diaminopurine derivatives, c) -[ -hydroxy- -(phosphonomethoxy)propyl]-adenine (hpmpa), and hpmpdap derivatives, d) guanidino analogues of pmpdap, e) -heteroalkyl substituted -amino- -guanidinopurines, and f) -amino- -(purin- -yl)propanoic acid derivatives. possible presence of lps in the stock solutions of the samples was checked and excluded using the chromogenic limulus amoebocyte assay. results: approximately compounds were found to activate the secretion of anti-hiv effective chemokines rantes and mip- a and cytokines tnf-a and il- . although these anps did not stimulate biosynthesis of no on their own, they substantially augmented production of no triggered by ifn-g. no clear-cut relationship between the chemical structure and biological effects of anps was observed. however, the most effective proved to be the n -cycloalkyl derivatives of pme-dap. the effects were produced in a dose-dependent fashion, exhibiting the immunostimulatory effects at as low concentrations as to mm. the remarkably enhanced secretion of chemokines was reached within - h of the cell culture. the effects were found to depend on activation of nf-kb. conclusions: it may be suggested that anps represent a new generation of antivirotics with combined antimetabolic and therapeutically prospective immunostimulatory properties. acknowledgements. the work was supported by the grant m . host related immune factors in childhood chronic hepatitis b and change of initial profile with ifn-a treatment needs to be clarified. sixteen patients were included, and million units of ifn-a treatment three times a week for months was initiated. before and after treatment: percentages of the il- and ifn-g in cd + t cells were assessed to determine intracellular t helper cell (th ) type cytokine expression. similarly, percentages of intracellular il- and ifn-g were detected to verify cytotoxic t cell (tc ) type cytokine expression in cd + t cells. percentage of th and tc type cytokine expression, (il- and il- ) were determined in cd + and cd + t cells, respectively. six ( %) of these were evaluated as having no response and the other half with partial/complete response. all patients had higher percentages of th cells with respect to healthy controls before treatment. tc percentages, both tc and tc , were significantly different between these groups, being higher in the patient group. when values of no responder group were compared with healthy controls, il- expression was higher and the percentages of th type cells were significantly low. il- expression in th and tc cells decreased after treatment in the unresponsive group. intracellular cytokine profiles of treatment responders and normal controls were not different. this has been the first study in children comparing baseline and post treatment intracellular cytokine profiles with healthy controls. the frequency ( , %) of high concentration of igg anti-oxldl antibodies in patients with hcv infection were significantly elevated in comparison to healthy subjects ( , % according to bibliographic data). the immune response was significant but it was not assosiated with the viral load. it is probable that humoral immunity plays a critical role and contributes in an immunoinflammatory reaction of hcv-infection. abstract withdrawn by author t. schwandt , f. juengerkes , b. schumak , g. gielen , j. kalff , p. knolle , b. holzmann , a. limmer university hospital bonn, institute of molecular medicine and experimental immunology, bonn, germany, university hospital bonn, department of surgery, bonn, germany, department of surgery, tu munich, munich, germany bacterial translocation is a possible risk of abdominal surgery and could be the cause of life-threatening consequences such as organ failure and septic shock. patients surviving septic shock often suffer from opportunistic infections as well as defects in adaptive immunity. here we investigated the influence of bacteremia and bacterial translocation on systemic adaptive immune responses using murine models. to mimic abdominal surgery, mice were subjected to intestinal manipulation (im). to study septic conditions, mice underwent colon ascendens stent peritonitis (casp) or received e.coli intravenously. we monitored the distribution of gut-derived bacteria by in vivo imaging (xenogen) and additional microbiological assays and determined antigen-specific ctl responses towards subsequent infection with recombinant adenoviruses (av). we detected comparable amounts of bacteria in lung, liver and spleen of mice that underwent casp or were injected i. v. with e.coli. in addition, mice infected systemically with av lacked an antigen-specific ctl response, whereas the ctl responses of locally av infected mice were not affected. in contrast, bacteria were detected in lung and liver but not in spleen of mice that were subjected to im or received e.coli by injection into the hepatic portal vein. here, the ctl response was not impaired. depletion experiments imply that kupffer cells as well as soluble mediators such as tumor necrosis factor alpha play an important role in trapping and clearance of translocated bacteria in liver and lung. our experiments demonstrated that translocation of bacteria did not cause immune suppression as long as they did not reach the spleen in high numbers. we suggest that liver and lung fulfill a filter function to prevent systemic distribution of gut-derived bacteria. failure of or bypassing these barriers might enable bacteria to access the spleen and thus cause systemic suppression of adaptive immunity, whereas induction of local immunity is not affected. objectives: varicella-zoster virus (vzv) is one of the most frequent pathogens for which a vaccine is available. tropism of vzv for skin is the most obvious manifestation of vzv infection, producing vesicular cutaneous lesions that are associated with varicella and herpes zoster. the striking difference in the clinical outcome of infection by rush inducing circulating virulent vzv strains and asymptomatic infection by the vaccine leads to the assumption that the virus interferes with cutaneous immunity. therefore, we comparatively investigated the impact of vzv clinical isolates and the vaccine on the reciprocal crosstalk of immature dendritic cells (idcs) and epithelial gd t cells. methods: skin punch biopsies of herpes zoster patients were analyzed by dual immunofluorescence microscopy. phenotypic changes of cutaneous dcs and monocyte-derived dcs after vzv infection were investigated by flow cytometry. the cytokine profile of vzv-infected dcs and epithelial gd t cells was determined through elisa. results: we observed that innate lymphocytes recognize dcs, which infiltrate herpes zoster lesions, after infection with vzv. strikingly, only the vaccine but not vzv clinical isolates could license the bidirectional crosstalk between idcs and gd t cells resulting in release of ifn-g and il- , the signature cytokines of antiviral immune responses. this is the first demonstration that virulent virus strains disrupt dendritic cell instruction whereas the corresponding vaccine does the opposite. we describe a novel immune evasion strategy in the skin, which provides the opportunity for efficient and symptomatic virus replication. this result is also of practical importance: future vaccine design has to ensure that candidate vaccines do not impair dc instruction in order to allow stimulation of powerful antiviral immune responses. a. jafarzadeh rafsanjan university of medical sciences, rafsanjan, iran, islamic republic of objective: it has been reported that the caga + h. pylori strains induce more severe gastric mucosal inflammation. the aim of this study was to investigate the association of the h. pylori virulence factor caga with serum levels of il- and il- in h. pylori-infected duodenal (du) patients and h. pylori-infected asymptomatic (as) carriers. methods: totally, h. pylori-infected du patients ( patients were positive for anti-caga antibody and patients were negative for anti-caga antibody), h. pylori-infected as carriers ( subjects were positive for anti-caga antibody and subjects were negative for anti-caga antibody) and healthy uninfected subjects (as a control group) were enrolled to study. a blood sample was obtained from all participants and the serum levels of il- and il- was measured by elisa method. the mean serum levels of il- in total du patients ( . pg/ml ± . ) was significantly higher than those observed in total as subjects ( . pg/ml ± . , p x . ) and healthy control group ( . pg/ml ± . , p x . ). in du group, it was found that the mean serum levels of il- in subjects with positive test for anti-caga ( . pg/ml ± . ) was significantly higher than those observed in subjects with negative test for anti-caga ( . pg/ml ± . ; p x . ). the mean serum levels of il- in du ( . pg/ml ± . ) and as groups ( . pg/ml ± . ) was significantly higher than those found in uninfected control group ( . pg/ml ± . , p x . and p x . , respectively). however, no significant difference was observed for mean serum levels of il- between du and as groups. moreover, in both du and as groups the mean serum levels of il- was not significantly differ in subjects with positive test for anti-caga and those were negative for anti-caga antibody. the results of the present study showed higher serum concentrations of il- and il- in h. pylori-infected subjects as compared with control group. in du group the expression of il- influenced by the bacterial caga factor. a. aral , , a. atak gazi university faculty of medicine, department of immunology, ankara, turkey, gazi university institution of health sciences, department of immunology, ankara, turkey objective: ebv is a human herpesvirus, which infects human b lymphocytes latently and immortalizes the cells due to transformation. ebv infection is asymptomatic in childhood while it may cause a self-limiting lymphoproliferative disorder called infectious mononucleosis (im) in adolescence. in immunodefective patients, the virus may lead to malignancies like burkitt's lymphoma, nasopharyngeal carcinoma and immunoblastoma. the virus can also cause latent infections. cytokine production in response to ebv infection differs according to the phase of the infection. neopterin, ifn-g and il- levels are elevated in acute ebv infection in vitro; but in chronic phase, particularly, il- elevation could not be detected. tnf-a enhances the effects of ifn-g on neopterin synthesis while neopterin enhances the secretion of tnf-a via various stimuli. ifn-g levels are elevated particularly in the acute phase of im. since the elevation of cytokine levels changes according to the phases of disease, it's thought that the association between host defense and viral replication depends on different parameters. ifn-g is the major stimulus for neopterin synthesis, which stimulates monocytes and macrophages primarily. increased production of neopterin in body fluids can be used to monitor the activation of cell mediated immunity. method: in order to analyze the effects of neopterin release and other cytokines, mononuclear cells have been isolated from peripheral blood samples of healthy donors and transformed via ebv. neopterin, ifn-g, tnf-a and il- levels have been measured with eia kits in culture supernatants. results: neopterin levels increased dependent on time and independent of ebv transformation. in ebv-transformated cell culture tnf-a levels increased beginning from the th hour and reached to maximum levels at the st week and decreased again at the rd week; however there were no significant differences between the levels among three weeks. likely tnf, ifn-g levels also increased at the st week and started to decrease at the rd week. il- reached to its peak at the rd week. conclusion: according to these results, neopterin levels, which increased dependent on time but independent of ebv transformation, may be a helpful marker for evaluating the acute response to viral infection but not for transformation. v. jurisic , m. jurisic university of kragujevac, school of medicine, kragujevac, serbia, university of belgrade, school of dentistry, belgrade, serbia tnf-alpha is a pleiotropic cytokine that is considered as a primary modifier of inflammatory and immune reaction in response to various inflammatory diseases and tumour. we investigated tnf concentration in radicular inflamed cysts and odontogenic tumours obtained from patients undergoing surgery, under local anaesthesia, and after aspiration of cystic fluid from non-ruptured cysts. further, we estimated the role of cyst wall on production of tnf-alpha in respect of presence of inflammatory cells, degree of epithelial proliferation and degree of vascularization. the concentrations of tnf-alpha in the cystic fluids were measured by elisa, while proteins analyzed after separation by two-dimensional gel electrophoresis. the presence of pericystic inflammed cells were analyzed in thick section for routine histological examinations and by immunohistochemisty for cd , cd and cd . tnf-alpha is elevated in both cysts fluid, but higher values were found in radicular inflamed cysts in comparison to odontogenic tumours. higher concentration of tnf-a were associated with higher protein concentration, higher presence of inflammatory cells in peri cystic tissues, cysts wall thickness and higher degree of vascularisation (mann-whitney u-test, p x . ) in radicular cysts. no correlation was found, based on these parameters in odontogenic tumours, but all sumple have detectable concentrations of tnf-alpha. objectives: interactions between the neuroendocrine and immune system play an important role in maintaining and restoring homeostasis. in susceptible individuals a dysfunction of the neuroendocrine system may be one of the risk factors involved in the pathogenesis of septicemia and bacterial infection at all. we will study prolactin role in defensive reaction of immune system to bacterial infection. as a type of this bacterial infection we have chosen septic status, where we are expecting the most significant alterations of immune reaction, and specially septic statuses in blood malignancies, where we are expecting deficiency of immune system. our idea is that prolactin takes part in this defensive reaction by its cytokine effects, and it has contraregulative role against activation of adrenocortical system. the aims of this study are to extend our knowledge about the activation of peripheral prolactin expression and by this way to contribute elucidation of its role in periphery. ) drawings blood from patients and healthy donors. blood of patients and healthy persons were sampled together with past histories after getting their acquainted approval. status of patient had to meet these conditions: a) the presence of systematic inflammatory response syndrome (sirs) according to standard clinical and laboratory criteria. b) positive hemoculture or determination of septic focus with demonstration of microbiologic source. ) detection of the gene expression of prolactin and tlr- in cd + peripheral blood monocytes from patients with septic status and from healthy controls has been performed by rt-pcr at the level of mrna and flow cytometry at the level of intracellular protein results: we have found statistical significant differences (p p . ) in expression profile between patient and control groups. these differences were at both levels of expression, mrna and protein. conclusion: septic statuses change tlr- and peripheral prolactin expression in cd + monocytes. the function of interferon (ifn)-induced immunoproteasomes (i-proteasomes) is at present almost exclusively acknowledged in connection with improved processing of mhc-class i antigens. the experiments performed here challenge this existing paradigm of i-proteasome function. we demonstrate in vivo and in cellulo that the key role of i-proteasomes resides in the protection of cells against the formation of protein aggregates, which is ultimately crucial for preservation of cell viability under ifn-induced oxidative stress. ifns up-regulate the ubiquitylation machinery and enhance the formation of oxidant-damaged, nascent, poly-ubiquitylated proteins, which essentially require i-proteasomes for their efficient degradation. i-proteasome deficiency results in the formation of aggresome-like induced structures and increased sensitivity towards apoptosis. enhanced degradation of poly-ubiquitin conjugates designed to protect cells, will therefore also increase the peptide supply for antigen presentation. thus, only by executing their physiological function in the maintenance of protein homeostasis i-proteasome induction also provides a mechanism for cellular immune-adaptation. background: revived by the description of new functions, b cells are considered to be essential in the genesis of autoimmune diseases. in strong support of this interpretation, baff would play a key role in their physiology. however, the correlation between circulating baff levels and disease activity is not clearly established. conflicting results concerning levels of baff and b-cell repopulation after rituximab treatment have been reported. in fact, basal serum levels of baff reported in literature vary according to the antibodies (ab) used in the elisa and the mode of calculation. the aim of this study was to understand these variations. material and methods: different anti-baff abs were tested to verify whether they recognize glycosylated baff purified from u culture supernatant. sera from patients with autoimmune diseases were also tested. a western-blot analysis of sera was performed to evaluate anti-baff abs specificity and the best combination of anti-baff abs validated for elisa. then, different commercial baff elisa-quantification kits were compared to our "in-house" elisa. results: unexpectedly, the binding of some anti-baff ab was restricted to glycosylated baff. however, both glycosylated and non-glycosylated forms of baff were found in sera from patients with autoimmune diseases. most of the kits commercially designed to quantify baff suffer from some limitations. some sera are indeed positive with a kit and negative with another and vice versa. furthermore, there seems that rheumatoid factors (rf) interfere and correlate with the optical density of the anti-baff elisa. conflicting results concerning levels of baff and disease activity or auto-abs titers should be reconsidered in light of the glycosylation status of baff. (table- ). when tb household contacts and healthy controls were compared, cfp and esat seemed to be more useful than tst in tb contacts for displaying ltb (table- ) . although cfp spot numbers were much more than esat spots at the beginning and follow up period, statistically there was no difference in terms of median values(p: , )( table- ). both esat and cfp spots were prone to decline during the follow up period. [ is some evidence to suggest that aspects of innate immunity (e. g. triggering of cytokine production by dendritic cells) may be impaired by hcv. to gain insight into some features of the innate immune response activated in vivo in the context of acute hcv replication, we analysed cytokine and chemokine levels in serum samples from chronic hcv patients undergoing liver transplantation. luminex multiplex assays and elisas were used to quantitate serum levels of g analytes immediately prior to liver transplantation and at sequential time points up to days post-transplantation. the increase in serum hcv rna levels associated with acute viral infection of the transplanted liver was found to be associated in most patients with elevations in serum levels of cytokines and chemokines including ifn-gamma, tnf-alpha, ip- , il- and il- . notably, the pattern and magnitude of systemic analyte elevations were very similar to those accompanying the acute burst of viral replication in primary hcv infection. high-magnitude elevations in systemic type ifn levels were not observed in either setting, which may reflect an in vivo impairment of plasmacytoid dendritic cell functions by hcv similar to that observed in in vitro studies. we suggest that the liver transplant setting can be used as a model to study aspects of the innate immune response activated in acute hcv infection. to test the hypothesis that virus interactions with sialic acid receptors may play a role in innate antiviral immunity, we used recombinant viruses and differentiated cultures of human airway epithelial cells (hae). the hemagglutinin of the pandemic virus a/hong kong/ / (h n ) (hk) differs from its putative avian precursor by amino acid substitutions. we generated the complete recombinant virus rhk and its ha variants with amino acid reversions back to the ancestral avian sequence (rhk- aa-i r, n d, k n, s n, g a, human ( - ); rhk-r -l q, s g and rhk- aa-i r, n d, k n, s n, g a, l q, s g, avian ( - )). among these variants, the double mutant rhk-r and the seven mutant (rhk- aa) had a typical avian-virus-like receptor-binding specificity owing to substitutions l q and s g.we infected hae cultures with the viruses and collected samples from the apical and basolateral sides of the cultures at different times post infection. virus titers were determined in mdck cells, and concentrations of about pro-and anti-inflammatory mediators and chemokines were measured using a multiplex bead assay.concentrations of most cytokines progressively increased at the apical side of the cultures in the course of the infection. many cytokines, including t-cell-attracting chemokines such as ip- and rantes, were induced to similar levels by different viruses. however, some mediators were induced significantly stronger by avian-like viruses rhk-r and rhk- aa as compared to rhk and rhk- aa. in particular, avian-like viruses stimulated a higher release of potent chemo-attractants of innate immune cells, such as g-csf and il- , shedded adhesion molecules (cd , vcam- , icam- ), and pro-apoptotic factors (trail). remarkably, the patterns of secreted cytokines differed between the apical and basolateral sides of the cultures. whereas avian-like viruses typically induced similar or higher levels of cytokines at the apical side than did rhk and rhk- aa, the human-like viruses were stronger inducers of basolaterally secreted mediators. these data provide the first direct experimental evidence that receptor specificity of influenza viruses can significantly affect patterns of innate immune responses in human airway epithelium. further studies are warranted to determine the role of the observed effects in the host range and pathogenicity of influenza viruses in humans. a total of newborn infants were enrolled in the study. forty-nine newborn infants (group i), who met the criteria of sepsis, had a routine sepsis evaluation as well as measurement of pct, il- , and neutrophilic cd levels, procalcitonin and il- were measured by elisa technique while, neutrophilic cd by single colour flowcytometric technique. of these "infected" infants, had positive blood culture (subgroup ia: culture-positive sepsis), and infants were diagnosed to have clinical sepsis with negative blood cultures (subgroup ib: culture-negative sepsis). another newborn infants classified as control group (group ii) . results: sensitivity, specificity, positive predictive value, and negative predictive value for diagnosis of sepsis were analyzed for pct, il- , cd , and crp. il- had the highest sensitivity and specificity, % and %, respectively, using cutoff n . pg/ml. for pct, the highest sensitivity and specificity, % and %, respectively, were at a cutoff value of n . pg/ml. neutrophilic cd had maximal sensitivity and specificity of % and %, respectively, at cutoff value of . %. combinations of different markers may improve the sensitivity and specificity of biomarkers such as the tests used in this study. we found that the best combination was il- and neutrophilic cd , which together provided sensitivity and specificity of % and %, respectively, and npv %. the combination of il- and crp had high sensitivity and moderate specificity, % and %, respectively. conclusions: il- and neutrophilic cd levels determination can be used as good tests for early detection of neonatal sepsis. procalcitonin measurement might be used as an additive parameter to improve the diagnostic efficacy of the other markers in neonatal sepsis, but it is not helpful as a single test. objectives: the assembly and activation of inflammasomes are essential processes in the immune response to many inflammatory stimuli. inflammasomes are typically formed by at least one member of the cytosolic innate immune sensor family, the nod-like receptors (nlr). the nlr family members nalp , naip or ipaf and the adaptor asc are involved in caspase- activation in response to bacterial infection, triggering the processing and secretion of il- b and il- . recent studies have demonstrated that tlr-dependent inflammasome activation in macrophages is modulated by autophagy, a homeostatic mechanism for the catabolism of cytosolic constituents. autophagosome biogenesis and maturation requires activation of the class iii pi k, vps and can be inhibited with the pi k inhibitors wortmannin and methyladenine ( ma). in contrast, activation of akt, via class i pi k, results in inhibition of autophagy. the aim of this study was to determine the nature of this inflammasome and whether autophagy influences il- b secretion in dendritic cells. methods: murine bone marrow-derived dendritic cells (bmdm) were treated with tlr ligands in combination with ma, wortmannin or an akt inhibitor. supernatants were analysed for il- b by elisa. results: ma enhanced il- b secretion by bmdc treated with the tlr and tlr ligands poly(i:c) and lps, but not with any other tlr ligands tested. similar results were obtained using wortmannin. this increase in il- b secretion was greatly reduced in bmdc from nalp -/mice compared to wild type c /bl controls. treatment with the akt inhibitor had no effect on lps-induced il- b secretion by bmdc. tlr-dependent secretion of il- a was also enhanced by treatment with ma. conclusions: these data demonstrate that il- b secretion by bmdc in response to treatment with pi k inhibitors, in combination with lps or poly(i:c), is dependent on the nalp inflammasome. this response is limited to tlr and tlr agonists. inhibition of akt had no effect on lps-induced il- b production, suggesting that the effect of wortmannin and ma on inflammasome activation is not dependent on the inhibition of akt activation by class i pi k. objectives: in the last few years, several evidences have shown the modulation of toll-like receptors (tlrs) by g-protein coupled receptors, i. e. our group has recently demonstrated the attenuation of tlr signaling by the inflammatory lipid mediator sphingosine -phosphate (s p) through receptors and in human monocytes-macrophages, which could explain some of the s p anti-atherogenic effects. since adhesion of monocytes to endothelial cells is considered an important event in atherogenesis, our goal was to investigate the putative implication of tlr-s p receptors crosstalk on the expression of adhesion molecules in endothelial cells. methods: for the study, in vitro cultured endothelial cells from arterial and venous origin were challenged with a combination of tlr ligands and s p, and later analyzed by flow cytometry. a pharmacological analysis of the s p receptor subtype involved in the response was also performed. results: data from flow cytometry experiments revealed that icam- expression was increased following lps and s p concomitant stimulation in both venous and arterial cells, suggesting that tlr and s p receptors cooperate in the expression of icam- . conversely, no cooperation was observed when tlr ligands were used. in order to elucidate which s p receptor subtype was involved in the increase of icam- expression, we used a pharmacological approach with s p receptor inhibitors and pertussis toxin. results showed differences between arterial and venous cells. while in arterial cells elevated icam- after lps and s p challenge was significantly reduced by blocking s p receptor and the effect was pertussis toxin-insensitive, in venous cells the response was pertussis toxinsensitive and not blocked with inhibitors of s p receptors and , which suggest that s p receptor might be involved in the effect. conclusions: altogether these data demonstrate that tlr and s p receptors can interact to increase adhesion molecules such as icam- in human endothelial cells, and the s p receptor subtype involved in the effect differs between arterial and venous cells. with ssc without pah) and a pool of sera of healthy controls (hc) were tested. results: in dimension immunoblot, serum igg from ssc patients, patients with ipah and hc tested individually reacted with - , - and - protein bands in a human vsmc protein extract with qualitative and quantitative differences between groups, respectively. in dimension immunoblot, igg of pools of patients with ipah, igg of pools of patients with ssc with or without pah, and igg of a pool of hc recognized ± , ± , ± and protein spots respectively. twenty one protein spots were recognized by more than % of igg of pools of sera in each group of patients and not by igg of hc. twenty seven protein spots were recognized by the great majority of igg of pools of patients with a higher intensity than igg of pools of hc. identified proteins were constituents of cytoskeleton, proteins involved in oxidative stress as stress-induced phosphoprotein and peroxyredoxin and proteins involved in regulation of cell energy metabolism as triosephosphate isomerise. we have identified anti-vsmc abs in the serum of patients with idiopathic and ssc-associated pah. these abs bind to cytoskeleton, oxidative stress and cell cycle antigens. objectives: this study aimed to verify that subcutaneous lymph node transplantation inducing lymphatic regeneration is possible in healthy adult rats, as obtained in other species. methods: this rat model was used to determine the effects of lymph node fragmentation as well as sheep erythrocytes and platelet-rich plasma injection on the regeneration of the transplanted lymph nodes. results: this rat model is adequate to study the regeneration of transplanted lymph nodes. lymph node fragmentation seems to affect transplant regeneration negatively. an immune challenge by injection of sheep erythrocytes in the drainage area of the transplanted lymph nodes does not improve fragment regeneration. however, injection of syngeneic platelet-rich plasma containing several growth factors resulted in an improvement in regeneration. conclusion: lymph node fragment regeneration, although still experimental, could be relevant for lymphedema prevention. acquired lymphedema has a high prevalence in developed countries as a consequence of the removal and/or radiotherapy of tumor-draining lymph nodes in cancer patients. this disease causes lifelong disability due to chronic swelling and increased risk of infections. it currently lacks an effective treatment. methods: patients suffering from different diseases were enrolled in our study. patients were suffering from bone diseases (osteomyelitis, necrosis, tumour) whereas of them were suffering from inflammatoty diseases (soft tissue inflammation, diabetic ulceration). blood specimens were collected before hyperbaric oxygen treatment and the serum levels of icam- and vcam- were assesed by an enzyme immunoassay (elisa). results: out of the patients ( , %) had elevated levels of the intercellular adhesion molecule. out of the patients suffering from bone diseases ( , %) had raised values (mean value ng/ml) whereas patients out of the suffering from soft tissue diseases and diabetes ( , %) had raised values (mean value , ng/ml). reference value for icam- was - ng/ml. vascular cell adhesion molecule's assesment revealed no elevated levels in our patients. conclusions: our study revealed a high rate of patients ( %) having increasd levels of icam- . high icam- levels were more prevalent in patients suffering from soft tissue inflammatory diseases and diabetes ( , %) than in patients with bone diseases ( . %). mean values were found , ng/ml and ng/ml accordingly. those findings verify the positive correlation between icam- and inflammatory diseases and tissue damage but not for vcam- . colorectal cancer (crc) was the first solid tumour to be successfully targeted with anti-angiogenic therapy in the clinic. tumour angiogenesis is critical for cancer progression in that it permits expansion of the tumour mass and fosters malignant dissemination. angiogenesis is a multistep process involving endothelial cells as well as numerous stromal components within the tumour microenvironment that also represent potential therapeutic targets. inflammation dependent-angiogenesis is increasingly recognised as a central force in tumour growth and progression, while use of anti-inflammatory drugs has been found to reduce incidence of crc carcinoma potentially through repression of tumour angiogenesis. we investigated the link between inflammatory angiogenesis and colorectal cancer in archival tissues across a range of pathologies that represent diverse steps in the progression of crc: cases of ulcerative colitis (urc), adenocarcinomas developed from preexisting tubular or tubulo-villous adenomas, tubular or tubulo-villous adenomas with low grade dysplasia, and infiltrating adenocarcinomas. immunohisto- objectives: to determine the effect from the administration of preoperative pravastatina to therapeutic dose in the expression of cd in the leucocitaria adhesion to endotelio vascular in the isquémico-reperfundido miocardico weaveal endotelio vascular en el tejido miocardico isquémico-reperfundido by the circulation extracorpórea (cec). methods: they were included in way randomizada double blind patients with intervened controlled hiperlipidemia of surgery coronary low circulation extracorpórea (cec). mg of pravastatina oral hours they were administered before the procedure (group study, n= ) or placebo (group placebo, n= ), and control (group control, n= ). samples of outlying veined blood were extracted to the hours. the separation of leukocytes was made in peripheral blood, to determine the expression of cd in such. in all the samples one quantified the intensity of the expression pattern and the percentage of leucocitarias cells. results: types of patterns were distinguished: cytoplasmic, of membrane and compound. the intensity of the expression was classified in degrees: degree . without expression. degree . weak; degree . moderate; degree . intense. in the group control: most of the samples they presented/displayed a mixed pattern (cytoplasmic and of membrane) with an intensity degree - . the placebo group: mixed pattern, degree - . group study ( mg. oral pravastatina): most of the cells they presented/displayed a predominance of membrane pattern: degree - . the percentage of cells that expressed cd was greater in the group study ( mg. oral pravastatina). the preoperative oral pravastatina to therapeutic unique dose ours study produces a greater expression cd answer induced by the cec; it seems that these molecules located in the leukocytes participate in the adhesion to the activated endoteliales cells, necessary for the extrusion of the lymphocytes through endotelio towards the inflammatory center and in quimiotaxis of the leukocytes towards the inflammation sites. several surface molecules on endothelial and epithelial cells undergo regulated cleavage by the disintegrin and metalloproteinases adam and adam . we recently identified transmembrane chemokines, junctional adhesion molecule-a (jam-a), and members of the proteoglycan family as novel substrates for these proteases. here we demonstrate that cell lines and primary cells of human endothelial or epithelial origin release considerable amounts of soluble jam-a and proteoglycan ectodomains. this release is enhanced by treatment with the proinflammatory cytokines ifng and tnfa. the enhanced release was not caused by an increased gene induction but rather associated with a reduction of the surface expressed molecules. both, constitutive and induced release required the presence of adam as demonstrated by specific inhibitors, lentiviral silencing experiments as well as treatment with the recombinant catalytic domain of adam . these data suggest that the proinflammatory cytokines ifng and tnfa induce enhanced proteolytic shedding of cell surface molecules on endothelial and epithelial cells. to investigate the physiological relevance of this induced shedding, mice were treated systemically with ifng/tnfa leading to increased presence of soluble jam-a in the blood serum. both cytokines also stimulated jam-a release from excised murine aortas with was associated with enhanced adam activity in the tissue. in the presence of the adam inhibitor induction of jam-a release was suppressed. in cultured epithelial cell lines enhanced shedding of jam-a or proteoglycans was not associated with increased mrna or surface expression of adams but rather with increased activity of cellular adam as shown by means of a synthetic substrate assay. our study demonstrates that the proinflammatory cytokines ifng/ tnfa upregulate adam -mediated shedding activity rendering the protease an important modulator of endothelial and epithelial surface molecules in inflammatory settings. rium, and the haplotype vegf- / vegf+ is associated with rcc risk ( p= , ), metastases ( p= , ), nuclear grade ( p= , ), tumor stage ( p= , ), and tumor size (p= , ). on the other hand, the polymorphism vegf - a/c is not associated with rcc risk and clinical parameters. our results shed a new light to the knowledge on the association between vegf polymorphism and rcc risk and development. these data could help to improve our understanding of the rcc pathogenesis and disease progression. pten is a lipid phosphatase, whose substrate is phosphatidylinositol , , -trisphosphate. therefore, pten is one of the main antagonists of the pi -kinase, which plays a major role in many important cellular functions, such as proliferation, migration or response to inflammatory stimuli. here we investigated the role of pten in collagen induced arthritis. we show that conditional deletion of pten under the lysm promoter (lysmcrepten flox/-) leads to a significant reduction in clinical severity of collagen induced arthritis (cia). histological analysis of cia, lysmcrepten flox/mice displayed significantly reduced joint inflammation as well as erosive bone destruction. total anti-collagen antibodies, however, as well as anti-collagen iggs were identical in both groups. upon analysis of inflammatory cytokines in serum after immunisation we found a significant reduction of il- as well as il- levels. furthermore, pten deficient macrophages and dendritic cells showed reduced induction of il- as well as il- and il- mrna upon stimulation with various tlr-ligands. since these cytokines play an important role in the induction of pathogenic th- t cells, we measured th- cytokines in lymph nodes after immunisation with collagen. although dendritic cell and macrophage recruitment to the draining lymph node was comparable in both groups, there was a slight reduction of il- and a strong reduction of il- mrna in the draining lymph node of immunized lysmcrepten flox/compared to wild-type mice. one of the mechanisms through which il- exerts its anti-inflammatory effects consists in promoting the release of anti-inflammatory molecules. in this context, particularly important is the production of il- ra, whose expression is induced by lps in human neutrophils and monocytes and significantly potentiated by the presence of il- . based on our previous observation that support a direct role of il- -activated stat in the enhancement of il- ra transcription induced by lps, we plan to characterize the transcriptional activators recruited to the il- ra promoter in vivo and responsible of the increased rate of transcriptional initiation upon exposure of lps-treated cells to il- . quantitative chromatin immunoprecipitation (chip) studies were employed to examine the in vivo binding of transcriptional activators to the il- ra promoter. crosslinked nuclear lysates were immunoprecipitated and min after il- addition with different antibodies and immunoprecipitated dna was analyzed by quantitative real-time pcr for the presence of target sequence located in the il- ra promoter. chip assays showed that the pol ii recruitment to the il- ra promoter induced by lps is significantly increased by il- , further strengthening the concept that the rapid enhancement of lpsinduced il- ra gene expression by il- initially occurs by targeting transcriptional events. as expected, real-time pcr of anti-stat immunoprecipitated dna showed statistically significant levels of stat binding to the il- ra promoter only in cells stimulated with lps in the presence of il- . surprisingly, anti-p and anti-p chip assays revealed enrichment of both p and p recruitment to the il- ra promoter when il- was added to lps-stimulated cells, suggesting that il- enhances the recruitment of nf-kb to the il- ra promoter. interestingly, when nf-kb is recruited to this promoter in lps + il- -treated cells, the overall nf-kb nuclear translocation (analyzed by western blot) and dna binding activity (detected by emsa analysis) were not modified with respect to cells stimulated with lps alone. the enrichment of nf-kb at the il- ra promoter site is dependent on il- -activated stat , since it is greatly reduced when stat activation by il- is impaired. the molecular mechanism through which il- -activated stat promotes the recruitment of nf-kb to the il- ra promoter is currently under investigation. major components of mast cell secretory granules are proteases. we could recently report that intracellular stored mast cell-produced cytokines regulate mc protease activities and provided evidence that il- acts as a specific negative transcriptional regulator of mouse mast cell protease- (mmcp- ). we examined the mechanisms underlying the repression of mmcp- gene expression. our data show that the "repressor" effects of il- on mmcp- promoter activity are still operating on the mmcp- bp long minimal promoter. moreover, il- deficiency in mast cells causes a specific dysregulated expression of the transcription factors c/ebpb and yy . furthermore, chromatin immunoprecipitation revealed that il- promoted specific reciprocal recruitment of c/ebpb but not yy to the mmcp- promoter. finally, il- deficient mast cells display a predominantly non-cpg methylated pattern of the mmcp- . thus, we proposed that the expression of mmcp- and possibly other immunoregulatory genes may be regulated by il- through epigenetic modification and by balancing the content and binding of c/ ebpb and yy in mast cells. i. nagy , k. filkor , a. vörös , l. kemény , a. szász bzaka, baygen, szeged, hungary, university of szeged, department of dermatology and allergology, szeged, hungary micrornas (mirnas) are evolutionary conserved small non-coding rnas that act as key regulators of gene expression at post-transcriptional level by targeting mrnas for translational repression and/or degradation. mirnas have been shown to have unique tissue-, developmental stage-and diseases-specific expression patterns. during the last years several studies highlighted that mirnas play critical role in the differentiation and function of the adaptive as well as innate immunity. little is known however, about the differential regulation of mirnas following the activation of pattern recognition receptors. in order to tackle this issue, we treated hacat keratinocytes with staphylococcus aureus-derived peptidoglycan (pgn) once or repeatedly, the latter mimicking persistent infection. after appropriate treatments we first analyzed the expression profile of mirnas mir- , mir- a and mir- , which are known to participate in immune processes of the skin. repeated pgn-treatment significantly decreased mir- expression; in contrast, pgn re-stimulation had no further effect on mir- a and mir- expression. next, we investigated the correlation between the expression of mir- and its two known direct targets: regulatory protein p and suppressor of cytokine signalling- (socs- ). although the gene-expression profile of neither p nor socs- changed, we found that the expression of mir- reversibly correlates with both p and socs- protein expression, a phenotype that we verified by two independent protein analysis methods (western blotting and immunofluorescent labeling). importantly, transfection of hacat cells with anti-mir- prior to pgn-treatment completely abolished both p and socs- down-regulation, revealing the involvement of mir- in pgn-induced transcriptional regulation. finally, methylation-specific pcr experiments unravelled the role of dnamethylation in regulating mir- expression upon pgn-treatment. taken together, our results strongly suggest that sets of mirnas may be differentially regulated during persistent infection. results: tgfb +/-had a lower incidence and burden of benign papillomas when compared to tgfb +/+ animals. however, more scc developed in the tgfb +/-mice. after acute and chronic promotion, tgfb +/-skin showed a reduced proliferative response with no increase in epidermal tgfb or nuclear p-smad compared to tgfb +/+ mice. tpa-induced pkca activity as well as phosphorylation of specific pkc substrates in keratinocytes correlated with tgfb gene dosage. further, pharmacological inhibition of alk suppressed tpa-mediated pkca activation suggesting that physiological levels of tgfb are required for maximal activation of pkc-dependent mitogenic responses. even though the tpa-induced inflammatory response was greater in tgfb +/-skin, tgfb +/+ papillomas had more tumor infiltrating neutrophils. tpa-induced proinflammatory gene expression was sustained in tgfb +/-skin and primary keratinocytes but it was elevated in v-ras ha -transduced tgfb +/+ but not tgfb +/-keratinocytes, indicating that tgfb switches from an anti-inflammatory cytokine in the skin to a proinflammatory factor in tumors dependending on an activated ras. despite this differential proliferative and inflammatory response to tpa and enhanced papilloma formation in the tgfb +/+ mice, there was no increase in conversion to scc in this genotype. conclusions: tgfb acts to promote benign tumors enhancing cell proliferation and inflammation through its ability to regulate pkc activation in skin, yet retains a suppressive function for malignant conversion. background: proto-oncogene survivin has been recently shown as a prognostic marker distinguishing patients with destructive rheumatoid arthritis (ra). in the present study we studied the relationship between survivin and urokinase (upa), a fibrinolytic serine protease being over expressed in the inflamed joints and exhibiting arthritogenic properties. material and methods: levels of survivin and upa were measured in the paired blood and synovial fluid samples of patients with ra, using elisa and compared to controls with non-inflammatory joint diseases. the ability of upa to induce survivin and requirement of upa receptor (upar) was studied in primary synovial fibroblasts and pbmc of ra patients, human monocytic (thp- ) and fibroblast (mrc- ) cell lines employing antibodies against upar, sirna technique, and synthetic inhibitors of intracellular pathways. the ability to prevent urokinase-induced arthritis by interruption of survivin expression was evaluated in mouse model of arthritis. results: in the present material of ra patients and controls the levels of survivin correlated to urokinase (upa) (r= . ), a plasminogen activator over expressed in inflamed joints and known to exhibit potent arthritogenic properties. we found that / ra patients had high circulating levels of survivin. these patients had erosive arthritis and were characterized by high levels of upa. in vitro studies showed that upa induced survivin in leukocytes and this process was dependent on signaling through upa receptor. in turn, survivin was required for expression of upar. additionally, survivin was essential for upa production in mrc- and synovial fibroblasts. down-regulation of survivin with sirna was followed by significantly reducion of upa synthesis. finally, treatment with downregulation of survivin by sirna in vivo efficiently abrogated upa-induced arthritis in mice model. these findings indicate that survivin is an essential mediator of arthritogenic properties of upa regulating its synthesis in synovial fibroblasts and upar expression in leukocytes. close correlation between survivin and upa in patients with ra supports the improtance of these interaction for the pathogenesis of arthritis. upon cell activation, ubiquitously expressed inositol , , -trisphosphate -kinase type b (itpkb) phosphorylates inositol ( , , ) trisphosphate (ins( , , )p ), a calcium-mobilizing second messenger with pleiotropic effects. itpkb inactivation leads to severe t cell deficiency, altered thymo-independent b cell responses and neutrophil hyperactivation. we here report that itpkb-deficient (itpkb -/-) mice also display profound alterations in mast cell development and function. indeed, while mast cell number, c-kit and fcepsilonri expression were comparable in itpkb-deficient and proficient mice, itpkb -/mast cells were almost completely devoid of granules. this phenotype could be partially reversed upon treatment with sodium cromoglycate. nevertheless, fcepsilonri or c-kit activation on mast cells led to increased ca + responses and to stronger erk phosphorylations. however, itpkb -/mice displayed an attenuated sensitivity to ige-mediated passive systemic anaphylaxis, correlated to the absence of fcepsilonri-dependant histamine release and to downregulation of h and h receptor expression due to high basal histamine concentrations. production of neosynthesized mediators remained normal. finally, itpkb deficiency also severely impaired scf-induced mast cell differentiation in vitro. taken together these results demonstrate that itpkb is a key regulator of mast cell activation. itpkb antagonists might thus be of therapeutic interest for programmed and progressive depletion of histamine stores. the large percentage of immune relevant genes that are alternatively spliced and the connections between splicing and disease, strongly indicate that alternative splicing plays a central role in the regulation and fine-tuning of physiological immune responses. il- b is an important proinflammatory cytokine produced by activated macrophages and monocytes. il- b is produced as an inactive cytoplasmic precursor that is proteolytic processed by the inflammatory caspase- to generate the mature secreted active form. caspase- is also synthesized as an inactive form that requires processing by the inflammasome to become active. we have used a subset of the trc lentiviral human library to generate loss-of-function phenotypes for most of the splicing factors and splicing regulators. we were able to silence the expression of genes involved in splicing with an average -fold coverage. after the primary screen and several rounds of phenotypic validation, we have identified genes that significantly affect the production of il- b by thp- cells after a h challenge with pfa-fixed e. coli, as measured by elisa. knockdown levels were analyzed by qrt-pcr for the most significant candidates to validate the phenotypes observed. exon array analysis are being performed to identify possible targets of the most significant splicing factor candidates obtained by the shrna screening in order to dissect their mechanism of action in the regulation of the inflammasome and il- b secretion. tissue transglutaminase (tg ) has a critical role in the pathogenesis of chronic inflammatory diseases such as celiac or neurodegenerative diseases. we have previously described the key role of tg in cystic fibrosis (cf), a genetic disease characterised by chronic lung infections and inflammation. in cf, mutation on the cftr gene results in an increased tg expression and activity leading to functional sequestration of the anti-inflammatory pparg and increase of the classic parameters of inflammation. here we tested whether in vivo inhibition of tg can reverse inflammation in chronic inflammatory diseases. to assess the importance of tg not just in cf but in chronic inflammatory diseases in general, we injected cystamine, a potent tg inhibitor, in a transgenic mouse model cf and in the taz transgenic mice that spontaneously develop autoimmune thyroiditis. intraperitoneal administration of cystamine had a significant impact on the lung epithelium in the cf model, where it decreased tg expression and activity. the treatment was also able to dampen all the classic inflammatory parameters as well as restoring normal cellular levels of functional pparg. interestingly, cystamine injections could also block inflammation in the taz tcr transgenic mouse model with chronic thyroiditis, highlighting the pivotal role of tg in generating inflammation in two very different pathologies. this work underlines the critical role of tg in inflammation and provides new opportunities to develop therapeutic strategies for sufferers of chronic inflammatory diseases. angiogenesis, the growth of new blood vessels, is a process that is essential during tissue repair, foetal development, and female reproductive cycle. angiogenesis is also a relevant process associated to many pathologic conditions including autoimmune diseases and tumors. we have shown that dendritic cells activated in the simultaneous presence of pro-and anti-inflammatory signals (alternatively activated dc, a-dc) display potent angiogenic activity in vivo which is mediated by the release of biologically active vegf-a. here, we investigates the molecular mechanisms leading to vegf-a secretion in lps+pge stimulated a-dc. preliminary results indicate no accumulation of hif- alpha in a-dc, therefore suggesting that vegf-a is induced by a non-classical, hif- alpha independent pathway. in addition, we found that vegf-a secretion depends on the activation of mapk p but not erk / or jnk. inhibitor studies, nascent transcript analysis and polimerase ii recruitment on the promoter show that the induction of vegf-a is largely due to new transcription and not to changes in mrna stability. chromatin immunoprecipitation studies aimed at the characterization of the modifications of vegf-a regulatory regions in a-dc and at the identification of transcription factors bound to vegf-a promoters are being performed. this will possibly allow the description of novel transcription factors involved in vegf-a activation in a-dc. wnt proteins are secreted palmitoylated glycoproteins with multiple functions in cell proliferation, migration as well as tissue organization. they are best known for their role in embryonic development and tissue homeostasis. deregulation of wnt signaling has been shown to promote carcinogenesis. recently we identified wnt signaling to be involved in the regulation of inflammatory processes: wnt a is induced in human macrophages in response to mycobacteria and conserved bacterial structures and contributes to the regulation of the proinflammatory cytokines il- and ifn-gamma. to gain deeper insights into wnt mediated modulation of inflammatory processes we now used murine bone marrow derived macrophages and analyzed the effects of the addition of exogenous wnt homologs. we monitored wnt-mediated activation of primary macrophages by measuring the activation of signaling pathways and transcription factors, analyzed the expression of target genes by real-time pcr and measured the secretion of inflammatory cytokines by elisa. exogenous wnt a -but not wnt a -was able to induce cytokine expression in primary macrophages. in infection experiments wnt a promoted the mycobacteria-induced macrophage activation and enhanced the expression of inflammatory mediators in murine macrophages. in contrast, addition of wnt a reduced the expression of inflammatory mediators upon mycobacterial infection. these data corroborate our previous findings and further support the notion that tlr/nf-kappab and wnt signaling, both being evolutionary highly conserved pathways, are functionally interconnected infection of immunocompetent mammals with t. gondii induces a chronic infection of the brain. t. gondii cysts persist in neurons and escape elimination by the immune system. in immunodeficient individuals, the infection can be reactivated resulting in a lethal toxoplasma encephalitis (te). in te, the parasite is primarily controlled by infiltrating t and b cells. also brain resident cells may contribute to control of the disease and however, the mechanisms of brain resident cells leading to the protection of the vulnerable brain in chronic te are largely unknown. in a previous study, we could show that expression of gp on astrocytes in mice is critical for survival of te. in the present study, we analyzed the function of neuronal gp in te. after infection with t. gondii, mice lacking neuronal gp (synapsin-cre gp fl/fl ) died significantly earlier in the chronic phase of infection than control gp fl/fl mice. death of synapsin-cre cre gp fl/fl was due to a severe encephalitis with larger inflammatory lesions and higher numbers of inflammatory leukocytes. additionally, te of synapsin-cre gp fl/fl mice resulted in a substantial apoptosis of neurons both in the vicinity of inflammatory lesions and also in brain areas without inflammation. in vitro, apoptosis of gp -deficient neurons was also significantly increased upon infection with t. gondii or stimulation with tnf as compared to gp expressing neurons. interestingly, the intracerebral parasitic burden was not increased in synapsin-cre gp fl/fl mice indicating that the immunoregulatory role of neurons is more important than their anti-parasitic function. t. objectives: persistent production of tnfa in many autoimmune diseases, including intraocular inflammation (uveitis), can lead to significant tissue damage. targeting tnfa with neutralising antibodies or tnf receptor fusion proteins is often, but not always, an effective therapy. high serum concentrations of tnfa, il- b, il- and il- have been detected in a spectrum of autoimmune diseases; while in contrast, the levels of il- , il- and tgfb are reduced. this suggests, indirectly, that failure to regulate an appropriate balance of inhibitory factors contributes to the pathogenesis or propagation of tissue inflammation in autoimmunity. thus, understanding the homeostatic control of tnfa by tgfb further may generate more effective therapies. as tnfa mrna ' untranslated region (utr) contains an au-rich element (are), which targets mrnas for degradation, we wished to test whether tgfb suppresses tnfa protein production by upregulating the rnabinding protein fxr , which can bind to tnfa mrna and inhibit translation. methods: using raw . cells and mouse bone-marrow derived macrophages stimulated with lps and tgfb , we assessed mrna expression by q-pcr and tnfa protein expression by flow cytometry. the 'utr of tnfa mrna was isolated and inserted into a luciferase reporter vector on a constitutive promoter. transfected raw cells were treated with lps and tgfb and luciferase expression was quantified. cells treated with lps and tgfb were also examined for fxr expression using pcr and western blot. following fxr knockdown using sirna, the influence of tgfb and lps on tnfa protein production was examined by flow cytometry. results: we find that while tnfa mrna expression remains constant, lps induced tnfa protein expression is suppressed by tgfb . using the luciferase-tnf- 'utr vector we show that tgfb targets the 'utr of tnfa. furthermore, tgfb and il- both upregulate fxr mrna and protein; and treatment with tgfb and lps can synergistically upregulate mrna expression, more than tgfb alone. following sirna inhibition of fxr , tgfb can no longer inhibit lps-induced tnfa production. comtb up-regulated mmp- and mmp- secretion from saecs, nhbes and fibroblasts to a peak of . +/- . ng/ml, at hours. interleukin- augmented comtb-stimulated up-regulation of mmp- and mmp- secretion from saecs and fibroblasts in a synergistic manner. in contrast, interleukin- down-regulated mmp- secretion from saecs by %. interleukin- up-regulated mmp- and mmp- secretion from fibroblasts but not from saecs. timp secretion from saecs was enhanced by interleukin- but there was no effect of interleukin- . mmp up-regulation by interleukin- and comtb was inhibited by the pi kinase inhibitor ly and on western analysis akt (protein kinase b) was phosphorylated at minutes. chemical inhibition of the p d isoform of pi kinase with ic abrogated the il- and comtb driven secretion of mmp- from the small airway epithelial cells. chemical inhibition of the tumour suppressor phosphatase, pten (phosphatase and tensin analogue on chromosome ) accentuated mmp- secretion. these inhibitory effects were confirmed with sirna. mmp- up-regulation was secondary to increased gene expression with promoter activity peaking h after stimulation. in summary, interleukin- and interleukin- drive transcription dependent mmp- and mmp- secretion from airway epithelial cells and fibroblasts. interleukin- also increases timp but down-regulates mmp- gene expression and secretion. this may contribute to the matrix degrading phenotype in tuberculosis. the pi kinase pathway is central in interleukin- driven tissue destruction in the context of m. tuberculosis infection. v. delgado-maroto , l.s. moreira , e. gonzalez-rey , m. delgado institute of parasitology and biomedicine lopez-neyra , csic, granada, spain, university of seville, sevilla, spain objectives: atherosclerosis is an inflammatory chronic disease characterized by the formation in the arteries of lesions that involve inflammation, lipid accumulation, cell death and fibrosis. over time, the rupture of these atherosclerotic plaques releases prothrombotic material to the blood and causes thrombotic occlusion at the site of disruption. atherosclerosis will probably become the most common cause of death within years. one of the initial hallmarks of the disease is the uptake of oxldl particles by macrophages, which leads to intracellular cholesterol accumulation and the formation of foam cells. t cells undergo activation after interacting with foam cells, which process and present local antigens including oxldl, generating a t helper response. cholesterol metabolism is regulated by factors such as pparg (proliferator activated receptor g), srb (a class b scavenger receptor), cd or abca , that can induce cholesterol exit from the macrophage which may help to solve the lesions. expression of these factors depends on intracellular camp. adrenomedullin (am), urocortin (ucn) and vasoactive intestinal peptide (vip) are novel neuropeptides synthesized by immune cells that have various characteristics to be considered as possible therapeutic agents for atherosclerosis. they are potent anti-inflammatory agents, which downregulate a broad spectrum of pro-inflammatory mediators, and inhibit th immune response. their mechanism of action involves binding to gpcr and adenylate cyclase activation with subsequent increase of camp in the cell, which is recognized as an anti-inflammatory response. methods: we investigate am/ucn/vip effect on bone marrow-derived macrophages stimulated with oxldl. we determine the levels of pparg , srb , cd , and abca . we also analyze the cholesterol metabolism of oxldl-stimulated macrophages after neuropeptides incubation using oil red o staining of lipids drops and tritium labelled cholesterol. objective: endovascular aortic repair (evar) is considered a minimally invasive procedure, and the patients are expected to be discharged after a day or two. however up to % develop a systemic inflammatory response syndrome (sirs), resulting in prolonged convalescence. as yet there is no satisfactory explanation to this severe response. previous studies have shown a high level of il- in the mural thrombus lining the aneurysm. the thrombus is manipulated during the procedure, but whether or not it is the source of circulating il- and/or other cytokines during and after the operation is unknown. methods: quantitative analysis of the pro-inflammatory cytokines il- , tnf-a, il- b, il- and il- , and the anti-inflammatory cytokine il- , in plasma from five patients, as of yet, was carried out by means of cytometric bead array, while analysis of plasma il- was performed using the luminex platform. the cytokine levels were compared to the clinical response, in terms of sirs. results: evar induced the production of il- and il- , and in some cases, of il- . the maximal plasma levels of il- and il- were found at hours and of il- between - hours. modest plasma levels of il- were also observed, with maximal production at various time points ( - hours). by contrast, production of tnf-a, il- b and il- did not occur to a significant extent, while production of il- occurred sporadically. although our preliminary data indicate that sirs is associated with enhanced cytokine responses, the production of il- , il- , il- and il- also took place in patients who did not develop sirs. conclusion: evar is associated with the sequential production of il- , il- , il- and il- , i. e. a mixed pro-and anti-inflammatory response, even in the absence of sirs; but the production seems to be exaggerated in patients developing sirs. further studies involving patients are in progress, and will clarify this. we hypothesize that sirs is elicited by il- , activated by manipulation of the mural thrombus. to reveal whether this is the case, studies involving immunohistochemistry of the thrombus, will be performed. il- is a novel il- cytokine family member that is expressed as an intracellular precursor (pro-il- ) and is thought to be cleaved by caspase- to yield a mature bioactive form of the molecule (mat-il- ). to date however, evidence of cell-associated proteolytic processing and caspase- dependent secretion of mat-il- has not been reported. here we show that pro-il- but not mat-il- is released from uvb-irradiated keratinocytes. we demonstrate binding of pro-il- to the il- r and also il- r-dependent bioactivity of pro-il- on mast cells. we propose a previously unrecognized role for pro-il- as a pro-inflammatory mediator and suggest a direct link between uvb-mediated epithelial cell damage and cutaneous mast cell activation. we have previously shown that induction of er stress and tlr signalling synergistically enhance il- p mrna expression in myeloid cells, and markedly increase secretion of il- , but not il- , by dendritic cells. the aim of this study is to investigate the mechanism of this synergy. we examined the il- promoter for potential binding sites for er stress induced transcription factors and identified a putative site for chop . chromatin immunoprecipitation (chip) assays using anti-chop and isotype control mab were performed using nuclear lysates from u cells and il- promoter dna measured by qpcr. chop binding on the il- promoter was detected following stimulation of u cells with lps or tp alone, but this was significantly enhanced when er stress and tlr stimuli were combined. il- promoter dna was not detectable following chip with the isotype control antibody. to confirm the role of chop in il- gene transcription, u cells expressing shrna's specific for chop or non-specific gene target were tested for their ability to express il- following tlr and er stress stimulation. u expressing three independent shrna targets for chop exhibited significant reductions in il- p mrna (up to % reduction of the response to lps+tp) compared to u expressing a control shrna. chop shrna expression did not affect the expression of other lpsresponsive genes, including il- , il- , ccl and sod . to identify if er stress induction of il- mediated by chop expression plays a role in a more physiological setting, we examined the role of chop in the induction of il- p gene expression following chlamydia trachomatis (ct) infection. infection of u cells with live but not g-irradiated ct induced expression of er stress response genes, including chop . u infected with live ct exhibited increased il- p mrna expression compared to u infected with nonviable bacteria. chop silencing significantly reduced the ability of live ct to induce il- p mrna, confirming the important role of chop in this response. these data suggest that er stress induction of chop could contribute significantly to the pathogenesis of diseases in which il- plays an major role, through induction of il- and il- producing cells. the clonal deletion of thymocytes by negative selection is an important process to ensure immunologic tolerance, even though the underlying molecular mechanisms are poorly understood. here, we show that gadd b, a regulator of mitogen-activated protein kinases, is critically involved in selection processes in the thymus. gadd b expression was inducible in different in vitro and in vivo models of negative selection. strikingly, only tcr-ligating peptides resulting in negative selection induced gadd b expression, while positively selecting ligands or dexamethasone, a tcr-independent apoptosis agonist, failed to do so. expression of gadd b maintained a sustained activation of p kinase and thereby promoted tcr-mediated apoptosis. in contrast, thymocytes from gadd b-deficient mice showed only transient p activation and reduced caspase activation. interestingly, we observed a switch to positive selection in gadd b-deficient mice since a higher percentage of single positive thymocytes was found. moreover, markers of positive selection as cd and cd were elevated on gadd b-deficient thymocytes. thus, we provide evidence that gadd b and a resulting persistent activation of p constitute a novel apoptotic pathway involved in negative selection. these results also provide evidence for the novel concept that not only the on-off switch of a signaling module but also its spatiotemporal regulation may crucially determine cell fate decisions. di santo institut pasteur, paris, france, monash university, victoria, australia > the thymus represents the ''cradle'' for t cell development, with distinct thymic stroma components providing multiple soluble and cellular membrane cues that foster in a step-wise fashion developing thymocytes. although il- is recognized as an essential factor for thymopoiesis, the nature of the thymic il- niche remains poorly characterized in vivo. > using a novel bacterial artificial chromosome transgenic mice in which yellow fluorescent protein (yfp) is under control of il- promoter, we identify a subset of thymic epithelial cells (tecs) that co-express yfp and high levels of il transcripts (il- hi cells). il- hi tecs arise during early fetal thymic development, persist throughout life, and co-express homeostatic chemokines (ccl , ccl , cxcl ) and cytokines (il ) that are critical for normal thymopoiesis. in the adult thymus, il- hi cells are found in cortico-medullary regions and display traits of both cortical or medullary immature tecs. interestingly, the frequency of il- hi cells decreases with age, suggesting a mechanism for the age-related thymic involution that is associated with declining il- levels. conversely, the frequency of il- hi cells is markedly increased under severe lymphopenia imposed by genetic mutations that cause an early and profound block in t cell development. this augment indicates that thymocyte-tec crosstalk may condition il- -expression by tecs. > together, our temporal-spatial analysis of il- -expressing cells in the thymus suggests that thymic il- levels are dynamically regulated under distinct physiological conditions. this novel il- reporter mouse provides a valuable tool to further dissect the molecular and cellular mechanisms that govern thymic il- expression in vivo. two lines of evidence have recently demonstrated that the pre-b cell receptor (pre-bcr) is associated with autoimmunity, through its surrogate-light-chain (slc) components l and vpreb. it has been shown that pre-bcrs are polyreactive for several self-antigens. the polyreactivity of the pre-bcr induces pre-bcr signaling and activation. furthermore, in human a self-reactive b cell subset was identified that co-expresses immunoglobulin light chain (ig lc) and the slc components. these vpreb + lc + b cells, found in healthy individuals, are potentially harmful as they express autoreactive antibodies associated with autoimmune diseases, like sle and ra. to elucidate the contribution of pre-bcr components to the development and activation of autoreactive b cells, we have recently generated a slc transgenic (slc-tg) mouse model in which all b cells express slc proteins. slc-tg mice exhibit spontaneous igm + plasma cell development. moreover, aging slc-tg mice have elevated anti-nucleosome igm levels, accompanied by immune complex deposition in the kidney, but do not display auto-immune pathology. nevertheless, vpreb + lc + b cells may induce pathology when self-tolerance mechanisms fail. to test this hypothesis, slc-tg mice were crossed on two autoimmune-prone genetic backgrounds: (i) em-bcl -tg mice with b cell-specific overexpression of the anti-apoptotic protein bcl and (ii) fcgriib-deficient mice. both in young slc-tg;em-bcl -tg double tg mice and in slc-tg;fcgriib -/mice spontaneous germinal center (gc) formation -which is associated with autoimmunity -was significantly enhanced, when compared with control em-bcl -tg and fcgriib -/mice. in slc-tg;fcgriib -/mice, numbers of splenic igg plasma cells and serum igg levels were˚ - fold increased. importantly, serum from young slc-tg;em-bcl -tg and slc-tg;fcgriib -/mice contained high titers of igg auto-antibodies in / and / cases, respectively. these values were increased when compared with control groups: / in fcgriib -/and / in bcl -tg. finally, we found that the collagen induced arthritis (cia) was significantly enhanced in slc-tg;fcgriib -/mice, compared to fcgriib -/mice. taken together, these findings demonstrate the slc components have the capacity to induce auto-antibody formation in the mouse and and to enhance autoimmune pathology in ra. t cells are generated from progenitor cells that enter the thymus from bone marrow via blood. these progenitor cells once within the thymus have little selfrenewal capacity. differentiation from hematopoietic stem cells to early t lineage cells proceeds through a series of intermediate precursor populations. however, it is largely unknown to what extent these cell populations contribute to t cell development in the presence of other precursor populations and how the earliest intrathymic t cell progenitors are generated from extrathymic precursors. to assess the relative contribution of potential precursors to t lineage differentiation we developed a strategy based on the depletion of well-characterized precursor populations rather than their enrichment and subsequent adoptive transfer together with an equal amount of congenic non-depleted bone marrow. thus, the physiological ratio of extrathymic precursors remained largely intact and we were able to address the question whether there is only one physiological t cell precursor or many. we showed that, under such competitive conditions, t lineage progenitors are confined to the cd + cd + fraction of bone marrow cells. notably, t lineage reconstitution was not restricted to either cd hi cells, representing multipotent progenitors, or cd + cells, representing common lymphoid progenitors, both of which contributed to t lineage differentiation with different kinetics. in conclusion, our data suggest that multiple physiological extrathymic t cell precursors exist, which are able to compensate for the loss of depleted populations. thus, our findings may have implications for devising strategies for improved t lineage reconstitution after hematopoietic stem cell transplantation. background: previous results from our group have demonstrated ephb and ephb expression on both thymocytes and thymic epithelial cells (tecs). we used chimeric models to determine that those molecules govern in an autonomous and non autonomous manner thymocyte and tec development, and how they regulate interactions between both cell types. objectives: in order to better define the importance in thymus of eph-ephrin b interactions we have analyzed the effects of the lack of ephrin b and/or ephrin b , the ligands of ephb and ephb receptors. this approach is specially interesting taking into account that eph-ephrin signaling is transmitted to both the two cells participating in the interaction and that the cell responses depend on the type of signals (reverse, forward or both), their direction and intensity. methods: for this purpose we have used cre-loxp recombination systems for deleting ephrinb or ephrinb genes specifically on either thymocytes or tecs. results: animals with ephrin b deficient thymocytes showed thymic hypocellularity and alterations on t-cell development whose severity depended on the background of the analyzed mice. in these mice only a few changes occurred in the cortical tec network. on the contrary, mice with conditioned deletions in tec, especially ephrinb /b double mutants, showed a more severe phenotype that began early in the ontogeny and resulted in very small thymi exhibiting an extremely compact cortical and medullary network, decreased numbers of cd + cells in the cortex, increased proportion of k +k + cells and high presence of cysts. in addition, t-cell development was partially blocked at the dn cell stage. conclusion: these data reveal an autonomous and non-autonomous role for ephrinb and ephrinb in the development of both t cells and tecs, confirming the importance of these molecules in the establishment of a crosstalk between the main two cell types of thymus. we discuss how eph-ephrin contacts modulate cellular homotypic and heterotypic interactions that take place during thymus organogenesis and in t cell differentiation. a. rolink , d. vanhecke university of basel, developmental and molecular immunology, basel, switzerland the importance of normal t lymphocyte development in the immune system is exemplified by the occurrence of inherited and acquired human immunodeficiencies where the development or functional maturation of t cells is defective. in order to identify molecules/genes and elucidate developmental processes that are essential for human t cell development we use a novel in vitro tool, the op -dl cell culture system ( ) . using this in vitro assay we obtain large numbers of human cycd + and cd + cd + double positive thymocytes starting from umbilical cord blood (ucb) derived cd + hsc. signals and molecules that are involved in t cell development are being addressed by using blocking antibodies and/or chemical inhibitors. similar as in mice we found an essential role for il- and notch mediated signaling in the development and survival of particular developmental stages of human thymocytes. among the molecules that are rapidly induced in cd + cells upon notch signaling is cd followed by cd . t cell specification is accompanied by the induction of cd a and loss of cd on cd + cd + cells. these cd -cd a + cd + cells become dependent on continuous il and notch signaling for sustained survival and further differentiation into cd + cd ab + dp thymocytes. we found that flt l is not essential for the differentiation of cd + cd + human thymocytes but that addition of exogenous flt l in the co-cultures increases the number of cd + precursors and consequently result in higher yields of developing cd + cd ab + dp thymocytes. finally few mature tcrab + t lymphocytes develop from the cycd + cd + cd ab + dp subset in this in vitro assay suggesting that op stromal cells lack the required selecting mhc-antigen complexes and/or costimulating molecules to induce and sustain positive selection of human thymocytes. this in vitro assay will allow us now, by using rna interference, to test additional genes for their role during human lymphoid development. from a clinical standpoint, a better understanding of the mechanisms controlling human t-cell development is a fundamental step towards the development of specific therapies for the treatment of primary and acquired immunodeficiencies as well as for the treatment of malignant t-cell disorders. brain derived neurotrophic factor (bdnf) promotes various neuronal functions such as survival, regeneration and synaptic plasticity. emerging evidence also indicates an essential role for bdnf in the immune system, e.g. in the b and t cell lineages. we therefore investigated the impact of bdnf on thymocyte development using bdnf knockout (ko) mice and conditional ko mice lacking bdnf specifically in t cells. in both models, we found reduced thymocyte numbers and a significant increase in double negative thymocytes. in contrast, the percentage of naturally occurring regulatory t cells and the expression of activation markers were unaltered. moreover, the lack of bdnf did not result in enhanced thymocyte apoptosis. the increase in double negative thymocytes was due to a partial block in the transition from the dn to dn stage, where bdnf and its receptor p are expressed as revealed by real-time pcr. the observed partial block in thymic maturation results in mild peripheral lymphopenia without affecting the activation status of peripheral t cells, their homeostatic proliferation and without compromising peripheral immune responses in general. in summary, our findings point to a critical role of t cell lineage derived bdnf in thymocyte development acting in an autocrine and/or paracrine manner. r. berga , c. lópez-rodríguez pompeu fabra university, barcelona, spain nfat is a transcription factor that belongs to the rel family (nf-kb and nfatc proteins). its expression in primary cells and organs is restricted to certain proliferative tissues, like activated t lymphocytes and thymocytes, where levels of nfat are relatively high and its subcellular distribution is predominantly nuclear. recent mouse models suggest that nfat participates in thymocyte development and also indicate its involvement in t cell proliferation and survival. nfat deficient mice present a t cell immunodeficiency consistent on lymphopenia, which is more accused for cd + lymphocytes. these observations are of substantial relevance as we and others have described that, in vivo, nfat -null mice are unable to mount cd +-and cd + -immune responses. data from our laboratory indicate that nfat -null mice suffer from hyperosmolarity in plasma (hypernatremia) as a result of the incapacity to induce an osmoprotective gene expression program at a systemic level. to selectively analyze the t-cell autonomous effects derived from the lack of nfat during the development of t lymphocytes, we developed mouse models that delete nfat at early (lck-cre + /nfat flox/flox ) or late (cd -cre + /nfat flox/flox ) stages of thymocyte maturation and that present isotonic plasma. our work indicates that nfat is expressed at all stages of t cell development. in addition, analysis of mouse models that lack nfat at different points of t cell development indicate that it participates at early stages of the ontogeny of t cells. objectives: apoptosis mediated by the tumor suppressor molecule p , is regarded as a major player in tumor prevention but this may not be its only role. we have investigated this by creating a mouse (m ¿ pro) lacking residues - of the proline-rich domain of p . methods: we compared the ability of various hemaptopoietic tissues from m ¿ pro mice and wild type mice to undergo apoptosis following irradiation or treatment with pro-apoptotic drugs. apoptosis was measured by staining with annexin v in vitro and by detection of caspase activation in vitro and in vivo. we also compared their ability to undergo cell cycle arrest using brdu staining. tumor development was monitored in cohorts of m ¿ pro, p null (p -/-) and wild type (p +/+) mice, with or without prior irradiation. results: apoptotic function was lacking in m ¿ pro mice, but they were able to arrest cell-cycle progression in hematopoietic tissues. m ¿ pro developed late-onset b-cell lymphoma, but not the thymic t-cell tumors found in p -/-mice. interestingly, m ¿ pro lymphomas were comprised of incorrectly differentiated b-cells. bcell irregularities were also detected in m ¿ pro prior to tumor onset, in which aged mice showed an increased population of inappropriately differentiated b-cells in the bone marrow and spleen. we propose that the apoptotic function of p has an important role in b-cell homeostasis, which, in turn, is important for prevention of b-cell lymphomas moreover, our data suggest that the apoptotic function of p is not important for preventing thymic t-cell tumors. s. myrczek , r. pardi , a. gessner microbiological institute-institute for clinical microbiology, immunology and hygiene, university hospital erlangen, erlangen, germany, vita-salute san raffaele university school of medicine, milano, italy jab is the catalytic subunit of the highly conserved cop signalosome. this complex plays a central role in various cellular processes as proliferation and cell cycle control. jab regulates the neddylation of ubiquitin ligases and thus contributes to degradation of many proteins. furthermore jab regulates the activity of ap transciption factors. to date jab is thought to be essential for every cell type as jab knock out mice are embryonic lethal and t cell development is blocked by t cell selective absence of jab . to investigate the function of jab in b cells we established a mouse strain deficient for jab selectively in b cells. mice with floxed alleles of jab kindly provided by r. pardi were crossed with a mouse strain expressing the cre recombinase under control of the mb -locus (m. reth, freiburg). ablation of jab expression resulted in an almost complete block of b cell development at the pro b cell stage. the absence of peripheral mature b and b cells and serum immunoglobulins resulted in chronic arthritis with high pathogen burden after experimental infection with borrelia burgdorferi. the observed block in b cell development is rescued by over expression of the anti apoptotic protein bcl under the control of the m enhancer. facs analyses revealed that all b cell subtypes analyzed in the jab -deficient, bcl -transgenic mice are present albeit at reduced numbers compared to wild type animals. serum immunoglobulin titers are detectable and after borrelia infection specific antibodies are produced. we confirmed the absence of jab in sorted spleen b cells by immunoblot analysis. in summary, we show for the first time that cells are viable and functional without jab when apoptosis is prevented. t. nitta , s. murata , k. tanaka , y. takahama university of tokushima, tokushima, japan, university of tokyo, tokyo, japan, rinshoken, tokyo, japan how self-peptides are generated and displayed in the thymus to select a useful and self-protective repertoire of t cells is largely unknown, whereas the role of thymic self-peptides in eliminating self-reactive t cells and thereby preventing autoimmunity is well established. a recently identified form of proteasome, termed thymoproteasome, is specifically expressed by thymic cortical epithelial cells (ctec) and is required for the optimum generation of cd t cells. here we show that ctec display a thymoproteasome-specific spectrum of class i mhc-associated self-peptides, which is essential for positive selection of major and diverse repertoires of class i mhc-restricted t cells. indeed, cd t cells generated in the absence of thymoproteasomes display a markedly altered tcr repertoire that is defective in both allogeneic and antiviral responses. these results demonstrate that thymoproteasome-dependent self-peptides are required for positive selection of a diverse repertoire of immunocompetent cd t cells. defects in helper t cell number or function causes susceptibility to infections and in some cases autoimmunity or allergy. our understanding of the genetic control of helper t cell differentiation into specific functional subsets is still far from complete. here we present the results to date from a genome-wide enu mutation screen for mice with inherited deficits in specific helper cell subsets. these deficits were detected by multi-colour facs analysis of peripheral blood samples, and by antibody production following immunization with heat-killed b.pertussis and cgg coupled with the hapten arsonate (aba) in alum, which induce internally polarized th and th antibody responses, respectively. using this screen, a number of new mutant strains have been isolated with complete or partial loss of cd + t cells or functional deficits that selectively interfere with th or germinal centre responses. in this talk i will present data from some of the first strains that have been identified including the first strains where we have been able to identify the causative mutation. systematic genetic analysis of helper t cell differentiation in the resulting strains will illuminate how t cell help is correctly polarized for immunity and to avoid immunopathology. intrathymic t-cell development provides a unique model system to study cell fate determination because of the well-defined cellular stages and the confined microenvironment of this process. in order to highlight the differences and similarities between fetal and adult t-cell development at the molecular level we performed a microarray study. labelled rna from facs purified fetal and adult dn c-kit high (etp), dn and dn thymocyte populations was hybridised to affymetrix mouse a- . genechips. the resulting data were grouped into four distinct gene clusters: cluster i contained genes over-expressed throughout adult development and included a large proportion of transcription factors ( out of genes), illustrating a significantly different transcriptional program acting during adult differentiation. conversely, cluster ii consisted of genes that were over-expressed in fetal progenitors and included signal transducers (out of genes) such as acvr , bmpr , fzd , chemokine receptors cx cr , cxcr and integrins a , a , a , ae and av, pointing to a difference in microenvironments. genes that showed uniform down-regulation during consecutive stages of fetal and adult development were restricted to cluster iii. amongst these were transcripts governing alternative developmental choices, therefore emphasising a common mechanism of lineage restriction during thymopoiesis. on the other hand, cluster iv was limited to genes that were homogeneously up-regulated during development. these included gata- , tcf- , notch- , rag- , rag- and pre-ta, which are indispensable for t cell development. interestingly, levels of expression of these genes were elevated in fetal progenitors, especially at the etp and dn stages, suggesting that the molecular program of t-cell development is more advanced in the early stages of fetal differentiation. discriminant analysis with the use of the support vector machine arrived at the same conclusion that demonstrated a nearby clustering of all fetal stages with the adult dn population, therefore implying a more committed state of fetal progenitors. finally, transcriptional signatures of each developmental stage were defined by "recursive feature elimination" with support vector machines. this approach can now be used to classify characterised and aberrant hematopoietic progenitors and thus construct an ontological scheme of hematopoietic development based upon transcriptional signatures of populations under normal and pathological conditions. tcrgd+ cells and tcrab+cd aa+ intraepithelial lymphocytes (iels) of the gut are unconventional t cells that reside in tissues and provide innate-like immune responses to "stressed-self". as these cells share common functional properties in the periphery, we have hypothesised a common mechanism of development in the thymus; their progenitors diverging from the conventional t cell developmental pathway based on tcr signal strength at the dn stage. the pre-t-alpha chain (pta) that pairs with tcrb to generate the pre-tcr, has two isoforms; pta a and pta b . both can form a functional pre-tcr with tcrb. ligand-independent signalling by the pre-tcr is a result of spontaneous oligomerisation (followed by internalisation), that is mediated through charged residues on the pta chain. pta b lacks out of of these essential residues and therefore, we speculate results in higher surface expression and different signalling capabilities. we have hypothesised that pta a and pta b permit differential signal strength through the pre-tcr at the dn stage, facilitating the divergence of the conventional and unconventional lineages of tcrab+ t cells. preliminary semi-quantitative pcr data suggest that pta a and pta b are differentially expressed in wt thymocytes at different stages of ontogeny. retroviral transduction of pta -/-e thymocytes with either pta a or pta b alone, followed by fetal thymic organ culture, confirmed the rescue of abt cell development by both isoforms. however the two isoforms appear to differentially regulate the kinetics of thymocyte development by - days of culture; pta b expression generates a greater percentage of tcrab+ cells while pta a expression results in the accumulation of isps. these results suggest different roles for the two isoforms of pta in the thymic development of abt cells. in order to determine the mechanism by which pta a and pta b may generate qualitatively different signals, site directed mutagenesis was used to produce mutant chains of pta a and pta b that lack the "dimerisation residues" necessary for internalisation of the receptors. in addition, bac transgenic mice that express singly either pta a or pta b under the pta promoter are being generated to fully characterise their role in conventional vs. unconventional lineage commitment. erythroid, myeloid and lymphoid cells are initiated in parallel in appropriate cytokine environments so that specific number of erythrocytes, myeloid cells, natural killer cells, thymocytes and t cells, and precursor of b cells can be detected and counted at day of culture. if needed for further functional analyses, long-term proliferating lines and clones of progenitor t and b cells can be established at this point of "in vitro" development. hence the "in vitro" differentiation of es cells to different hematopoietic cell lineages and their progenitors can be quantified. it allows for testing the efficiencies for hematopoietic development of genetically or epigenetically different es or ips cells. aim: increasing evidence includes wnt proteins inside the group of master-signalling pathways which govern immune and non immune differentiation systems. although their precise functions in bone marrow and thymus are still controversial, numerous studies show that wnt signalling is able to control the proliferation of hsc and thymic progenitors and might also affect both their cell-fate decisions and subsequent maturation. in the present work we analyse the effect of transient stimulation of canonical wnt pathway in the differentiation potential of lin -cd + cd ahuman thymic progenitors, a multipotent and heterogeneous cell population which has the capacity to develop into t cells, nk cells, monocytes, conventional dendritic cells (cdc) and plasmacytoid dcs (pdcs). methods: human thymus samples from patients aged month to years undergoing corrective cardiac surgery were obtained and used according to the declaration of helsinki. transient b-catenin stabilization was triggered culturing purified thymic lin -cd + cd precursors with recombinant wnt a ( ng/ml) or with licl ( mm) for hr. active b-catenin, was detected by flow cytometry using anti-human active b-catenin mab ( e ) under conditions of phosphatase activity inhibition. wnt a or licl pre-treated precursor were assayed in chimeric human-mouse ftoc, in il- and scf-supported cultures for generation of nk cells and in co-cultures with murine bone marrow stromal st cells suplemented with il- and flt l. phenotype of recovered cells, apoptosis and cytokine receptors were analysed by flow cytometry. expression profile of transcription factors was analysed by real-time quantitative rt-pcr . our results demonstrate that giving a boost to canonical wnt signalling triggered by transient exposure of thymic progenitors to wnt a or licl, change their differentiation capacity enhancing nk cell production. on the contrary, wnt a or licl pre-treated thymic progenitors generate a significant lower number of myeloid lineage cells, monocytes and cdc, as well as reduce their capacity to differentiate into pdc lineage. as a possible mechanism for this effect we show that wnt pre-treated progenitors change their expresssion of receptors for cytokines pivotal for their expansion and differentiation, such are il- and flt l and modify the transcription factor profiles of cd + cd thymocytes mainly increasing hes- and id expression levels. human th clones and circulating th cells showed lower susceptibility to the anti-proliferative effect of tgf-beta than th and th clones or circulating th oriented t cells, respectively. accordingly, human th cells exhibited lower expression of clusterin, and higher bcl- expression and reduced apoptosis in the presence of tgf-beta, in comparison with th cells. umbilical cord blood naï ve cd (+)cd (+) t cells, which contain the precursors of human th cells, differentiated into il- a-producing cells only in response to il- beta plus il- , even in serum-free cultures. tgf-beta had no effect on constitutive rorgamma t expression by umbilical cord blood cd (+) t cells but it increased the relative proportions of cd (+) t cells differentiating into th cells in response to il- beta plus il- , whereas under the same conditions it inhibited both t-bet expression and th development. these data suggest that tgf-beta is not critical for the differentiation of human th cells, but indirectly favors their expansion because th cells are poorly susceptible to its suppressive effects. m. irla , w. reith university of medecine, pathology and immunology, geneva, switzerland objectives: medullary thymic epithelial cells (mtecs) are specialized for inducing central immunological tolerance to self-antigens. to accomplish this, mtecs must adopt a mature phenotype characterized by expression of the autoimmune regulator aire, which activates the transcription of numerous genes encoding tissue-restricted self-antigens. the mechanisms that control mature aire(+) mtec development in the postnatal thymus remain poorly understood. however, the generation of mutant mice exhibiting blocks in thymocyte differentiation at different stages, together with studies on embryonic development of the thymus, have demonstrated that reciprocal interactions between developing thymocytes and tec control not only t cell development but also the differentiation and organization of tec, a phenomenon designated as 'crosstalk'. the aim of the project outlined here is to elucidate the cellular and molecular mechanisms by which thymocytes control the numbers of mature mtec, key mediators of central tolerance. we have demonstrated by generating different transgenic mouse models, that although either cd (+) or cd (+) thymocytes are sufficient to sustain formation of a well-defined medulla, expansion of the mature mtec population requires autoantigen-specific interactions between positively selected cd (+) thymocytes bearing autoreactive t cell receptor (tcr) and mtecs displaying cognate self-peptide-mhc class ii complexes. these interactions also involve the engagement of cd on mtecs by cd l induced on the positively selected cd (+) thymocytes. conclusion: this antigen-specific tcr-mhc class ii-mediated crosstalk between cd (+) thymocytes and mtecs defines a unique checkpoint in thymic stromal development that is pivotal for generating a mature mtec population competent for ensuring central t cell tolerance. q. qiu , i. ravens , g. bernhardt hannover medical school, institute of immunology, hannover, germany cd is originally identified as human poliovirus receptor (pvr) and as rodent tage , which is overexpressed in rodent colon carcinoma. cd is also known as necl- , a particular notable nectin-like molecule belonging to immunoglobulin superfamily, owning its unique expressing frofiles. cd expression is very low in most adult organs, but is abundant in the developing or regenerating liver. in addition, cd is overexpressed in transformed cells and promotes the cell cycle. thus, cd seems to be an oncofetal protein that functions in embryonic development and cancer progression. t-cell development is characterized by the progression through several phenotypically distinct stages, defined as double negative (dn), double positive (dp) and single positive (sp) based on expression of the co-receptors cd and cd ; the dn subset is further subdivided into four stages (dn - ) by differential expression of cd and cd . thymocytes at different stages of development occupy distinct spatially restricted domains in the adult thymus, indicating that differentiation occurs concomitantly with a highly ordered migration. during their final maturation in the medulla, semi-mature sp thymocytes down-regulate activation markers and subsequently exit into periphery. while semimature cd + sp are sensitive to negative selection, it remains elusive when negative selection occurs in the cd lineage. here we show that the frequency of terminally matured cd + sp cells but not that of cd + sp present in thymus varies depending on age. in mice lacking expression of the adhesion receptor cd , a selective deficiency of mature cd + sp thymocytes was observed emerging first in adolescent animals at the age these cells start to accumulate in wild type thymus. evidence is provided that the mature cells emigrate prematurely when cd is absent thus cutting short their retention time in the medulla. moreover, in unmanipulated wild type mice semi-mature cd + sp thymocytes are subjected to negative selection as reflected by the diverging t cell receptor repertoires present on semi-mature and mature cd + t cells. in cd deficient animals, a shift in the tcr repertoire displayed by the pool of cd + sp cells was found demonstrating that cd is involved in negative selection. in the adult, steady-state, homeostatic conditions, lymphohematopoietic cell lineages display high rates of cell turnover. yet, the frequencies of simultaneously cycling cells are small, except in intermediate cellular stages of transit-amplifying precursor cell stages. the analysis of the molecular targets controlling these proliferation rates may provide relevant information to understand differentiation pathways along the ontogeny as well as mechanisms of leukemic transformation (passegué et al. j. exp. med. , , . during development, hematopoietic stem cells and their derived cell lineages need to expand to cope with continuously-increasing somatic demands. by using complementary, quantitative analyses (brdu labelings, hoescht , propidium iodide), we are dissecting the proliferation rates of hematopoietic cell lineages and their differentiation stages along the whole mouse gestation from e (e, gestational day) on, in yolk sac, splanchnopleura/agm, blood, liver, spleen and bone marrow. we have observed that around half of cd + ter + erythroid and cd + cd b + myeloid cells are simultaneously cycling (s/g /m) in the post-gastrulation mouse embryo (e - ). the peak of lymphohematopoietic cell proliferation occurs at e in a sort of wave-like pattern. these high-proliferation frequencies are present not only in immature, but also in mature cells, the latter thus displaying a different behaviour from the one present in the adult. later on, the proliferating cell subsets are restricted to fetal liver, whereas the equivalent cells become arrested in the periphery. interestingly, nucleated erythroid cells suddenly go into quiescence - hours before they enucleate, suggesting that this cell arrest is required for the enucleation process. we are also analysing the proliferation state of the first b and t lymphoid progenitors emerging at e - that give rise to perinatal lymphocytes and, in some cases, to innate-like lymphocytes displaying self-renewal in the adult. we attempt to dissect the mechanisms regulating proliferation and death in the embryo versus those of adult lymphohematopoietic precursors, which may influence the functional activities of the mature cells. objectives: the role of cd -cd interactions in t cell activation of antigen presenting cells and b cells is known, but a role for this receptor-ligand pair in hematopoiesis control has not been described. following an initial discovery that b lineage cells in the bone marrow (bm) as early as pro-b cells express cd , we hypothesised a role for cd -cd interactions in the control of b cell haematopoiesis. the objectives of this study were to investigate this hypothesis further. methods: flow cytometry was used to investigate cd expression by precursor b cells using b cell specific markers. reverse transcription of bm stromal cell rna and pcr were used to assess the presence of cd message and cell lineage specific mrna. irradiation and bone marrow transplantation (bmt) in both directions between cd -/-and wt mice was used to assess potential functional contributions of stromal or haematopoietic cd on reconstitution of b cell numbers following depletion. we show that cd is expressed by pro-b cells, and these cells proliferate in response to cd signalling in vitro. pcr identified a source of cd , negative for cd eta, in the bm of wt mice showing this cd is not provided by activated re-circulating t cells. we have shown that when cd -/-mice are recipients, but not donors of bmt, b cell recovery after irradiation is significantly delayed regardless of the donor cell source. in the in vitro experiments we found that the pta gene is expressed from the dn (cd -/cd -/cd + /cd -) to the dp (cd + /cd + ) stage, whereas no yfp expression could be observed in the b lineage. the in vivo analysis of thymocytes confirms the appearance of yfp positive cells during t cell development from the dn stage on. in the bone marrow we found yfp + /b + and yfp + /b populations. thus these pta expression analyses show closely similar pattern to those observed with hucd preta-reporter transgenic mice (gounari f. et al. , martin et al. . the bac pta reporter system can be used together with specific markers of other hematopoietic lineages and their progenitors to trace lymphopoiesis. gounari f et al., nat. immunol. , - ( ) martin c. h. et al., nat. immunol. , - ( . the individual functions and the reason for the tightly regulated expression of igm and igd during b cell development are poorly understood. our data show, that igd requires stronger stimuli than igm to induce b cell activation and that this silences autoreactive vdj recombination products when expressed as igd. in agreement with this, mhc and dhc, the respective heavy chains of igm and igd, differ dramatically in pre-bcr signaling, which represents the prototype of an autoreactive receptor. together with published data, our results reveal a novel role for igd and suggest that the differential expression of igm and igd is important to raise the activation threshold of mature b cells, thereby avoiding hypersensitivity and ensuring tolerance towards self-antigens. p. d. rymkiewicz , g. klein zmf (center for medical research), section for transplantation immunology and immunohematology, tübingen, germany thymic conduits which are exclusively found in the medullary region of the thymic lobules have been recently identified. the core of the conduits consist of fibrillar collagen bundles and is surrounded by a basement-membrane-like structure which contains the typical basement membrane components such as laminins, collagen type iv, nidogens and perlecan. a marker molecule for the conduits in the human thymus is the laminin isoform lm- which is synthesized by the medullary thymic epithelial cells (tecs) which tightly surround the conduits. functionally the conduits are too small to transport cells but they are able to transport small molecules x kda.mmp- , a secreted member of the matrix metalloproteinase superfamily, is a protease capable of digesting lm- . in the human thymus medullary, but not cortical thymic epithelial cells strongly express mmp- . by western blotting the zymogen and the activated form of mmp- can be detected in whole thymus lysates, whereas in lysates from isolated tecs mainly the activated form is present. an in situ zymographic analysis revealed an increased proteolytic activity in the medullary region of the thymus. using confocal laser scanning microscopy double immunofluorescence staining showed that lm- and mmp- can be found in close neighbourhood, but they do not exactly co-localize. why activated mmp- which can be secreted by medullary tecs does obviously not destroy the surrounding basement membrane of the conduits has not been solved so far. two natural inhibitors of mmp- , timp- and timp- , are found in the thymic medulla, but they are not expressed and secreted by tecs. whether mmp- plays a role in processing medullary chemokines which are produced by the thymic epithelial cells is presently under investigation. to study the process of t cells differentiation in more detail, we intend to establish an inducible gene expression system (tet-on system) in primary t cells. the tet-on system comprises two retroviral vectors. the response vector contains an inducible modified minimal cmv promoter which per se is unable to induce expression of the gene of interest (goi). the second vector encodes a transactivator which is constitutively expressed and undergoes conformational changes upon binding of doxycycline. in this state, the transactivator enables the minimal cmv promoter to transcribe the gene of interest. therefore, co-transduction of both vectors is required to achieve transcription of the gene of interest. to date we have tested two tet-on systems (revtet system and retro-x tet-on advanced inducible expression system) that differ in the sequence of their inducible promoters. to monitor successful transfection in retrovirus-generating phoenix cells and transduction in t cells, respectively, we have cloned the reporter gene gfp under the control of a constitutive cmv promoter, into the response vector of the revtet system. this allowed identification of transduced gfp-positive cells via facs. however, when we used a red fluorescent protein, tomato, as a surrogate goi, we detected considerable leakiness of the promoter irrespective of the presence of the transactivator or doxycycline. in contrast, we found comparably low leakiness when using the retro-x tet-on advanced inducible expression system. here, co-transfection of phoenix cells with the transactivator and supplementation of doxycycline yielded an induction of - % compared to only % basal rate. therefore, the retro-x tet-on advanced inducible expression system appears suitable for our studies. future experiments will aim at establishing this system in primary t cells. although a number of different experimental approaches has been used to elucidate impact of basal levels of adrenal gland-derived glucocorticoids (gcs) on t-cell development, and thereby t-cell-mediated immune response, their relevance for these processes is still far from being understood. the study was undertaken to explore relevance of basal levels of gcs for t-cell differentiation/maturation. eight days post-adrenalectomy in adult male rats thymocyte yield, apoptotic and proliferative rate and relationship among major thymocyte subsets defined by tcrab/cd /cd expression were examined using flow cytometry analysis. it was found that adrenal gc deprivation affects: i) thymocyte apoptosis, producing thymic hypercellularity and ii) kinetics of t-cell differentiation/maturation leading to an overrepresentation of the cd +cd + double positive (dp) tcrab low cells entering selection, and their cd +cd + dp tcrab-immediate precedents followed by underrepresentation of the selected cd +cd + dp tcrab high and the most mature cd -cd + and, particularly, cd +cd -single positive (sp) tcrab high cells. the study suggests that withdrawal of adrenal gcs produces alteration in thymocyte selection processes that may affect diversity of functional t-cell repertoire and generation of potentially self-reactive cells as indicated by the reduced proportion and number of cd -cd -double negative tcrab high cells. in addition, it indicates that gcs influencing post-selection maturation of thymocytes play a regulatory role in controlling mature cd +cd -/cd -cd + sp tcrab high cell ratio. in the thymus a specific subset of thymic stromal cells -medullary thymic epithelial cells (mtecs) -express a highly diverse set of tissue-restricted antigens (tras) representing essentially all tissues of the body, which is known as promiscuous gene expression (pge). this allows self-antigens, which otherwise are expressed in a spatially or temporally restricted manner to become continuously accessible to developing t cells. the scope of central tolerance is to a large extent dictated by the pool of promiscuously expressed genes. thus, even lack of a single tra can result in spontaneous organ-specific autoimmunity. promiscuously expressed gene which have no structural or functional commonality display two prominent features, they are highly clustered in the genome and show a preference for tras. for better understanding these features, we set out to precisely define the genomic organization of this gene pool. in particular, we probed to what extent and according to which rules predefined genomic clusters of tras are transcribed in mtecs. our analysis proceeded from the bioinformatic definition of tra clusters via gene expression analysis in mtecs using whole genome arrays to the in depth analysis of selected tra clusters by rt-pcr at the population and single cell level. patterns emerging from these studies will hopefully yield insight into evolutionary mechanisms responsible for selecting this gene pool. conceivably, positional cues in the genome and/or particular properties of self-antigens (e. g. immunogenicity) could have been driving forces during the co-evolution of pge and adaptive immunity. although catecholamines have been shown to influence thymocyte proliferation and differentiation, long-lasting b-adrenoceptor (ar) blockade failed to show any significant effects on thymic cellularity. bearing that in mind, the present study was undertaken to explore: i) a -ar expression on thymic lymphoid and nonlymphoid cells and ii) putative role of a -ar-mediated mechanisms in modulation of thymic cellularity and t-cell development. for this purpose a -ar expression on thymic cells was assessed using both immunocytochemistry and flow cytometric analyses, while their putative modulatory role in thymopoiesis was estimated by analyses of thymocyte proliferation and apoptosis, as well as expression of major thymocyte differentiation antigens (cd / cd /tcrab), in adult wistar rats subjected to -day-long treatment with a -ar blocker urapidil ( . mg/kg body weight/day s. c.). the a -ar immunoreactivity was found in both thymocytes (mainly less mature cd and cd low cells) and thymic nonlymphoid cells (thymic epithelial cells located mainly at cortico-medullary junction and cortical ed -postive cells, which comprise macrophages and dendritic cells). chronic treatment with urapidil increased thymic weight and caused the organ hypercellularity. the thymic hypercellularity reflected, at least partly, increased frequency of proliferating thymocytes, which was followed by diminished thymocyte apoptosis. in addition, in these rats changes in distribution of major thymocyte subsets delineated by cd / cd /tcrab expression were observed. these changes comprised of an increase in the percentage of cd + + tcrabthymocytes, which was accompanied by the reduction in that of cd + + tcrab low cells in urapidil-administered rats, and divergent changes in the percentage of the most mature single positive tcrab high thymocytes. compared with saline-administered controls, the percentage of cd + -tcrab high thymocytes in urapidil-administered rats was increased, while that of the cd - + tcrab high was reduced. in addition, the percentage of cd + t regulatory and cd +tcrab+ nkt cells was increased. collectively, this study clearly showed the expression of a -ar on both lymphoid and nonlymphoid thymic cells, and indicated that a -ar-mediated mechanisms may be implicated in modulation of multiple steps of t-cell development. we have recently shown that the thymus is a common target for mycobacterial infections. of notice, while bacterial growth is arrested in the spleen around weeks post infection with mycobacterium avium, it takes several weeks longer within the thymus to reach a bacterial plateau. this observation suggests that a specific immune response occurs in the thymus, although this seems to be distinct from that occurring in the spleen. since t cell differentiation occurs, to a large extent, in the thymus, and depends, among other factors, on the antigens encountered within the thymus and on the cytokine milieu of the organ, we decided to characterize the pattern of the thymic immune response against mycobacteria and investigate possible consequences on the normal function of this organ. methods: c bl/ mice infected with m. avium ( cfus, iv) were sacrificed at different time points after infection ( , and weeks). non-infected animals were used as controls. bacterial load was assessed in the spleen and thymus and cytokines (such as ifn-g, tnf, il- ) were quantified by rt-pcr in tissues (normalized for hprt expression) or by elisa in the supernatants of cultured thymocytes and splenocytes. statistical significances were determined by anova. we observe increased levels of ifn-g in infected thymi at weeks post infection. this increased expression of ifn-g is concordant with the late bacterial growth arrest within the thymus. at weeks post infection this difference is still present. throughout the course of infection no significant differences are found in the expression of tnf and il- in this organ. in the spleen, ifn-g reaches a peak of expression earlier ( weeks post infection) and this is accompanied by increased tnf expression. conclusion: our cytokine analysis of the thymus and spleen confirm that an immune response against mycobacteria is mounted within the thymus, although different in timing and pattern from the one in the spleen. since the cytokine milieu influences t cell differentiation within the thymus, our observations raise the question on the consequences of such response on the normal function of this organ. such implications should next be investigated. precise regulation of eukaryotic gene expression requires interactions between distal cis-acting regulatory sequences with the looping out of the intervening dna, but how trans-acting regulatory proteins work to establish and maintain dna loops during gene activation remains largely unexplored. lps-induced transcription of the mouse igx gene in b lymphocytes utilizes three distal enhancers and requires the transcription factor nf-xb, whose family members include rela and c-rel. using chromosome conformation capture technology in combination with chromatin immunoprecipitation, here we demonstrate that lps-induced igx gene activation creates chromosomal loops by bridging together all three pair-wise interactions between the distal enhancers and rna polymerase ii, the apparent molecular tie for the bases of these loops. rela and actin polymerization are essential for triggering these processes, which do not require new transcription, protein synthesis or c-rel. we have thus identified both essential and non-essential events that establish higher-order chromatin reorganization during igx gene activation. this investigation was supported by grants gm and ai from nih and grant i- from the robert a. welch foundation to wtg, and by grants hl and hl from nih to lst. allelic exclusion of immunoglobulin (ig) genes supports burnet's clonal selection theory. the recognition that m-chain expression is sufficient for the maintenance of the silenced allele status by a process of feedback inhibition is yet not enough to explain the earlier monoallelic activation by the rag complex. attempts to prove the probabilistic or epigenetic nature of monoallelic v(d)j recombination were insightful and favor the epigenetic hypothesis, mainly by the observation that, like autosomal imprinted or x-chromosome inactive genes, ig genes are differentially marked at the chromatin level, and replicate asynchronously in virtually any cell of the mouse organism ever since the embryonic life (mostoslavsky, singh et al. ) . we are testing this hypothesis, i. e., that an epigenetic event has previously marked, on each b cell progenitor, which of the ig alleles is going to be activated for rearrangement and expression. for this, we are generating b cell clones from b cell progenitors of (c bl/ x balb/c)f mice, because the original strains have ig heavy chain (igh) alotypes distinguishable by monoclonal antibodies. we are analyzing if individual heterozygous clones show biased expression of a particular igh allele, and we expect to map the cell stage at which the (epigenetic) allelic marks are fixed. we have already shown, for the igh gene, a clear segregation of monoallelic expressors among b cell clonal lines that were generated from the common lymphoid precursor, but no allele bias was observed among multi-potent progenitor or hematopoietic stem cell b cell clones. this result, although suggesting epigenetic silencing starting at the common lymphoid precursor stage, does not favor the prevailing epigenetic hypothesis in its original formulation. we are currently exploring this result by the analysis of the igh silenced allele rearrangement status in sorted fractions of igm+ b cell clones. we are also testing the same epigenetic hypothesis for the ig kappa light chain gene (igk), using f mice in which the igk constant region from both alleles can be distinguished by antibodies. a. giniewski , s. lang , m. stein , t. winkler friedrich-alexander university, erlangen, germany vdj recombination is considered to be regulated by lineage and stage specific changes in accessibility of the loci to rag recombinase. accessibility is expected to correlate with certain histone modifications such as acetylation (e. g. h ac) or methylation of h on lysine (h k me / ). previous studies in our lab revealed three regions in the intergenic part of the distal v h cluster (ivars), which are associated with high levels of active chromatin marks (h ac and h k me / ) only in pro b cells but not in pro t cells. it is also known that vdj h recombination is accompanied by sense and antisense transcription, however, little is known about the function and origin of the antisense transcripts. since one ivar (ivar # ) shows promoter activity in antisense orientation, it was analysed in more detail. we found three transcription start sites by 'rlm race at the ivar # element. background: t-all is a malignancy of the lymphoblast committed to the t-cell lineage with translocations between tcr genes and oncogenes as a genetic hallmark. these translocations are thought to be driven by v(d)j-recombination mechanisms. we believe that these mechanisms only partly facilitate the occurrence of tcr translocations and that the accessibility of involved genes plays an intrinsic role in promoting these events. the lmo locus, is thought to be accessible only during the double-negative (dn) and thymocyte stages based on mrna expression, implying that translocations between lmo and tcr genes can only occur within these stages. gene expression as a readout for accessibility can not elucidate the involvement of other oncogenes such as tlx (hox ), which are not expressed in thymocytes, as being accessible for translocations to occur. objectives: we aimed ) to evaluate lmo and tlx breakpoint-site accessibility during thymocyte development; ) to determine in which stage of development there is an increased chance for lmo or tlx translocation to occur based on this accessibility. methods: dna of immunologically "healthy" sorted thymocytes was isolated using faire (formaldehyde-assisted isolation of regulatory-elements). this dna was used to quantitatively assess lmo accessibility during thymocyte development at both the transcription start site (tss) and negative regulatory element (nre), and within different in t-all documented breakpoint-sites of both the lmo and tlx loci. results: quantitative analysis on the tss showed a correlation with mrna expression, with the dn and dn development stages showing the highest accessibility. the nre, showed an inverse pattern of accessibility to the tss region. analysis of breakpoint-sites revealed the highest accessibility levels within the earliest stages of development, dn , dn , dn and immature single positive (isp) stages for both lmo and tlx . conclusion: our findings show that both the lmo and tlx loci are accessible during thymic development irrespective of gene expression and that this accessibility is not restricted to dn and dn stages, suggesting that these loci are much more active than assumed, thus increasing the opportunity for translocations to occur. we addressed this issue, by building a model able to account for of v-ja gene rearrangements observed experimentally during thymus development of mice. we developed, based on experimental data, a numerical model on the whole tra/trd locus to estimate va and ja genes accessibility to rearrangements. the progressive opening of locus to v-j gene recombinations is modeled through windows of accessibility of different sizes and with different speeds of progression. furthermore, the possibility of successive secondary v-j rearrangements was introduced in the modeling. the model points out some unbalanced v-j associations resulting from a preferential access to gene rearrangements and from a non-uniform partition of the accessibility of the j genes, depending on their location in the locus. the model shows that to successive rearrangements are sufficient to explain the use of all the v and the j genes of the locus. finally, the model provides information on the kinetics of rearrangements and on the frequency of each v-j association. the model accounts for the essential features of the observed rearrangements on the tra/trd locus and may provide a reference for the repertoire of the v-j combinatorial diversity. the genetic programs of b-cell differentiation and the first dj h gene rearrangements appear in the post-gastrulation mouse embryo (e - ), shortly after the first multipotential hematopoietic progenitors do emerge. these dj h joints represent the unselected baseline of the ig repertoires. we have undergone a systematic sequencing of embryo dj h joints obtained from normal balb/c embryos and heterozygous embryos obtained from rag -/mothers mated to balb/c males (to discard any mother-derived contribution), as well as newborn and adult control groups. the embryo dj h s displayed unexpected mechanisms of diversity, including short stretches of non-templated n nucleotides in one-third of the studied sequences (in the absence of tdt expression) and frequent dj h s with large nucleotide deletions, as a consequence of ligation to joint-distal microhomology sites. because the dna polymerase m (polm), a highly-homologous tdt member of the x dna polymerase family, showed an increased expression in the embryo, we analysed the dj h s of polm -/mouse embryos. we observed that polm was mainly responsible for introducing n nucleotides at the mouse embryo dj h joints. also, and based on its dna-dependent polymerization ability, polm filled-in small sequence gaps at the coding ends, and ligated highly-processed ends by pairing to internal microhomology sites, although at the cost of germline sequence losses and the generation of "useless" gene products. we think that, more than attempting to increase diversity, polm acts as a "connector" in the embryo, subsequently participating in the repair of rag-induced double-strand breaks, to preserve genomic stability and cellular homeostasis in cells with high proliferation rates. along the end of gestation, further selective pressures acting over these first v-dj h products will contribute to establish the differential neonatal ig repertoires. although mortality from infectious diseases peaks during infancy, many vaccines are ineffective in early life. most children with infantile bronchiolitis are under months of age, and most cases are due to respiratory syncytial virus (rsv) infection, for which vaccines continue to be elusive. we now show that, compared to adults, the antibody response to rsv infection is very poor in neonatal mice and is unaffected by cd cell depletion. however, cd depletion in infancy led to a remarkable boosting of antibody responses during adult re-challenge. to test the possibility that poor antibody boosting is caused by rsv-specific cd t cells killing rsv-infected b cells, we sorted cells from the lungs of infected neonates. viral copy number was high in neonatal b cells, but viral load in surviving b cells was unaffected by cd cell depletion. in addition, fas ligand (fasl) deficient gld mice responded to rsv infection in the same way as normal mice, indicating that fasl is not required for the inhibition of antibody responses. this new mechanism of regulation of b cell responses by cd t cells has important implications for vaccine development against neonatal infections. ( ) showed that irf knockout mice had significantly reduced numbers of pre-pro-b cells in marrow and a phenotype similar to agammaglobulinemia. these results prompted us to consider icsbp/irf as a candidate gene in the pathogenesis of defective early b cell development. therefore we decide to undertake direct sequencing of the gene encoding icsbp/irf in a small cohort of patients with autosomal recessive agammaglobulinemia. methods: eight patients affected by agammaglobulinemia were included in this study. all patients were under regular ig replacement therapy. informed consent was obtained from all patients. genomic dna was extracted from whole blood and amplified with specific primers designed on the flanking regions of every exon. direct gene sequencing of the eight exons of icsbp/irf were obtained using abi prism sequencer. results: seven of the eight patients result wild type while only one patients present a synonymous snp in exon v, yet documented as rs . conclusions: although recent findings indicated that irf function is essential for early b cell development, our data in a small cohort of patients affected with autosomal recessive agammaglobulinemia did not evidence any mutations in icsbp/irf that may be responsible for this disorder. the hh/ptch signaling system is known to control the development and neoplastic transformation of several cell types. however, the role of hh/ptch for the differentiation of b and t lymphocytes from hematopoietic stem cells (hsc) has not been assessed so far. to analyze the function of hh/ptch for lymphopoiesis in vivo, we have employed a genetically engineered mouse mutant in which the ptch gene can be conditionally inactivated by virtue of the cre/loxp recombination system. we show that targeted disruption of ptch in the adult organism results in a dramatic specification and differentiation defect of the lymphoid lineage leading to rapid disappearance of newly generated b and t lymphocytes from peripheral lymphoid organs. the developmental block occurs at the level of the common lymphoid progenitor cell (clp), which defines an early branching point of hsc differentiation and lineage commitment. in contrast to the lymphoid lineage, development of cell types of the myeloid lineage from common myeloid progenitors (cmp) appears normal. our data identify hh/ptch-induced signaling as a key regulator for proper development of immunocompetent lymphocytes. hence, the progression of tumors, which are initiated upon oncogenic hh/ptch mutations, may be further promoted due to impaired tumor surveillance by a compromised immune system. l. calderon dominguez , t. boehm max-planck institute for immunbiology, developmental immunology, freiburg, germany in many organ systems of animals and plants, specialized niche microenvironments maintain and specify stem and progenitor cells. the ability to modify or artificially create such niches in vivo and in vitro has many implications for stem cell research and therapy. by analysis of several mutant mouse strains and subsequent transgenesis in the mouse, we disentangle and individually modulate niche functions responsible for collection, maintenance and specification of multipotent thymocyte progenitors. we demonstrate how an epithelial niche, rendered functionally inactive by disruption of the foxn transcription factor, can be specifically rebuilt in a modular and combinatorial fashion to only attract, or attract and maintain, or attract, maintain and specify progenitor cells into the b and t cell lineages, respectively. the strategy of engineering niche functions in a modular fashion might be applicable to other progenitor cell systems. silencing of dc-sign using lentiviral rna interference revealed its critical function for pd-l expression on dcs after m. tuberculosis infection. as a counterpart to expression of its ligand, we showed that cd and cd t cells from tuberculosis patients highly express pd- when compared to healthy uninfected individuals. in addition, analysis of pd- expression in lung biopsies from tuberculosis patients revealed that pd- is expressed on cd and cd t cells confined to lung granulomatous lesions. finally, blocking of the pd- /pd-l axis using monoclonal antibodies abrogated the down-modulation of t cell proliferation and ifn-g production induced by manlam, a mycobacterial cell wall glycolipid and ligand for dc-sign. taken together, our results suggest that the pd- /pd-l pathway is involved in the exhaustion of t cell responses to m. tuberculosis. the inflammatory canonical nfkb pathway is critically involved in virtually all aspects of inflammation in general. yet, the role of the alternative, non-canonical nfkb pathway in inflammation and adaptive immunity remains largely elusive. the alternative pathway is primarily mediated through the nfkappa-b inducing kinase (nik) which in turn leads to the phosphorylation and the cleavage of p to p . among the receptors engaging nik is the ltbr, which is also required to form the anlage for secondary lympoid tissues (slts). due to a point mutation within nik, alymphoplasia (aly) mice do not develop slts and are highly immunodeficient. however, while the immunodeficiency of aly mice is widely held to stem from their developmental malformation, it has been overlooked, that the mutation of nik itself could potentially lesion the development of immune responses. to verify this notion, we generated a series of bone marrow chimeric mice (bmc) in which the absence of slts was disconnected from the hematopoietic loss of nik function. we generated mice, which lack all slts, but are equipped with a normal systemic immune system (wt aly), and conversely, mice with normal slts, but lacking nik in all leukocytes (aly wt). surprisingly, we discovered that nik is vital for the development of autoimmune disease, while slts (ie. lns, spleen etc.) are essentially dispensable for cell-mediated immunity. we found that nik is required for the polarization of effector t cells and that th and th cells cannot be generated in the absence of nik. preliminary data implicate the involvement of nik in a discrete and novel pathway required for the formation of cell-mediated immune responses. the family of nfat (nuclear factor of activated t-cells) transcription factors is indispensable for t cells, for example playing an important role in cytokine gene regulation. in peripheral cd + t cells, nfatc and c are predominantly expressed. nfatc is synthesized in six isoforms which have partly opposing functions regarding activation and apoptosis. here we address the functional difference of the short isoform nfatc /a, which is highly induced upon t cell activation, and the long constitutively expressed isoform nfatc /c. as demonstrated by y h screen and co-ips, nfatc /c-specific c-terminus can be highly sumoylated. confocal microscopy studies revealed that upon sumoylation nfatc /c -but not the unsumoylated nfatc /a -translocates to promyelocytic leukemia-nuclear bodies (pml-nbs). this leads to interaction with hdacs followed by deacetylation of histones (co-ips), which in turn induces transcriptionally inactive chromatin (chip and confocal microscopy). as a consequence, multiple expression studies revealed sumoylation dependent suppression of the nfatc target gene interleukin- . other lymphokines like ifng and il are reversely regulated. interestingly, ntreg cells which do not express il exerted only nfatc /c, but no nfatc /a expression (qrt-pcr). these findings demonstrate that the modification by sumo converts nfatc from an activator to a site-specific transcriptional repressor, revealing a novel regulatory mechanism for nfatc function. therefore, especially ntreg cells and anergized cd + t cells might be regulated by the long sumoylatable isoform nfatc /c. lnk/sh b and aps/sh b , two members of the lnk/sh b family of adaptor proteins, play an important role as negative regulators in b cell lymphopoiesis. they possess several protein-protein interaction domains and motifs that allow their interaction with different signalling effectors. mice deficient for these proteins demonstrated that lnk inhibits expansion of pro/pre-b cells while aps controls mature b- cell population, suggesting specific roles for these adaptors during b cell development. however, the molecular mechanisms underlying their regulatory function in these cells, have not been identified. to address this question, we used primary and b cell lines at different stages of differentiation as our cellular system. analysis of lnk/aps expression pattern showed that lnk is expressed at all developmental stages, while aps is only detected in immature and mature cells. we then first examined the role of lnk in il- signalling in pre-b cells overexpression of lnk dramatically inhibits il- -dependent growth demostrating that lnk negatively regulates il- pathways. furthermore, we showed that il- stimulation induces lnk phosphorylation and its subsequent association with important signalling effectors, notably the e ubiquitin ligase cbl. we next analyzed the role of aps in mature b cells by imaging and biochemical techniques. our results showed that aps colocalizes with the bcr complex after bcr triggering. interestingly, lnk is not recruted to the bcr signalosome in these cells, suggesting that interaction of the adaptors with the receptor complex regulates their function at different development stages. moreover, we showed, for the first time, that aps can associate, upon bcr stimulation, with the signalling molecules cbl and vav . to address the functional implications of these interactions, we examined specific b cell responses, notably bcr trafficking and cytoskeleton remodelling. we demonstrated that overexpression of aps enhances ligand-induced endocytosis of bcr, possibly through interaction with cbl and affects the kinetics of bcr-induced cell spreading. our results therefore suggest a regulatory function of aps in bcr internalization and cytoskeleton dynamics. altogether, our findings demonstrate that lnk and aps display sequential specific regulatory roles during b cell development that are important for maintaining b cell homeostasis. signaling through the t-cell receptor (tcr)-cd complex is a critical event in adaptive immunity. it is still not clear how ligand binding to the tcr is communicated across the plasma membrane and leads to phosphorylation of the cytoplasmic domains of the cd complex. it is widely accepted that dimerization or multimerization of tcr is required for tcr triggering. in our model t-cell activation is initiated by recognition of monomeric mhc/peptide complexes on the surface of antigen presenting cells (apc). critical to tcr triggering is the movement of the t-cell across the apc. engagement of a mhc/peptide complex on the surface of the apc will change the mobility of the tcr leading to partitioning with lipids of lower mobility that are enriched in signaling molecules critical for t-cell activation. furthermore, the change in mobility will lead to dislocation of the itams from the plasma membrane so that they become accessible to tyrosine kinases. to test the hypothesis we established a new approach where we created a soluble bifunctional complex composed of a pmhc and a fab that recognizes an epitope tag that we express on the t-cell surface. binding of the fab to the expressed epitope tag will constrain the lateral mobility of the tcr that is engaged by the pmhc arm of the same complex. the bifunctional complexes induced activation and proliferation as well as ca influx and cytokine production in human cd + t-cell clones that displayed the epitope, but not in t-cells that did not display the epitope. activation required interaction of the fab with its epitope on the t-cell surface because no activation was observed when soluble epitope peptide, which acts as a competitor for the fab binding site, was added. these results demonstrate that a monomeric copy of a pmhc is sufficient to trigger tcr and that formation of a tcr dimer is not an obligatory step in t-cell activation. the bifunctional complex we generated may also have a great immunotherapeutic impact. exchanging the fab with a fab or cytokine directed to a surface molecule may allow an antigen specific stimulatory or inhibitory modulation of t-cell responses. adaptor proteins are crucial in signal transduction, cell cycle regulation, apoptosis and stress response. adaptor proteins containing characteristic sh or sh domains known to mediate protein-protein interactions are key players in these processes. sly (sh domain protein expressed in lymphocytes ) was identified as a putative adaptor protein containing a sh and a sam domain as well as a bipartite nls. sly belongs to a family of three molecules: sly , sly and sash .in humans, the sly gene is located on chromosome , in mice on chromosome . sly is widely expressed for example in immune tissue as well as in hematopoietic cells, brain, lung and pancreas. subcellular fractionation showed that the sly protein is located in the cytoplasm and the nucleus and to a lesser extend in the plasma membrane.to elucidate the function of sly we searched for possible interaction partners by yeast two hybrid screening with a mouse t cell lymphoma library. this approach identified sin -associated polypeptide p (sap ) as a putative interaction partner of sly . sap is a conserved member of the sin a-hdac corepressor complex that contains histone deacetylase (hdac ) and histone deacetylase (hdac ) and acts as a transcriptional repressor for a variety of genes. we confirmed this interaction by implementing coimmunoprecipitations with lysates from transiently transfected t cells. in addition, we could show a direct interaction between sly and hdac . to investigate the functional impact of this molecular interaction, we performed hdac enzymatic activity assays. we were able to show that sly increases the activity of hdac in whole cell lysates and, more precisely, in nuclear extracts of t cells. the interaction of sly with sap and hdac indicates a transcriptional function of this protein. within the sin a-hdac corepressor complex sly might act as a switch for the activity of hdac . cd -cyt and cyt are co-expressed in human t cells and undistinguishable from the cell surface. in order to determine their specific role in t cell activation, we have expressed chimeric proteins consisting of the extracellular domains of cd (b cell marker) fused to the transmembrane and intracellular domain of cd -cyt or cyt in primary t cells. we show that these two isoforms differently control human t cell function. specific cyt coengagement controlled il- secretion, while cyt coligation inhibited ifng production. moreover, our preliminary data suggest that cd -cyt inhibits the phosphorylation of several molecules known to be activated by cd stimulation. these data suggest that these two isoforms act as molecular switches for t cell activation, either promoting or turning off t cells. they demonstrate for the first time the distinct roles of cd cytoplasmic isoforms in primary human t cell activation. this also suggests that the modulation of their expression and/or activation might provide new therapeutic avenues. nck is a ubiquitously expressed adapter protein that is almost exclusively built of one sh domain and three sh domains. nck connects receptor and non-receptor tyrosine kinases to the machinery of actin reorganisation. in t cells, nck participates in different and interdependent signalling pathways linking t cell activation and effector function with actin remodelling proteins that in turn initiate changes in cell polarity and morphology. we previously showed that nck directs the death factor fasl to the cytotoxic immunological synapse when t cells encounter putative target cells. we now performed a systematic screening for interaction partners of the four individual interaction modules of nck in primary and leukemic t cells. we precipitated putative binding partners from untreated or pervanadate-treated pha blasts, jurkat and hut cells with gst fusion proteins containing full length nck, the three sh domains or the individual sh and sh domains. binding proteins were excised from gels after staining with coomassie, silver or flamingo pink and processed by tryptic in gel digestion for mass spectrometrical analysis. as expected, we observed major differences in nck binding proteins precipitated from resting versus activated t cells. we not only verified established interactions (e. g. with the tcr signalling components slp and cd epsilon, the actin-regulatory proteins wasp and wip and the nuclear protein sam ) but also identified novel nck-interacting proteins. the interaction with the actin-binding protein hip once more underscores the fundamental role of nck in tcr-mediated actinreorganization. the identification of the nuclear proteins sfpq/nono points to novel, yet unknown functions of nck that might be associated with the recently reported nuclear translocation/localization of nck. accordingly, employing laser scanning microscopy, we clearly detected nck within the nucleus also in human t cells. the present data highlight that nck serves versatile functions in t cells, which include the different interdependent pathways of tcr-induced actin reorganization but also novel, yet poorly defined protein networks that are associated with a nuclear translocation of nck. cytotoxic t lymphocytes (ctl) mediated killing is tightly regulated according to the strength of t cell receptor signal. killing is regulated by the delivery of perforin-containing lytic granules moving along microtubules towards the centrosome, which polarizes and docks at the central supramolecular activation complex (csmac) within the immunological synapse. although much has been learnt about the mechanisms controlling the strength of tcr signal and the mechanisms required for release of the lytic granules, little is known about how the strength of the tcr is able to control the degree of ctl-mediated killing so finely. here we examine how the strength of tcr signal controls polarization of the secretory apparatus leading to ctl-mediated killing using tcr transgenic ot-i ctl. decreasing the tcr signal by reducing the concentration of ova peptide or using the weak agonist peptide, g , results in a slight reduction in the number of ctl target cell conjugates formed, and the number of conjugates in which a csmac (visualized by a patch of lck-staining at the immunological synapse) was formed. tcr signals result in reduced or absent (in the case of g ) staining with psrc and perk antibodies in the immunological synapse and reduced or absent (g ) degranulation as measured by cd a assays. the centrosome docks at the csmac of the immunological synapse even with relatively weak tcr signals, but the lytic granules require a certain threshold of signaling to successfully polarize to the immunological synapse. inhibitors support a role for pi k in granule polarization. together these data demonstrate that the strength of tcr signal controls the level of ctl mediated killing at the single cell level by controlling, the number of conjugates formed, the formation of the csmac and the accumulation of psrc and perk at the synapse. the centrosome polarizes to the csmac even with relatively weak tcr signals, but granule recruitment requires a higher threshold of signaling. these findings reveal how ctl can fine tune the degree of killing in response to tcr signals at the single cell level. cytotoxic t cells play an essential role in the immune system, particularly in the elimination of tumor and virus-infected cells. cytolytic t-cell activity is mediated through the pore-forming molecule perforin allowing granzymes to enter the target cell and to initiate apoptosis. perforin and granzymes are stored in specialized secretory granules, called secretory lysosomes. they are capable of undergoing regulated secretion in response to a t cell receptor engagement which involves binding to a cognate mhc class i-peptide complex. the intracellular transport of lysosomal proteins from the golgi to the lysosomes is mediated by the cationindependent mannose- -phosphate receptor which exhibits structural and functional similarity to the vps p-receptor sortilin. sortilin was characterized predominantly in neuronal cells where its function in protein sorting was identified. in the secretory pathway, sortilin is putatively involved in trafficking of proteins in the constitutive and regulated pathway. to explore whether sortilin has a broader functional relevance, we asked if sortilin might act as an alternative receptor for the cation-independent mannose- -phosphate receptor in cytotoxic t cells. first, we demonstrate that sortilin is expressed in t cells. to examine its function during an adaptive immune response, we analysed sortilin-deficient cytotoxic t cells derived from a knockout mouse strain. in strong contrast to the results reported from neuroendocrine cells, we obtained a reverse phenotype in sortilin-deficient cytotoxic t cells. whereas the regulated release of secretory lysosomes was enhanced, the constitutive release of interferon-g was found to be decreased. the enhanced release of cytotoxic molecules from sortilin-deficient cytotoxic t cells translated into an increased cytotoxicity in vitro. thus, the deletion of sortilin imposed a specific phenotype in cytotoxic t cells which could not be compensated for by other sorting receptors. our localisation studies of sortilin in t cells were consistent with the results previously described in neuronal cells which indicated that sortilin acts as a sorting receptor during the anterograde transport of lysosomal hydrolases from the trans-golgi-network to endosomes and lysosomes. taken together, we suggest that sortilin might play a modulatory role in the regulation of the adaptive immune response through the control of the constitutive and regulated secretory pathway. there is growing interest in the soluble splice variant of ctla- (sctla- ) as an immune inhibitor secreted by t cells, because genetically determined variation in its production is associated with susceptibility to autoimmune disease. however, little is known of the biology of sctla- in immune responses. using a specific anti-human soluble ctla- monoclonal antibody, jmw- b that selectively binds the soluble isoform but not membrane bound ctla- , or cleaved fragments of it, we demonstrate that sctla- plays a vital role in regulating antigen-specific immune responses. we used antibody blockade to show that antigen-specific t cell responses are strongly enhanced upon blockade of sctla- , secreting increased amounts of cytokines including interferon-g, il- and tnf-a, but lower amounts of il- . soluble ctla- was also prepared from sera for use in experiments by antibody based affinity purification techniques. addition of sctla- induced secretion of the immunoregulatory cytokine il- by human pbmcs both in an antigen-selective and dose-dependent manner, while antibody blockade abrogated that effect. the immunosuppressive indoleamine , dioxygenase enzyme cascade was also initiated by sctla- . it is clear that the importance of this natural soluble molecule has been overlooked and like membrane-bound ctla- it is crucial to t cell inhibition. membrane-bound ctla- exists as a homo-dimer on t cells but sctla- is usually considered to be monomeric in form, implying its functional capacity is diminished because of an inability to cross-link b ligands on antigen presenting cells. a third important observation from this study is that sctla- exists both in serum and culture supernatants as a natural kda homo-dimer, and not as a monomer. this goes some way to explaining why this molecule has such potent immunoregulatory effects on antigen-specific immune responses. together, these results lead us to reappraise sctla- , concluding it to be a mediator of negative feedback, secreted as a recall regulatory t cell response to antigenic stimulus, rather than a product of resting t cells. this work also raises the possibility that where il- dependent regulation is most critical, boosting sctla- secretion by regulatory t cells could be a novel therapy for immune mediated diseases. recently, we identified a new adaptor protein, swiprosin- /efhd- , in lipid rafts of b cell lines that undergo apoptosis after b cell receptor (bcr) stimulation. swiprosin- /efhd is expressed in immature b cells of the bone marrow, in resting and activated splenic b cells, in t cells, macrophages, mast cells and some nonlymphoid tissues. ectopic expression of swiprosin- /efhd- in the immature murine b cell line wehi enhanced spontaneous and bcr-induced apoptosis. in contrast, shrna-mediated down-regulation of swiprosin- /efhd- impaired spontaneous and bcr-elicited apoptosis, but not bcr-induced g cell cycle arrest. to understand how swiprosin- /efhd enhances pro-apoptotic bcr signals, we analyzed whether swiprosin- /efhd is involved in proximal bcr signalling. in fact, ectopic expression of swiprosin- /efhd enhanced bcr-induced calcium flux in wehi cells, whereas shrna-mediated down-regulation of swiprosin- / efhd impaired bcr-elicited calcium signals. concomitantly, gst-pulldown experiments revealed that swiprosin- /efhd interacts with phospholipase cg (plcg ) and with the tyrosine kinase syk (splenic tyrosine kinase), both of which are important for bcr-induced calcium flux. the interaction of plcg and swiprosin- /efhd was further established by co-immunoprecipitation. reconstitution of bcr-elicited calcium signals through complementation of swiprosin- /efhd silenced wehi cells with swiprosin- /efhd- was inhibited by the syk inhibitor bay - . in analogy, swiprosin- /efhd regulated syk activity positively. moreover, swiprosin- /efhd re-expression accelerated tyrosine phosphorylation of several proteins, specifically tyrosine phosphorylation of plcg and of syk tyrosine residue , which is involved in syk activation. finally, reconstitution of swiprosin- /efhd knock-down cells with swiprosin- /efhd mutants revealed that the n-terminal putative sh -binding site, the first ef-hand, and to a lesser extent, the second ef-hand and the c-terminal coiled-coil domain, are important for bcr-induced calcium flux in wehi cells. interestingly, swiprosin- /efhd re-expression in swiprosin- /efhd -silenced cells induced already in unstimulated cells raft partitioning of syk, plcg and the bcr, which was reversed after min of bcr stimulation. in summary, swiprosin- /efhd is an accelerator of proximal bcr signalling and acts through syk and plcg by assembling a syk-dependent calcium initiation complex in lipid rafts. this might be relevant for memory b cell signalling or central b cell tolerance. to test the biological relevance of cbl-b e ligase activity, these mice were analyzed for t cell proliferation, susceptibility to autoimmunity, in vivo t cell tolerance responses, and tc tumor rejection. results: when stimulated, t cells from rf mutant mice hyperproliferate compared to wild type t cells, even in the absence of cd co-stimulation. preliminary data also suggest that rf mutant mice are more susceptible to autoimmunity. in addition, rf/p mice die within hours after a second challenge with p peptide, indicating a severe defect in t cell tolerance induction. more importantly, cbl-b e ligase dead mice can spontaneously reject tc tumors. conclusion: cbl-b e ligase dead mutant mice phenocopy total body cbl-b knock out mice, thus indicating that cbl-b e ligase activity is indispensable for its regulatory in vivo functions. intriguingly, our data suggest that its inactivation could be sufficient to confer anti-tumor activity. to further elucidate the cellular mechanism of cbl-b mediated tumor rejection we have now generated the conditional cbl-b e ligase dead mutant mice to for the first time study the cbl-b ubiquitination function in a tissue specific and temporal fashion. our research is also currently focused on identifying the relevant in vivo cbl-b ubiquitination substrates. interferon alpha (ifn-a) has been broadly used in the treatment of specific malignancies and chronic viral diseases. for a long time it was thought that the direct inhibitory effects on malignant or virus infected cells were the major mechanisms involved in the response to ifn-a therapy. however, recent studies in mice have revealed that ifn-a/b also exerts effects on several host immune cells. ifn-a has been shown to enhance cd t cells (ctls) responses against soluble antigens in mice. this immunostimulatory activity of ifn-a results at least partly from its direct ability to induce maturation of dendritic cells. several studies have recently demonstrated that ifn-a/b also acts directly on murine ctls, inducing clonal expansion and differentiation into effector and memory cells. to date, little is known about the effects of ifn-a on human ctls. to approach this issue, magnetically sorted untouched human cd + cd ro -t cells (mainly naï ve cells) were unstimulated or stimulated with human ifn-a and gene expression profiles were compared using an affymetrix human array. interestingly, ifn-a stimulation of highly purified human ctls without any other concomitant signals remarkably enhanced the expression of several molecules involved in death receptor signalling (trail) and chemotaxis (ip and itac). in a second genome-wide array analysis, we analyzed the effects of ifn-a on human ctls responding to antigen (signal ) and co-stimulatory signals (signal ), provided by beads coated with anti-cd /cd antibodies. gene expression patterns were compared for cells stimulated with anti-cd /cd beads alone or along with ifn-a. ifn-a regulates the expression of a number of genes that promote proliferation, activation and survival of ctls, tcr stabilization, chromatin remodelation, and, importantly, enhances the expression of genes involved in ctls effector functions (granzyme-b, ifn-g, trail, fasl) and chemotaxis (ip , itac). the enhanced expression of granzyme-b, ifn-g, trail and ip were further confirmed at the protein levels by flow cytometry analysis and/or elisa. enhancement of granzyme-b-and trail-mediated cytolitic functions was also found by functional assays using anti-cd -coated p cells and trail-sensitive caki-i cells as targets. our results show that ifn-a provides a strong signal- to human ctls leading to their differentiation into effector ctls. t cell activation is an important process of the adaptive immune system, which requires recognition of mhc-associated antigens by antigen presenting cells (apcs) via the t cell receptor (tcr). to induce a productive t cell response the interaction of t cells with apcs needs to be stabilized by adhesion molecules. junction adhesion molecules (jams) are a recently discovered group of immunoglobulin (ig) superfamily proteins, which are involved in the regulation of various inflammatory and vascular events. the third member of the jam protein family, jam-c, is highly expressed in platelets and endothelial cells, whereas expression in t cells is largely unknown. to investigate the regulation of jam-c in t lymphocytes, we determined jam-c gene expression in quiescent and activated human t cells. treatment with the polyclonal t cell activator phytohemagglutinin (pha) increased surface and total jam-c expression in t cells time-and dose-dependently, as determined by flow cytometry and immunoblot analysis. by contrast, no up-regulation of jam-a in activated t cells was detectable. the highest level of jam-c up-regulation by pha was observed in cd + foxp + and cd + cd high t cells. moreover, t cell receptor activation with combined anti-cd and anti-cd stimulation induced jam-c expression in t cells. jam-c induction occurred at the mrna level suggesting a transcriptional regulatory mechanism of jam-c expression. accordingly, we studied the regulation of the human jam-c gene promoter in transiently transfected t cells. luciferase activity of a jam-c promoter gene construct with three potential consensus sites for the transcription factor nfat was markedly induced in activated t cells. finally, pretreatment with two pharmacological inhibitors of calcineurin, cyclosporin a and fk- , but not with mapk inhibitors, blocked jam-c induction in activated t cells. in summary, the present data indicate that jam-c is induced in activated human t lymphocytes via a transcriptional mechanism and suggests a major regulatory function of jam-c for the t cell response. hiv- infection leads to immune dysfunction owing to a successive loss of the cd + t cell compartment. the molecular mechanisms underlying this depletion are not well-understood but may involve the viral nef protein. nef is a multifunctional accessory protein that is required for full hiv- virulence and the maintenance of high viral loads. nef enhances viral infectivity and replication by downregulating cell surface receptors, e. g. cd and mhc class i, and modulating signal transduction pathways. the latter is thought to raise the cellular activation level and in this way may increase the infected cell's susceptibility to apoptosis. in this study we identify a signaling complex assembling at the n-terminus of nef, which contains the kinases lck and pkcv. formation of this complex, termed nakc for nef-associated kinase complex, led to activation of lck, as assessed by in-vitro kinase assay, and recruitment of pkcv to membrane rafts, as detected by discontinuous sucrose density gradient ultracentrifugation. recruitment of pkcv to membrane rafts is a hallmark of t cell activation and has been associated with activation of the nfxb transcription factor. however, contrary to our expectations, nef-mediated nakc formation did not activate nfxb. instead, it led to a strong induction of erk / . this correlated with a nakc-mediated increase in hiv transcription that was demonstrated by luciferase reporter assays suggesting that erk / directly targets hiv transcription, possibly via induction of transcription factors. to our surprise, however, the effect of nakc on hiv transcription was found to be independent of ap- , nfat and nfxb suggesting an alternative mechanism of nakc-mediated enhancement of hiv transcription. on the basis of our previous results we propose that nef enhances hiv transcription via removal of inhibitory factors and thus derepression of the hiv promoter. how erk / is involved in this mechanism and whether nakc targets other cellular promoters, which may enhance the cellular activation level and thus sensitize the cell to apoptosis, remains to be determined. p. otahal , t. brdicka , v. horejsi institute of molecular genetics as cr, praha, czech republic aims: c-terminal src kinase (csk) and cd are key regulators of src-family kinases in leukocytes. while cd is a transmembrane phosphatase, csk is localized mostly in cytosol. however, a fraction of csk is found at the cell membrane and in lipid rafts where it inhibits signaling by phosphorylating inhibitory tyrosine of src-family kinases. currently, it is accepted that sh domain of csk binds phosphotyrosine of transmembrane adaptor protein pag and via this interaction is recruited to the cell membrane and lipid rafts. however, pag knock-out mice still have cell membrane-associated csk and do not show any apparent dysregulation of signaling which would be expected due to the low levels of membrane csk. thus, the mechanisms of membrane targeting of csk remain unclear. to analyze the role of membrane and lipid raft targeting of csk on lymphocyte signaling we targeted csk to different membrane compartments by fusing csk with transmembrane domains of lat, lax, cd and n-terminal part of src kinase. methods: csk chimeras containing n-terminal membrane targeting motif and c-terminal orange fluorescent protein were cloned into retroviral vector pmxs. jurkat t cells expressing individual constructs were subsequently prepared and analyzed for the inhibitory effect of these csk chimeras on t-cell receptor (tcr) signaling by measuring calcium flux and cd upregulation. the efficiency of inhibition depended on the membrane targeting motif, while lat-csk chimera completely inhibited tcr signaling and src-csk chimera inhibited the signaling only partially; lax-csk and cd -csk chimeras showed almost no inhibition of tcr signaling despite efficient presence at the plasma membrane. conclusions: our data demonstrate that the function of csk strongly depends on its targeting to the specific areas of plasma membrane. it also strongly supports the idea that membrane compartmentalization is critical for regulation of t-cell signaling. peripheral cd t cell tolerance can be generated outside lymphatic tissue in the liver. however, the course of events leading to tolerogenic interaction of hepatic antigen presenting cells with circulating t cells is unclear. here, we demonstrate that systemically circulating antigen was preferentially taken-up by liver sinusoidal endothelial cells (lsec) and not by other antigen presenting cells in the liver or spleen. uptake and cross-presentation of circulating antigen was followed by rapid antigen-specific naï ve cd t cell-retention in the liver but again not in other organs. using bone-marrow chimeras and tie- kb mice, we could show that antigen cross-presentation by lsec was both essential and sufficient to cause antigen-specific t cell-retention under non-inflammatory conditions, which was followed by cd t cell proliferation and expansion, but ultimately led to the development of t cell tolerance. our results show that cd t cell tolerance towards circulating systemic antigens is predominantly generated in the liver by lsec, which preferentially take-up and cross-present circulating proteins to cd t cells, leading to their rapid local antigen-specific retention and subsequent tolerisation. these insights broaden our understanding not only of physiological immune regulation towards circulating antigens but also of therapeutic manipulation of cd t cell responses. alphapix is a rho gtpase guanine nucleotide exchange factor domain-containing signaling protein that associates with other proteins involved in cytoskeletalmembrane complexes. it has been shown that pix proteins play roles in some immune cells, including neutrophils and t cells. in this study, we report the immune system phenotype of alphapix knockout mice. we extended alphapix expression experiments and found that whereas alphapix was specific to immune cells, its homolog betapix was expressed in a wider range of cells. mice lacking alphapix had reduced numbers of mature lymphocytes and defective immune responses. antigen receptor-directed proliferation of alphapix deficient t and b cells was also reduced, but basal migration was enhanced. accompanying these defects, formation of t-cell-b-cell conjugates and recruitment of pak and lfa- integrin to the immune synapse were impaired in the absence of alphapix. proximal antigen receptor signaling was largely unaffected, with the exception of reduced phosphorylation of pak and expression of git in both t cells and b cells. these results reveal specific roles for alphapix in the immune system and suggest that redundancy with betapix precludes a more severe immune phenotype. s. merluzzi , s. parusso , b. frossi , g. gri , c. pucillo university of udine, dstb, udine, italy in this study, we investigated whether primary mcs could modulate the activation and proliferation of primary b cells. we performed co-culture assays using mouse splenic b cells and bone marrow-derived mcs. naï ve and activated b cells proliferation could be induced by nonsensitized mcs while an increase in b cell proliferation was observed when mcs are activated. moreover, b cell proliferation was partially abolished when mcs and b cells were separated by the transwell membranes suggesting that cell-cell contact is important in this event. using both il- -/-mcs and anti-il- receptor antibody, we demonstrated that in co-culture of primary b cells and mcs, il- derived from activated mcs is a key cytokine implicated in the b cell proliferation. moreover, we showed that activated mcs can influence the surface expression of costimulatory molecules as cd on naï ve b cells and the interaction of cd on b cell surface and cd l on mcs is important for the further differentiation of b cells to plasmacells. indeed, we presented for the first time evidence that cytokines produced by activated mcs and interaction between cd l e cd on mc and b cells respectively can contribute to differentiate mature b cells to iga secreting cells. in conclusion, in the present report, we showed a novel role of mcs as promoter of both the survival and activation of naive b cells and of the proliferation and further differentiation of activated b cells through soluble factors production and cell-cell contact, suggesting that mcs can contribute to the regulation of specific immune response. e. fourmentraux-neves , n. bercovici , a. caignard inserm u , paris, france inhibitory killer ig-like receptors (kir dl - / ) which bind to hla-c molecules are expressed by human natural killer cells and effector memory cd + t cell subsets. these receptors suppress cd + t cell activation through recruitment of the src homology domain-containing protein tyrosine phosphatase (shp- ). to further analyse the yet largely unclear role of inhibitory kir receptors on cd +t cells, kir dl transfectants were obtained from a cd + t cell line and primary cells. the transfection of cd + t cells with kir dl dramatically increased the t cell receptor (tcr)-induced production of il- independently of ligand binding, but inhibited tcr-induced activation after ligation. kir-mediated tcr activation requires intact itim motifs, involves kir dl -itim phosphorylation, shp- recruitment, zap- and pkc-v phosphorylation. synapses leading to activation were characterized by an increase in the recruitment of p-tyr, shp- and p-pkc-v but not of shp- . in contrast, the kir dl /hla-cw interaction led to a strong synaptic accumulation of kir dl and the recruitment of shp- / , inhibiting tcr-induced il- production. kir dl may induce two opposite signaling outputs in cd + t cells, depending on whether the kir receptor is bound to its ligand. these data highlight unexpected aspects of the regulation of t cells by kir dl receptors. b cell receptor (bcr) binding by antigen initiates activating signaling cascades and facilitates the exposure of specific b cells to powerful co-stimulatory signals, such as t cell help or toll-like receptor ligands. the role of bcr binding in modulating the access to these second signals is complex and varies between stimulatory conditions. by quantitative tracking of b cell responses in vitro we can measure which signals affect b cell proliferation or differentiation, or both, and thereby establish a novel understanding of how b cells respond appropriately to different combinations of stimuli. we utilised hel-specific bcr transgenic sw hel mice to assess the effect of a specific antigen signal on b cell responses to the t-independent mitogen lipopolysaccharide (lps). the presence of antigen renders a greater proportion of cells responsive to lps stimulation and profoundly influences effector cell differentiation. antibody secreting cell formation is dramatically inhibited by hel, but we found that isotype switching to igg is strongly upregulated. both of these alterations to differentiation outcomes occur independently to the proliferative effects induced by antigen. when b cells are exposed to antigen for a limited period of time, switching to igg still occurs but some capacity to differentiate to antibody secreting cells is recovered, leading to effective secretion of igg antibody during these conditions. the observed igg switching behaviour mimics that of b cells responding to lps and il- , but is mediated by a different, stat -independent pathway. these data are indicative of the important role specific antigen signals play in regulating b cell responses in stimulatory environments. a. quintana , c. schwindling , m. pasche , c. junker , c. kummerow , u. becherer , e.c. schwarz , j. rettig , m. hoth saarland university, biophysics, homburg, germany, saarland university, physiology, homburg, germany the adaptive immune response requires the interaction between antigen-presenting cells and t cells. this cell-cell interaction, called the immunological synapse (is), facilitates the activation of t cell receptor-mediated signalling cascades including a rise of cytosolic calcium through the activation of crac/orai channels. to allow sustained activity of crac/orai channels, the calcium-dependent inactivation of the channels through local calcium microdomains has to be prevented. objectives: the purpose of the study was to analyze local and global calcium signals in t cells and to test the hypothesis that the is controls these signals through mitochondrial positioning. methods: we used different microscopy techniques including very fast wide-field microscopy with subsequent deconvolution, total-internal reflection microscopy, and confocal microscopy in combination with electrophysiological techniques in primary human t helper cells and cell lines. to test the statistical significance of our data, we used two-sided student t-tests or non-parameterized tests. results: following is formation, we found that mitochondria translocated to the is in a calcium-dependent way. the distance between mitochondria and the plasma membrane at the is was lower than nm. following accumulation at the is, mitochondria limited calcium entry to the orai channels localized right at the is by preventing their calcium-dependent inactivation. in contrast, no calcium influx was observed at sites where no mitochondria were accumulated as orai channels were inactivated at these sites. mitochondrial positioning at the is thus induced local calcium influx at the is without the necessity to enrich orai channels at the is. mitochondria took up calcium at the is distributing it further into the cytosol by releasing it at different sites, which kept the local domain at the is low enough to prevent calcium dependent orai inactivation and to prevent excessive calcium clearance by the calcium aptases in the plasma membrane, which could inhibit an efficient t cell activation. conclusion: mitochondria positioning at the is controls local calcium entry through orai channels. mitochondria prevent orai inactivation and excessive calcium clearance at the is to facilitate calcium-dependent t lymphocyte calcium signalling. we aimed to determine the functional correlates of cd + t cell tolerance and immunity in vivo. ovalbumin (ova)-specific transgenic cd + t cells were adoptively transferred into syngeneic mice immunized with soluble ova protein ± lipopolysaccharide (lps) by the i. v. route, and analyzed for a variety of immunological parameters over a period of days. under tolerogenic conditions (ova alone), cd + t cells showed substantial early activation, but their activation profile differed markedly, both in magnitude and quality (icos, - bb), from t cells activated by ova+lps. this difference in activation also translated into differing cd + t cell expansion and contraction kinetics in the early phase of the t cell response (days - ). in the late phase of the primary response (days - ), under immunizing conditions, the large majority of transgenic cd + t cells in the spleen developed into mature effectors with a prominent capacity to secrete il- , ifn-g, and il- a, and only few ova-specific foxp + regulatory t cells ( x %) were observed. germinal centers were prominent and ova-specific ig of all isotypes were generated. in contrast, under tolerizing conditions, antigen-specific cd + t cells failed to migrate into the b cell follicles, but production of ova-specific igm was nevertheless observed. in these animals, the proportion of splenic ova-specific regulatory t cells ( %) was substantially increased. on day , both groups of mice were re-challenged via the airways with ova+lps to functionally assess their immune status. in tolerized animals, the transgenic t cell population in the lung infiltrate was composed of ova-specific regulatory t cells ( %) or t cells with a reduced capacity to secrete effector cytokines. in contrast, in immunized animals, this population almost exclusively consisted of cd + effector t cells with a pronounced inflammatory cytokine profile (ifn-g, il- a). with this model we provide a comprehensive analysis of the many functional correlates of "immunity" versus "tolerance" to soluble protein antigen in vivo. we identify and characterize a number of the key players (cell surface molecules, cytokines, cell subsets) representing the decision between immunity and tolerance in the immune system. mast cells (mcs) are well-recognized as key effector cells in immunoglobulin e (ige) -associated immune responses and as prototypic regulators of innate immunity. the characteristics, importance, and molecular requirements for interactions between mast cells (mc) and cd t cells (tc) remain to be elucidated. using myelin/oligodendrocyte glycoprotein (mog), we demonstrated that mcs induce antigen-specific cd tc activation and proliferation. the antigen crosspresentation by mcs induces the secretion of interleukin- , interferon-g and macrophage inflammatory protein- by cd tc. in vivo evidence that mcs modulate t cell responses has been obtained so far in the murine experimental autoimmune encephalomyelitis (eae), the standard animal model for multiple sclerosis, in which both cd tc and mcs are now recognized as key players. one of the main central nervous system (cns) antigens recognized by autoreactive tc in eae is the myelin oligodendrocyte glycoprotein (mog). to investigate the in vivo-relevance of the identified mc-cd tc interactions, we have employed the eae as a model of organ specific autoimmune disease in wild type mice and mc-deficient w/w sh mice. wt and w/w sh mice were immunized with the mog - protein. our results provide direct evidence that mc contribute to cd -specific priming in eae and show that the tc proliferation failure is specific for cd tc from mog - -immunized w/w sh mice. the role of mc-cd tc interaction in induction of autoimmunity will be further investigated in eae. in summary, we provide the first evidence that mcs regulate antigen-specific responses of primary cd tc in vitro and in vivo. our study further supports the emerging concept that mcs, protagonists of innate immunity are also important regulators of adaptive immune responses and corresponding cd tc responses. this newly uncovered mc function might be of great biological relevance in situations where effector cd tc are critically involved, e. g. viral infections or infections with intracellular pathogens and/or autoimmune diseases such as multiple sclerosis. activation of resting t cells in vitro is triggered by combined t cell receptor (tcr) and cd engagement and can be modulated by simultaneous ligation of various other surface receptors. although the fasl is best known for its capacity to initiate cell death in fas-bearing cells, it has recently been implicated in the regulation of t cell activation. thus, a crosstalk between the tcr and fasl is likely, but far from being biochemically elucidated. we report that fasl engagement by immobilized but not soluble fasfc fusion protein and anti-fasl antibodies blocks the activation of primary human peripheral t cells even in the presence of cd costimulation at the level of an early signal initiation. inhibition is thus associated with a reduction of tyrosine phosphorylation of a number of key elements in tcr signal transduction and also with a lowered calcium response. the data presented stress the importance of the fas/fasl-system for signal initiation via the tcr/cd complex and provide further arguments for a retrograde signaling capacity of fasl or a crucial role of fas as a costimulatory molecule. golgi network (tgn). moreover, trim specifically associates with the cytoplasmic tail of ctla- , but not via any conventional motifs in this region. overexpression of trim augments ctla- surface expression, whereas down-regulation of trim expression by shrna results in disturbed ctla- localisation, mainly restricted to the tgn. ctla- vesicles and surface expression were significantly reduced but not abolished, suggesting that other factors are involved in ctla- trafficking. here, we identify additional transmembrane adapter protein (trap) family members as novel binding partners and regulators of ctla- expression. although there is some redundancy amongst traps, our results highlight the importance of this family of proteins in ctla- transport to the cell surface. it is imperative to reveal the mechanisms by which ctla- is transported to the cell surface, given that minor changes in expression can have major effects on t-cell function and in the development of autoimmunity. natural killer t (nkt) cells are found within the liver and are known to exhibit immune regulatory function. upon recognition of glycolipids presented on cd molecules, nkt cells are activated and release cytokines, including ifn-g, il- and il- . nkt cells are efficiently recruited to the liver via cxcr -dependent chemotaxis toward cxcl and constitute a large proportion of the liver-resident lymphocytes. we have previously shown, that liver sinusoidal endothelial cells (lsec) can scavenge circulating soluble antigens, and can cross-present these antigens to naive cd t cells. cross-presentation leads to initial t cell activation and expansion, but ultimately these cd t cells are rendered tolerant. as both naive t cells and nkt cells come into close contact with lsec in the hepatic sinusoids, we investigated whether nkt cells can modulate cd t cell tolerisation via interaction with lsec. to this end we analysed cd d expression on lsec and their ability to activate nkt cells by presentation of the cd d-binding glycolipid a-galactosylceramide (agalcer). we found that lsec express functional cd d, as agalcerpresenting-lsec were capable to induce tnf-a, il- , il- and ifn-g production in nkt cells in vitro. the interaction of agalcer-presenting-lsec with nkt cells led to the upregulation of cd and b -h on lsec. as naï ve cd t cell tolerisation by lsec critically depends on b -h , we hypothesise that hepatic nkt cell activity may contribute to the immunological capabilities of the liver by regulating the tolerogenic function of lsec. improved antibody responses by class-switched memory b cells require enhanced signaling from their antigen receptor (bcr). however all bcr classes on naïve and antigen-experienced b cells utilize the canonical iga/igb subunit for signaling. we identified the signal amplification mechanism of the igg-and ige-bcr. for these isotypes tyrosine-based signaling is not confined to iga/igb but extends to a conserved tyrosine residue in the cytoplasmic segments of immunoglobulin heavy chains. the phosphorylated immunoglobulin tail tyrosine recruits the adaptor grb in order to sustain protein kinase activation and generation of second messengers causing robust cellular proliferation. hence membrane-bound igg and ige not only recognize antigen but also exert bcr-intrinsic costimulation to render memory b cells less dependent on t cell help for activation. objectives: the majority of circulating human gd t cells harbor tcr containing vg , jg . , and vd gene products. they recognize nonpeptide antigens like (e)- hydroxy- -methylbut- -enyl pyrophosphate derived from pathogenic microbes and isopentenyl pyrophosphate (ipp) in malignant cells. recently, we and others found out that gd t cells express a variety of costimulatory molecules including icos, and pd- . one of the inhibitory receptors, pd- , is a member of cd /ctla- family and contains a single ig v-like domain in its extracellular region. pd- can bind to two b homologue molecules, pd-l and pd-l . it has been reported that interaction of pd- with its ligands resulted in peripheral immune regulation and tolerance in ab t cells. in this study, we show that pd- is expressed on activated human gd t cells and regulates the effctor functions of gd t cells. methods: peripheral blood mononuclear cells were resuspended in yssel's medium and stimulated with -methyl- -butenyl- -pyrophosphate plus il- to obtain gd t cells. pd-l + and pd-l -human tumor cell lines were established from cancer patients. in order to prepare anti-pd-l mabs, the pd-l extracellular domain was expressed in e.coli as inclusion bodies and refolded in the standard arginine-based buffer. mice were immunized with the refolded protein and mabs were established. to determine the function of pd- in gd t cells, we determined cytokine production and cell mediated tumor lysis by activated gd t cells in the presence of inhibitors of pd- /pd-l interaction. results: gd t cells expressed pd- upon simulation with nonpeptide antigens and many tumor cell lines expressed pd-l . we first examined whether or not the engagement of pd- receptor could modulate the cytotoxic activity of gd t cells. pd-l -expressing tumor cells tempered cytotoxic activity of pd- + gd t cells, and cytokine production such as tnf-a was down-regulated by pd- engagement. in addition, inclusion of anti-pd-l mab reversed cytotoxic activity and cytokine production when pd-l -expressing tumor cells were challenged by pd- -expressing gd t cells. conclusion: pd- delivers inhibitory signals in gd t cells upon engagement with pd-l . peripheral tolerance plays an important role in preventing t lymphocyte responses to self or harmless antigens. one of the mechanisms that contribute to this form of tolerance is anergy, which is characterized by a lack of proliferation and il- production by t cells in response to antigenic challenge. the acquisition of the anergic phenotype is an active process, with negative regulators of t cell signalling being induced. among these are the e ubiquitin-protein ligases which recognize target proteins for ubiquitination and catalyse the transfer of ubiquitin to them, directing them to the proteasome or to the endosome-lysosomal pathway, and hence downregulating their activity. the e ubiquitin-protein ligases cbl-b, itch and grail have been shown to be upregulated in anergy and to ubiquitinate and downregulate tcr signalling elements. our objectives were to determine the expression of cbl-b, itch and grail in antigen-specific cd + t cells in both the induction and maintenance phases of anergy, in vitro and in vivo, and to investigate their functional signalling role(s) in the maintenance of the tolerance phenotype. in order to accomplish these objectives we induced priming or tolerance of ovalbumin (ova - peptide)-specific t cells from do . tcr transgenic mice in vitro or, following adoptive transfer of near physiologically relevant numbers of such cells into recipients, in vivo and correlated functional outcome (via proliferation and cytokine readout assays or antibody production) with e ubiquitin-protein ligases expression and the ubiquitination status of the tcr signalling machinery. cbl-b, itch, grail and target ubiquitination status, in terms of tissue, cellular and subcellular protein expression, modification and localisation, were assessed by a combination of immunoprecipitation and western blotting studies. moreover, we have performed quantitative analysis at the single cell level by tracking such antigen-specific cells in vitro and in vivo by using laser scanning cytometry. our current work focuses on the functional consequences of adenoviral transfection of such antigen-specific t cells by mutant e ligase-, signal target-and ubiquitin-constructs. collectively, these approaches have facilitated the dissection of the potential differential roles of ubiquitin signalling in priming and tolerance of antigen-specific t cells. s. j. keppler , p. aichele immh, university freiburg, immunology, freiburg, germany interleukin (il- ) is produced by cells of the innate immune system during infection and plays an important role in controlling various pathogens. it was postulated recently that il- has a direct influence on cd + t cells in vitro, enhancing expansion and the development of effector functions as a third signal, additionally to tcr engagement (signal ) and costimulation (signal ). we analysed direct il- signaling to cd t - signaling exhibited normal degranulation activity, cytolytic functions, ifn-g and tnf-a production. however, cd t cells lacking il- signaling failed to up-regulate klrg and to down-regulate cd in the context of listeria but not viral infections. thus direct il- signaling to cd t cells determines the cell fate decision between short-lived effector cells (slecs) and memory precursor effector cells (mpecs), dependent on the pathogen-determined local cytokine milieu. cd + t lymphocytes are required for effective host defense against pathogens but also for mediating effector responses against uncontrolled proliferating self tissues. we could now reveal that individual cd + t cells are tightly controlled in their effector functions by cd (ctla- ). we demonstrate that signals induced by cd reduce the frequency of interferon-gamma (ifn-g) and granzymeb expressing cd + t cells. for this novel function cd specifically represses the transcription factor eomes, but not t-bet. a cd mediated induction of the inhibitory transcription factor ckrox has been ruled out. ectopic expression of eomes reversed cd -mediated inhibition of effector molecule production. additionally, enhanced cytotoxicity of individual cd + t cells differentiated in the absence of cd signaling could be demonstrated in vivo. the novel insights that cd -mediated signal transduction in vivo indeed alters cd + t cell cytotoxicity qualitatively at the single cell level and not only quantitatively by enhancing expansion extend the understanding how to selectively modulate immune responses of cd + t cells. objectives: atp constitutes a damage associated molecular pattern (damp) and contributes together with pathogen associated molecular patterns (pamp) to the efficient priming of the innate immune system. atp is a ubiquitous extracellular messenger, which activates plasma membrane receptors for extracellular nucleotides termed p receptors. p x - receptors open to non-selective ion channels, whereas p y , , , , - are g-protein coupled receptors, which bind preferentially adp, udp, utp or udp-glucose. as the role of p receptors in the control of b cell activation has been poorly investigated, aim of the present study is to understand better the mechanisms of intracellular atp production and release by human b cell subsets. methods: intracellular atp measurement has been performed using a bioluminescence assay while extracellular atp has been measured by hplc. storage and release of atp by b cells have been elucidated using confocal and tirf microscopy, to study vesicles distribution and dynamics near the plasma membrane. results: in both human naive and memory b cell we observed a prominent increase of atp synthesis upon tlr but not bcr stimulation. glycolytic pathway rather than oxidative phosphorilation was involved in atp synthesis. p x antagonists inhibited both proliferation and differentiation to plasma cells of human b cells thus suggesting that atp is released in the pericellular space. labelling of resting and activated human memory b cells with quinacrine, a nucleotide binding component, revealed a typical vesicular pattern of atp, confirmed with subcellular fractionation on sucrose equilibrium gradients. tirf imaging showed a fluorescently labelled vescicle underwent fusion with the plasma membrane after stimulation with anti-ig and this event was ca( +)-dependent. conclusion: these data provide evidence that atp is produced by b cell preferentially by glycolytic pathway and vesicular exocytosis is a key mediator of atp release in human b cells. atp released in the pericellular space might act as an autocrine and paracrine signalling molecule that regulates the functions of b cells. o. ballek , a. brouckova , d. filipp institute of molecular genetics as cr, laboratory of immunobiology, prague, czech republic two src family tyrosine kinases lck and fyn are critical for the proximal t-cell signaling. we have previously demonstrated that induced lck activation outside lipid rafts (lr) results in lck translocation to lr. central in this sequence of events is the rapid translocation of kinase active lck to lr, yet the mechanism underpinning this process is unknown. the main aim of this study is the characterization of molecular mechanisms and its functional elements regulating the early recruitment of signaling molecules to lr and forming immunological synapse. we have recently characterized the c-terminal yqpqp sequence as a novel cis-acting component essential for partitioning of lck to lr. here we report that the expression of the c-terminal truncate of constitutively active lck ( ¿ fqpqp) in nih t cells failed to phosphorylate several proteins detected in the presence of untruncated kinase active y flck. comparative -d gel analyses followed by ms/maldi identified rack as a candidate protein for interaction with the c-terminal tail of lck. co-expression in nih t cells of ha-tagged rack with either a wild type lck or constitutively active y flck revealed a significantly enhanced complex formation between y flck and rack compared to that of wtlck. ectopic expression of y flck with its domain-inactivating mutations showed that lck-rack interaction depends on functional sh , sh and the c-terminal tail sequence of lck. lck-rack interaction is readily detectable also in primary cd + lymph node t cells. upon their activation, only the pool of lck molecules associated with high molecular weight complexes can translocate to lipid rafts. co-purification of rack with these fractions further suggests that it plays a role in the translocation of lck to lr. in addition, lck and rack co-redistribute to both forming immunological synapse and to antibody-mediated capping clusters. moreover, the importance of interaction between activated lck and rack in the context of lck translocation to lr is further strengthen by the observation that rack is associated with elements of cytoskeleton. these results are the first to characterize rack as a candidate molecule involved in the regulation of lck translocation to lr through linking the c-terminal sequence of lck to cytoskeletal network. human b cells are currently not known to produce the pro-apoptotic protease granzyme b (grb) in physiological settings. we have discovered that b cell receptor stimulation with either viral antigens or activating antibodies in the context of the acute phase cytokine interleukin (il- ) can induce secretion of substantial amounts of grb by human b cells. grb response to viral antigens was significantly stronger in b cells from subjects recently vaccinated against the corresponding virus as compared to unvaccinated subjects. both, naï ve and memory b cells differentiated into grb-secreting cells, which featured a homogeneous cd +cd +cd -cd -igd-phenotype, improved survival and enhanced expression of co-stimulatory, antigen-presenting and cell-adhesion molecules. b cellderived grb was enzymatically active and its induction required activation of similar signaling pathways as in cytotoxic t cells. our findings suggest grb-secreting b cells play a role in early anti-viral immune responses, thereby contributing to the elevated serum grb levels found in various viral diseases. further studies will elucidate whether b cell-derived grb induces cytotoxicity towards virus-infected cells or exhibits other functions. results: transfer of otii cells augments the wild type th response to alumova inducing large early germinal centres and massive plasma cell formation with more than % of these switching to igg . the plasma cells up-regulate cxcr , but not cxcr , a chemokine receptor that attracts plasma cells to inflammatory sites. oti cells respond to alumova by producing ifng, a th -associated cytokine. when both oti and otii cells are transferred switching is diversified with plasma cells being igm (˚ %), igg a (˚ %), igg b (˚ %) or igg (˚ %). in addition to cxcr , some % of these plasma cells strongly express cxcr . the induction of cxcr in these plasma cells correlates with their increased expression of the transcription factor t-bet, which has been linked with igg a switching during th responses. this is functionally significant for oti-dependent cxcr expression, as well as induction of switching to igg a, are dependent on t-bet expression by the responding b cells, although t-bet-deficient b cells still switch to igg b. t-bet is known to be induced in b cells exposed to ifng or tlr stimulation. these two hypothetical mechanisms are currently being tested in mice injected with blocking anti-ifng antibodies or mice deficient in myd . objective: a successful immune response against malaria has to be tightly controlled. the early production of pro-inflammatory cytokines is required to control the growth of intraerythrocytic parasites but the same cytokines are also involved in the induction of severe malaria in both humans and mice. activation of t lymphocytes through tcr signalling takes place within the context of numerous other cell surface protein interactions. to prevent unnecessary activation of t cells the immune system has developed an intricate balance between positive and negative costimulatory signals. costimulatory signals determine whether antigen recognition by t lymphocytes leads to full activation or to anergy. in contrast negative costimulators expressed by t cells seem to mediate the regulation of immune responses and thus play a pivotal role in the maintenance of peripheral tolerance. recently, btla (b and t lymphocyte attenuator, cd ) was described as a novel negative costimulatory receptor. btla is predominantly expressed on t and b cells and dampens t cell activation. in this study, we analyzed the function of btla during experimental malaria infection. to study the function of btla we employed a mouse model of blood-stage malaria using p. yoelii nl infection of btla-deficient and hvem-deficient mice. p. yoelii provokes a high parasitemia in infected mice that is cleared within three weeks from time of infection. immunity in this model depends on cd + t cells and hence the role of negative costimulators that modulate t cell function can be studied using this model. results: peak parasitemia of p.yoelii-infected btla-deficient and hvem-deficient mice was much lower compared to wild type mice. the increased immunity of btla-deficient mice depends largely on cd + t cells. we found that btla::hvem interaction regulates the size and the cytokine-production of the responding t cell pool. however, in contrast to the ctla- pathway, the manipulation of btla::hvem interaction triggers no pathology during infection. the hvem::btla interaction dampens the protective immune response during experimental malaria and thus manipulation of this pathway is an attractive target for therapeutic interventions. . so far, the contribution of actin regulatory proteins to this process remained largely unknown. here we demonstrate that the actin-bundling protein l-plastin is indispensable for segregation of lfa- and cd in the psmac of untransformed human t-cells. in marked contrast, tcr/cd accumulation in the csmac is not dependent on l-plastin. the relocalization of l-plastin in the immune synapse occurs within seconds of t-cell/apc contact formation and relies on actin polymerization. importantly, binding of calmodulin to l-plastin is required for the maintenance of l-plastin in the immune synapse and inhibition of calmodulin prevents psmac formation. thus, receptor segregation in the immune synapse is a consequence of the combined activities of the actin-bundling protein l-plastin and calmodulin. protective t cell responses are based on expansion and persistence of clones with optimal affinity for antigen. presently, it is unknown which mechanisms guard the selection and expansion of the highest affinity clones from the very diverse naï ve pool. rapid cell division creates shifts in selective pressure, which is a basic biological prerequisite for elimination. therefore we hypothesized that apoptosis might play an important role during this phase of t-cell biology. here we show that the balance between the pro-apoptotic protein noxa and its antagonist mcl- regulates interclonal t cell competition during acute and chronic immune activation. we found p -independent noxa gene induction and mcl- downregulation upon t cell activation. concomitant we observed the release of death-inducing factor bim from mcl- , which was delayed in noxa -/cells. using ot- cells and altered peptide ligands we observed that the level of mcl- downregulation in activated t-cells depended on the antigen affinity of the t-cell receptor. since mcl- -/mice are embryonic lethal, noxa -/mice were used to study the functional implications of this mechanism in vivo. at a young age noxa -/mice have a normal lymphoid compartment, but accumulate effector t cells over time. upon acute influenza infection, normal levels of effector cells were generated. however, the quality of the antiviral (np ) response was impaired in these mice as many subdominant clones persisted in the effector t-cell population of noxa -/mice at the peak of infection. this increased diversity correlated with exacerbated pathology and a reduced rate of viral clearance. in a model of chronic immune activation, effector t cells rapidly accumulated in the noxa -/mice and infiltrated the peripheral organs, culminating in severe multi-organ pathology and premature death. these results establish a novel role for the noxa/mcl- axis during immune responses and suggest that the formation of a high-affinity effector population of restricted clonal diversity depends on a darwinian selection of t-cells during the expansion phase based on antigen affinity, with survival of only the fittest clones. cytotoxic t lymphocytes (ctls) are essential for immunosurveillance, a process that requires the presentation of virus-or tumor derived antigenic peptides in context of antigen presenting cells. insight into intracellular mechanisms facilitating lytic granule release and formation of the immunological synapse as a prerequisite for target cell destruction was primarily obtained from loss-of-function mutations in hereditary human diseases and gene-mutated mice. here, we refer to estrogen receptor-binding fragment-associated antigen (ebag ) as a negative regulator of secretory lysosome release. in gene deleted mice we show that loss of ebag confers ctls with enhanced cytolytic capacity, in vitro and in vivo. here, we show that ebag , which was previously identified as a snapin-interacting protein in neuronal cells, interacted with the adaptor molecule g -adaptin in t cells. both interactions suggested an involvement of ebag in endosome-lysosome related organelle biogenesis and membrane fusion. efficiency of granzyme b sorting towards secretory lysosomes was improved, which was consistent with the enhanced kinetics of cathepsin d proteolytic processing. while the formation of the immunological synapse remained unaffected in ebag -/-ctl, relative size distribution of lytic granules revealed a shift towards smaller granule diameters and volumes in ebag -deficient ctls. these data imply a role for ebag in regulating the formation of mature ctl granules and identify ebag as a tunable inhibitor of ctl-mediated adaptive immune response functions. finally, ebag defines a novel negative regulator of secretory lysosome release in ctls. thus, the elucidation of the ebag -related pathway might provide a fresh impuls on therapeutic approaches in the treatment of autoimmune disorders. the liver is known to induce tolerance, rather than immunity, through tolerogenic antigen presentation or elimination of effector t cells. recently, we could show that liver sinusoidal endothelial cells (lsec) inhibit activation of naive cd t cells by antigen-presenting dc. this regulatory effect of lsec on dc function was mhc-independent and not limited to soluble mediators, but required physical contact. interestingly, interaction with lsec led to reduced dc expression levels of cd / and il- . in addition to indirect inhibition of t cell activation by de-licensing of dc, we now detected another influence of lsec in form of direct inhibition of t cell priming. in the presence of lsec, stimulation with acd /acd or pma/ionomycin could not significantly activate cd and cd t cells. thus, lsec did not only inhibit t cell priming triggered by tcr activation but also after elicitation of ca + influx into the cytoplasm. furthermore, we found that ifn-g secreted by t cells in the early phase of activation is crucial for licensing the inhibitory function of lsec. taken together, these data indicate that the inhibitory effect of lsec is mediated by a machinery induced at the early phase of t cell activation, however, interferes with late events in the t cell activation cascade. we propose a model of "inducible inhibition", where on the one hand naive t cell priming is directly inhibited by lsec, and on the other hand tolerogenic priming by antigen-presenting lsec is still allowed. taken together, these results reveal a novel principle, operative in hepatic tolerance induction, in which lsec not only tolerize t cells themselves, but also inhibit the responsiveness to local activation stimuli. m. almena , s. carrasco , i. merida centro nacional de biotecnología csic, inmunología y oncología, madrid, spain self-tolerance acquisition is essential for the immune system to control its own response. t cells achieve self-tolerance trough thymic selection and anergy, two processes where rasgrp -ras-erk signal intensity is critical to determine the final cell outcome. rasgrp is a gef for ras that is activated in a diacylglycerol (dag)dependent manner. dag is generated by plcg after tcr stimulation and is consumed by diacylglycerol kinases (dgk). dag generation, as a result of the concerted regulation of these two enzymes, activates ras, providing a mechanism to translate the strength of the stimulus into a quantitative cell response. . analyze the impact that dag metabolism plays in t cell tolerance in vivo, using transgenic mice where dag generation is impaired. . develop a method to sense dag production and localization, in both thymocytes and peripheral t cells, and its correlation with the strength of the stimulus used. methods: we generated transgenic mice expressing a constitutively active dgk in t cell lineage. this protein was anchored to the plasma membrane, thus diminishing the lipid levels in this specific location after tcr stimulation. ot-i cd cells expressing gfp-c domains were used with peptide-pulsed apcs to study dag generation and dynamics by confocal microscopy. results: transgene expression was obtained in thymic and peripheral t cells. no major defects were observed in t cell subsets but analysis of peripheral t cells demonstrated important defects in t cell activation. we are currently studying thymic selection breeding our transgenic with h-y mice, in order to check if t cell populations are being properly selected. using t cell-apc conjugates with peptides with different tcr binding affinities we found a clear correlation between the strength of the stimulus, dag production and ras-mapk activation. conclusion: our data demonstrate that dag generation not only activates c domain containing proteins but regulates a mechanism by which t cells sense the magnitude of the stimulus received, translating it into the intensity of the generated response, a process essential in t tolerance. future experiments will help to define the exact contribution of the lipid to tcr signaling pathways and to t cell homeostasis. the inducible costimulator (icos, h , cd ), a cd -like costimulatory molecule, has an important role in the development of efficient t cell responses. early data showed that icos costimulation produced th biased responses and high production of the anti-inflammatory cytokine il- , and was essential to the development of germinal centres. however, icos can also help in the il- -dependent differentiation of inflammatory th cells. these different functions could be due to differences in the apcs bound by icos-expressing t cells and/or because of the intervention of distinct molecules binding the cytoplasmic domain of icos. icos shares with cd a yxxm cytoplasmic motif that can bind, upon tyr phosphorylation, the regulatory p subunit of class ia pi- kinases. these can complex with one of the three kda catalytic subunits (p a, p b, and p d) expressed by leukocytes that generate pip affecting cell growth, cell cycle progression, survival, intracellular traffic, cytoskeletal changes and migration. there is also evidence that the regulatory and catalytic class ia pi k isoforms fulfill specific functions in macrophages and lymphocytes. we have used proteomic and immunochemical approaches to identify molecules binding the phosphorylated or unphosphorylated cytoplasmic domain of icos, and particularly the presence of distinct pi kinase isoforms. then, the functional importance of these molecules has been analyzed by using pharmacological inhibitors specific for downstream mediators of icos activation. pull down of t cell lysates using phosphorylated or unphosphorylated synthetic peptides covering the cytoplasmic domain of icos was carried out. proteomic and immunoblot analysis of bound proteins showed that phosphorylated icos bound the different pi -k regulatory (p a, p b, p a) and catalytic (p a, p b, and p d) pi -kinase subunits expressed by leukocytes. these data were confirmed in icos immunoprecipitates from pervanadate-activated cells. icos bound regulatory and catalytic subunits in the order p a g p a g g p b and p a g p d g g p b, in agreement with quantitative rt-pcr and immunochemical estimation of subunit abundance in the t cells and t cell lines used. the use of specific pi -kinase inhibitors has confirmed the relative importance of the catalytic isoforms in icos function, including reorganization of actin cytoskeleton induced by icos ligands, or costimulation of tcr/cd -induced secretion of il- and il- . during the process of antigen recognition between t-cell and antigen-presenting cell (apc), structural and spatial changes take place at the cell-cell contact, where the molecules involved in the formation of the immune synapse (is) reorganize, displaying a segregated localization. in this context, the translocation of the microtubule-organizing center (mtoc) is an early event that occurs during the formation of the is, bringing with it the golgi apparatus, thus providing the basis for a polarized secretion. however, the molecular mechanisms involve in the localization of the mtoc at the contact area between the t cell and the apc are not completely understood yet. we have studied the possible role of scaffolding protein akap , a member of the a-kinase anchoring protein (akap) family that localizes at the centrosome and interacts with pka regulatory subunit and other signalling molecules, in mtoc polarization and immune synapse formation. either the overexpression of gfptagged c-terminal cg-nap/akap construct that acts as a dominant negative, or sirna knockdown of endogenous akap expression in t cells prevents the correct organization of cd z and pkcv to the is and mtoc reorientation towards t cell-apc contact area in antigen and superantigen-dependent human models, resulting in a disorganized is; lfa- localization was also analyzed to assess p-smac architecture and, interestingly, confocal d reconstruction revealed that lfa- ring was not clear in the akap -disrupted cells. moreover, akap was required for tcr signalling since the knock down with specific sirna and overexpression of c-terminal of akap decrease the phosphorylation of molecules such as lat, plcg and pkcv. these defective activation events as reflected in a reduction of il- production. together, our results underscore a key role for akap in the organization of the immune synapse and in the antigen-specific reorientation of the mtoc. the tcrbeta/ptalpha pre-tcr complex signals the expansion and differentiation of developing thymocytes. functional properties of the pre-tcr rely on its unique ptalpha chain, which suggests the participation of specific intracellular adaptors. in fact, we have recently identified cms, a member of the cin /cms family of adaptors, as a ptalpha-binding protein that specifically interacted with the human ptalpha cytoplasmic domain via its sh domains, and to the actin cytoskeleton via its c-terminal region. we found that cms co-localized with polymerized actin in pre-tcr clusters at the pre-tcr activation site, and also in the ptalpha endocytic compartment. since actin polymerization plays a critical role in regulating signalling through the alpha/beta tcr in mature t cells, we decided to investigate the potential function of cms as a regulator of actin polymerization and pre-tcr signalling in pre-t cells. using pre-t cells expressing a mutant pre-tcr lacking the cms-binding motif in the ptalpha tail and short hairpin irna-based gene silencing, we demonstrate that binding of cms to ptalpha contributes to cytoskeleton dynamics and pre-tcr-mediated signalling in human pre-t cells. cms-deficient cells specifically showed defects in pre-tcr-induced ca + mobilization and cell activation involving the pi k, nfat, plcg and erk signalling pathways, together with defects in actin polymerization and cell motility. cms therefore links cytoskeleton dynamics with the function of discrete pre-tcr signalling components, suggesting the functional implication of cms in human t-cell development in vivo. abstract withdrawn by author j objectives: most signaling pathways engaged after bcr activation have been described. however, several negative regulators of these pathways are unknown. the characterization of these regulators is important to understand the control of transduction pathways in adaptative immunity. carabin (tbc d c) has been recently described as a negative regulator of tcr signaling. it interacts with calcineurin and inhibits the formation of calcineurin/calmodulin complex, blocking nfat nuclear transport. moreover, carabin maintains ras protein under an inactive form, thus inhibiting ras-mapk cascade. expression of carabin is finely regulated following tcr signaling, and its knockdown (kd) enhances t cell activation. considering the important molecular similarities of antigen receptor signaling pathways in t and b cells, we studied the role of carabin in b cell. could carabin play a role of negative regulator of b cell function? methods: we studied by quantitative rt-pcr ) the expression of carabin in different purified subsets of bone marrow and splenic mouse b cells, as well as ) the kinetic of expression of carabin in bcr stimulated murine splenic mature b cells. ) we then studied the phenotype of carabin kd (shrna expressing) a b cells after bcr stimulation. ) the expression of carabin is significant in murine b cells, with an increase during b cell development, from bone marrow pro/preb to immature, to splenic t tot b cells and to follicular mature b cells. ) the kinetic of expression of carabin in bcr stimulated murine mature b cells suggests a fine regulation of carabin expression. ) bcr simulation, but not lps stimulation, of carabin kd a b cells shows an acceleration of ras target erk / phosphorylation, without any for the phosphorylation of mapk jnk, which is not targeted by ras. conclusion: carabin is expressed in murine b cells in a developmental regulated manner, with the highest expression in mature compartment. bcr stimulation leads to a fine regulation of carabin expression in wild-type mature b cells, and to a faster activation of erk / pathway in carabin kd b cells. altogether, these results strongly suggest a role of carabin as a negative regulator of b cell function toll like receptors are pattern recognition receptors, which recognize invariant pathogen associated molecular patterns. toll like receptor (tlr ) binds doublestranded rna, a nucleic acid frequently associated with viral replication. we observed that freshly isolated human cd + t cells express tlr and respond to the well characterized synthetic tlr ligand polyinosinic-polycytidylic acid [poly(i:c)]. the expression of activation markers and cytokine production by cd + t cells upon t cell receptor (tcr) stimulation is enhanced in response to co-stimulation via tlr . tlr stimulation on its own had no effect on expression of activation markers and cytokine production. to elicit the molecular basis of a potential cross-talk between tcr and poly(i:c) induced signaling, we used jurkat cells to perform luciferase assays. we observed that costimulation with poly(i:c) in comparison to tcr stimulation alone enhanced nf-kb but not nfat activation in jurkat cells. similarly to jurkat cells, tcr stimulation activated nf-kb in primary cd + t cells. this effect was further enhanced by additional poly(i:c) stimulation as shown by real-time-pcr and western blot analysis. on the other hand, we observed that poly(i:c) stimulation on its own activated the transcription factor interferon regulatory factor (irf ) as revealed by realtime-rcr analysis of ifn b and irf , whose transcription depends on the activity of irf . combined tcr and poly(i:c) stimulation further enhanced the transcription of these two genes. these results indicate that tlr signaling modulates tcr-driven responses and vice versa both in jurkat cells and in freshly isolated cd + t cells. this study was supported by dfg spp "innate immunity" (ka / - ). the initiation of protective t cell responses requires the recognition of mhc-bound peptides from pathogen or tumor antigens by the t cell receptor (tcr). how this signal is transmitted across the t cell membrane to the cytoplasmic signaling motifs is still unknown, and is the focus of this project. in textbooks, the cytoplasmic domains are depicted as flexible chains in the cytoplasm, but biochemical studies show that the cd e cytoplasmic domain (cd e cd ) binds to synthetic lipid vesicles that contain acidic phospholipids. this binding is predominantly due to electrostatic interactions between basic residues of cd e cd and acidic phospholipids. in the cell, acidic phospholipids are enriched in the inner leaflet of the plasma membrane. phosphatidylserine in particular is concentrated on the inner leaflet of the plasma membrane due to active transport mechanisms, explaining how such charge-charge interactions are generated. to study the interaction of the cd e cd with the membrane in live cells, we have developed a fluorescence resonance transfer (fret) assay which measures the proximity between a fluorescent protein (tfp) attached to the c-terminus of cd e cd and a fluorescent membrane dye (r ). with this assay, we show that the cd e cd is membrane-bound in resting cells and that binding is abrogated by introduction of mutations that disrupt lipid binding in the biochemical assay. additionally, in vitro analysis confirm functional domains for cd e cd lipid binding and conformational change. finally, nmr spectroscopy analysis reveals key features in membrane binding dynamics of cd e cd to lipid bicells. membrane binding by the cd e cd could thus be subject to dynamic regulation during the engagement of the tcr and further activation of the t cell. m. xydia , y. ge , u. quitsch , p. beckhove german cancer research center (dkfz), heidelberg, germany in peripheral tissues and the factors affecting their proliferation. cd + t cell help is believed to contribute to optimal cd + memory expansion via cd l on cd + t cells binding cd on dendritic cells. however, a few reports suggest that cd l-cd engagement may mediate direct cell-cell contacts between cd + and cd + t cells. in this study, we investigated the importance of cd -cd co-operation and cd l-cd interactions for t em proliferation. methods: we isolated human cd + and cd + t em cells from peripheral blood of healthy donors by facs or macs sorting. separated or mixed cd + and cd + populations were activated in vitro using anti-cd /cd beads. proliferation was measured by [ h]-thymidine incorporation, in some experiments after irradiation of one t em subset and/or incubation with blocking mabs against cd or cd l. furthermore, facs staining was used to assess cell-surface markers. statistical comparison was performed by student's t test. results: upon activation mixed t em populations showed a highly better proliferative response than separated cd + or cd + t em cells, demonstrating that optimal t em expansion requires direct cd -cd interactions. surprisingly, not only cd + but also cd + t em cells proliferated much more in mixed populations compared to the separated ones, indicating that optimal cd + t em proliferation depends on signals from cd + t em cells. activation induced the expression of cd on both populations and cd l on subsets of cd + and cd + t cells. blocking of cd l on cd + t em cells impaired significantly cd + t em proliferation, which confirms that the improved expansive potential of cd + t em cells in mixed populations depends on cd l co-stimulation by the cd t em subset. conclusions: our data demonstrate for the first time that activated cd + t em cells deliver help to the cd + t em subset via cd l-cd signalling and may play an important role for cd + t em expansion upon stimulation. the t cell surface glycoprotein cd , a member of the scavenger receptor cysteine-rich (srcr) family of proteins, targets to the immunological synapse upon t cell binding to antigen presenting cells (apc). however, it has not been established whether this translocation is due to the binding of a ligand expressed in the apc, or to intracellular interactions with signaling molecules or components of the cytoskeleton, that may control cd localization upon t cell:apc conjugation. we have questioned which domains of cd mediate the localization within the is, and for this we have expressed cd mutants as gfp fusion proteins in human t lymphocytes. we have also used jurkat cell lines expressing different cd mutants. t cells were incubated with superantigen-loaded raji b cells, and following the establishment of stable interactions between the cells, we analyzed the localization of cd by immunofluorescence and confocal microscopy. interestingly, our results show that the translocation of cd depends on sequences within the cytoplasmic domain, as a cd deletion mutant lacking most of the cytoplasmic tail, cd .k stop , is randomly distributed through the whole cellular surface, even in sustained t-apc interactions. the cytoplasmic domain relevant to cd translocation was mapped within amino acids glu and his since the cd .h stop mutant, just short of aa is still able to translocate to the is, whereas cd .e stop , that lacks important tyrosine residues, is no longer transported to the is upon t cell: apc interactions. although these studies do not exclude a role for the extracellular domain binding to an elusive apc-expressed ligand, they suggest that a major mechanism of regulation of cd translocation is dependent on molecular association of a short stretch of its cytoplasmic region (glu -his ) to intracellular signaling effectors. however, in hodgkin's lymphoma (hl) ebna- is missing, but lmp- is still expressed. using a hl derived cell line, we have shown that the cytokine il- can induce lmp- expression in vitro and can replace ebna- . we have investigated the molecular events for this mechanism. stat proteins bind to the palindromic ttc(n) x gaa sequence, where × is , or . a high affinity stat binding site is spaced by nucleotides. we found three potential stat binding sites in the lmp- promoter, which we named lrs, tr and edl . they were spaced by , and nucleotides, respectively. electrophoretic mobility shift (emsa) experiments were performed with nuclear extracts prepared from il- -treated or non-treated kmh -ebv cells. dna binding activity was analyzed using a double stranded oligonucleotide corresponding to the germline (gl) epsilon promoter, which is known to contain a high affinity stat binding site, or lrs-stat . a stat complex binding to the gl-epsilon promoter and lrs-stat was induced by il- . the specificity of the stat complex was shown by supershift experiments with anti-stat , but not anti-stat antibodies. when gl-epsilon or lrs-stat was used as cold competitors in a -fold excess, both unlabelled probes could compete out the labeled probe, providing evidence that the lrs-stat contains a functional stat binding site. oligonucleotides, corresponding to lrs in which the stat site had been mutated, could not compete for stat binding. interestingly, the unlabeled lrs-tr with nucleotides as spacer could also function as competitor. however, when ttc/gaa palindrom was spaced by nucleotides (lrs-edl ), it could not compete. thus, expression the transforming protein lmp- can be induced directly by the t cell derived cytokine il- in a stat dependent manner. it is likely that this mechanism operates in vivo as well and determines expression of the ebv encoded protein lmp- and thus the pathogenesis of ebv carrying hls. established knockout/ knockin mice with a fasl deletion mutant that lacks the intracellular portion (fasl ¿ intra). co-culture experiments confirmed that the truncated fasl protein is still capable of inducing apoptosis in fas-sensitive cells. preliminary immune histochemistry data suggest that, in contrast to published data, the absence of the intracellular fasl domain does not alter the intracellular fasl localization in activated t cells. we are currently investigating signalling and proliferative capacity of b-and t-cells derived from homozygous fasl ¿ intra mice. our data point to a rather inhibitory role of fasl reverse signaling during immune responses. during an immune response numerous receptor-mediated signals delivered to t cells direct their proliferation, survival and differentiation. we are using a quantitative model and in vitro methods to assess the "calculus", or decision-making algorithms t cells use to process these multiple signals. previous experiments with ot-i cd t cells revealed that tcr affinity regulated both the frequency of cells responding and the average time taken for cells to reach their first division (ji (ji . : . furthermore, affinity was the sole regulator of the rate of cell death in subsequent divisions. here we examine the same question for cd t cells. again we find that lower affinity peptides stimulate t cells to divide rapidly, however, a high proportion of cells die within each division round, revealing an important potential mechanism for affinity maturation and selection of dominant clones over time. in contrast varying the number of dendritic cells used to stimulate cd + t cells primarily affect the proportion of cd + t cells going into division rather than affecting division time or cell death in subsequent divisions. currently we are using these quantitative methods to measure the effect of cytokines and co-stimulatory molecules cd , cd and cd on parameters of cd + t cell proliferation to inform quantitative models of the immune response under different conditions. our goal is to develop quantitative models of t cell behaviour that can accommodate information at the molecular, cellular and population level. interaction between cd , a member of the tumor necrosis factor receptor superfamily constitutively expressed on antigen-presenting cell as b cells, and cd l, a member of the tumor necrosis factor family transiently expressed on activated t cells, are essential for the development of humoral adaptative immune response. various studies have shown that dual stimulation of b cell through antigen binding on bcr and cd leads to an enhancement of ig and cytokine production. the current dogma postulates that these signals are necessary and sufficient to drive naive b cell proliferation and differentiation to ig secreting plasma cells. however, recent evidence suggests that the innate immune responses could regulate humoral adaptive immune response. indeed, b cells can be activated through engagement of a variety of innate immune receptors, including toll-like receptors (tlrs). soluble cd l is unable to induce murine b cell proliferation. however, we and others have shown that recombinant mouse cd l (rmcd l) can increase proliferation induced by tlr (poly ic) and tlr (lps) agonists. by contrast, we never observed any synergy between rmcd l and tlr / (pam csk ) or tlr / (pam csk ) agonists. to go further in the study of cd l/tlr agonist synergetic effect, we have developed trimeric synthetic molecule to mimick cd l, named mini-cd ls, based on a c -symmetry core holding cd -binding motif lys-gly-tyr-tyr. in surface plasmon resonance experiments, mini-cd ls bind to immobilized human cd and compete with the binding of cd l homotrimers and diplayed effector functions that matched those of the much larger recombinant cd l homotrimers as maturation of mouse dendritic cells and activation of in vivo immune response in a mouse model of trypanosoma cruzi infection. as soluble cd l, mini-cd ls synergize tlr (lps), tlr (poly ic) and tlr / (r ) agonist-induced murine b cell proliferation but no synergy was observed between mini-cd ls and tlr / (pam csk ), tlr / (pam csk ) and tlr (odn ) agonists. synergy between cd l and tlr agonist provide the ground to use such a combination as adjuvant in vaccination strategy. however, to reach this goal, evaluation of cd l/tlr combinations on murine and human b cell activation and differentiation in antibody producing cells are under investigation. interaction of naïve cd + t cells with immature dendritic cells (idc) expressing self-peptides can result in their abortive activation (aa), which leads to the induction of cd + t cell tolerance. we have defined a phenotypic profile for cd + t cells undergoing such aa. these cells undergo limited proliferation which is associated with lack of ifn-g production, low cell surface expression of cd and cd , and high levels of expression of cd l and ly c. whereas, cd + t cells undergoing productive activation (pa), following encounter with mature dc, form effector ctl which is evidenced by extensive t cell proliferation, high levels of ifn-g, cd and cd , and loss of cd l and ly c expression. ly c is a gpi-anchored cell surface glycoprotein expressed on cells of hemopoietic origin: however, its role in peripheral tolerance induction is not understood. in this study, we show that mab-blocking of ly c in vivo and in vitro results in pa rather than aa. we hypothesize that the interaction of ly c, expressed on naïve cd + t cells, with its ligand on idcs, may be vital in controlling the induction of peripheral tolerance amongst self-reactive cd + t cells. objectives: organophosphorus compounds (opcs) are commonly used in the manufacture of insecticides and pesticides. exposure to opcs is associated with neurological toxicity but the effect on the immune system remains ill-defined. in this study, we used a subchronic exposure model to investigate the effect of the organophosphorus compound, paraoxon, on the murine immune system. methods: balb/c mice were injected i. p. daily with saline (control group) or paraoxon (experimental group) for weeks. during the treatment, animals were weighed and blood was collected weekly for determination of acetylcholinesterase activity in red blood cells. at the end of treatment, mice were sacrificed and spleen cells analyzed by flow cytometry. spleen cells were also cultured in the presence or absence of mitogens and supernatants were analyzed for cytokine content by elisa. for in vivo survival studies, mice were treated as described above and then orally infected with a virulent strain of s. typhimurium. animal survival was followed for up to days after infection. results: daily injection of paraoxon induced g % reduction in acetylcholinesterase activity by the end of the first week of treatment, a level which was thereafter maintained during the remaining weeks of treatment. mice exposed to paraoxon exhibited g % reduction in the rate of body weight gain over the treatment period in comparison with control group. at the end of treatment, ex vivo analysis of spleen cellularity and function revealed no significant differences between control and experimental groups. to analyze the status of the immune system in vivo, mice were infected with a lethal dose of a pathogenic strain of s. typhimurium and followed for survival. unexpectedly, paraoxon-treated mice exhibited a significant degree of resistance with % of mice surviving the infection compared to % in control group. protection in paraoxon-treated group was dependent on the reduced acetylcholinesterase activity as it was abrogated by coadministration of a reactivator of cholinesterase. conclusion: our data demonstrate that a reduction in the level of acetyl cholinesterase rendered mice more resistant to a virulent infection. this suggests a hitherto novel function of the neurotransmitter acetycholine in modulating the immune response to infection. t cell-dependent (td) and t cell-independent (ti) igg autoantibodies have been described in the context of the autoimmune disease systemic lupus erythematosus (sle). however, their different roles in autoimmunity are unknown. here we show that ti antigens induce anti-inflammatory igg antibodies and protect from antigen-specific immune pathology. administration of antigen-specific anti-inflammatory igg antibodies was sufficient to mediate this effect independent of the igg inhibitory receptor fcgammariib. ti but not td igg autoantibodies were further associated with inhibition of pro-inflammatory th and th cells and disease in mice deficient for fcgammariib, a spontaneous model for sle. the data suggest a novel immune regulatory function for ti immune responses through the generation of anti-inflammatory igg antibodies. objective: class i phosphoinositide -kinases (pi k) constitute a family of enzymes that generate -phosphorylated polyphosphoinositides at the cell membrane after stimulation of protein tyrosine (tyr) kinase-associated receptors or g protein-coupled receptors (gpcr). the class i pi k are divided into two types: class ia p /p heterodimers, which are activated by tyr kinases, and the class ib p g (p gamma) isoform, which is activated by gpcr. although the t cell receptor (tcr) is a tyr kinase-associated receptor, previous studies showed that p g deletion affects tcr-induced t cell stimulation. mice lacking p g show a partial defect in t cell differentiation, activation and survival. p g participates in signaling pathways that regulate pre-tcr dependent differentiation and cd +/cd + t cell lineage commitment. in the mrl/lpr mouse model of systemic lupus erythematosus, administration of a pi kg-specific inhibitor causes a reduction in the number of cd + memory t cells that mediate renal injury. similarly, pi kg deletion in p pi k transgenic mice also reduces the numbers of cd + memory t cells. there is therefore evidence that pi kg has an important function in tcr-mediated t cell activation, although the mechanism by which pi kg regulates this process is not well understood. we studied the specific role of p g in t cell activation. methods: we studied whether the tcr activates p g and the consequences of interfering with p g expression or function on t cell activation. results: we found that after tcr engagement, p g interacts with and forms a complex with ga q/ , lck and zap . tcr stimulation activates p g, which affects -phosphorylated polyphosphoinositide levels at the immunological synapse. we show that tcr-stimulated p g controls rac activity, f-actin polarization, and the interaction between t cells and antigen-presenting cells (apc). we show that p g deletion affects the activation of many pathways downstream of tcr crosslinking, as well as the interaction between t cells and apc; these findings could explain the defective activation of p g-/-t cells. our observations clarify the activation mechanism and mode of action of p g in the control of t cell activation, confirming a crucial role for p g in tcr-induced t cell activation. we investigated mechanisms controlling central location of lytic granules and kinetics of their release within immune synapses formed by cytotoxic t lymphocytes (ctl). we show that cytolytic granules in ctl can be delivered to the secretory domain via two different pathways -"short" and "long". the choice between these pathways is regulated by the kinetics of early tcr signaling which depends on the strength of tcr/pmhc/co-receptor interactions. meanwhile, the molecular hardware used to deliver the granules remains the same. we conclude that the difference in temporal and spatial coordination of the two principal events, i. e., granule movement toward microtubule organizing center (mtoc) and the mtoc polarization, accounts for two different pathways of granule delivery to the secretory domain that influence efficiency of ctl cytolytic response. our findings reveal a mechanism of well-documented flexibility in t cell responsiveness that is derived from differential use of the similar set of immune receptors, signaling proteins and intracellular effector molecules. objectives: in addition to specific immune cytokines, lymphocyte activation and immune response are modulated by universal mediators like acetylcholine. nicotine was shown to suppress both cellular and humoral immune responses. previously we found that two nicotinic acetylcholine receptor (nachr) subtypes, a b and a , expressed in mouse b lymphocytes regulate their development within the bone marrow. the aim of the present study was to evaluate the roles of these two nachrs in b lymphocyte activation. methods: b lymphocytes were magnetically separated from the spleens of c bl/ j mice. they were stained with fluorescently labeled igm-, cd -or cd specific antibodies in the presence/absence of unlabeled nachr subunit-specific antibodies to be examined by flow cytometry. b lymphocyte activation was studied by h-thymidine incorporation upon stimulation with anti-cd and nachr-specific agonists or antagonists. the antibody response of mice immunized with cytochrome c with or without a nachr antagonist methyllicaconitine (mla) was studied by elisa. results: antibodies against a or b nachr subunits inhibited binding of igm-and cd -specific antibodies but facilitated that of cd -specific antibody. in contrast, antibody against a subunit prevented binding of anti-cd but not of anti-igm or anti-cd suggesting that a nachrs are located close to cd , while a b ones are close to bcr/cd . consequently, anti-cd -induced b lymphocyte proliferation was increased by mla much stronger than by a b -specific antagonist dihydro-b-erythroidine. it was also increased when cells were incubated with the inhibitor of acetylcholine synthesis hemicholine- . in contrast, proliferation of b lymphocytes from mice consuming nicotine was significantly weaker than that of control mice. mice co-injected with cytochrome c and mla responded with igm antibodies faster than those injected with cytochrome c alone, while the secondary / igg responses were similar. the cd -mediated b lymphocyte proliferation, but not the igm-igg switch or memory b cell activation, is negatively controlled by either endogenous acetylcholine or consumed nicotine through a nachrs. therefore, acetylcholine may be regarded as an auto/paracrine regulator of lymphocyte activation.this work was supported by philip morris usa inc. and philip morris international. binding of cd + t h -lymphocytes to antigen presenting cells or of cd + cytotoxic t-lymphocytes (ctl) to their target cells lead to a tight contact between these two cells, called immunological synapse (is). formation of the is induces calcium signaling, rearrangement of the actin cytoskeleton, and the recruitment of various molecules to the is, all of which are crucial for t-cell functions such as cytokine release or target cell killing. objectives: using primary human t-lymphocytes, none of the proteins involved in either calcium influx, cytokine release, actin cytoskeleton rearrangement nor in killing of target cells can be analyzed by knock-out strategies. for testing protein functions, down-regulation by rnai technology is thus an important tool. we used short interfering rnas (sirnas) to analyze the role of proteins involved in calcium influx and proliferation (stim and trpc ), and to analyze snare proteins which were shown to accumulate at the is and are good candidates to play a role in cytotoxic granule fusion and exocytosis to kill target cells. methods: to validate down-regulation of different mrnas quantitative rt-pcr was used. down-regulation of proteins was confirmed by immunocytochemistry, western blotting and various functional assays depending on the potential role of the protein of interest (calcium imaging, proliferation, cytokine release, killing assay). results: transfection efficiency of sirnas in t-lymphocytes was about %. down-regulation of stim was confirmed by qrt-pcr and by calcium imaging, but only for early time points following activation of cd + t h -lymphocytes, probably because of stability problems. to increase stability of sirnas within t-lymphocytes we used modified sirnas published by mantei et al. (eji, ) . we show that these sirnas down-regulate various snare proteins in ctls more efficiently than non-modified sirnas. the optimal sirna concentrations for transfection in primary human t-lymphocytes was found to be - pmol, which is lower than the concentrations reported in other cell types. conclusions: following optimization, down-regulation of mrnas by sirna is a powerful tool to investigate the role of different proteins involved in the activation of t-lymphocytes in primary human cells. chemical modifications increase the lifetime and efficiency of the sirnas in primary human t-lymphocytes. stress-inducible heat shock protein (hsp ) has gained plenty of attention because of its potent adjuvant capability to induce antigen-specific cd + cytotoxic t-lymphocyte (ctl) and cd + t-helper cell (th ) responses. in this study, we investigated the behavior of t-cell subsets stimulated with endotoxin-free recombinant hsp with respect to proliferation, cytokine expression, cytotoxicity against allogeneic b-lymphoblastoid cell line (b-lcl) and k cells as well as targetindependent cytotoxicity. cd + cells exhibited a strong increase in proliferation after stimulation with hsp , with rates of up to %. in the presence of target cells, a -fold up-regulation of granzyme b mrna was observed after stimulation of cd + t-helper cells with hsp in combination with il- , - and - . the target cell-independent secretion of granzyme b by cd + cells was greatly augmented after stimulation with hsp plus il- or il- , - and - . in this study, we have shown that hsp is capable of inducing a cytotoxic response of t-helper cells in the absence of lps or any other pamps. the granzyme b secretion and the cytolytic activity of cd + t cells is induced in a target-independent way, whereas the cytotoxic activity of cd + and cd + t cells can be further enhanced in the presence of the target cells. our data provide novel insights into the role of extracellular hsp on t-cell immune response concerning the induction of target-independent t-helper cell cytotoxicity. jun n-terminal kinases (jnk) have been shown to play controversial role in regulation of cell fate. cd , which is responsible for germinal centre formation in lymph nodes, trigger jnk activation. the role of other b cell co-receptor molecules that may be involved in antigen-driven differentiation were not clarified. the aim of this study was to find out whether cd receptor contributes to jnk activation in mature human b cells. protein expression and phosphorylation were studied by western blot analysis. protein associations were evaluated by immunoprecipitation and gst-pull down assays. hpk overexpression in a model system was achieved by transfection. pjnk / expression in primary hrs cells was assessed by immunohistochemistry. ligation of cd on resting (dense) and activated (buoyant) human tonsillar b cells lead to jnk , but not jnk activation. cd ligation on primary tonsillar b cells also resulted in jnk activation. however, bcr crosslinking did not affect the level of jnk / phosphorylation. cd -mediated jnk activation was independent from sh d a/sap adaptor protein expression, and was demonstrated for all studied b-lymphoblastoid, burkitt's lymphoma and hodgkin's lymphoma (hl) cell lines of b cell origin. we were searching for serine/threonine kinase that could coprecipitate with cd and link this receptor with jnk pathway. using immunoprecipitation and gst-pull down assays we found that hematopoietic progenitor kinase (hpk ) was associated with cd in primary b cells as well as in b cell lines. cd -hpk association was independent from cd tyrosine phosphorylation and sh d a expression. overexpression of hpk in a model system significantly enhanced cd mediated jnk phosphorylation. it is known that tnf family receptors such as cd , cd , rank trigger survival signals in hrs cells. we observed the expression of pjnk / in hrs cells of primary classical hl. cd could be involved in sustained jnk activation in primary hrs cells, and this may reflect the role of cd receptor as well as other receptors in the regulation of hrs survival. overall, it was shown that jnk is activated via cd in primary b cells and in all studied cell lines of b cell origin. serine-threonine kinase hpk is involved in cd -mediated jnk activation. objectives: cd has been shown to act as a negative regulator of tcr signaling during thymocyte development. however, the molecular mechanisms involved in this process remain elusive. one potential key molecule involved in the downmodulation of tcr signaling is c-cbl, a ubiquitin ligase that physically associates with cd upon tcr crosslinking in thymocytes. the objective of this study was to determine which sequences within the cytoplasmic tail of cd are involved in c-cbl phosphorylation and association. methods: el thymoma cell line was stably transfected with wild-type human cd or hcd cytoplasmic tail mutants: cd .k stop (maintaining only a pseudo itim); cd .h stop (lacking the distal s and y in the carboxy-terminal region); cd . ¿ e -l stop (lacking the pseudo-itam, putative site for c-cbl association). phosphorylaton of y in c-cbl was analyzed, which is required for vav recruitment and c-cbl dependent degradation by the proteasome. stable clones were stimulated with anti-murine cd in combination or not with anti-human cd biotinylated antibodies and phosphorylation of c-cbl was detected by flow cytometry after intracellular staining anti-phospho c-cbl (py ) antibody. murine thymocytes were used as positive control. data was analyzed using flowjo software. unpaired two-tailed student t test was used to calculate statistical significance (p x . ). in murine thymocytes, co-crosslinking of cd with cd induces an increase in c-cbl phosphorylation compared to cd alone. analysis of the el- transfectants showed that mutants cd .k stop and cd .h stop lost the ability to costimulate cd -mediated phosphorylation of c-cbl. in contrast, cd . ¿ e -l stop mutant, was able to efficiently costimulate cd -mediated c-cbl phosphorylation, similarly to the hcd wt. our results indicate that the absence of the pseudo itam in cd does not interfere with c-cbl phosphorylation in response to cd plus cd crosslinkiing on the other hand, sequences present in the carboxy-terminal region of cd appear to be important for c-cbl phosphorylation. therefore, c-cbl phosphorylation might not require physical association with the cd cytosplasmic tail, but rather, may indirectly associate with cd through the interaction with other sh -sh domain-containing molecules, that may be recruited to cd through its carboxy-terminal region. l. kolly , s. narayan , j. tschopp , a. so , n. busso chuv, rheumatology, lausanne, switzerland, unil, biochemistry, epalinges, switzerland apoptosis-associated speck-like protein containing a caspase recruitment domain (asc) is an adaptor protein that is essential for the recruitment of pro-capase- into inflammasomes and thus plays a key role in regulating capase- -dependent il- b and il- production. despite recent evidence implicating asc in adaptive immunity against infections, hyperresponsiveness and vaccination, the cellular and molecular basis for asc involvement in adaptive immune responses remains largely unexplored. to investigate the impact of asc on t cell activation and subsequent effector function. asc +/+ and asc -/-t cells or purified cd + and cd + t cells were activated in vitro through anti-cd stimulation and their proliferative potential and cytokine profiles characterized. proliferative responses by asc -/-t cells were significantly inhibited two-fold following tcr-cd ligation when compared to asc +/+ t cells. furthermore, cytokine analysis revealed that anti-cd activated asc -/-t cells predominantly displayed a more th phenotype, producing more il- ( vs. pg/ml; asc +/+ vs. asc -/-t cells respectively; p= . ) and less ifn-g ( , vs. , pg/ml; asc +/+ vs. asc -/-t cells respectively; p = . ). when asc +/+ and asc -/-t cells were purified into cd + and cd + t cell fractions and activated individually using anti-cd , no inhibition in proliferation was observed amongst activated asc -/-cd + and cd + t cells. interestingly, the activated asc -/-cd + t cell fraction produced significantly more il- when compared to activated asc -/-cd + t cells and asc +/+ cd + and cd + t cells (asc -/-cd + t cells = pg/ml il- ; asc -/-cd + t cells = undetectable il- ; asc +/+ cd + t cells = pg/ml il- ; asc +/+ cd + t cells = undetectable il- ). cd + and cd + t cell mixing experiments revealed that asc -/-cd + t cells are able to inhibit the proliferative ability of asc -/-cd + t cells, asc +/+ cd + and cd + t cells in vitro and that this suppression appears to be mediated by a soluble factor secreted by activated asc -/-cd + t cells. collectively, these results demonstrate that the absence of asc drives cd + t cells towards a suppressor cell phenotype, suggesting that asc might play an important role in determining the fate of cd + t cells. various members of the eicosanoid family derived from arachidonic acid participate in inflammatory reactions and may act as potent regulators of the immune response. in particular, e-series prostaglandins, pge and pge suppress some t-cell functions including proliferation, activation and cytokine production. pge signals through four types of gpcrs called the ep receptors. at low concentrations, pge is believed to be necessary for t cell function, whereas at higher concentrations, pge inhibits t cell proliferation. these effects are largely governed by various cell specific stimuli and tissue microenvironment. objectives: to delineate, compare and contrast the effects of pge and ep receptor antagonists on t cell activation. methods: flow cytometry, proliferation assays, migration assays. we have observed that pge diminishes expression of early, intermediate and late t cell activation markers. in contrast, pre-treatment of cd + t cells with ep receptor antagonists was found to impair cell surface expression of cd , cd , cd and ox but not cd . suppression of t cell proliferation by pge has already been widely studied. however, blocking ep receptors in cd + t cells by the use of ep antagonists prior to activation surprisingly caused a defect in t cell proliferation. migration of cd + t cells to the chemokine sdf- b was also found to be reduced due to pre-treatment with ep antagonists. in order to study the physiological relevance of these findings we studied the trafficking of basal and activated t cells to regional lymph nodes during inflammation in the presence and absence of ep receptor antagonists. this model revealed that the use of ep antagonists causes a reduction in the amount of cd + cd + adoptively transferred t cells in the regional lymph node following the induction of a local inflammatory response. conclusions: in our study we show for the first time that ep receptors are required for expression of activation markers and activating proliferation in murine cd + t cells. our results also suggest that considering pge -mediated camp signaling in cd + t cells, it will be absolutely necessary to distinguish between transient increases, which have potentiating effects, and sustained increases, which have inhibitory effects in t cell activation. objective: our objective was to investigate how ros affect the different stages of t cell activation. because activation is initiated by changes in intracellular calcium concentration, we addressed whether and how ros affect calcium signalling. the experimental results were obtained using a combination of fluorescence microscopy, patch-clamp, t-cell activation assays and molecular biology. results: we show by direct measurement of ros that t-cells are exposed to high concentrations of oxidants when they are in close vicinity of activated phagocytes. the effect of ros on calcium signalling in jurkat t-cells as well as in primary naï ve and effector cd + human t-cells was examined. oxidation affects several ca + signalling pathways by altering the activity of ip receptors, trp channels and store operated ca + channels in a concentration dependent manner. interestingly, calcium signalling is differentially affected in naï ve and effector t cells. thiol reducing agents were able to significantly reduce the effects of oxidation implicating thiol oxidation as a major player in the regulation of ca + signalling in t-lymphocytes. cysteins are the main carrier of thiol groups in proteins and we show that orai ion channels contain reactive cysteine groups that mediate ros effects on the calcium influx pathway. conclusion: ros regulate the calcium dependent t-cell activation in a complex way, affecting all three major calcium signalling pathways. by mutational analyses of the orai proteins, we are able to pinpoint molecular targets of regulation. the activation of t cells during an immune response is a crucial but tightly regulated event. to make the grade, the t cells upregulate costimulatory but also inhibitory receptors upon antigen recognition. this enables the t cell to be stimulated for proliferation to keep pace with pathogens infection, but also to become dampened upon successful defense against the pathogens via negative feedback mechanisms. in this study we present data of the signaling mechanisms underlying the potent t cell inhibitory receptor cd (emmprin, basigin) , a member of the ig-family. previous studies reported that lymphocytes from cd knockout mouse possess enhanced mixed lymphocyte reactions and cd monoclonal antibodies can interfere with t cell activation. these observations already pointed to a negative crosstalk of cd signals with the t cell antigen receptor or co-stimulatory signals. consistent with these studies, we found that rna interference (rnai) with cd in jurkat t cells augments the secretion of the t cell growth-factor interleukin- (il- ) upon t cell activation. this up-regulation is at least partially due to an increased activity of the nuclear factor of activated t cells (nfat), which resulted in an enhanced il- promoter activity. by reconstituting the rnai-mediated knockdown with various truncated rnai-resistant forms of cd , we identified the immunomodulatory sub-domain of cd . supported by the gen-au program of the austrian federal ministry of science and research. mirnas play a critical role in the control of hematopoiesis. the goal of this project is to determine whether mirnas function also during the antigen-induced activation of mature b lymphocytes. therefore, we determined mirna profiles in primary splenic b cells before and after polyclonal activation with either lps (simulates t cell-independent activation) or a combination of anti-igm, anti-cd and il (simulates t cell-dependent activation). microarray assays identified about mirnas in unstimulated b cells. of these were downregulated and one was upregulated upon stimulation. in silico analyses with various mirna target prediction programs revealed an interesting and promising set of transcripts whose translation/stability could be controlled by mirnas during the antigen-induced activation phase of mature b cells. among these targets are bcr signalling molecules and transcription factors that control proliferation, igh class switch as well as differentiation in antibody-secreting plasma cells. one of these transcripts codes for the interferon regulatory factor- (irf- ). the graded expression of this important transcription factor has been shown to coordinate isotype switching with plasma cell differentiation. first results indicate that the expression kinetic of irf- transcripts differs from that observed for irf- protein abundance after b cell stimulation. further analysis identified the irf- transcript as a target whose expression is obviously fine-tuned by a mirna upon antigen stimulation. we are in the process to biochemically verify potential targets for each of the differentially regulated mirnas and determine the effect of ectopic and retrovirally mediated expression of mirnas on b cell differentiation. the work was in part supported by the izkf erlangen, the dfg graduiertenkolleg gk and the dfg forschergruppe for . objective: transforming growth factor-b (tgf-b) signals through type i (tgfbri) and type ii (tgfbrii) tgf-b receptors and receptor regulated smad proteins. tgf-b exerts predominantly anti-proliferative and pro-apoptotic effects which are frequently lost in cancer. the mechanisms of resistance against tgf-b have not been fully elucidated. our aim is to describe how b cell lymphoma cells respond to tgf-b compared to normal peripheral b cells, to create an overview of the different signaling pathways involved, and to characterize the mechanisms behind the loss of sensitivity to tgf-b. methods: proliferation assays were performed on different b-cell lymphoma cell lines and normal peripheral b cells to screen for tgf-b-induced effects. western immunoblotting analysis was conducted to characterize protein expression and phosphorylation related to tgf-b signaling pathways. facs analysis was used to measure tgf-b receptor surface levels. cells were treated with demethylating agents to examine changes in gene expression levels. s. manthey , f. hauck , i. berberich , f. berberich-siebelt , gk -immunomodulation institute for virology and immunobiology, university of wuerzburg, würzburg, germany, university of wuerzburg, department of molecular pathology, würzburg, germany the transcription factor ccaat/enhancer-binding protein b (c/ebpb) can not act only as a transcriptional activator but also as a transcriptional repressor. in murine cd + t lymphocytes, the transcription factor is predominantly expressed in t helper (th ) compared to t helper (th ) cells. in contrast, by binding to the c-myc promoter(s), c/ebpb represses c-myc expression thereby arresting t cells in the g phase of the cell cycle. both, transactivation and repression depend on the n-terminal transactivation domain of c/ebpb. blimp- encoded by prdm is a transcription factor necessary for terminal differentiation of b cells to plasma cells. furthermore, blimp- is expressed in differentiated effector t cells where it is higher in th than th cells. the regulation of the blimp expression is not fully understood. interestingly, we found that c/ebpb can bind to the prdm promoter and activates blimp- expression in t cells. as c/ebpb is also expressed in b cells, we hypothesize that this transcription factor might as well influence the expression of blimp- in b cells. so far, we were able to show a similar expression profile of c/ebpb and blimp- in b cells using cre recombinase. moreover we found a new putative blimp- isoform lacking exon . currently, we analyze the expression of c/ebpb and blimp- in primary b cells and b cell lines after various stimulations. to get more insights into the function of c/ebpb in b and t cells, we are generating mice carrying a b as well as a t cell-specific deletion of c/ebpb. engagement of antigen receptors on lymphocytes leads to rapid increases in intracellular free calcium concentrations via phosphorylation of phospholipase c gamma (plcy) and plays an important role in activation of cells. by screening cvid patients with a flow cytometric assay we demonstrate that calcium flux is significantly reduced in b and t cells isolated from the peripheral blood of patients in the group ia of the freiburg classification as compared to non-ia patients and healthy donors (hd). ia patients are characterized by the expansion of an unusual cd low b cell population in which calcium mobilization is strikingly lower than in other b cell subsets. common subpopulations like naï ve and mz-like b cells as well as cd + t cells but not transitional b cells or cd + t cells also revealed significantly decreased calcium peaks. the cytometric data correspond to a semiquantitative rt-pcr assay and functional data showing reduced induction of the calcium dependent macrophage inflammatory protein- a (mip- a), and abrogated activation and proliferation, respectively. preliminary data on b cell receptor (bcr) mediated phosphorylation of plcy revealed constitutively high background levels in cd low b cells of ia patients. since phosphorylation in the other b cell populations as well as calcium flux upon ionomycin were the same for patients and healthy donors, we postulate an abrogated amplification or altered inhibitory pathway targeting the signalling events downstream of plcy and upstream of internal store release, thus resulting in defective calcium signalling. the underlying mechanism yet remains to be elucidated and is part of our work in the future. c. balas , v. courtois , k. de luca , r. sodoyer sanofi pasteur, marcy l'etoile, france the presence and relative abundance of cytokines at different stages of infection is relatively well documented, but their involvement in immune status, pathogenesis or disease progression is still unclear. a potential explanation to the difficult interpretation of the results obtained might be related to the intrinsic weakness of the analytical techniques. for instance monitoring of the expression level of cytokines, such as il- , il- or il- could lead to misinterpretation if molecular isoforms are not detected by antibodies currently used to measure them. the analysis of the human transcriptome is a way to access the subset of genes involved in the immune response upon infection by various pathogens. such an analysis might be completed and enriched by the analysis of the relative expression of some cytokine splice variants. methods: genetic tools (primers and qpcr probes) capable of discriminating and quantifying alternatively spliced messenger rnas from il- , il- and il- . furthermore, the recognition by several commercial antibodies of the different cytokine isoforms (expressed as recombinant proteins) has been investigated. the genetic tools have been validated on in vitro models as well as on biological samples (please refer to the abstract no a- - - ). conclusion: implication of such kind of analysis in diagnostic application and disease progression survey will be discussed. in a different context, the same kind of analysis could be applied to the monitoring of the immune response upon vaccination or more generally for new antigens or adjuvant screening. parasitic helminths affect about one third of the world population. therefore the mechanisms, which are involved in the persistence or the expulsion of the parasite, are of special interest. from other parasitic infections it is known, that the regulatory receptor cytotoxic t lymphocyte antigen- (ctla- ) plays a crucial role during infections. here, we use the strongyloides ratti infection of mice as an experimental system to investigate the role of ctla- during nematode infections. we employed a quantitative real-time pcr (qtpcr) analysis to quantify the migrating larvae (il ) in the tissue and the released eggs and first stage larvae (l ) in the feaces. the cytokine response of lymphocytes, prepared from the spleen and the mesenteric lymphnodes (mln) upon stimulation with polyclonal a-cd and s. ratti antigen was determined. additionally the humoral response was analysed in the primary and the secondary infection. to investigate the role of ctla- during the infection, a neutralysing antibody (a-ctla- ; f ) was administered intraperitoneally ( mg) two hours before subcutaneous infection with s. ratti il . the in vivo neutralisation of ctla- -signalling by applying a-ctla- during s. ratti infection led to an altered cytokine response, compared to infected mice treated with a control antibody. we detected an increase in th cytokines, such as il- and il- and a reduction of the proinflammatory cytokines ifn-g and il- . the investigation of the humoral response showed a remarked increase of the igg -titer in the serum during secondary infection in mice that had been treated with a-ctla- during primary infection. furthermore, the blockade of ctla- resulted in a diminished worm burden as indicated by reduced release of l in the faeces. these results suggest that the blockade of ctla- during s. ratti infection induces an activation of the appropriate effectors of the immune system that are beneficial for the host defence. in particular the transition of the t cell cytokine profile towards a th response supports this hypothesis and might be the reason for the reduced worm output in the primary infection. the strong increase of igg during secondary infection also reflects the induction of a potent th response. objectives: cd is a class b scavenger receptor, which has been shown to be involved in the pathogenesis of atherosclerosis as well as in the clearance of apoptotic cells by macrophages. this clearance is important in regulating the immune system to avoid autoimmune reactions, as seen in systemic lupus erythematosus (sle). it was recently described that cd is highly expressed also on the marginal zone b cell subtype. we therefore set out to investigate the role of cd on the regulation of b cell in the setting of apoptotic cell clearance and autoimmune activation. we used a mouse model for sle where apoptotic cells were injected repeatedly in order to study the auto-reactive antibody response that follows. elisa was used to measure antibody levels and flow cytometry to study cell activation as well as cd expression. cd knock-out (ko) and wild type mice were used. results: preliminary in vivo data show a tendency for a higher antibody response towards ds-dna and the common self-antigen pc in cd ko compared to heterozygous mice. since reduced levels of cd are expressed in heterozygous mice we are currently repeating this experiment using wild type mice as controls for comparison. in support of the in vivo findings, the immunosuppressive effect of injected apoptotic cells seen in wild type mice after in vitro stimulation of splenocytes with lps is gone in cd ko mice. after one injection of apoptotic cells, cd ko b cells are activated while wild type b cells are not. after four injections a break of tolerance is seen and apoptotic cells do no longer have an immunosuppressive effect and we show that cd on b-cells are involved in setting this threshold. conclusion: our data suggest that cd is involved in the early regulation of b cell response towards apoptotic cells and production of autoreactive antibodies. it does so by being involved in regulation of the tolerance effect exerted by apoptotic cells. successful t cell immunity requires lymphocytes to be at the right time at the right place. the co-receptor cd acts as a major check-point of immune responses, but the mechanism by which cd controls peripheral t cell responses is unknown. the consequences of cd signaling on murine th cell migration were analyzed using chemotaxis assays in vitro and radioactive cell tracking in vivo. the genetic and serological inactivation of cd in th cells reduced migration towards ccl , cxcl and ccl . crosslinking of cd together with cd and cd stimulation on activated th cells increased expression of the chemokine receptors ccr and ccr , which in turn enhanced cell migration. sensitive liposome technology reveals that mature dendritic cells but not activated b cells were potent at inducing surface cd expression and cd -mediated migration-enhancing signals. importantly, migration of cd positive th lymphocytes in in vivo experiments increased, as compared to cd negative counterparts, showing that indeed cd orchestrates specific migration of selected th cells to sites of inflammation and antigenic challenge in vivo. these data show that cd signaling does not just silence cells, but selects individual ones for migration. this novel activity of cd adds to the already significant role of cd in controlling peripheral immune responses by allowing t cells to localize correctly during infection. it also suggests that interference with cd signaling provides a tool for altering the cellular composition at sites of inflammation and antigenic challenge. here we analyzed the role of cd signaling on the longevity of cd null t cells. using a sensitive staining method for cd , we show that human cd + cd null and cd + cd null t cells rapidly express surface cd . serological inactivation of cd using specific fab fragments or blockade of cd ligands using ctla ig in cd + cd null and cd + cd null t cells reduces the number of non-apoptotic cells in a fas/fasl-dependent manner. cd crosslinking on activated cd null cells prevents activation-induced cell death (aicd) as a result of reduced caspase activity. apoptosis protection conferred by cd is mediated by pi 'k dependent activation of the kinase akt resulting in enhanced phosphorylation and thereby inhibition of the pro-apoptotic molecule bad. we show that signals triggered by cd act directly on activated cd null t lymphocytes and, due to its exclusive expression as a receptor for cd /cd on cd null t cells, prevention of cd mediated signaling is likely a major target mechanism taking place during therapy with ctla ig. objectives: cd is a transmembrane protein tyrosine phosphatase (ptp) expressed in all nucleated leukocytes. it activates src family kinases (sfks) by dephosphorylating inhibitory tyrosines in their c-terminal tails. in cd -/mouse t cell signaling and development is severely impaired, while other leukocyte populations seem much less affected. at least in part, it is due to the activity of another transmembrane tyrosine phosphatase cd (ptprj, dep- ) which acts as a positive regulator of sfk in cd -/-b cells and macrophages and can compensate for cd deficiency in these cells. indeed, combined deficiency of cd and cd in mice results in defective macrophage and b cell signaling and development, a phenotype much more severe than the loss of either protein alone. naïve murine t cells do not express cd and its expression is increased only after activation. accordingly, no defects in t cell development and signaling in cd -/mice were reported so far. however, in human t cells the role of cd may be different since naive human t cells express cd at a level comparable to b cells. using cd -/-/cd -/human t cell line (jurkat-derived js- cells) we tested the ability of cd to complement cd deficiency in t cells. we used retroviral transduction to express human cd or cd in js- cells and tested their ability to reconstitute major signaling pathways. we also employed substrate trapping mutant of cd to identify direct substrates of this phosphatase. in agreement with previously published data, defective t cell receptor (tcr) signaling was observed in js- cells. expression of wild type cd or cd in js- cells resulted in more rapid calcium mobilization, enhanced tyrosine phosphorylation, and increased cd upregulation after tcr cross-linking. moreover, the carboxy-terminal tyrosine of lck, major t cell sfk, was hypophosphorylated in js- cells expressing cd when compared to control cells. finally, cd substrate trapping mutant expressed in js- cells interacted with lck in vivo suggesting that lck is a direct substrate of cd in js- cells. the results suggest a level of redundancy between cd and cd in human t cells not appreciated so far. during the past decades, great efforts have been made to get insights into the complex process of antigen-induced t cell activation and the underlying signal transduction pathways. the t cell antigen receptor signaling cascade is initiated by phosphorylation of itam-tyrosine residues through the t-cell specific src protein tyrosine kinase family member lck. during t cell activation, lck is supposed to undergo structural changes from a closed inactive to an open active conformation followed by phosphorylation of the itam-motifs. in order to resolve conformational changes of lck in living cells with high spatio-temporal resolution, we designed biochemically active conformational-sensitive förster resonance energy transfer (fret) biosensors using cyan and yellow fluorescent proteins inserted at special positions of the complete kinase backbone. for the live-fret imaging and biochemical assays we complemented lck-deficient jurkat t cells (jcam . ) with the biosensors. by introducing point mutations affecting the two major regulatory tyrosines tyr and tyr we found a dramatic decrease and increase, respectively, of intramolecular fret efficiency compared to the wild type biosensor. these results correspond to unfolding of the biosensor to its active conformation on the one hand and condensation of the kinase structure to its inactive form on the other hand. thus, our biosensor is able to detect phosphorylation modifications of key residues. however, we could not detect any overall change in fret and thus conformation of membrane-associated lck molecules during t cell activation indicating that other mechanisms, presumably reorganization of localization, underlie lck regulation. furthermore, we observed a contribution of intermolecular fret, which indicated homophilic interaction of lck. indeed, by performing single molecule analysis and native d immunoblotting we found lck dimers and higher order oligomers. together, these advanced imaging studies in the live cell context provide a novel picture of the function and regulation of this key kinase in signaling via the t cell antigen receptor. it has been reported that mitochondria accumulate under the immunological synapse (is) in response to tcr (t cell receptor) stimulation. this process seems to be required to allow proper tcr-induced calcium influx in t cells in contact with antigen presenting cells (apcs), because mitochondria can sequester calcium and thus keep crac (ca + release-activated ca +) channels open. however, antigen-induced calcium signaling is very fast, and clearly much faster than mitochondrial translocation toward the is. thus, we speculated that other signals are involved in recruiting the organelles to the contact region between t cells and apcs. we found that the adhesion molecule lfa- (leukocyte function-associated antigen ) induces localization of mitochondria at the is. this process is antigenindependent and is enhanced by the presence of chemokines in the t cell environment. however, tcr triggering stabilizes mitochondria at the synapse and it is important to sustain their recruitment in time. our data suggest that, by recruiting mitochondria to the cell-cell contact region, lfa- prepares and facilitates tcr signaling. we are performing experiments to understand the signalling pathways involved in mitochondria translocation at the is. burkitt lymphoma (bl) is a high grade b cell malignancy (non-hodgkin lymphoma (nhl)) derived from germinal center b cells, that harbours a chromosomal translocation juxtaposing the protooncogene myc next to the regulatory elements of one of the immunoglobulin loci. however, the precise contribution of myc to the pathogenesis of this tumour is poorly understood. based on the definition of a distinguishing gene expression signature for the molecular burkitt lymphoma (mbl) with myc as one hallmarking signature gene (hummel et al. ) we describe a non-viral vector based approach (vockerodt et al. ) to express myc in primary human gcb cells from pediatric tonsils. comparative whole genome gene expression profiling was performed in independent preparations. our data reveal a global change in gene expression in lymphoma precursor cells by myc giving new insight into potential changes of the gene expression program of gcb cells on the accidental way to bl in addition as a first step the function of selected signature genes in bl is accomplished. in a representative cell line with a mbl signature and with a non-mbl signature rnai directed inhibition of elements of the cd signaling cascade was conducted. after activating this particular signaling cascade (cd ) we analysed respective gene expression profiles of ikks, trafs and mapk deficient cells. based on these different rnai-mediated ge-profiles a comparison between both lymphoma types is performed. first attempts are made to reconstruct the topology of the respective signaling pathway by using the nested effects bioinformatic model, which has been described recently (markowetz et al. ). a rat thymic epithelial cell (tec) line (r-tnc. ) was established from a long-term tec culture. this line was characterized as a type of rat cortical tec with nursing activity (tnc). very little is known about molecular mechanism of the tnc/thymocyes interaction. in our previous studies we investigated molecular mechanisms involved in the binding and emperiopolesis of resting thymocytes by r-tnc. cell line in vitro. it was found that a number of adhesion molecules, such as cd , cd , cd , cd a, cd , cd , cd was involved in these processes. objectives: a main goal of this study was to define the adhesion molecules involved in the interaction between r-tnc. line and activated thymocytes. methods: experiments was performed on inbred ao rats. monoclonal antibodies (mabs)-mediated modulation of thymocyte binding and emperiopolesis was tested by adhesion and engulfment assay, respectively, using a coculture of cona and il- activated syngeneic thymocytes and unstimulated or ifn-g stimulated r-tnc. cells. we found that both the adhesion ( min and h) of activated thymoytes were partially blocked by mab to cd and cd molecules (ifn-g unstimulated and ifn-g stimulated r-tnc. cells). early adhesion was inhibited by mab to cd , abtcr, mhc class i molecule (ifn-g stimulated r-tnc. cells) and cd molecule (ifn-g unstimulated r-tnc. cells). after prolonged incubation, significant inhibition was obtained using anti-mhc class i mab (ifn-g unstimulated r-tnc. cells). almost all mabs which were inhibitory in the binding assay were inhibitory in the engulfment assay ( h), namely mab to cd , cd , cd , cd molecule (ifn-g unstimulated and ifn-g stimulated r-tnc. cells) and mhc calss i and mhc class ii molecule (ifn-g unstimulated r-tnc. cells). our results also suggest the involvement of cd a/cd dependent -cd independent pathway in adhesion and cd a/cd dependent -cd dependent pathway in emperiopolesis. the obtained results imply that adhesion, deadhesion and emperiopolesis of activated thymocytes by r-tnc. cell line are tightly regulated processes in which multiple adhesion molecules are involved. the crucial roles of cytokines in shaping t cell responses have been documented in both healthy and disease conditions. interleukin- (il- ), a recently described cytokine, has been shown to exhibit both pro-and anti-inflammatory properties. il- favours naï ve cd t cell differentiation into th cells to the detriment of th or th differentiation. the il- receptor (il- r) is a heterodimer composed of tccr, which confers ligand specificity, and gp , a signal transducing chain that is utilized by several other cytokines. il- has been demonstrated to promote cytotoxic lymphocyte functions of mouse cd t cells, but the potential impact of il- on human cd t cells has not been elucidated. our goal is to investigate the impact of il- on human cd t cell functions. we used peripheral blood mononuclear cells (pbmc) from healthy donors, either exvivo or after short term in vitro activation to perform our analyses. we first assessed whether the il- r is detectable on ex-vivo t cells using flow cytometry. we observed a greater proportion of cd than cd t cells expressing the complete surface il- r (gp +tccr). however, we detected high amounts of intracellular tccr in both, cd and cd t cells, but only polyclonal activation (anti-cd ) of cd t cells led to an actual increase of il- r surface expression. purified cd t cells from healthy donors were shortly stimulated in vitro and then analyzed using flow cytometry-based functional assays. il- activated stat and stat signalling with rapid kinetics in both cd and cd t cells, indicating the capacity of il- to signal through these molecules. addition of il- to anti-cd activated cd t cells led to a significant dose dependent increase of proliferation (as measured by cfse-based assay) and ifn-gamma and granzyme b production (determined by intracellular staining). these results demonstrate a pro-inflammatory impact of il- on human cd t cells. defects in immune regulation could result in the breakdown of immune tolerance leading to development of multiple sclerosis (ms). the pd- /pd-l pathway is associated with production of the immunoregulatory cytokine il- , the suppression of t lymphocytes proliferation by inhibition of akt phosphorylation (pakt), and the elicitation of apoptosis of antigen-specific cells; an impairment in this pathway could play a pathogenetic role in ms. we analysed by flow-cytometry the surface expression of pd-l and pd , as well as myelin basic protein (mbp)-stimulated il- production, pakt inhibition, and apoptosis (annexin v), in ms patients with relapsing-remitting disease. twenty-six patients were diagnosed as being affected by acute disease (ams); had a diagnosis of stable disease (sms). results showed that: ) pd-l -expressing cd + and cd + cells are reduced in ams compared to sms individuals (p= . ); and ) pd expression is increased in cd + t cells of sms individuals and is comparable on cd + t cells of ams and sms patients. this is associated with a significant decrease in il- production by mbp-stimulated cd + and cd + cells of ams patients (p= . ). additionally, cd + anexin v+ (av+) cells were diminished and cd + pakt+ cells were higher in ams compared to sms patients, while similar percentages of cd +av+ and cd + pakt+ were observed in both groups of individuals. data herein show that the impairments of the pd-l /pd- pathway seen in ams patients result in a reduced mbp-specific il- production by cd + and cd + cells as well as in a reduced apoptosis (annexin v) and an augmented proliferation (pakt) of mbp-specific cd + t. the pd /pdl pathway plays an important role in the pathogenesis of multiple sclerosis. monitoring of the expression of these proteins could be a novel diagnostic tool. anti- - bb in cd cells. this difference could be due to down regulation of cd by activated lymphocytes and possible preferential response of cd cells to anti- - bb costimulation. moreover, increase in ifn-g concentration in costimulated cultures also may enhance the suppressive function of mscs which again could explain the inability of costimulation in proliferation recovery. likewise, reducing tgf-ß by costimulation is not sufficient to abolish suppressive effect of mscs. in overall, these results suggest that lack of costimulation expression by mscs is not the mechanism of msc suppression and other mechanisms are involved. cytotoxic t lymphocytes (ctls) kill target cells by secretion of cytotoxic components contained in lytic granules at the contact zone between the target cell and the ctl, the immunological synapse (is). t cell receptor (tcr) enrichment at the is is one of the early and key events of is formation. objectives: soluble nsf attachment receptor (snare) proteins are required in almost all fusion events in cells. in the present study we tested if the snare protein syntaxin (stx ) is part of the is and whether it serves as a key player of is formation and/or the fusion process itself. methods: pcr-techniques, cell transfection, immunocytochemistry and different microscopic techniques like confocal microscopy and total internal reflection microscopy (tirf) were used on primary human ctls to test the function of stx . rna interference technique was also used to down regulate stx expression in primary human ctls. results: we identified stx in ctls by pcr and immunocytochemistry. stx accumulates at the is after ctl/target cell contact. when stx function was blocked by overexpression of a dominant negative stx mutant (deletion of the transmembrane region), functional studies with tirf showed a reduced accumulation and fusion of lytic granules at the is. furthermore, confocal studies showed a loss of tcr accumulation at the ctl/target contact side. conclusion: these results imply that the snare protein stx is present at the is and moreover is required for is formation in ctls. the observed block of lytic granule release is probably caused by disturbing an upstream process such as vesicle transport, recycling or sorting. objectives: despite the years history of mouse t h and t h subpopulations, relatively little is known about the differences in their signaling mechanisms and the membrane organization of critical receptors and signal transducing molecules. we have developed mouse t h hybridomas to study these differences between polarized t h cells. the in vitro established hybridomas were first characterized as t h , t h or t h phenotypes, based on their cytokine production (il- , ifng or il- ). a comparative analysis of t-bet, ifng and il- mrna levels was also done on quiescent and activated t h hybridomas. in the present study, the ca +response, membrane raft expression/organization, k + -and ca + -ion channel expression/function and sensitivity to apoptosis (aicd) were compared in these hybridomas. expressions and molecular localizations were investigated by flow cytometry and confocal microscopy, respectively. ion channnels were functionally analyzed by patch-clamp technique. apoptosis was analyzed using three markers (mitochondrial membrane potential, caspase activation, dna fragmentation) and flow cytometry. results: expression level of plasma membrane rafts/gangliosides (assessed by cholera toxin b-staining) showed the following rank: t h g t h g t h , although the membrane cholesterol level (detected with anti-cholesterol ab, ac ) was similar in the three cells. in connection, tcr displayed stronger colocalization with rafts and appeared more polarized in t h cells upon activation than in t h cells. t h cells produced a more sustained calcium response with higher amplitude than t h cells to the same tcr-mediated triggering signal. interestingly, this does not coincide with the expression of cav . and kv . ion channels, major functional determinants of the sustained calcium influx. t h cells expressed the highest levels of these two ion channels. there were also marked differences in their sensitivity to activation induced apoptosis (aicd) as assessed by three different markers of apoptosis. the results suggest that a different membrane compartmentation/organization rather than the differential expressions of certain receptors, ion channels and/or other upstream signaling molecules of these t h hybridomas may be responsible for the observed differences in their functional characteristics. objectives: bone morphogenetic proteins (bmps) belong to the tgf-b superfamily, which plays a central role in controlling cellular processes like proliferation, differentiation, apoptosis and migration. whereas tgf-b is well established as one of the most potent negative regulators of hematopoietic cells, the role of bmps in b lymphoid cells remains more elusive. in this study we investigated the effects of bmps on mature human b-cells. methods: b cells were isolated from peripheral blood of healthy donors using cd -dynabeads. cd + isolated cells were facs sorted into cd + cd naïve b or cd + cd + memory b cells. dna synthesis was measured by h-thymidine incorporation, immunoglobulin (ig) levels in cell supernatants were measured by elisa and phospho-protein levels were measured by western immunoblotting analysis. results: all bmps significantly suppressed anti-igm-induced proliferation of cd + cd naï ve b cells, of which bmp- and - were most efficient ( % suppression). similarly, all bmps suppressed cpg-induced proliferation of cd + cd + memory b cells by - %. to induce differentiation, both naï ve and memory b cells were stimulated with cd l and il- . this increased the production of igm, iga and igg - -fold compared to medium control, whereas addition of bmps inhibited the production of all ig classes. all bmps highly induced phosphorylation of smad / / in cd + b cells. the mechanisms for how bmps mediate their inhibitory effects are currently being explored in more detail. conclusion: bmps have prominent inhibitory effects on anti-igm-and cpg-induced proliferation of naive and memory human b cells, respectively. they also suppress cd l/il- -induced production of igs in mature human b cells. s. gutenberger , k. warnatz university medical centre freiburg, freiburg, germany background: signals through the b cell receptor (bcr) and co-receptors are essential for the survival, differentiation and effector function of b cells. the stimulation of the bcr initiates several independent but interrelated signaling pathways. one important pathway leads to the activation of mitogen activated protein kinases (mapk) and especially the phosphorylation of extracellular signal-regulated kinases and (erk / ). in a subgroup of patients with common variable immunodeficiency (cvid) we have previously demonstrated intrinsic defects in the activation of b cells revealed by the insufficient cd upregulation and proliferation after b cell receptor (bcr) stimulation. therefore we assessed signaling pathways downstream of the bcr in order to identify defects in the activation of b cells. methods: pbmc of hd and cvid patients were stimulated by anti-igm. different igm expressing b cell subsets were analyzed separately for erk / phosphorylation by intracellular flow-cytometry using phospho-specific antibodies to erk / . to increase the signal intracellular phosphatases were inhibited by h o . as markers of activation and initiation of proliferation, cd and ki expression were measured after days of in vitro stimulation. k. theil , p. aichele immh university freiburg, immunology, freiburg, germany type i interferons are homone-like molecules that are produced early after viral and bacterial infections. they signal via the type i interferon receptor (ifnar) and have pleiotropic effects on different cells of the immune system. their best known function is the antiviral activity. to test the direct effect of type i interferons on cd t cells in vivo we adoptively transferred lcmv glycoprotein specific tcr transgenic p cd t cells that are deficient in type i interferon receptor (ifnar-/-) into wild-type b -recipient mice and compared their expansion with wild-type (wt) p t cells after viral infection. we could demonstrate a severe impairment in the capacity of p t cells lacking type i ifnr (ifnar-/-) to expand after lcmv infection. following infection of recipient mice with recombinant vaccinia virus, recombinant vsv (vesicular stomatitis virus) or recombinant listeria monocytogenes expressing lcmv glycoprotein, p t cells expansion was considerably less dependent on type i ifnr expression. therefore direct type i ifn signalling is essential for cd t cell expansion and survival only after lcmv infection. our experiments showed that the lcmv generated cytokine milieu is responsible for the failure of expansion of ifnar-/-t cells during lcmv infection. a suitable model for elucidating the impact of the lcmv generated cytokine milieu is the transfer of p t cells into h mice. h mice ubiquitously express the lcmv immunodominant glycoprotein-epitope gp - . therefore the antigen-presentation can be uncoupled from the lcmv induced cytokine milieu when the h mice are infected with lcmv . . this is a lcmv variant that has got a point mutation in gp - and consequently cannot be recognized by the p t cells. s. frischbutter , r. baumgrass deutsches rheuma-forschungszentrum, signal transduction, berlin, germany antigen-specific stimulation of t helper cells induces activation of the main transcription factors nfat, nf-kb and ap which are important for expression of cytokines such as il- , ifng and il . it is known that the immunosuppressive drug cyclosporin a (csa) blocks the activity of the ser/thr phosphatase calcineurin and thereby the activation of the transcription factor nfat. however, we and others observed that this drug also inhibits the activation of nf-kb. to detect targets of calcineurin within the nf-kb pathway we analyzed phosphorylation and degradation levels of different nf-kb signaling proteins in the presence of csa and other calcineurin inhibitors. we found that phosphorylation of the signaling protein bcl- was prolonged in cells treated with inhibitors. our data do not indicate an enhanced bcl- phosphorylation but rather an inhibition of bcl- dephosphorylation. furthermore, calcineurin and bcl- co-precipitated with each other. interestingly, this interaction was observed only in t cell receptor-but not in tnfa-stimulated cells. in our proposed model, we hypothesize that calcineurin interacts with the carma/bcl- /malt signaling complex and dephosphorylates bcl- and, thus, promotes nf-kb activation. therefore, calcineurin is not only a hub for nfat but also for nf-kb activation. a. t. fulop , j. lamoureux , c. fortin université de sherbrooke, medicine, sherbrooke, canada objectives: aging is accompanied by a decrease in immune functions, called immunosenescence. the exact cause is still not known. changes in t cell subpopulations, thymic involution were invoked. we have demonstrated that the signal transduction is altered with aging. in the present work we studied the negative regulatory molecules in the t cell signaling to explain the altered activation of t cells with aging leading to decreased clonal expansion. methods: healthy young and elderly subjects were studied. lymphocytes were separated by fycoll-hypaque. the molecules participating in the negative control loop of lck were studied by western blot and confocal microscopy. the surface expression of ctla- has been studied by facscan. the translocation of the molecules in the membrane lipid rafts (mlr) was also studied by western blot. the activity of phosphatases was also determined. results: we found that the phosphorylation of pag was altered with aging explaining the decreased release of csk from mlr and the decreased lck activation. the activation of fynt was also altered. the phosphatase activity studies showed an increase in their activities with aging. the ctla- expression was higher after stimulation in t cells of elderly. there was differences between cd and cd t cells with aging. conclusion: these results suggest that the negative regulation is preponderant in t cells with aging on the positive activation and as such explaining the defect in t cell functions with aging. this opens new therapeutical avenues in the future. in contrast to other members of the tumour necrosis factor superfamily, fas ligand (cd l) contains a cytosolic proline-rich domain (prd) that enables interactions with sh and ww domain proteins. since fasl surface expression is regulated by adam -mediated ectodomain shedding and fasl might be subsequently released into the cytosol by regulated intramembrane proteolysis (riping) through the secretase-like enzyme sppl a, we are interested in defining interactions involving the generated intracellular fragment of fasl. employing a monoclonal antibody directed against the intracellular domain of fasl, we observed that previously described fasl-interacting proteins of the pch family selectively bind to the full length molecule but not to n-terminal fragments (ntfs). in order to identify other sh domain proteins that potentially interact with the riped fasl prd, we used a sh domain phage display library containing all sh domains expressed in humans. the screen confirmed several previously identified interactions but also revealed numerous new and interesting candidate binding proteins includig non-receptor tyrosine kinases and adaptor proteins or enzymes implicated in membrane, organelle, and actin cytoskeleton dynamics. selected interactions were verified biochemically and by laserscanning microscopy in transfected cells. it could be demonstrated that tec kinases known to be involved in immune receptor-associated signal transduction as well as members of the snx family, which are crucial regulators of endocytic and endosomal dynamics and trafficking, join the list of known fasl-interacting proteins. of note, in contrast to pch proteins, the snxs bound both ntfs and unprocessed fasl, indicating that individual interactors might influence different facets of fasl biology. in conclusion, the present data provide substantial evidence for a selective binding of individual interaction partners of fasl to the full length protein or ntfs. this more detailed glance at the fasl interactome will facilitate focussed strategies to clarify unanswered questions regarding reverse signalling and functional conse- optimal t cell activation requires the engagement of the t cell receptor (tcr) by the specific mhc/antigen complex and costimulatory signals as the interaction of b family members on antigen-presenting cells with cd on t cells. remarkably, whereas classical glucocorticoids (gcs) effectively suppress solely tcrtriggered t cell activation in vitro, additional cd co-stimulation leads to gc-resistance. in this study, we compared the non-steroidal selective glucocorticoid receptor agonist (segra), compound , with classical gcs regarding their suppressive effect on cd -costimulated t cells. human primary t cell subpopulations and jurkat cells were stimulated in vitro with plate-bound anti-cd and anti-cd , and proliferation, cytokine secretion as well as phenotypic activation parameters were determined. remarkably, a clearly improved inhibition of ifn-gamma secretion was observed in cd -costimulated human memory/effector cd + t cells by compound than by classical gcs. interestingly, apoptosis and activation antigen expression were similarly regulated. improved inhibition of lymphokine secretion by compound was also seen after pma / ionomycin stimulation of human primary t cells and jurkat cells. when investigating the in vivo effects of compound and prednisolone in acute and subacute dnfb-induced contact hypersensitivity models in mice, we observed comparable efficacy for inhibition of t cell-dependent skin inflammation when treating before hapten challenge. in contrast, however, when treating around hapten sensitization markedly stronger effects were demonstrated for compound than prednisolone. when evaluating possible mechanism for the increased activity of compound in inhibition of t cell activation we got hints for a specific inhibition of the calcineurin pathway by compound which was not prevented by the partial gc receptor antagonist, ru- , in vitro. moreover, in vivo we observed less induction of il- beta and tnf-alpha by pre-treatment with compound than with prednisolone. our data indicate that the non-steroidal segra, compound , may represent a promising drug candidate for the treatment of t cell-dependent inflammatory diseases where therapy with classical gcs is hampered by t cell resistance. influenza a infection of b mice elicits robust cd + t cell responses, with virus-specific cells showing a distinct pattern of cytokine production: tnfa+ cells always express ifng; and il- + cells are contained entirely in the ifng+tnfa+ subset. interestingly, the co-expression of ifng and tnfa varies for different epitope specificities. almost all ifng+ pa -specific cells also express tnfa, but only about half of the ifng+ np -specific cells co-express tnfa. this was originally linked to the avidity of the responding population for the specific peptide/mhc complex, with the ifng+tnfa+ phenotype representing cd + t cells with higher avidity and a more differentiated phenotype. however, the same cytokine pattern is seen in adoptively transferred cd + t cells expressing a clonal tcr, implying avidity alone cannot control development of cytokine profiles. co-expression of ifng and tnfa by adoptively transferred cfse-labelled ot-i cells following infection with influenza a virus expressing ova - peptide shows a close correlation with division in vivo. early after antigen encounter ( - divisions) the vast majority of cells express only tnfa. after - divisions cells begin to co-express ifng and tnfa. the emergence of an ifng+tnfa-phenotype increases with subsequent divisions ( - divisions), indicating cytokine profile is closely linked to cell cycling, as described previously for both b cells and cd + t cells. titration of adoptively transferred ot-i cells, which controls the level of expansion in vivo, reveals that more cd + t cells develop an ifng+tnfa-phenotype with increased expansion. thus we conclude that while tcr avidity and co-stimulation can impact the differentiation of cd + t cells, expansion plays a very important role in the regulation of cd + t cell effector function. in addition to its chemo-attractant function, sdf- a (stromal-cell derived factor- a, cxcl ) has been described to costimulate cd + t cell during tcr triggering. our objective is to clarify the mechanism regulating this costimulatory activity. tcr-driven proliferation of human cd + t cells was increased by immobilized sdf- a to a level similar to that obtained with the costimulatory molecule cd . as visualized by real time confocal microscopy, t cells entering in contact with sdf- a formed a tether and displayed an active scanning activity. since sdf- a induced a similar activity in t cells stimulated with a sub-optimal dose of anti-cd mabs, it is conceivable that the sdf- a-driven scanning may favour productive tcr engagement. to test this hypothesis, we are studying the effect of sdf- a on tcr internalization, calcium mobilization, mapk activation and actin cytoskeleton reorganization. we are also studying the role of sdf- a in the context of cd + t activation by antigen-presenting cells secreting sdf- a. this study should help us to better define how sdf- a modulates cd + t cell activation beyond its chemo-attractant function. background: propolis, an ancient herbal medicine, is well known for the management of respiratory diseases. caffeic acid phenethyl ester (cape), an active component in propolis, is known to have anti-tumor, anti-inflammatory, and antioxidant properties. in this study, the effect of cape on the functions of t cells, which play the major role in chronic airway inflammation of asthma, was evaluated. method: cd + t cells isolated from human peripheral mononuclear cells by automacs were stimulated with anti-cd and anti-cd antibodies and cape for days. cytokine levels were dertermined by elisa and lymphoproliferation was analyzed by h-thymidine incorporation method. signaling pathway of t cells was studied by western blot. result: it was found that cape significantly inhibited ifn-g and il- production and lymphoproliferation in cd + t cells stimulated by anti-cd /cd . cape could inhibit nuclear factor-kb (nf-kb) activation, but not mitogen-activated protein kinase (mapk) family phosphorylation in t cells. cape could also inhibit akt phosphorylation. conclusion: these results indicated that cape inhibits cytokine production and lymphoproliferation of t cells which might be related to the nf-kb and akt signaling pathway. this study also provided a new insight into the mechanism of cape in immunology and the rationale for propolis in the treatment of allergic disorders. objective: upon activation, cd t cells express a variety of molecules on their surface, such as mhc-class ii, cd , cd , cd , whose ligands are constitutively expressed on resting t cells. whereas these molecules are physiologically expressed on antigen presenting cells, their function on t cells is not understood. we tested the hypothesis that activated cd t cells might induce t cell proliferation and differentiation from cd resting t cells through interaction of activationinduced surface molecules and their constitutively expressed ligands. methods: cd t cells from the peripheral blood of healthy donors were co-cultured with fixed activated t cells from the same donor. after days of co-culture, the phenotype of the resulting cells was analyzed by assessing their surface molecules and production of cytokines. results: cd memory t cells but not naive t cells proliferated in response to contact with activated t cells. these cells showed a mild activated phenotype assessed by the expression of cd , cd , and cd . analysis of the cytokine profile of these cells revealed the differentiation of il- -and ifn-g-double-producing cells in response to contact with th effector cells, and il- -producing cells in response to contact with th effector cells. the levels of produced cytokines were, however, significantly lower than those produced by activated cells in response to anti-cd /cd stimulation. whereas neutralization of ifn-g or il- during culture did not diminish the frequency of the arising cytokine-producing cells, separation of the responder cell population from effector cells by a transwell system led to a significant decrease of cytokine secretion. blocking particular receptor/ligand interactions by neutralizing antibodies against hla-dr, cd , cd , and cd could not prevent cytokine production induced by t-t cell interaction. however, simultaneous addition of all antibodies significantly inhibited cytokine production to - %. conclusion: interaction of cd memory t cells with activated t cells resulted in the production of the cytokines il- , il- , and ifn-g. given the immunomodulatory capacity of il- and il- , these findings might indicate a novel potential negative feedback mechanism to control t cell-driven immunity. a. nasir , s. thompson , j.j. murphy king's college london, division of immunology infection and inflammatory disease, london, united kingdom the murine bcl leukaemia cell line can be induced to undergo plasmacytoid differentiation in vitro with cytokines il- and il- and this is characterised by a marked reduction in proliferation and production of large amounts of secreted igm. these cells were observed to express significant levels of the zinc fingercontaining protein zfp l by western blot analysis. this protein is reported to act in post-transcriptional regulation of gene expression by binding to au rich elements (ares) of mrnas of certain genes and consequently promoting mrna degradation. at a cell functional level, zfp l has been described to have roles in apoptosis, proliferation and differentiation in different cellular contexts. cytokine-induced bcl differentiation was observed to be associated with downregulation of zfp l protein. in an attempt to determine whether zfp l downregulation was directly linked to bcl differentiation, a zfp l shrna expressing lentivirus (psicor) was employed to knockdown zfp l expression. this reagent downregulated zfp l expression very effectively . shrna infected cells proliferated less well than either control virus infected cells or wild-type cells with or without cytokines. zfp l shrna infected cells also produced more secreted igm per cell than either control virus infected cells or wild-type cells in the presence or absence of cytokines. these results are consistent with a role for zfp l downregulation in promoting bcl plasmacytoid differentiation. vidual lysates of peripheral blood lymphocytes (pbl) of patients with igg multiple myeloma and healthy controls were investigated for the expression of sialic acid (sa), galactose (gal) and n-acetylglucosamine (glcnac), the sugars known to specify the glycoforms of human serum igg. the degree of glycosylation and signaling status of all isolated myeloma igg bcrs were correlated and compared with the glycosylation of the igg paraproteins isolated from sera of the same patients. it was shown that bcr igg in myeloma is more heavily sialylated when compared with normal controls, that the increased sialylation of igg bcr is associated with higher levels of tyrosine phosphorylation (signaling activity) of the igg bcr supramolecular complex and that bcr igg and serum igg paraprotein from the same patient differed in all cases in the levels of terminal sugar expression. the results suggest that the development of the malignant clone in mm from postswitch b cells expressing igg bcr at their surfaces to plasma cells secreting igg paraprotein may be followed by permanent glycosylation changes in the igg molecules. caused by thapsigargin-induced release of calcium from the endoplasmic reticulum was insensitive to tpen. conclusion: the signal with fluorescent probes for the detection of calcium ions in response to thimerosal is entirely due to zinc release, and no indication for a calcium signal was detected. in light of these observations, zinc may also contribute to calcium signals caused by mercury containing compounds other than tms, and a potential involvement of zinc release in the immunomodulatory effects of these substances should be considered. although best known for its pro-apoptotic function, it seems clear now that cd (fas, apo- ) also exerts anti-apoptotic effects associated with costimulation and the induction of proliferation. we investigated effects of fas co-ligation during tcr/cd /cd -triggered activation of freshly isolated human t-lymphocytes. to this end, tcr-triggered cells were incubated in presence or absence of different ligand concentrations of anti-apo mab, faslfc or faslstrepfc fusion proteins, or leucin zipper (lz-)cd l. interestingly for all ligands tested, we could clearly demonstrate a correlation between ligand concentration and t cell response: low doses drastically augmented proliferation in the sense of costimulation, whereas high doses completely blocked tcr-induced cell proliferation without inducing cell death. the positive costimulatory effect of fas at low concentrations is associated with elevated il- and ifng production, upregulation of activation markers, adhesion molecules and cell-cycle regulating cdks and cyclins. in addition, we observed an increased activation of important signalling molecules including mapk and caspases. using pharmacological inhibitors, we demonstrate that fas is internalized upon ligation. we also observed an increased tcr internalisation following fas co-incubation potentially resulting in the generation of larger signalling platforms that allow optimal t cell activation. in stark contrast, most fas ligands at high concentrations almost completely inhibited cell proliferation of tcr-triggered lymphocytes. in this context, crucial events associated with t cell activation, i. e. tyrosine and erk / phosphorylation, the expression of various activation markers, the il- production and caspase activation were almost completely abrogated. these findings highlight that fas-triggering accelerates or blocks t cell activation, depending on the strength of the stimulus. in addition, we provide further evidence for an anti-apoptotic function of fasl during signal initiation in human t lymphocytes. sponsored by the dfg (sfb ) and the medical faculty kiel (to oj) it has been shown that glycosylation of cell surface proteins controls critical t cell processes, including homing, thymocytes maturation, activation, and cell death. plant lectins have been long used to study changes in cell surface carbohydrate structures, to identify leukocyte cell subsets, and as surrogates for authentic t cell activation stimulus. the galb , galnac-specific lectin from amaranthus leucocarpus (all) shows a differential binding pattern to murine thymocytes and peripheral cd + and cd + t cells. in addition, mitogenic stimulus increase -fold the all binding to cd + t cells. previous studies in human pbmc showed that all binds to human cd + t cells and all-binding increased after a mitogenic stimulus using total cell cultures as murine studies. these data suggest that all detects selectively activation-related changes in cd + t cell surface carbohydrate but none study has been performed to examine the all effect on human t cell activation. to examine the effect of all on human t cell activation, we analyzed the anti-cd -dependent activation of purified cd + t cells from pbmc in presence or absent of all by measuring proliferation using cfda-se staining, expression of the surface activation marker cd and calcium influx by flow cytometry. results showed that all did not induce significantly t cell proliferation or cd expression, but enhanced the anti-cd -dependent proliferation and cd expression of purified cd + t cells. analisis of calcium influx showed that all enhanced anti-cd dependent calcium influx. our findings indicated that all alone does not affect t cell activation but suggested that all induces a costimulatory effect on human cd + t cells by up-regulating t cell activation mediated by anti-cd stimulus, as further studies have to be performed to elucidate all-induced costimulatory effect. financed in part by papiit-unam (in ) a. the adaptor protein lat (linker for activation of t cells) has a prominent role in the transduction of intracellular signals elicited by the tcr/cd complex. upon tcr engagement, lat becomes tyrosine-phosphorylated and thereby recruits to the membrane several proteins implicated in the activation of downstream signaling pathways, leading to tightly equilibrated programs of activation and survival or induced cell death. the balance between cell survival and cell death is critical for normal t cell development and activation, and is maintained by signals through lymphocyte antigen receptors and death receptors such as cd receptor. it has been previously demonstrated that cd ligation in t cells induces the proteolytic cleavage of several adaptor proteins, including gads, slp- , slap- and lat. given the dual role of lat as a transducer of activation and negative signals in t cells, we have analyzed the role of the lat cleavage in t cell functions and studied the proteases responsible for this cleavage. objective: the study is designed to explore preliminarily the need of t cells for cytokines during the culture in vitro, which are associated with the activation, proliferation and apoptosis of t cells, and by detecting the expressions of il-rs, co-stimulatory molecules and apoptotic receptors/ligands onto human peripheral blood lymphocytes (hpbls). the results may lay a theoretic and experimental basis for developing the condition media qualified especially to t cell culture. methods: pbls were isolated , and cultured in different media. both immunocytochemistry staining and cell enzyme linked immunosorbent assay (celisa) were used to detect the expressions of il- r, il- r, il- r, il- r, il- r, il- r, cd , cd , cdw ( - bb), cd (fas) and cd (fasl) on hpbls in different cultured time, i. e. d, d, d, d, d, d, d and d. using typan blue staining, the living cells, dead cells and total cells of each cultured group were counted, then their cell growth curves were drawn out. to evaluate the cellular activity, growth situation and cell cycle of t cells, both mtt and fcm analysis were also performed separately. . the expressions of several membrane immune molecules on the lymphocytes in different cultured conditions. ) the expressions of membrane immune molecules before cultured. ) expressions of the membrane molecules on hpbls during culturing. % fbs rpmi group ( group), il- group, pha group... ( ) mtt assay. ( ) proliferative times and growth curves of hpbls... . during cultured in vitro, there are expression changes of the il- rs (il- ra, il- ra, il- rg, il- r, il- r, il- r, il- r), co-stimulatory molecules (cd , cd , - bb) and apoptosis associated molecules (fas/fasl) on hpbls in different time and cultured media. the expression patterns of the most molecules checked are similar in group, il- group and pha group, but the rests are different. . our data also suggest that the hpbls cultured in cd mcab+cd mcab+il +il a group has a great proliferative potential compared with the other groups. using this condition medium, may have a practical prospect to tumor therapy. . celisa will become probably an effective test to detect the expressions of membrane receptors or molecules quantitatively on a large scale. f. beceren-braun , r. tauber zentralinstitut für laboratoriumsmedizin und pathobiochemie, berlin, germany l-selectin is a leukocyte cell surface glycoprotein involved in carbohydrate-specific ligand binding which mediates tethering of leukocytes to the endothelial surface during inflammation. apart from its role in adhesion, l-selectin functions as a signal transduction molecule. crosslinking of l-selectin with antibodies or ligand binding to the receptor have been shown to elicit a wide range of cellular responses. in addition to process signals coming from outside of the cell, the intracellular part of l-selectin (lscyto) is also able to conduct intracellular signals, e. g. activates tyrosine kinase p lck and the ras/rac signalling pathway ( ) followed by mitogen-activated protein kinases ( ) and c-jun n-terminal kinase ( ), which leads to an enhanced binding of l-selectin to soluble ligands ( ). in our previous work we described an association of lscyto with isozymes of the pkc family which phosphorylate the receptor on serine residues ( ). here we show that the protein phosphatase a inhibitor phapii is a novel direct interacting partner of the lscyto. we propose a model in which the l-selectin mediated signalling is regulated by the interaction of pkc, pp a and phapii: phapii binds to the unphosphorylated lscyto. upon l-selectin crosslinking lscyto is phosphorylated, pha-pii dissociates and inhibits the phosphatase pp a. in addition we have started structural analysis to investigate ligand binding induced conformational changes of the cytoplasmatic domain of l-selectin. v. heissmeyer , e. glasmacher helmholtz center munich, molecular immunology, munich, germany during self-antigen recognition, roquin dependent posttranscriptional downregulation of icos prevents t cell help to b cells and autoantibody production. the molecular mechanism by which roquin interferes with icos translation remained unclear. we have identified two critical regions in roquin. the amino-terminus is required for rna binding and can be functionally replaced by conserved sequences from its paralog mnab. the carboxy-terminus mediates p body localization and has specialized in roquin for efficient repression of icos in t cells. using knockout cells of dicer or ago - genes, we prove that roquin mediated repression of icos occurs in the absence of mirisc formation. instead, roquin function required intact p bodies, and was impaired after knockdown of lsm and rck or expression of dominant-negative gw . interestingly, roquin activity is blocked through induced mirisc formation implicating the mutual regulation of different mechanisms of posttranscriptional gene silencing in immune responses. s objectives: upon encountering their antigens, naï ve t cells are activated and driven to clonal expansion and differentiation into armed effector cells. according to the two-signal hypothesis, the induction of an optimal cd + t-cell immune response requires both antigen-specific and co-stimulatory signals. in contrast, stimulating naïve cd + t cells with specific antigens and costimulatory signals is insufficient to induce optimal clonal expansion and effector functions. thus, cd + t cells require additional signals for full activation and further differentiation into effector cells. methods: in this study, we adopted an in vitro approach to dissect the cellular and molecular requirements for cd + t-cell activation and differentiation. naïve cd l hi cd lo cd + t cells were sorted and stimulated by anti-cd and anti-cd antibodies. results: firstly, we show that the activation and differentiation of cd + t cells require il- provided by activated cd + t cells at the initial priming stage after stimulation. secondly, this critical il- signal is delivered through il rbg of cd + cells and is independent of il- ra. besides promoting cell proliferation, il- stimulation increases the amount of ifng and granzyme b produced by cd + t cells. conclusion: therefore, our studies demonstrate that a full cd + t-cell response is elicited by a critical temporal function of il- released from cd + t cells, providing mechanistic insights into the regulation of cd + t cell activation and differentiation. most antigenic peptides recognized by cd t lymphocytes are produced through degradation of intracellular proteins by the proteasome. however, some antigenic peptides are produced by a proteasome-independent pathway, which is poorly characterized. mage-a - is a tumor antigenic peptide presented by hla-a and widely used for vaccination of melanoma patients. we observed that proteasome and tppii inhibitors failed to block presentation of the antigen by tumor cells. however, processing of this peptide occurred in the cytosol because tap inhibition prevented its presentation. to characterize the cytosolic peptidase producing mage-a - we setup an in vitro digestion assay using a -mer precursor peptide encompassing the sequence of the antigenic peptide. we observed that only the cytosolic fraction was able to produce the antigenic peptide from this precursor. this production was abolished by treating the cytosolic fraction with o-phenanthroline, a broad-spectrum inhibitor of metallopeptidases. this inhibitor also blocked the presentation of mage-a - by tumor cells. by electroporating hla-a cells with a precursor peptide blocked at the c-and the n-terminus, we could exclude the involvement of exopeptidases in the processing of this peptide, and conclude to a major role of a cytosolic metalloendopeptidase. one such enzyme is insulin-degrading enzyme (ide). we observed that depletion of ide abolished the capacity of a cytosolic fraction to produce the antigenic peptide. furthermore, recombinant ide was able to produce the peptide in vitro from the precursor peptide. lastly, silencing of ide with sirna reduced presentation of the peptide by tumor cells. with tppii, ide is the second example of a proteasomealternative pathway in the production of class-i restricted peptides. antigen-specific t cell based tumor immunotherapy, though extensively studied, has only been of limited clinical success so far. immune escape, due to impairment of hla dependent tumor epitope presentation is believed to be one major reason for this failure. to identify novel mechanisms by which tumors can become refractory to immune elimination, human melanoma cells of different donors expressing the transmembrane mart- /melan-a tumor antigen were exposed to two or three rounds of brief co-culture with mart- /melan-a - specific cytotoxic t lymphocytes (ctls). immune selected melanoma cell clones, being resistant to lysis by mart- /melan-a - ctls due to impaired epitope processing were further investigated. our results show that in addition to previously described immune evasion mechanisms like down regulation of mhc class i and mart- expression, the ifn-gamma independent endoplasmic reticulum associated degradation (erad) pathway is crucial for mart- /melan-a - epitope generation. moreover, deregulation of several erad components is essentially responsible for the observed immune escape of the immune selected melanoma cells. in support, re-expression of down-regulated erad components in ctl-resistant melanoma cells completely restored immune recognition by mart- /melan-a - ctls. thus, our studies demonstrate for the first time that erad not only plays a central role in the production of cd + t cell epitopes from membrane proteins but also contributes to tumor escape mechanisms by cancer immunoediting. studies of t cell responses to hen egg lysozyme suggest that several conformers of peptide-mhc class ii complexes can be generated for a single peptide epitope and that distinct cd t cell repertoires known as type a and type b recognise these different conformers (lovitch and unanue, immunol rev : - , ) . type a t cells recognise peptide-mhc complexes generated from intact proteins after intracellular antigen processing under h -m (dm) control, where as type b t cells respond to synthetic peptides in the absence of dm editing, but fail to respond to processed intact protein. type b t cells escape thymic deletion in mice (petersen et al., immunity : - , ) , with implications for autoimmunity. so we studied whether type a and type b recognition patterns occur in t cell responses to autoantigens such as the rheumatoid arthritis (ra)-associated proteoglycan aggrecan, and whether naturally occurring extracellular ligands that activate type b t cells are found in inflamed joints. lymph node cells from aggrecan-immunised balb/c mice proliferated in response to intact aggrecan and to the immunodominant peptide - , whereas peptide-immunised mice responded to peptide, with low or absent responses to intact aggrecan. t cell hybridomas generated from - peptide-immunised mice either recognised peptide only (the majority) or peptide and intact aggrecan (the minority), a pattern consistent with type a and type b t cell recognition. responses to staggered and alanine-substituted peptide sets showed that type a and b t cell hybridomas recognized the - epitope in the same register, consistent with this peptide epitope binding to mhc in distinct conformers. type b t cell hybridomas recognised aggrecan fragments in supernatants from cartilage degraded by stimulation with proinflammatory cytokines that induce raassociated aggrecanases. our data suggest that inflammation generates extracellular peptides that activate type b t cells. we are also characterising human type b t cell responses as well as searching for type b t cell ligands in synovial fluid from ra patients. we propose that extracellular cartilage degradation generates ligands that induce autoreactive type b t cell responses which participate in the pathogenesis of autoimmune arthritis. a to cope with mhc i antigen presentation hcmv encodes for several post-translational strategies which have been extensively studied in transfected cells. in this study we analysed the plc in naturally hcmv-infected cells and monitored the composition of the plc throughout hcmv replication. metabolic labeling experiments revealed the absence of tapasin incorporation into the plc. in contrast, western blot analysis demonstrated only a slow decline of tapasin steady state levels in infected cells, suggesting a blocked synthesis rather than degradation. tapasin mrna levels were found to be continuously downregulated during infection, however, the tapasin transcripts were stable and long-lived. taking advantage of a novel method, in which newly transcribed rna is selectively labeled and analysed (dölken et al, ), we found, after an initial induction at hrs p. i., a strong inhibition of tapasin transcription at hrs p. i. furthermore, also reduction of tap and tap transcription was observed contrasting to the elevated levels of erp and mhc i transcripts. importantly, ectopic expression of tapasin restored the incorporation of tapasin into the plc in hcmv-infected cells. the data indicate that hcmv controls mhc i antigen presentation also on a transcriptional level and show for the first time the regulation of tapasin transcription as a viral immune evasive function. most peptides presented by mhc class i molecules are produced by the proteasome during degradation of intracellullar proteins. two main proteasome types have been described, differing in their content of catalytic subunits. the standard proteasome comprises catalytic subunits ß , ß and ß , which are replaced by their ifng-inducible counterparts ß i, ß i and ß i in the immunoproteasome. the thymoproteasome represents a third proteasome type, where catalytic subunit ß i is replaced by a thymus-specific subunit ß t. the standard proteasome is present in most tissues, the immunoproteasome in found in cells exposed to ifng and in dendritic cells, while the thymoproteasome is found exclusively in the thymus. we produced a panel of novel antibodies that recognize subunits ß i, ß i, ß i and ß in their native form. using these antibodies for successive immuodepletions performed on tumor lysates, we identified two new proteasome types that are intermediate between the standard proteasome and the immunoproteasome, i. e. they contain only one or two the three catalytic subunits of the immunoproteasome. one comprises ß ,ß and ß i (single intermediate proteasome), and the other comprises ß i, ß and ß i (double intermediate proteasome). we quantified these intermediate proteasomes in a series a tumor lines of various origins, and found that they represent - % of the total proteasome content of those tumor cells. they are also present in dendritic cells, where they represent about % of the proteasome content. we characterized the activity of these intermediate proteasomes, not only on fluorogenic substrates but also on actual antigenic peptides recognized by anti-tumor ctl. with respect to antigens known to be processed differently by the standard and the immunoproteasome, the intermediate proteasomes often behaved like the immunoproteasome. importantly, we identified two tumor antigens that are processed exclusively by either the single intermediate proteasome ( tapasin is a multi-functional protein dedicated to mhc-i biosynthesis; it serves as a structural component in the so called mhc-i peptide loading complex (plc), as a chaperone putatively acting as an active peptide editor and mhc-i quality control mechanism, as an er retention signal for immature mhc-i, and as a chaperone stabilizing tap expression and increasing tap-performance. furthermore, tapasin has been found outside the er, where it has been suggested to regulate retrograde transport of escaped immature mhc-i back to the er from the trans-golgi compartment. the role of tapasin as an active peptide-editor has been debated and we here set out to study the effect of tapasin on binding of peptides of both high-and low-affinity to a human mhc-i allele (hla-a* ) using protein interaction-and peptide-competition assays. specifically we wanted to in detail compare the binding of two peptides of the same affinity. at high concentrations all of the tested hla-a* binding peptides (tap-transported high-affinity peptide (ttp-ha), signal-peptide of high affinity (sp-ha), tap-transported mediumaffinity peptide (ttp-ma)) induced dissociation of hla-a* from tapasin, but only ttp-ha dissociated hla-a* from tapasin at lower concentrations. using peptide-competition assays against ttp-ma, a peptide of lower affinity, we could show that ttp-ha, one of the two peptides of equally high affinity was a significantly more efficient competitor than peptide sp-ha. however, analysis of mhc-i peptide loading in the tapasin-negative cell line lcl- . -a showed no competitive advantage of ttp-ha compared to sp-ha supporting a role for tapasin as a selective facilitator of mhc-i peptide binding. in conclusion, we here show that peptides of different affinities dissociate hla-a* from tapasin in a dose-dependent manner, and that tapasin facilitates ttp-ha, but not sp-ha replacement of a lower-affinity peptide (ttp-ma). together these data strongly suggest a role for tapasin as a selective facilitator of peptide binding to mhc-i. importantly, this study implies that criteria in addition to peptide-affinity determines whether tapasin will promote peptide binding to hla-a* . m. basler , , c. lauer , m. groettrup , biotechnology institute thurgau, kreuzlingen, switzerland, university of constance, division of immunology, department of biology, konstanz, germany two lmp -dependent antigens have been described that relied on the 'structural presence' of lmp in the proteasome but not on the activity of lmp . here we have investigated processing of the h- d b -restricted uty - epitope of the male minor antigen uty reported to be lmp -dependent. using splenocytes from lmp -/-, lmp -/and mecl- -/mice we found that the uty - epitope requires lmp and lmp but not mecl- . curiously, a selective lmp inhibitor did not interfere with uty - presentation. objective: we investigated why the deletion but not the inhibition of lmp interferes with uty - presentation. we hypothesized that the 'structural' requirement for lmp is based on replacement of the caspase-like activity of b in the proteasome. methods: it was determined if t a mutants of lmp and/or b can rescue the uty - epitope. we used a b -selective inhibitor to determine if the inhibition of the caspase-like activity of b preserves the epitope. finally we determined by mass spectrometry if the uty - epitope embedded within a mer precursor peptide is differentially cleaved by lmp -deficient and proficient immunoproteasomes in vitro. results: we found that t a mutants of lmp and b rescue presentation of uty [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . also inhibition of cells with a b -selective inhibitor preserves uty [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] presentation. an aspartate in position of the uty - sequence wmhhnmdli is preferentially used as a cleavage site by lmp -deficient but not half as frequently by lmp -proficient immunoproteasomes. the generation of the uty - epitope relies on the replacement of the caspase-like activity of b by lmp because the b activity destroys the uty - epitope. this is the first example for the 'structural' requirement of lmp for generation of an epitope. eliminating the activity of their constitutively expressed homologous subunits may explain the requirement for immuno-subunits of the proteasome also for the generation of other antigens. thus we have discovered a so far unrecognized mechanism how lmp and perhaps also lmp and mecl- exert their function in antigen processing. a. linnemann , a. musiol , r. lindner hannover medical school, cell biology, hannover, germany, hannover veterinary school, graduate school for biomedical sciences, hannover, germany objectives: mhc i molecules are constitutively endocytosed and recycled to the cell surface. this process is required for the turnover of aged molecules and for some forms of cross-presentation of exogenous peptides on mhc i. in fibroblasts, mhc i is known to internalize via a clathrin-independent, arf -regulated pathway that is highly sensitive towards the cholesterol-sequestering drug filipin. although this observation suggests that membrane rafts are involved in the internalization of mhc i, no evidence for an association of mhc i with membrane rafts has been found in this cell type. methods: a novel detergent extraction protocol was used to investigate the association of mhc i with membranes rafts. endocytosis of mhc i was measured with a biotinylation-based biochemical assay and with a cell biological assay employing confocal laser scanning fluorescence microscopy. for characterization of mhc i internalization pathways, dominant negative mutants of gtpases (dynamin and arf ) were overexpressed in t fibroblasts. we show that antibody-mediated oligomerization of mhc i in t fibroblasts shifted this molecule from soluble fractions to detergent-resistant membranes. this change in detergent resistance coincided with a switch to a novel internalization pathway: oligomerized mhc i internalized faster and more completely and arrived at different endocytic organelles. the two mhc i internalization pathways differed in their sensitivity towards dominant negative arf : endocytosis of oligomerized mhc i was not affected, whereas non-oligomerized mhc i endocytosed more slowly and changed its subcellular distribution. unlike transferrin receptor internalization, none of the mhc i endocytosis pathways was affected by overexpression of dominant negative dynamin suggesting internalization mechanisms independent of clathrin, caveolin and rhoa. conclusion: we propose that mhc i switches from an arf -regulated to a novel, arf -independent internalization pathway in response to a change in membrane environment induced by oligomerization of mhc i. since mhc i is one of the cellular receptors for sv virus and since sv binding triggers mhc i oligomerization, this novel pathway may be involved in sv uptake. antigen cross-presentation in dendritic cells is a complex intracellular membrane transport process, but the underlying molecular mechanisms remain to be thoroughly investigated. in this study, we tested the effect of sirna-mediated knockdown of rab gtpases, the key regulators of membrane trafficking, on antigen cross-presentation. twelve rab gtpases were identified to be associated with antigen cross-presentation, and among which rab b, c were found to be colocalized with mhc class i molecules at perinuclear tubular structure. tracing with fluorescence protein tagged beta -microglobulin demonstrated that the mhc class i molecules were internalized from plasma membrane to rab b and rab c postitive compartment. moreover, the recycling ligand transferrin was enriched in the rab b or c positive vesicles. furthermore, the rab b, c positive compartment were colocalizd with a fraction of rab a at a juxtaposition of phagosomes. together these data demonstrate that rab b and rab c positive vesicles is involved in and may constitute the recycling compartment of exogenous antigen crosspresentation. introduction: while the proteasome is thought to generate most of the hla peptidome, other proteases were also proposed to be significant for this process. both t cell based assays and proteasome inhibitors were used in the past to follow presentation of specific model hla peptides. the hla-peptidomes presented at the cell surface depend on the rate of peptide generation within the cells, their transport from the cytoplasm and loading in the er, binding stability at the cell surface and retrograde uptake of the hla molecules back into the cytoplasm. objectives: the role of the proteasome in hla peptide presentation was evaluated using proteasome inhibition, while following the turnover rates of the entire hla peptidome. the peptidomes of both the authentic membranal and a recombinant soluble form of the hla molecules were collected for analysis at different time points after the inhibition of the proteasomes. the turnover rates of the hla-peptides were followed using pulse-chase analysis with stable-isotope labeled amino acids concurrently with epoxomicin treatment. the hla molecules were immunoaffinity purified and the peptides were analyzed by capillary chromatography and orbitrap tandem mass spectrometry. both the endogenous membranal and soluble mhc molecules were studied in parallel from the same cells. peptides were identified by their ms/ms fingerprints and the turnover rates were determined by the shift from the 'light' to the 'heavy' leucine of each peptide. results: a few thousands hla-peptides were identified, and for a large portion of them, the turnover rates could be defined. proteasome inhibition did not affect the complexity of the hla peptidomes or reduced significantly the amounts of membranal hla molecules. many peptides were labeled relatively rapidly with heavy leucine, indicating that the hla peptidome contains also the products of newly synthesized and rapidly degrading proteins. the source proteins of the hla peptides seemed to have similar biological functions and cellular origins in both the inhibited and untreated cells. the centrality of proteasomal degradation in hla-peptide presentation is put into doubt and the role of the proteasome in the generation of each peptide and each cleavage site can be defined. epstein-barr virus (ebv) is a ubiquitous y-herpesvirus, infecting over % of adults worldwide. it can cause mononucleosis and several lymphomas and carcinomas, reflecting the tropism of the virus for b-lymphocytes and epithelial cells. ebv persists for life despite the presence of virus-specific adaptive immunity, indicating that it has evolved strategies to counter the host immune response. one such strategy is the persistence of the virus in the latent phase of its life cycle, where expression of viral proteins is minimized. however, for ebv replication and dissemination to occur, it must enter the lytic phase. here, over viral proteins are expressed, creating many potential antigens for presentation to cytotoxic t -lymphocytes. ebv can circumvent possible eradication by cd + t lymphocytes during the lytic phase by interference with antigen processing and presentation through hla class i in the infected cell. the viral proteins bnlf a and bglf have been shown to achieve this by impairing peptide-loading of hla class i and inducing the degradation of mrnas encoding hla molecules, respectively. a third ebv lytic phase protein, the g-protein coupled receptor (gpcr) bilf , has now been found to down-regulate cell surface hla class i expression (zuo et al, plos pathogens ). this represents a novel function for a virally-encoded gpcr. bilf is expressed early in the ebv lytic cycle and is localized predominantly at the cell surface. there it can interact with hla class i molecules, resulting in their internalization and lysosomal degradation. this has a profound effect on the ability of cytotoxic t-lymphocytes to recognize cells displaying antigens derived from ebv proteins. interestingly, bilf displays a differential effect on distinct hla class i haplotypes. furthermore, we have shown that the intracellular c-terminal tail of bilf is required for its effect on hla class i expression. however, the ability of the gpcr to activate intracellular signaling pathways is dispensable in this regard. thus, by reducing the cell surface expression of hla class i molecules, ebv bilf can hinder the recognition of virally-infected cells by cytotoxic cd + t lymphocytes, thereby facilitating the evasion of adaptive immune mechanisms. t lymphocytes mature in the thymus, generating a non-dangerous t cell repertoire. for the adquisition of tolerance, thymocytes suffer positive and negative selection processes. during t cell maturation, tcrs contact with different mhc-peptide complexes on the surface of pressenting cells, allowing tolerization against self proteins. to obtain a non-self-reactive t cell repertoire, it is of most importance that pressenting cells in the thymus express a repertoire of mhc-peptides complexes representative of the proteins that t cells will found in periphery, including tissue restricted antigens (tras)-derived peptides. in the last decade, transcription of tras in thymus has been well-reported. furthermore, the expression of many genes codifying for tras are dependent.on the expression of the autoimmune regulator (aire). aire is mainly expressed in medullar thymic epithelial eells (mtecs), which are involved in negative selection. so far no systematic study have been made to describe the peptide repertoires associated to hla molecules in the thymus. in addition, although many data of tras transcription in thymus have been reported, much less work has been performed at biochemical level, and to our knowledge, no hla ligand arising from any tra have been reported in thymus. in this report we present the results of analyzing the hla-dr-associated peptide repertoire from whole tissue samples of different human thymi by mass spectrometry. we describe natural ligands, including two peptides derived from semenogelin- , a tissue restricted antigen expressed mainly in the prostate, and present in semen. using qpcr we demonstrate that semg is transcribed in thymus from both male and female individuals. finally, we detected the semg mrna expression in a fraction enriched in stromal cells, but not in the thymocyte fraction of the thymi. the proteasome is the major protease complex for non-lysosomal protein degradation in eukaryotic cells, which generates most peptides for mhc class i antigen presentation. vertebrates express two sets of catalytic subunits, constitutive (beta , beta , beta ) and immuno-subunits (beta i, beta i, beta i). deficiency in beta i results in profound reduction of mhc class i expression, demonstrating the significance of this subunit for efficient antigen presentation. currently, this is attributed to the specific proteolytic activity of the beta i subunit, its role in the maturation of immunoproteasomes or both. however, re-expression of catalytically inactive beta i subunits is capable to rescue antigen presentation suggesting that the proteolytic activity of this subunit is not limiting in this process. here, we show that following infection with listeria monocytogenes induction of beta i expression increases the cellular proteasome content in the infected organs. our results indicate that this is due to the high chaperone activity of its propeptide which drives proteasome neosynthesis and thus enhances the overall proteasome quantity. further, mhc class i antigen presentation on beta i-deficient cells could be restored by treatment with d t, which increases the amount of proteasomes independent of beta i via induction of mixed proteasomes containing beta i, beta i and beta . consequently, not the lack of the specific proteolytic activity of beta i or immunoproteasomes, but the reduced proteasome quantity in beta i deficient cells is the major limiting factor for mhc class i cell surface expression. . we have previously shown that lc, in contrast to ddc, do not express cell surface tlr , and , which results in their inability to respond to both gram-positive and gram-negative extracellular bacteria in terms of maturation into immuno-stimulatory cells and production of inflammatory cytokines. therefore, the question remained what the role is of lc in class ii mhc-mediated activation of anti-bacterial t cells. we determined the capacity of ddc and lc to internalize and process whole bacteria and present bacterial antigens to cd + t cells. in vitro generated lcs and ddcs were cocultured with gfp-expressing bacteria and subsequently analysed by clsm and facs for their uptake capacities. furthermore we investigated their capacity to stimulate autologous bacteria-specific t cell lines as a measure for antigen presentation. results: we found that lc are principally able to internalize bacteria, but far less efficient than ddc. moreover, visualisation of bacterial uptake by em revealed different uptake mechanisms by lc and ddc. both in lc and ddc internalized bacteria were detected in the endosomal and lysosomal compartments of the mhcii processing route. nevertheless, presentation of bacterial antigens by lc on mhcii was inefficient compared to that of ddc, as indicated by a low capacity to activate autologous bacteria-specific cd + t cells. the presence of exogenous tlr and tlr ligands did not overcome the differences between lc and ddc, indicating that the impaired capacity to internalize and process bacteria and activate bacteria-specific t cells is not due to the lack of tlr signalling or insufficient expression of co-stimulatory molecules, but could be an intrinsic characteristic of lc. conclusion: we propose that the epidermis of the skin is an immune-privileged site where lc play a minor role in anti-bacterial immunity and may play a role in inducing tolerance to the bacterial skin flora by steady-state presentation of antigens from commensal skin bacteria. e. james , i. bailey , t. elliott university of southampton, cancer sciences division, southampton, united kingdom regulatory t cells (tregs) play a pivotal role in the suppression of tumour specific t cell responses. depletion of tregs in balb/c mice results in a robust immunity to the normally poorly immunogenic ct colon carcinoma. this response is long lasting and mediated by both cd and cd t cells. importantly, the treg depleted ct specific immunity is cross-protective; capable of mediating rejection of tumour lines of different histological origins (a , c , bcl , renca) implying a broader repertoire of response. we have characterised one of these cross-protective antigens, gsw , which is h -d d restricted. analysis of the generation of gsw in ct revealed that the peptide is susceptible to over-processing by the er-resident aminopeptidase eraap. inhibition of eraap in ct cells substantially increased the amount of gsw present, observed by increased t cell responses to the tumour in vitro and hplc analysis. this increase was in spite of an overall reduction of mhc class i molecules at the cell surface. to investigate whether the increase in immunogenicity following knockdown of eraap would protect mice, we generated stable eraap knockdown (kd) ct and immunised balb/c mice. greater than % of mice injected with eraap kd ct were found to reject the tumour. analysis of t cell responses revealed the presence of gsw -specific t cells, however, these responses were small ( . - %). this compared to a much larger response to ct (˚ %). preliminary results indicate that the majority of the t cell responses (non-gsw -specific) in these mice are directed toward unstable peptide/mhc complexes, possibly indicating presentation of n-terminally extended peptide antigens. this highlights manipulation of the peptide repertoire as a potent tool for the generation of t cell responses in vivo. minor histocompatibility antigens play important roles in the outcome of stem cell and organ transplantation as they are involved in the development of graftversus-host-disease and in the graft-versus-tumor reactivity in hla-identical stem cell transplantation [ ] . the di-allelic hla-a restricted minor histocompatibility antigen ha- locus codes for the highly immunogenic ha- his and the non-immunogenic ha- arg nonapeptides, differing in one amino acid. the only difference that could explain the absence of the ha- arg immunogenicity was the estimated numbers of cell surface presented copies i. e. /cell for ha- his and less than / cell for ha- arg [ ] . as ha- his/arg is hematopoietic system specific and shows additional expression on epithelial cancer cells while absent on the normal epithelial cell counterpart, the ha- his allele is currently used for boosting the graft-versus-tumor responses after hla matched ha- mismatched stem cell transplantation. to elucidate the mechanisms underlying the differential cell surface presentation of the ha- allelic peptides, we investigated the impact of the ha- his/arg polymorphism on molecular and cellular processes involved in the intracellular generation and stable cell surface presentation of hla class i-bound peptides. therefore, proteasome-mediated digestion experiments, tap translocation analyses, and hla-dissociation assays with ha- his and ha- arg peptides were performed. moreover, the crystal structures of hla-a in complex with either ha- his , ha- arg or a ha- variant with a citrulline residue at position were determined in order to obtain atomic level insights into the conformation of the hla-a /ha- peptide complexes. our results exclude a role for antigen processing in preventing ha- arg to be presented at the cell surface and both the structural and hla-dissociation data clearly show that the lack of cell surface expression essentially results from an increased instability of the ha- arg allele in the hla-a peptide binding groove [ ] . they provide a rationale for the lack of ha- arg peptide immunogenicity essential for the choice of tumor peptides for stem cell based immunotherapeutical application. proteasomes play an important role in mhc class i antigen processing. exposure of cells to proinflammatory cytokines such as tnfa or ifng leads to the expression of three facultative catalytic proteasome subunits (i. e. immunosubunits) that replace the constitutively expressed subunits in the cellular proteasome population. immunoproteasomes generate many pathogen-derived cd t cell epitopes with high efficiency and thereby shape the specificity of the pathogen-specific cd t cell response. on the other hand, immunosubunit expression is not essential for development of cd t cell-mediated protective immunity, thus the physiological relevance of these cytokine-induced proteasome subunits remains unclear. we observed that mice that lack the immunosubunits lmp (ib ) and mecl- (ib ) develop a variety of autoimmune responses, including a latent form of t d (or insulin dependent diabetes mellitus, iddm), following irradiation and bone marrow reconstitution. iddm development in these mice is characterized by inflammation of the islets of langerhans, glucose intolerance and increased water consumption, and is dependent on the presence of cd but not cd t cells. a cd t cell epitope, encoded by the islet beta cell-expressed "islet-specific glucose- -phosphatase catalytic-subunit-related protein" (igrp) mrna, was identified as an important target of the cd t cell response. this epitope, like many other known diabetes-associated epitopes, binds its presenting mhc class i molecule with low affinity. as t cells specific for low affinity binders most likely can escape central and peripheral tolerance while t cells specific for high affine binders do not, we postulate that inflammation-induced immunoproteasome expression primarily functions to replace self-peptides that are derived from tissue-associated antigens and bind mhc class i molecules with low affinity, by a higher affine peptide species towards which t cell tolerance exists. thus, the inducible proteasome subunits may play an important role in immune regulation, by removing the targets of potential auto-immune cd t cells that enter inflamed tissues. endocrine epithelial cells, targets of the autoimmune response in thyroid and other organ-specific autoimmune diseases, express hla-ii molecules with compact conformation and are therefore expected to stably bind autologous peptides. the role of these molecules is not known but they could be involved in the maintenance and regulation of the in situ autoimmune response. to study in situ t cell responses without characterizing self-reactive t cells, we have identified natural hla-dr-associated peptides from autoimmune organs that will help finding peptide-specific t cells in situ. here we report the first analysis of hla-dr natural ligands from ex-vivo graves' disease-affected thyroid tissue. using mass spectrometry, autologous peptides were identified from hla-dr-expressing cells, including thyroid follicular cells, some corresponding to predominant molecules of the thyroid colloid. most interestingly, eight of the peptides derived from a major thyroid autoantigen, thyroglobulin. cell-free in vitro binding assays were performed with the thyroglobulin peptides and some other thyroid-eluted peptides as controls, to identify to which hla alleles were these peptides associated in vivo. all but two of the thyroglobulin peptides showed low binding with the corresponding alleles. the two peptides with relatively high binding affinity were presented in the context of dr and dr . analyzing the digestion patterns used for the generation of the thyroid peptides, a preferentially cleavage after a lys and arg was observed for all of them, independent of the restricting allele. our data demonstrate that although the hla-dr-associated peptide pool in autoimmune tissue mostly belong to abundant ubiquitous proteins, peptides from autoantigens are also associated to hla-dr in vivo and therefore may well be involved in the maintenance and the regulation of the autoimmune response. the t cell response generated following herpes simplex virus type (hsv- ) infection is known to be crucial in the clearance of replicating virus and in limiting the severity of infection. despite this, the relative contributions of cd + and cd + t cells in hsv- immunity have yet to be clearly elucidated. to better understand the role of hsv- -specific cd + t cells in immune control we have identified a amino acid epitope derived from glycoprotein d of hsv- . following flank infection, gd-specific cd + t cells were first detected in the draining brachial and axillary lymph nodes (ln) -days post-infection (pi), peaking at day and declining thereafter. gd-specific cd + t cells were first recovered from the spleen, skin and dorsal root ganglia (drg) at day pi and peaked at day . while hsv-specific t cells were first observed in the draining ln at day pi, hybridoma assays showed ex vivo presentation of the gd epitope by brachial ln cells as early as days pi, with peak activity days pi before declining to background by day . however presentation of the gd epitope was much more prolonged in vivo as proliferation of transgenic gdspecific cd + t cells was observed up to days post-infection in the brachial ln. ex vivo analyses suggest that only cd c + cells were involved in gd antigen presentation at days , and post-infection. subdivision of dendritic cells (dcs) populations indicated that both skin-derived dcs and cd a + dcs can present the gd antigen to cd + t cells at day pi, whereas by day pi the skin-derived dcs were the predominant population presenting the gd epitope. together these data show that following hsv- infection, antigen presentation is initiated rapidly and persists well after clearance of replicating virus. furthermore, we present evidence that different dc populations have distinct roles in the presentation of viral antigens and that they may vary during the course of infection. complementary zippers induced complete dimer formation, whereas identical zippers impaired stable interactions of the tagged peptidases. we also verified that the zippers did not influence the substrate "preferences" of the respective erap. our results from in vitro digestions suggest that the stabilised heterodimer is significantly more efficient in the production of a model epitope than the mix of monomeric erap and erap unable to form dimers. this observation is not due to mere thermodynamic stabilisation but involves positive cooperative effects in the heterodimers. conclusion: allosteric interaction of erap /erap in heterodimeric complexes enhances the global efficiency of precursor peptide trimming in the human er. during the biogenesis of class i molecules, newly synthesized heavy chains fold and acquire disulfide bonds while interacting with the lectin-chaperone calnexin (cnx) and its associated thiol oxidoreductase erp . upon assembly of the heavy chain with b m, the class i molecule enters a peptide loading complex (plc) that consists of the tap transporter, tapasin, the calnexin homologue calreticulin (crt) plus associated erp . both crt and erp are required for efficient assembly of peptide-loaded class i molecules and their subsequent expression at the cell surface. we examined functional sites on crt and erp to gain insights into their mechanisms of action in class i biogenesis. for crt, its lectin function is thought to be crucial for its association with class i molecules. however, when crt mutants lacking lectin function were expressed in crt-deficient cells, they completely complemented all class i biosynthetic defects. thus polypeptide-based contacts either mediated through erp or directly between crt and the heavy chain are sufficient to effect the chaperone and quality control functions of crt in class i biogenesis. we also tested the notion that erp must be recruited by cnx or crt to function on class i molecules. we found that the rates of heavy chain disulfide formation were normal in cells lacking cnx, crt or both chaperones. furthermore, an erp point mutant that fails to bind to cnx or crt was just as effective as wild type erp in normalizing rates of disulfide formation. we conclude that erp does not require recruitment by cnx or crt and likely acts directly on class i heavy chains to promote disulfide formation. furthermore, in cells expressing the erp point mutant, class i heavy chains, crt and the tapasin-erp disulfide conjugate were present at normal levels in the plc, indicating that the interaction between erp and crt is not required for plc assembly. finally, we show that mutations that destroy the enzymatic function of erp have no effect on plc stability or class i surface expression, suggesting that erp plays a structural as opposed to catalytic role in plc function. autoimmune pancreatitis (aip) underlies - % of cases of chronic pancreatitis and is characterized by prominent lymphocytic infiltration. a strong association of aip with the hla-drb * /dqb * haplotype has been reported, but identification of the predisposing hla gene(s) has been precluded by strong linkage disequilibrium. here, we show that hla-dr* transgenic ab nod mice suffer from aip and additional pneumonitis after sublethal irradiation and adoptive t cell transfer from syngenic donors, leading to complete pancreatic atrophy. pancreas histology is characterized by destructive infiltration of the exocrine tissue with cd + and cd + t cells, b cells and macrophages. mice with complete pancreatic atrophy have reduced serum lipase activity, develop fat stools and loose weight on regular chow. hla-dr* transgenic mice (cd + t cell competent) develop aip even unprovoked, similar to ab nod mice (cd + t cell deficient), while hla-dr* , hla-dq or hla-dr* /dq (double-) transgenic controls all remain normal after same treatment. we conclude that hla-dr* fails to protect from aip, likely due to defects in the induction of cd + regulatory t cells. our results identify hla-dr* as a prominent risk factor for aip on the hla-drb * /dqb * haplotype. this humanized mouse model should be useful to study mechanisms that underlie the hla association of autoimmune diseases, but also immunopathogenesis, diagnostic markers and therapy of human aip. s. khan , c. britten , h. overkleeft , g. van der marel , k. melief , d. filippov , f. ossendorp leiden university medical center, section tumorimmunology, leiden, netherlands, leiden university, biosynthesis group, leiden, netherlands objective: we have targeted peptide antigens to dendritic cells by the use of synthetic peptides chemically coupled to synthetic tlr ligands to study the impact on mhc class i and class ii antigen presentation. the potency of the vaccine was addressed by monitoring antigen presentation, priming of t-cells and tumor protection. results: our data show that this type of targeting of peptides greatly improves antigen presentation and t-cell priming compared to free peptide. vaccination of mice with the tlr-ligand peptide conjugates induced high numbers of functional cd and cd t-cells that could protect mice for aggressive melanoma. this potency relies on tlr signaling since peptide coupled to a non-functional tlr ligand was unable to support induction of specific t-cells. these data indicate that simultaneous encounter of antigen and a maturation signal are crucial for optimal t-cell activation by dendritic cells, and show the potency of tlr-l peptide conjugates as a vaccine modality. y. shi , , x. hu , , a. kawana- tachikawa objectives: nef protein of human immunodeficiency virus (hiv) holds some important immunodominant ctl epitopes. two overlapping -mer and -mer epitopes (rypltfgwcf (nef - ) and rypltfgw (nef - )) were found to be presented by hla-a* and some immune escape mutants of these two epitopes have also been found in some patients, e. g. y f, y w, t c, f l, w r, f r, f y etc. or their combinations. it's important to study the molecular basis of the peptide being displayed on the cell surface, through which we can analyze the mechanism of immune escape of hiv. methods: refolding method was used to attain the soluble protein pmhc. crystals are grown using hanging drop vapor diffusion method and x-ray diffraction technology is used to determine the structure. we have determined six peptide-mhc(pmhc) structures containing nef - (wild type) and its four mostly common immune escape mutants (y f, t c, y f&t c, f l), and also nef - (wild type). we found that there was little difference between the nef - (wild type) and nef - (y f) when they were displayed in the peptide-binding groove of mhc molecule, except water molecule distribution near the anchor residue y or f . interestingly the central bulge region of the peptide was becoming very flexible for the nef - (t c) and nef - (y f&t c), which may affect the binding of peptide and the recognition of t cell receptor. for nef - (f l), the side chain of l was more flexible compared to the nef - (wild type). alignment of the nef - and nef - showed that the nef - became flat and the side chain of f was not solvent-exposed due to shortening of the length of the peptide. conclusion: as the peptide nef - was featured, while the peptide nef - was featureless, so the different topology of these two epitopes indicates that they have different tcr repertoire diversity in hiv-specific responses. different immune escape mutants of nef - was using different strategies to avoid the killing of host ctls, which indicates that the therapy strategy based on the cellular immune response should be diversity. for the in vivo or ex vivo activation of antigen-specific t cell responses long synthetic peptides are used to activate both cd + and cd + t cells. in this study we investigated the efficiency and mechanism of cross-presentation of these long synthetic peptides in mhc class i. we observed a large variation in the effectiveness of activation of specific t cells by the extended peptides corresponding to different epitopes, indicating a difference in the efficiency of processing and presentation of these peptides. for the hla-a restricted cmvpp derived nlv epitope specific t cells were most efficiently activated by n-terminally extended variants of the minimal epitope, while the use of c-terminally extended variants resulted in a - log reduction of activation efficiency. this pattern was seen for / epitopes tested in different hla restrictions. furthermore, for all epitopes tested, extending both the c-terminus and n-terminus led to - log less efficient activation of the specific t cells, compared to the minimal peptide. exchange of the c-terminal sequence of the c-terminal extended hla-b restricted cmvpp rph peptide with the c-terminal extended nlv peptide led to the enhancement of t cell activation by the exchanged nlv peptide, indicating a role of the extended peptide sequence in the efficacy of processing and presentation of the peptide. tap-deficient t cells loaded with extended nlv peptides efficiently activated nlv-specific t cells, indicating that the route of presentation was tap-independent. addition of lactacystin did not affect activation of specific t cells, illustrating that crosspre-sentation was proteasome-independent. primaquine reduced the activation of specific t cells by extended nlv peptides, but not by the minimal nlv -mer peptide, suggesting that cross-presentation was dependent on endosomal recycling. these data suggest that long synthetic peptides can be processed by peptidases in endocytic compartments and presented by recycling mhc class i molecules. not all immunogenic epitopes that have been selected in vivo for efficient processing and presentation by the classical pathway may be presented efficiently by cross-presentation. therefore, a rational design of peptides is crucial for efficient activation of cd + t cells in approaches of vaccination, adoptive transfer and immune monitoring. antigenic peptides presented by mhc class i molecules are fragments that are usually excised from intracellular proteins while these are degraded by the proteasome. recently, three antigenic peptides were found to result from the splicing of segments that are not contiguous in the parental protein. for two of these peptides, splicing was found to occur in the proteasome by a mechanism of transpeptidation resulting from the nucleophilic attack of an acyl-enzyme intermediate by a free peptide fragment. one of them is derived from melanocytic protein gp and requires excision of a four-amino acid intervening segment. the other peptide is derived from protein sp , and requires splicing in the reverse order of two segments initially separated by six amino acids. the first spliced antigenic peptide described was derived from fibroblast growth factor- (fgf- ) and was recognized by human cytotoxic t lymphocytes directed against kidney cancer cells. it is made of two spliced fragments, which are initially separated by a long segment of amino acids. the splicing mechanism of this peptide has not been worked out. the length of the intervening segment made the transpeptidation model more difficult to account for the splicing of this peptide. we therefore evaluated the role of the proteasome in the splicing of this peptide. we observed that the spliced fgf- peptide was produced in vitro after incubation of proteasomes with a -amino acid long precursor peptide. we evaluated the mechanism of the catalytic reaction by incubating proteasomes with several peptide precursors in a pair wise manner. the results confirmed the transpeptidation model of splicing. we further compared the production of the fgf- spliced peptide by cells transfected with mutant constructs encoding fgf- proteins where the intervening segment was shortened from amino acids to , or residues. we observed an increase in the production of the spliced peptide that was proportional to the reduction in length of the intervening segment, as predicted by the transpeptidation model. finally, using the spliced gp peptide model, we observed that splicing did not occur at a significant level between fragments of two distinct proteins in the cell. the polymorphic residues within the peptide binding cleft of hla class i molecules not only diversify the range of peptides presented to cytotoxic t lymphocytes but also influence the pathway of antigen presentation. in order to acquire high affinity peptides, some class i allotypes, such as hla-b* , are heavily dependent upon tapasin and other molecules comprising the peptide loading complex (plc). other class i molecules, like hla-b* , appear to largely bypass this complex but are consequently loaded suboptimally with peptide. hla-b* and b* are naturally occurring allotypes that differ by only a single amino acid, making this difference in behaviour all the more remarkable. we have previously speculated that such tapasin-independent class i molecules may have been selected in response to viral inhibitors that target the plc, such as the human cytomegalovirus us protein. to address this hypothesis, us was stably coexpressed in b lymphoblastoid cell lines expressing hla-b* or hla-b* . in the presence of us , the surface expression of hla-b* was substantially reduced whereas hla-b* expression was relatively unaffected. although us was able to form complexes with both hla class i allotypes, only hla-b* was retained intracellularly in an immature form whereas hla-b* was transported to the cell surface. accordingly, in the presence of us , hla-b* , but not hla-b* , constitutively presented a hla-b restricted alloantigen to reporter t cells, suggesting that us binds hla-b* without interfering with peptide loading. us has been reported by others to bind the plc but surprisingly we have not detected such us -plc complexes in our system. rather, in the presence of us we identified a pool of class i molecules distinct from the plc and only present in us expressing cells, implying that us may act independently of the plc. these findings demonstrate how hla class i polymorphism not only impacts upon the t cell repertoire and diversifies determinant selection, but also serves to evade the impact of viral inhibitors on antigen presentation. c. massa , b. seliger martin-luther-university halle-wittenberg, institute of medical immunology, halle, germany in the attempt to optimize vaccine dc, modifications have been proposed both in the antigen loading and in the maturation protocols. for dc loading "whole antigens" are now preferred to peptides. therefore, it is important to consider not only the costimulatory properties of the vaccine dc, but also their antigen processing abilities. this is even more important since there is the trend to stimulate dc with tlr ligands combined with ifn-y in order to induce dc not only able to correctly migrate, but also secreting the bioactive il p . since ifn-g is known to influence the expression of multiple proteases involved in antigen processing, aim of this study was to compare the various maturation cocktails for the consequences on the antigen processing capabilities of the dc in parallel to their costimulatory potential. for this purpose monocyte-derived dc were stimulated for h with the gold standard of maturation (tnfa, il b, il and pge ) or a combination of ifn-g and different tlr ligands. the dc obtained exhibit a similar expression of costimulatory and adhesion molecules together with the ability to induce proliferation of allogeneic pbmc, but differ for the pattern of proteases expression as evaluated by real time pcr. with the exception of the downregulation of the tripeptidyl peptidase ii (tppii), no dramatic differences were observed for endo-and aminopeptidase between immature and "gold standard" mature dc. in response to the "ifn-gcontaining" cocktails there was a similar tppii downregulation, but also the induction of many other enzymes. the cytosolic leucine aminopeptidase- (lap ) had a more than -fold increase in transcription levels, whereas the mrna expression of the aminopeptidases of the endoplasmic reticulum erap and erap and of the immunoproteasome subunits lmp and lmp was enhanced between and -fold under these culture conditions. with regard to the different tlr ligands used in combination with ifn-g, there was a reproducible higher mrna induction in the presence of the tlr ligand mpla in comparison to the tlr- and / ligand polyi:c and r . these data suggest that the maturation cocktail of dc may alter the peptide repertoire presented by hla class i surface antigens. it has been suggested that mast cells might serve, under certain circumstances, as antigen presenting cells for t cells. however, whether cognate interactions between mast cells and class ii restricted cd + t cells actually occur, is still an open question. we addressed this question using peritoneal cell-derived mast cells (pcmc) as an antigen presenting cell model. our results show that in vitro treatment of pcmc with ifn-g and il- induced surface expression of mature mhc class ii molecules and cd . when ifn-g/il- primed pcmc were used as antigen presenting cells for cd + t cells they induced activation of effector t cells but not of their naive counterparts as evidenced by cd up-regulation, induction of proliferation and cytokine production. confocal laser scanning microscopy showed that helper ot-ii t lymphocytes form with pcmc functional immunological synapses, characterized by pkcq enrichment and ifn-g polarized secretion towards the antigen-presenting mast cells. finally, upon cognate interaction with ot-ii t cells, mast cells lowered their threshold of activation via fceri. our results show that mast cells can establish cognate interactions with class ii restricted helper t cells, implying that they can actually serve as resident apc in inflamed tissues. h the vast majority of peptide ligands presented by mhc class i molecules is thought to be produced by cytosolic degradation of source proteins by the proteasome. although, next to cytosolic and nuclear proteins, proteins targeted to the endoplasmic reticulum (er) can also be degraded through this pathway following retrograde transport into the cytosol, antigen processing of er proteins remains little characterized. studying processing and presentation of er-targeted and cytosolic forms of proinsulin (pi), an autoantigen playing a pivotal role in triggering of cellular autoimmune responses in type -diabetes, we found that er-targeting of this model antigen has profound effects not only on how pi is degraded, but also on regulation of its synthesis. as expected, proteasome inhibition inhibited degradation of cytosolic pi as well as presentation of the epitope insulin b - to specific cd + t cells. in contrast, prior exposure of cells to proteasome inhibitors strongly reduced production of er-targeted pi (pre-pi) through induction of er stress, both in cells infected with a recombinant vaccinia virus and in cells transfected with a tetracycline-regulated expression system. experiments using conditions permissive for pre-pi expression showed that er-targeting modified proteolytic processing of pi for mhc class i presentation. these experiments suggested that two proteolytic pathways contribute to degradation of er-targeted pi, with their relative contribution depending on the stability of the protein. while degradation of unmodified pre-pi was partially dependent on the proteasome, removal of one or several disulfide bridges increased the role of the proteasome in processing of pre-pi for presentation, while introduction of a site for n-glycosylation had the opposite effect. these findings imply that er-targeting together with structural features can have profound effects both on antigen production and on the pathway of proteolytic antigen degradation and presentation. cd + t cell immune response to exogenous antigens relies on cross presentation by dendritic cells (dcs) in secondary lymphoid organs. recently, in several infectious murine models, it has been shown that in addition to dc located in tissues, de novo differentiating dc participate in the protective th immune response. the role of de novo differentiating dc in cross presentation is however poorly documented, and difficulties of human immunology prevent the accurate identification of the apc subsets patrolling for exogenous ag. a prerequisite for cross presentation is a moderate ag degradation rate in the endocytic pathway, allowing the generation of antigenic epitopes and their binding to mhc molecules. this prerequisite is of special importance considering dc precursors (such as monocytes), which are not yet dcs and may take up antigen before differentiating into dcs. the objective of our in vitro study is to evaluate whether ex vivo purified human blood monocytes are able to cross present long antigenic peptides to cd + t cells and whether they are able to sustain this cross presentation while differentiating into dcs. we have previously shown the unique property of dendritic cells to maintain for several days the capacity to stimulate cd + tumor-specific t cell clones when pulsed with long antigenic peptides (that need to be processed before presentation to cd + t cell clones, faure, , eur j immunol ( ): - ). in the present study, we address the question of the mechanisms of long peptide cross-presentation by blood monocytes along the course of their in vitro differentiation into dcs. we have shown that despite their high degradative capacity, ex vivo purified monocytes pulsed with long peptides are able to stimulate cd + t cells after their in vitro differentiation into dc, days following their antigenic pulse. the delineation of apc subsets able to sustain ag cross-presentation and t cell stimulating potential might be of clinical relevance in immunotherapy using synthetic long peptides. viral genomes contain alternative reading frames (arfs) encoding for mhc-i restricted epitopes (arf-epitope). in the siv/macaque model, ctl responses directed against arf-epitopes participate in controlling viral replication. we previously described that hiv- genome contains arfs within gag, pol and env genes encoding for a panel of hla-b* restricted epitopes. qprsdthvf (q vf/ d) is one such epitope but its parental epitope qprsnthvf (q vf/ n) has a significant higher frequency among hiv- isolates. strikingly, q vf/ d-or q vf/ n-specific ctls recognize apcs infected with hiv strains encoding for q vf/ d (e. g. hiv lai ). in contrast, hiv strains (e. g. hiv nl-ad ) encoding for q vf/ n do not activate ctl responses raising the possibility that q vf/ n epitope is not presented by infected cells. we asked whether introducing mutations within q vf might be a mean for the virus to escape ctl responses directed against this arf-encoded epitopes. we dissected the mechanism responsible for the lack of q vf/ n mhc-i presentation. we modified hiv lai to introduce a d to n mutation in q vf. introducing this single amino-acid mutation abrogated ctl recognition indicating that this asparagine (n) alters q vf mhc-i presentation. we performed in vitro proteasomal digestions of mer peptides encompassing q vf/ d or q vf/ n and cleaved polypeptides were analyzed by mass spectrometry. the asparagine (n) in q vf/ n is a preferential proteasomal cleavage site. thus suggesting that proteasome cleavages within q vf/ n might be responsible for its lack of mhc-i presentation. we then sought in hiv-infected patients for the presence of proviruses encoding for q vf/ d or q vf/ n, and ctls responses directed against these epitopes. far thus, two out of three donors tested recognized the q vf/ d peptide. we cloned and sequenced hiv- genomes from the three donors. surprisingly, out of hiv proviral genomes isolated from pbmcs of q vf/ d reactive donors, we could not find any virus bearing the q vf/ d sequence. the isolated hiv sequences either encoded for q vf/ n or had a stop codon within the epitope. in contrast, viruses encoding for q vf/ d were isolated from pbmcs of the q vf/ d nonreactive patient. altogether, our data suggest that ctls exert a selection pressure on viral arfs. hiv- seems to escape immune surveillance by introducing mutations altering processing of arf-derived epitopes. i. e. flesch , y. wang , d.c. tscharke the australian national university, biochemistry and molecular biology, canberra, australia vaccinia virus (vacv) was the live vaccine used to eradicate smallpox and some strains are now being used as vectors for recombinant vaccines. cd + t cells recognizing viral peptides in association with mhc class i molecules on infected cells play a crucial role in the defence of viruses. despite the large number of possible mhc class i-peptide combinations, cd + t cells only recognize a small number of epitopes, a phenomenon called immunodominance. using recently defined cd + t cell epitopes for vacv in mice, we have investigated how heterozygosity of mhc class i molecules influences immunodominance patterns in h- bxd f mice compared with their inbred parent strains. we find that the immunogenicity of vacv peptides defined using inbred mice is variable in f progeny, with some peptides being almost equally immunogenic in f and inbred mice, while others elicit responses that are reduced by more than % in f mice. during acute infection as well as memory responses, the dominance hierarchy in inbred mice did not predict the epitopes that would be poorly immunogenic in f mice. in line with these findings, a multiepitope construct expressed by a recombinant vacv was less immunogenic in f mice than would be predicted from its performance in parent strains. in terms of mechanism, we find evidence of altered tcr repertoires including in the case of one epitope, the loss of many diverse tcr vb clones and outgrowth of cd + t cells with a restricted vb usage in f mice. these data have implications for our interpretation of experimental vaccine work done in inbred mice and for our understanding of how mhc diversity can alter the range of epitopes that are immunogenic in outbred populations. objective: tlr ligands are being exploited as potential adjuvants, and have impact on the antigen processing and presentation by dendritic cells (dc). therefore we aimed to study the efficacy of a tlr agonist, s-[ , -bispalmitoyiloxy-( r)-propyl]-r-cysteinyl-amido-monomethoxyl polyethylene glycol (bppcysmpeg), a synthetic derivative of the mycoplasma macrophage activating lipopeptide (malp- ), as an adjuvant for cross-priming against cellular and soluble antigens. malp- has been characterized as an effective mucosal adjuvant and synthesis of bppcysmpeg further improved solubility and pharmacokinetic features of the adjuvant. methods: dc isolation, in vitro and in vivo t cell stimulation, intracellular cytokine staining, in vivo cytotoxicity assays. results: systemic administration of bppcysmpeg induced maturation of cd + and cd -dc in the spleen resulting in enhanced cross-presentation of intravenously co-administered soluble antigen in mice. in addition, administration of bppcysmpeg and cell-associated ova resulted in generation of an effective ctl response against ova in vivo in a t-helper cell-dependent manner, but independent of ifna. delivering antigenic peptides directly linked to bppcysmpeg led to superior ctl immunity as compared to giving antigens and adjuvants admixed. in contrast to other tlr ligands such as cpg, systemic activation of dc with bppcysmpeg did not result in shutdown of antigen presentation by splenic dc subsets, although cross-priming against subsequently encountered antigens was reduced. we provide evidence that bppcysmpeg stimulation of dc via tlr / results in the generation of an effective ctl response and that delivering antigenic peptides linked to bppcysmpeg is a promising strategy for vaccination. while bppcysmpeg-matured dc retain their antigen uptake and presentation capabilities, cross-priming against subsequently encountered antigens is inhibited, indicating that mechanisms beyond down-regulation of macropinocytosis and phagocytosis contribute to shut-down of cross-priming after tlr-mediated dc maturation. altogether our study promotes synthetic lipopeptides as potential adjuvant for specific applications (e. g. viral infections, cancer) for the reason that they can be chemically engineered to carry specific antigenic peptides which allows targeting of antigens and simultaneous activation. tumor immunevasion. to verify whether the loss of erap expression could confer a survival advantage on tumor cells and enhance tumor progression, we stably knocked down expression of eraap (murine erap ) in a murine t lymphoma cell line, rma. we used a method that allows an efficient and continuous expression of mirnas that directly silence eraap and obtained several eraap-deficient rma clones with different levels of eraap expression (up to % of reduction at the protein level). microsomal aminopeptidase activity and mhc class i surface expression were decreased in all clones proportionally to eraap expression. moreover, low expression of eraap affected the stability of mhc class i molecules as evaluated after acid and brefeldin a treatment. de-regulated er peptide trimming also drastically affected the tumor formation of rma cells and host survival. eraap-deficient rma clones with different levels of eraap, and % as compared to control rma cells, were injected s. c. in the flank of c bl/ syngenic mice, and analysed tumor growth. all mice injected with control rma cells developed a tumor but survived up to days after injection. all mice injected with rma clone with a % level of eraap expression developed a tumor and died within days after injection. surprisingly, any animal injected with rma clone with a % level of eraap died or showed a visible tumor. thus, knockdown of eraap expression appears differently to affect the immunogenicity of rma cells, depending on the eraap silencing level. hemophilia a is an x-chromosome-linked bleeding disorder caused by the absence or dysfunction of clotting factor viii (fviii). treatment consists of regular administration of fviii, but is complicated by the formation of inhibiting antibodies against fviii. both genetic and treatment-related factors play a role in the etiology of inhibitor development in patients with hemophilia a. the development of inhibitory antibodies in hemophilia a patients has been shown to be a cd + t-cell driven process. therefore, in order to better understand the process of inhibitor formation, we aim to identify the epitope specificity and phenotype of t cells against fviii in hemophilia a patients using mhc class ii tetramers. cd + t-cell responses of two monozygotic twins with severe hemophilia a were analyzed. one of these subjects developed a high titer inhibitor ( bu/ml) following intensive factor viii (fviii) treatment. high dose immune tolerance therapy together with anti-cd therapy resulted in eradication of the inhibitor. in contrast, his twin brother developed a low titer inhibitor ( . bu/ml) which declined rapidly after tolerance induction. fundamental differences in the twins' antibody responses were further suggested by elevated and persistent igg levels in the subject with the high titer inhibitor. in order to gain a better understanding of processes leading to inhibitor formation versus tolerance, we investigated drb * -restricted t-cell responses of the high titer inhibitor subject, using fluorescent mhc class ii tetramers loaded with -mer synthetic fviii peptides to stain epitope-specific cd + cells.cd + t-cells from the high-titre inhibitor subject recognized three peptides corresponding to the fviii a domain: fviii - , fviii - and fviii - , as well as the c domain peptide fviii - , but not any c domain peptides. the c domain peptide contains a sequence that was reported as a promiscuous t-cell epitope (jones td et al., j thromb haemost. : - , ). analysis of t cells from the lower titer inhibitor subject is expected to reveal differences in the epitope specificity and phenotypes of t cells that may underlie the discordant immune responses of these twins to infused fviii. m. forloni , s. albini , m.z. limongi , l. cifaldi , d. fruci ospedale pediatrico bambin gesù, rome, italy neuroblastoma (nb) is a pediatric tumor that derives from neural crest. the most aggressive forms are characterized by amplification of the mycn oncogene and severe reduction of hla class i expression. mycn has been claimed to hinder hla class i expression through affecting the expression of the transcription factor p nf-kb subunit. since in many human tumors the expression of hla class i molecules is positively co-ordinated with that of er aminopeptidases, erap and erap , we wondered whether in nb cell lines mycn may impair expression of these aminopeptidases. to explore this possibility, nb cell lines that differ in mycn expression were quantified for expression of mycn, erap , erap and hla class i heavy chains by western blotting and for surface hla class i expression by flow cytometry. we found that mycn negatively correlates with expression of hla class i, erap and erap . this negative correlation was confirmed in a nb cell line expressing a tetracycline repressible mycn transgene. then, by the use of tnfa (a nf-kb nuclear translocation stimulator), sulfasazine and ikba mutant (two nf-kb nuclear translocation inhibitors) and knockdown of p nf-kb subunit, we demonstrated that nf-kb is involved in erap and erap expression in nb cell lines and that mycn does not affect nf-kb expression. furthermore, we showed that mycn and nf-kb are recruited to the promoter regions of erap and erap and that mycn affects the recruitment of nf-kb binding to these promoter regions. in conclusion, the present results indicate that an enhanced mycn level, linked or not to mycn amplification, represses erap , erap and hla class i expression in nb cell lines by affecting the recruitments of nf-kb binding to their promoters. s. brosch , s. tenzer , h. schild , e. von stebut-borschitz uniklinik mainz, mainz, germany infection of inbred mouse strains with the intracellular protozoan parasite leishmania major either leads to self-healing cutaneous disease (resistant phenotype; e. g. c bl/ mice) or systemic disease (susceptible phenotype; balb/c mice) depending on the genetic background of an individual. healing of leishmania infections is based on th immunity, whereas ifng secretion of both cd + th and cd + tc cells is critically important for protection by inducing oxidative radicals in macrophages, which enables them to kill the parasite. stimulation of antigen-specific effector t cells is driven by l. major-infected dendritic cells (dc) in an il- -dependent manner. proteasome/immunoproteasome-dependent antigen processing is necessary for clearance of viral or intracellular parasitic diseases to induce effective cd + t-cell responses via the mhc class i. here, we analysed the role of the ifng inducible immunoproteasome for the priming of cd + t cells in l. major infections. using an in vivo model, we show that the functional knock-out mouse in the chymotrypsin-like catalytic domain of the immunoproteasome lpm (lmp -/-) does not exhibit an altered course of infection (lesion development, parasite loads, cytokine profiles) in intradermal, low dose infections with l. major mimiking natural transmission of the parasite as compared to wild type c bl/ mice. in addition, ex vivo co-cultures with infected dc from either lmp -/or wild type mice together with antigen-specific t cells from infected wild types showed no differences in tc cell ifng secretion and the dc restimulatory capacity of cd + t cells. furthermore, significant differences in the proliferation of antigen-specifically restimulated (with soluble leishmania antigen; sla) cd + t cells, isolated from low dose infected c bl/ wildtype or lmp -/mice, were not detected. in summary, our data indicate that despite the fact that cd responses in l. major infections are important for disease outcome, processing of antigen and thus priming of cd + t cells against l. major is independent of the lmp subunit of the immunoproteasome. studies have defined an essential requirement for autoantigen-specific b cells as antigen presenting cells in rheumatoid arthritis. however, the cellular mechanisms involved in antigen processing and presentation of joint-derived autoantigens by b cells are unknown. in this study we have developed a system to investigate how antigen-specific b cells recognise and present the proteoglycan aggrecan, a major component and candidate autoantigen of joint cartilage. we have utilised these cells to characterise the mechanisms by which aggrecan-specific b cells could induce autoimmunity. we have constructed plasmids encoding an aggrecan-specific b cell receptor and have transfected them into the b cell line a , generating b cell lines that specifically recognise and target aggrecan for presentation to t cells. in addition, we have established conditions for a panel of aggrecan-specific t cell hybridomas to recognise aggrecan pulsed b cells following fixation, to allow the kinetics and mechanisms of aggrecan processing to be studied. we used inhibitors of mhc class ii transport, endosomal ph and enzymes involved in aggrecan degradation. we found that aggrecan-specific b cell lines presented the major arthritogenic cd + t cell epitope ( - ) from the g domain of aggrecan , times more efficiently than non-specific b cells and over times more efficiently than the macrophage line j . however, despite this highly efficient aggrecan capture, processing and presentation of the - epitope took at least hours, comparable to the time required for presentation of aggrecan by j . treatment of aggrecan-specific b cells with ammonium chloride to raise endosomal ph or brefeldin-a to disrupt golgi transport inhibited presentation of the - epitope, suggesting a requirement for low endosomal ph and presentation by newly synthesised mhc class ii. interestingly, aggrecan presentation by antigen-specific b cells was also reduced by phenanthroline, an inhibitor of the aggrecan-degrading metallo-proteinases that are found in abundance in the arthritic synovium understanding the mechanisms of antigen processing and presentation by autoantigen-specific b cells may explain their role in the pathogenesis of diseases such as rheumatoid arthritis. tapasin is an mhc-dedicated chaperone that facilitates peptide loading and optimization of the peptide cargo of mhc class i molecules within the peptide loading complex (plc). class i molecules differ in their dependence on tapasin for efficient cell surface expression, dependence that is determined by the nature of amino acids at positions , and at the peptide binding groove. position also determines the strength of tapasin binding and influences peptide specificity, but its precise effect is probably context dependent. the mhc class i antigen b is strongly associated to ankylosing spondylitis (as) and other spondyloarthropathies. hla-b subtypes differ in their dependence of tapasin for cell surface expression and incorporation into the plc. tapasin also modulates b folding but not maturation and although tapasin optimizes the constitutive peptide repertoire of b* , peptide loading is relatively independent of this chaperone. we analyzed the effect of b subtype polymorphism on tapasin binding and the correlation of this feature with the affinity of the peptide repertoires, the maturation kinetics and the folding efficiency of b subtypes. the association of b heavy chain with tapasin was analyzed in c r cells transfected with hla-b subtypes and mutants by pulse-chase analysis and co-immunoprecipition with the monoclonal antibody pasta- , which recognizes human tapasin. we also analyzed the global thermostability, as a measure of the stability of the peptide cargoes, and the optimization of the b peptide repertoire with thermostability assays, by pulse-chase analysis and immunoprecipitation with the me monoclonal antibody that recognizes b properly folded b /peptide complexes. the formation of fully assembled b molecules was analyzed by pulse-chase analysis and immunoprecipitation either with the monoclonal antibody hc , which recognizes mhc class i free heavy chains (hc), or with me . maturation was analyzed by pulse-chase analysis, immunoprecipitation with me and treatment with endoglycosidase h (endo h). hla-b polymorphic positions other than , both at the a and c/f pockets modulate tapasin binding and the optimization of the peptide cargo. the stability of the peptide repertoires critically influences the folding efficiency of b subtypes. from as early as the initial phases of infection, hiv is coated with complement (c) fragments and following seroconversion, the circulating virus forms immunecomplexes with igg and complement. recent in vitro experiments revealed differences with respect to productive infection of immature dendritic cells (idcs) with differentially opsonized hiv. the opsonization pattern of hiv may additionally have profound consequences for the outcomes of the antigen-presenting capacity of dcs and their ability to mount an adequate immune response. in this context, we compared the impact of differential hiv-opsonization on the antigen-presenting capacity of dcs and found that c-opsonized hiv triggered ctl responses, while igg-coated virus did not. these in vitro generated ctls showed an enhanced ifn-g secretion and recognized the help independent ctl epitope slyntvatl. c-generated ctls also degranulated upon stimulation with specific hiv peptides and were able to elicit antiviral activity against hiv-infected cd + t cells. our results indicate that c-opsonization of hiv drives the virus towards the mhc class i pathway in dcs, thereby promoting a more efficient stimulation of naïve cd + t cells. this ctl-stimulating property of c could be exploited when searching for a novel approach against hiv. igg isolated from patients with high titers of anti-ccp antibodies showed a cross-reactivity with hcit peptides. vaccination experiments supported a triggering role of hcit for the development of arthritis in mice model. conclusions: diamination process is significantly increased in patients with ra while carbamylation is suppressed. production of specific antibodies against diaminated residues in ra patients may have a modulating role for the development of autoimmune arthritis. the classical pathway of mhc class i antigen presentation involves cytosolic degradation of viral proteins by the proteasome. peptides generated entry the endoplasmic reticulum through the transporter associated with antigen processing (tap). previous reports have shown that viral epitopes are presented to ctl independently of tap in smaller viruses. we hypothesized that presentation of vacv by mhc class i might proceed by alternative pathways. the aim of this study was to characterize these alternative pathways in tap-deficient mice. our results show that ctl derived from c bl/ mice immunized with vacv, recognized tapdeficient dendritic cells infected with the virus. approximately % of vacv global presentation in the context of h- b was independent of tap. in addition, vacv infection induced a virus-specific ctl response in mice deficient in tap. dendritic cells (dc) initiate robust ctl immunity via the presentation of antigen-derived peptides by surface major histocompatibility complex class i molecules (pmhc). two major dc subtypes have been described, cd + and cd -dc, which differ in their mhci antigen presentation capacities. cd + dc are the major dc subset responsible for cross presentation (presentation of exogenous antigen by mhci), while cd -dc display little cross presenting capacity. here, we examined the mhci antigen presentation pathway of cd + and cd -dc in more detail. first, turnover (half-life) of total mhci at the cell surface of cd + and cd -dc was determined. surprisingly, cd + dc exhibit rapid surface mhci turnover compared to cd -dc (following culture in the presence of brefeldin a). following activation of dc with cpg, mhci levels at the surface of both cd + and cd -dc were stabilized and no longer underwent rapid turnover. this suggests that cd + and cd -dc differ in their regulation of surface mhci turnover and that this is subject to regulation by antigen-associated signals. second, we examined the ability of cd + and cd -dc to generate pmhci complexes containing cross presented antigen. we utilized the model antigen ovalbumin (ova) and an antibody that can detect h- kb loaded with the ova-derived peptide, siinfekl. cd + and cd -dc isolated from ova-expressing mice (actin-ova transgenics) displayed abundant kb-siinfekl complexes at their cell surface. in contrast, in response to exogenous soluble ova protein, only the cd + dc, but not the cd -dc, displayed kb-siinfekl complexes at the cell surface. similarly, when dc were pulsed with ova-coated splenocytes, kb-siinfekl complexes were only detected on the surface of cd +, and not cd -dc. this data further validates the role for cd + dc as the major cell type responsible for cross presentation and provides insight into the mechanisms that prevent other dc subsets from accessing the important cross presentation pathway. objectives: the action radius of matrix metalloproteinases or mmps is not restricted to massive extracellular matrix (ecm) degradation but extends to the proteolysis of secreted cytokines and membrane-bound receptors and adhesion molecules. although many instances exist in which cells disintegrate, often in conjunction with induction of mmps, the intracellular mmp substrate repertoire or degradome remains relatively unexplored. the aims of the present study were to identify novel intracellular mmp targets and to answer the question whether the proteolytic modification of intracellular proteins alters the immunogenicity of released intracellular contents. methods: multidimensional degradomics technology was developed by the integration of broadly available biotechniques and applied to thp- cytosol using gelatinase b/mmp- as a model enzyme. in the first dimension, ion exchange chromatography separated the thp- proteins by their net charge and/or isoelectric point (pi) followed by cleavage of the proteins by mmp- . in the second dimension, potential substrates were separated by molecular weight on sds-page. to evaluate the effect of proteolysis by mmp- on the immunogenicity of the intracellular protein pool, mice were immunized twice with thp- cytosol in complete freund's adjuvant. lymph node t cells were isolated and stimulated with mmp- -cleaved or intact thp- cytosol. proliferation was assessed by measuring incorporated hthymidine. results: - mmp- candidate substrates were isolated, of which were identified, revealing many novel mmp- (candidate) substrates from the intracellular matrix (icm), such as actin, tubulin, stathmin,... about / of the identified substrates were described as systemic autoantigens in one or multiple autoimmune conditions. remarkably, a significantly lower t cell proliferation was observed in the presence of cleaved vs. intact cytosol. conclusion: multidimensional degradomics technology is a valuable tool for high-throughput identification of novel mmp substrates. proteolysis by mmp- decreased the immunogenicity of the intracellular contents, suggesting that mmps may contribute to the dampening of inflammation by the clearance of toxic and immunogenic burdens of intracellular (matrix) proteins released after extensive necrosis and tissue injury. a preference for hla-a versus -b molecules; e - k did not detectably associate with hla-c molecules under identical conditions. this locus specificity may provide a functional advantage to ads by inactivating t-cell receptors, while avoiding activation of nk receptors. finally, we showed that residue in hla-a and residue in e - k are highly critical for association of both proteins. this defines a putative interaction surface between e - k and class i molecules. conclusions: our studies provide novel insights into the functional relationship between e - k and the class i antigen presentation pathway. moreover, because soluble e - k can differentiate between polymorphic gene products encoded in the mhc, our results may contribute to define paradigms for how class i substrate specificity is established for er retention. overall, our studies represent an important step towards a molecular understanding of the strategy evolved by ads to establish life-long persistence in host cells. objectives: the transporter associated with antigen processing (tap) belongs to the abc transporter superfamily and is a heterodimer consisting of the two subunits tap and tap . tap transports peptides yielded by proteasomal degradation from the cytosol into the endoplasmic reticulum (er) and is thus a key element of the mhc class i antigen processing machinery (apm). methods: target-specific tap knock downs were generated by shrna technology. the resulting transfectants were subsequently analysed regarding mhc class i surface expression using flow cytometry, whereas mrna and protein expression levels of tap and tap were analysed by rt-pcr and western blots, respectively. furthermore, the protein stabilizing effect of tap on tap was investigated in the presence of two distinct proteasome inhibitors. results: previous findings obtained with rare tap mutants suggested that the lack of tap protein expression is associated with a strong reduction of tap protein levels, which could be restored by tap gene transfer, whereas no such regulation is found vice versa. to investigate this stabilizing effect of tap on tap different shrna plasmids specifically targeting tap or tap , respectively, were stably transfected into constitutively tap and tap expressing hacat keratinocytes and colo melanoma cells. in both cell types the shrna-mediated tap and tap inhibition resulted in a significant downregulation of the respective transcript and protein expression levels. the knock down of tap caused not only an almost complete loss of tap , but also a strong decrease of tap protein expression. in contrast, the tap knock down exhibited no influence on the tap expression. specific inhibition of the proteasome prevented the degradation of tap in the tap knock down variants. the results of our study emphasize that an unidirectional stabilisation of tap on tap protein expression is not restricted to rare tap mutants, but rather suggest a common regulatory mechanism for the tap complex. uv injury profoundly affects the skin immune homeostasis by promoting strong inflammation and cellular immuno-modulation. in this study, we characterized the inflammatory cell subsets that emigrate in the epidermis the days following uv exposure. therefore, the buttock skin of healthy volunteers was exposed twice to . minimal erythema dose of uv. blister roofs were then collected before and , and days after uv-exposure from un-exposed and exposed skin and the resulting epidermal cells were analysed by flow cytometry. we demonstrated that, along with the rapid activation and migration of langerhans cells (lc), uv skin radiation exposure promotes the infiltration into the epidermis of a monocytic cd + cd + and of a macrophagic cd -cd + cell subsets that emerge and days post exposition, respectively. more importantly whereas classical cd a hi cd + lc are the unique dendritic cell (dc) subset found in the epidermis of unexposed skin, we detected two new subsets of epidermal dc namely cd a low cd and cd a low cd + that emerged and days post irradiation, respectively. these two distinct populations of epidermal dc (edc) differ from classical epidermal lc by their activation/maturation profile as assessed by the strong expression of cd and hladr. finally, days post-exposure, we observed that lc represented almost the only haematopoietic cell population in the epidermis. these results suggesting that the uv-recruited edc and monocytic/macrophagic subsets participate to the progressive recovery of the epidermal immune cell network homeostasis. i. bailey , e. reeves , t. elliott , e. james university of southampton, school of medicine, cancer sciences division, southampton, united kingdom there is accumulating evidence that cd + cd + regulatory t cells (treg) play an important role in anti-tumour immunity by preventing effective t cell responses to tumour antigens. tregs have also been shown to inhibit development of organ-specific autoimmune diseases suggesting they inhibit immune responses to tissue-specific self-antigens. the depletion of tregs prior to challenge with the murine colorectal tumour, ct , stimulates a robust, protective t cell response which is also protective to challenge with other tumours of different histological origins, such as b cell lymphomas and a renal cell carcinoma. this cross protection has not been seen with other tumour cell lines. we have identified a ct -derived cross-protective antigen, gsw , which was found to be encoded within the ectotropic murine leukaemia virus (emv- ) envelope protein, gp . this protein has previously been shown to encode ct -specific cd and cd antigens, implicating it as a 'hot-spot' for ct tumour antigens. interestingly, we have identified a truncated version of gp which may be responsible for generation of gsw . expression studies have revealed increased gp expression in ct compared to other tumour cell lines, indicating the ability to cross-protect is related to the quantity of antigen (gsw ) generated. the current knowledge of hla class ii antigen presentation and peptide binding is mainly based on studies of hla-dr molecules. they contain a large hydrophobic p pocket, which can accommodate large hydrophobic amino acid residues and is the most important pocket in selecting and binding peptides, while p , p and p tune the peptide repertoire that can be presented by individual hla-dr alleles. the same rules and requirements do not necessarily exists for peptide binding to hla-dp molecules, however. the present study adresses this issue. we have expressed and affinity purified soluble recombinant hla-dp molecules from drosophila melanogaster cells and studied its binding of a number of peptides known to bind hla-dr, -dq or dp molecules. unexpectedly, the immunodominant epitope in multiple sclerosis (ms), the myelin basic protein derived peptide, mbp - , bound to hla-dp with high affinity ( - nm). binding studies of mbp - derived peptides containing single alanine substitution at each position revealed that only three of the peptides (f a, f a and k a) were affected, and only by a - fold reduction in affinity for hla-dp . the observation that none of the substitutions resulted in a complete loss of binding to hla-dp indicates that ) hla-dp binding to peptides does not depend on a large hydrophobic residue accomodated in p , or ) mbp - can bind in more than one register. we will present data addressing this issue. the hla-dp peptide binding capacity was increased at neutral as compared to acidic ph, and by the presence of n-butanol, a small organic mhc loading enhancer (mle). in summary, the hla-dp molecule binds the immunodominant epitope in ms, mbp - , possibly in more than one register. additional studies are required to resolve the hla-dp peptide binding properties, and to determine whether expression of hla-dp affects the disease course in ms patients. results: depletion of mncs for cd + cells abrogated the tg-induced cytokine production and proliferation of cd + t cells, indicating a primary role for monocytes as apcs. however, the encounter of t cell with antigens presumably occurs in b cell-rich compartments such as lymph nodes or lymphoid tissue in inflamed organs. to mimic the conditions prevailing there, we depleted pbmcs for g % of the monocytes (without significant loss of t-cells), and compared the tgelicited t-helper cell responses in the presence and absence of b cells. the tg-induced cd + t cell proliferation was significantly reduced in cd /cd -depleted mnc cultures, as compared to cultures depleted for cd + monocytes alone. the same applied to the production of il- , il- and tnf-a. production of ifn-g and il- was generally not observed. our data indicate that normal b cells are capable of inducing a pro-inflammatory cytokine response in mnc-cultures, where monocytes and monocyte-derived cells are not preponderant. studies addressing the relative contributions to this cytokine production, by b cells themselves and by t cells (following antigen-presentation by b cells), are in progress. j. kyosiimire-lugemwa , , p. pala , g. miiro , j. todd , p. kaleebu , , n. imami , f. gotch , mrc uganda, basic science, entebbe, uganda, imperial college, london, united kingdom, mrc uganda, entebbe, uganda background: hiv- -specific t-cell responses are preserved in hiv- infected individuals with non-progressing hiv- disease. "long term non progressors" (ltnps) were defined as art naï ve individuals infected with hiv- for g years, maintaining cd + t-cell counts g , and with minimal cd + decline over time. we tested the hypothesis that gag-specific t-cell responses are inversely correlated to disease progression whereas nef-specific t-cell responses are not. methods: art naï ve hiv- infected patients from the entebbe cohort in uganda were recruited and stratified by cd + t-cell count, cd + decline slopes, and time of enrolment, into groups - ltnp and rapid progressors (rp). all patients were women reflecting the patient base at the entebbe cohort. we measured plasma viral load, current cd t-cell count, and ifn-g, il- and il- elispot responses to pools of to peptides ( -mers overlapping by aa). peptides were based on consensus sequences of gag and nef from hiv- clades a , a and d. medians and inter-quartile ranges were calculated and comparisons between groups were performed using the mann-whitney u test. correlations were presented using spearmann's linear correlation coefficients. results: some gag-specific ifn-g and il- responses were significantly higher in the ltnp than in rp (p= . , ifn-g responses to gaga pool ; p= . , il- responses to gaga pool ). il- responses were low and not significantly different between ltnp and rp. there was a positive correlation between il- responses to gaga pool and cd t-cell counts (r = . , p= . ), but no correlation between either il- or ifn-g responses and viral load. cytokine responses to nef peptides were not significantly different between the ltnp and rp. conclusion: overall, gag hiv- specific responses were higher in ltnps than in rps confirming previous results. non-specific il- responses were high possibly reflecting baseline th responses to helminths a common environmental exposure in the study population. objectives: in human and murine tumors and in in vitro oncogene-transformed cells defects in the expression of components of the hla class i antigen processing machinery (apm) have been described, which were associated with a reduced antigenicity of these cells. so far, the molecular mechanisms of such defects have not been elucidated in detail. to investigate whether impaired apm component expression was due to altered transcription and associated with cell growth properties murine her /neuand her /neu + fibroblasts were employed. methods: using tapasin as a model molecule its cell cycle-dependent expression was analysed in a time kinetics upon serum starvation followed by stimulation with complete culture medium over time. cells were harvested at different cell cycle phases and expression of tapasin was analyzed by qrt-pcr and western blot. flow cytometry was employed for determination of the distinct phases using -aad for dna analysis and specific antibodies directed against the proliferation marker ki- , the m-phase specific phistone h as well as for the h- l d surface antigens. in addition, chromatin immunoprecipitation (chip) experiments with an antibody directed against rna polymerase ii were performed to investigate the transcriptional levels of tapasin in her /neuversus her /neu + cells. results: serum starvation and subsequent stimulation with complete culture medium led to the enrichment of cells at the g /g -, s-and g /m-phases of the cell cycle, which was associated with an altered tapasin transcription during the cell cycle. tapasin mrna level decreased during cell cycle progression, whereas an inverse protein expression was observed with low expression levels at the g /g -phase, which continuously raised and peaked within the s-phase. however, h- l d surface antigen expression was not altered in her /neucells during cell cycle progression. in contrast to her /neufibroblasts the her /neu + transfectants exhibit a decreased tapasin transcription, which was accompanied by an altered h- l d surface expression. this was confirmed by a reduced promoter activity and decreased accessibility of the rna polymerase ii to the tapasin promoter. conclusion: these findings lead to an improved understanding of immune escape mechanisms demonstrating a cell cycle dependent and oncogene-mediated tapasin regulation that may provide novel targets for therapeutic intervention. recent studies suggest that dendritic cells (dcs) are key players in shaping the respiratory syncytial virus (rsv) specific immune response. before, dcs within the airway epithelium were characterized as langerhans cells. in this study, in vitro counterparts of langerhans cells expressing langerin (cd ) and ccr were cultured from cd + stem cells under the influence of tgf-b (tgf-b-dcs) and compared to cd + derived dcs, which passed through a monocytic stage . after infection with rsv, both types of dcs generated viral rna and viral-proteins. although tgf-b-dcs expressed higher levels of viral proteins as revealed by flow cytometry and fluorescence microscopy, more than hundredfold more viral particles were released by il- -dcs. the increased expression of viral proteins is most likely responsible for the pronounced inhibition of t-cell functions by tgf-b-dcs. since there is evidence that langerhans cells are expressed in airway epithelium not before the age of one year, the results may indicate, that an inhibition of rsv replication is characteristic of a more mature answer against rsv. the occurrence of inhibitory antibodies against exogenous factor viii (fviii) remains the major concern of fviii replacement therapy in patients with hemophilia a. initiation of the immune response implies the endocytosis of fviii by professional antigen presenting cells (apcs): b lymphocytes, dendritic cells (dcs) and macrophages (mØ). the organ where the anti-fviii immune response is initiated and the type of apcs involved in this process have not been investigated. we hypothesized that the spleen, which is the principal filter for blood-born antigens, is the principal organ where apcs interact with fviii to initiate the anti-fviii immune response. we first administered radiolabeled fviii at therapeutic doses to fviii-deficient mice. fviii was found to preferentially accumulate in the spleen and liver of the mice. levels of fviii in the spleen remained stable for up to min following fviii administration, while they rapidly decreased in the liver. unlabelled fviii was then administered to fviii-deficient mice that had been splenectomized or sham operated and the anti-fviii humoral responses were compared. removal of the spleen resulted in significantly reduced levels of anti-fviii igg. using flow cytometry, fviii was found to preferentially accumulate with splenic mØ than dcs and b cells. elimination of apcs by treatment of the mice with clodronate-containing liposomes prior to fviii administration resulted in a drastic reduction of the anti-fviii igg response, as compared to control mice treated with pbs-containing liposomes. taken together, our results suggest that the spleen is the principal organ in the initiation of the anti-fviii immune response and that splenic mØ have an important part in this process. the interactions between antigen presenting cells (apc) and t-lymphocytes are a relevant current issue. the area of contact between an antigen presenting cell and a t-lymphocyte is termed immunological synapse (underhill et al, ) . the present work started, in experiments with leukocyte of healthy individuals from the finding that under certain experimental conditions, cell-cell association with closely contact between monocyte-derived macrophages and human autologous lymphocytes are produced when the cells are harvested from total leukocyte cell cultures. in this way, such cells selective forming rosettes with a central macrophage and adherent lymphocytes. objectives: as central hypothesis it was postulated that the phenomenon would be due to antigen presentation made (performed) by macrophages to lymphocytes and that would be t cells. methods: autologous total human leukocyte cultures, from samples of healthy blood donors were harvested at various times and centrifuged and performed as previously reported (cabral and novak, , ) . cytopreparations of each experiment were performed. statistical analysis: regression model. results: experimentally, it was found a) phagocytosis of autologous antigens by macrophages, stimulates the formation of rosettes, b) a linear relation between rosettes formation and culture-time occurs, (p x , ), anova for regression, c) the cell-cell approximation is very important and was performed by centrifugation of the cells to form pellets, d) the forming rosettes lymphocytes are t-cells, cd +, e) purified macrophages and lymphocytes produced few rosettes, however if antigens were added, the phenomenon was stimulated, f) if inhibitors of the antigen processing and antigen presentation, such as chloroquine or brefeldin a, were added, rosettes were not formed, g) monoclonal antibodies anti-human mhc ii precluded the formation of rosettes, h) gangliosides diminishes rosettes formation. conclusion: taken together, the findings suggest that the model of rosettes formation might be useful to study cell-cell interactions during antigen presentation in immunological synapses and other immunologic aspects on the cells involved, in short time assays. objective: to investigate if patients with multiple sclerosis (ms), without the typical increase of antibodies in csf, are less likely to develop neutralizing antibodies (nabs) against ifnb-treatment, and whether the absence of such an immunological response might reflect a difference in their antigen presenting ability due to a distinct genetic background. methods: overall, patients were obtained from the swedish multiple sclerosis registry and the swedish nab registry, and treatment information was available for of them. for of these patients hla-drb data was available. results: a significant correlation between lack of antibodies in csf and nab-negativity was found (p= . ). patients without csf antibodies were to a lesser extent nab-positive when treated with the ifnb- a preparations, whereas no differences were shown for ifnb- b. an association between hla-drb * and nab-negativity was detected (p= . ). the known associations between hla-drb * and csf-positive ms and hla-drb * and csf-negative ms were confirmed. conclusion: we show for the first time that patients without antibodies in csf have a different propensity to induce nabs compared to csf-positive patients, indicating an extended immunological difference between the two ms sub-groups. hla-drb potentially contributes to this, which indicates that it might have something to do with differences in antigen presentation. in csf-negative patients the reaction against ifnb- a molecules, possibly through a t-cell dependent pathway, is lower than for csf-positive patients. however, reaction against ifnb- b, which might also be activated through a t-cell independent pathway, shows no difference in seroprevalence between the groups. abstract withdrawn by author tyrosinase-derived epitope was confirmed by five independent assays: flow cytometry on multiple melanoma lines generated from patients, confocal microscopy immuno-staining of melanoma lines, frozen sections staining of authentic melanoma tissue from patients, cytotoxicity assays using tyrosinase-specific ctls, and finally mass spectrometry analysis of peptides isolated from a melanoma cell line. there was no correlation between the level of antigen presentation and mrna expression levels for the three antigens; however, our data suggest that tyrosinase protein stability may play a major role in the high level presentation of this antigen. measurement of the half lives of these proteins revealed a hierarchy in protein stability, with mart- and gp more stable than tyrosinase. by the use of the cofactor dopa, which stabilizes the tyrosinase protein, significant decrease of hla-tyr complexes presentation was achieved. in addition to the study of antigen presentation, these tcr-like antibodies can also actively participate in immunotherapy as targeting molecules, considering their high affinity and specificity. by generating a whole igg antibody, tumor cell lysis was achieved by antibody-dependent cell-mediated cytotoxicity (adcc). with the addition of point mutations in the fc fragment, which increased the affinity of the fc to the fc receptor, enhanced tumor cell lysis was achieved. g. schiavoni , s. lorenzi , f. mattei , f. spadaro , l. gabriele istitituto superiore di sanità, cell biology and neurosciences, rome, italy cross-presentation is a crucial mechanism for generating cd t cell responses against exogenous antigens (ag), such as dead cell-derived ag, and is mainly fulfilled by dendritic cells (dc), particularly cd a + dc. however, apoptotic cell death occurring in steady-state conditions is largely tolerogenic, thus hampering the onset of effector cd t cell responses. type i ifn are a family of cytokines induced upon infection and acting as danger signals by stimulating multiple arms of the immune response. in particular, type i ifn have been shown to promote the cross-priming of cd t cells against soluble or viral antigens, partly through the stimulation of dc. in this study we evaluate the role of type i ifn to affect dc capacity to capture and cross-present apoptotic cell-derived ag. by using uv-irradiated ova-expressing eg thymoma line, we show that type i ifn promote the ability of cd a + dc to capture apoptotic eg cells and to undergo phenotypic activation, both in vitro and in vivo. remarkably, ifn-treatment prolongs the survival of ag-bearing cd a + dc and the persistence of apoptotic eg -cell ag within the phagosomal dc compartment, a process that is known to facilitate the recruitment of ag into the mhc-i presentation pathway. accordingly, type i ifn-treatment increases cross-presentation of apoptotic eg -derived ova ag by dc, as revealed by higher expression levels of siinfekl peptide in association to mhc-i molecules on cell surface of phagocytic cd a + dc. as a result, eg -loaded dc become competent at inducing ot-i cd t cell proliferation and activation both in vitro and in vivo. our data indicate that type i ifn promote the cross-presentation of apoptotic cell-derived ag by cd a + dc and suggest that these cytokines may act as a switch signal for cross-presenting dc, thus skewing the immune response from tolerogenic to immunogenic. ( ). we have investigated the mechanisms of cross-presentation of soluble antigen in freshly purified splenic dc subsets. using biochemical methods, we show that only cd + dc efficiently transfer soluble antigen to their cytosol. the amount of antigen detected in the cytosol increased up to ten-fold after a short exposure to tlr ligands cpg, poly i:c or pam csk , and this correlated with enhanced cross-presentation. the increase in antigen accumulation within the cytosol was not due to increased uptake of antigen. measurement of the proteasome activity at different times after exposure to tlr ligands revealed that tlr signalling induced transient inhibition (maximum at two hours) of the proteasome in cd + dc but not cd -dc, thus promoting accumulation of exogenous antigen in the cytosol of cross-presenting dc. this correlated with formation of aggresome-like structures only in cross-presenting dc exposed to tlr ligand. by limiting the degradation of transferred proteins during early activation, when endogenous proteins are being stored in aggresome-like structures, this mechanism could favour the loading of exogenous antigen peptides over endogenous peptides, promoting cross-presentation. to our knowledge this is the first report of a direct, immediate effect of tlr activation on proteasome activity. exosomes are nano-sized membrane vesicles of endosomal origin which can exert both immune stimulatory and tolerance inducing effects depending on their cellular origin. they are currently being investigated for use in vaccination and immune therapy strategies, but their physiological role has not been elucidated. here we explore whether exosomes of different origin can selectively target different immune cells. we compare the binding of exosomes from human breast milk, monocyte derived dendritic cells and b cells to peripheral blood mononuclear cells. flow cytometry, confocal laser scanning microscopy and multispectral imaging flow cytometry (imagestream) reveal that exosomes derived from human dendritic cells and human breast milk preferably associate with monocytes, whereas exosomes from an epstein-barr virus (ebv) transformed b cell line selectively target b cells. our data suggest a highly selective association between cells and exosomes which can be a way to direct their functional effects. one of the hallmarks of cancer cells is the resistance to cell death. it has been suggested that cancer cells also have the capacity to evade the surveillance by the immune system. the proteasome and macroautophagocytosis are attractive effector mechanisms for the immune system, because they can be used to degrade foreign substances, including pathogenic protein, within cells. here, we investigated that dm , which is a saponin derivative isolated the stem bark of dracaena mannii induced autophagocytosis on a human lung cancer cell line. methods: dm induced cell cytotoxicity was measured by mtt assay and propidium iodide staining on a cells. we examined the morphological change using optical microscope. and gfp-lc punctation was measured using confocal. the protein expression was measured by western blot analysis and the mrna expression was measured using reverse transcription poly chain reaction (rt-pcr). results: dm was showed high cytotoxicity on various cancer cell line, especially a cells. dm treated a cells did not show regular dna fragmentation and caspases activation. we also analyzed protein expression of apoptotic marker, but protein level didn't change. as a result, we hypothesized that this non-apoptotic cell death is mediated autophagocytosis. we checked lc and beclin- protein and mrna expression and inhibitory effect of autophagocytosis inhibitor. the expression level of lc was increased concentration and time-dependently. beclin- also was increased. and then, we examined gfp-lc punctation on a / gfp-lc stable cells using confocal. a cells were formed gfp punctuation after dm treatment. to confirm these data, we measured cell death ratio using -methyladenine ( -ma), an autophagocytosis inhibitor. after -ma treatment, dm induced cell death was decreased comparing with dm treatment. conclusion: dm did not induce apoptotic cell death. but dm showed several characteristics of autophagic cell death. taken together, dm induced autophagocytosis on a cells. these finding indicated that therapeutic potential of dm by triggering autophagic cell death. s. b. rasmussen , s. r. paludan university of aarhus, department of medical microbiology and immunology, aarhus, denmark during viral infections, different pattern recognition receptors detect specific pathogen associated molecular patterns (pamp)s. in the case of herpes simplex virus (hsv), detection is, among others, conducted by toll like receptor (tlr) , which is a transmembrane receptor located in the endosomes where it detects unmethylated cpg rich dna of extracellular origin, e. g. viruses. upon binding to dna, tlr initiates downstream signalling cascades resulting in induction of antiviral cytokines, interferon (ifn)a and ifnb being some of the most essential ones. the exact route of hsv to the endosomes and thus tlr recognition is not clear-cut. the endocytosis pathway is believed to be a central mechanism in which viruses translocate to the endosome, but recently the role of autophagy in tlr mediated viral recognition has been drawn in to focus. we have found indications of an autophagy dependent ifn expression during hsv- infection. by use of an entry defect glycoprotein l and glycoprotein h deficient hsv- , we found that tlr dependent ifn regulatory factor activation was abrogated. in addition, inhibition by -methyladenine of phosphoinositol -kinase class iii, which is crucial in autophagosome formation, abrogates ifnb induction. these findings points to a role for autophagy in tlr dependent recognition. in the ongoing project we are examining how hsv- triggers formation of autophagosomes. especially the role of endoplasmatic reticulum stress and doublestranded rna-dependent protein kinase will receive attention. also the newly found involvement of ubiquitin and acetylation in autophagy execution and regulation will be investigated. j. zovko , i. berberich , gk -immunomodulation universität würzburg institut für virologie und immunbiologie, würzburg, germany members of the bcl- family control the integrity of mitochondria and thereby influence survival and death of cells. most bcl- family members can localize to intracellular membranes via hydrophobic sequences within their c-terminal portion. murine a and its human homologue bfl- are anti-apoptotic members of the bcl- family. a is expressed in small amounts in the bone marrow and immature b cells, but in high amounts in mature b cells. thus the protein seems to be important for b cell maturation. we analyzed the function of the c-terminus of a . unless the c-terminal ends of other bcl- proteins the tail of a does not function as a strong membrane anchor. nevertheless, the last amino acids of a are important for the protein. in fact, the c-terminus of a serves a dual function by being required for the instability and the anti-apoptotic potential of the protein. we show that a undergoes proteosomal degradation controlled by its c-terminus. interestingly, binding to the proapoptotic bcl- factor bimel results in increased stability of a . this is due to reduced ubiquitination of a after binding of bimel. we conclude that the cterminus of a /bfl- serves as a docking site for e ubiquitin ligase(s) that control the stability of a by targeting the protein to the proteasomal pathway. very recently, we have identified a putative e ubiquitin ligase that interacted with a in a yeast two-hybrid screen. currently, we are trying to confirm this interaction in mammalian cells. suppressors of cytokine signaling (socs), initially identified as negative regulators of cytokine signal transduction, have recently emerged as multi-functional proteins regulating inflammation, survival, differentiation, and apoptosis of immune cells. here we describe novel function of socs- in the suppression of rosmediated apoptosis and associated mechanisms using tnf-alpha induced t cell apoptosis as a model system. both in jurkat t cells and primary splenocytes, socs- is induced under tnf alpha-induced apoptosis conditions. the tnf-induced apoptosis was mediated by generation of ros, and the over-expression of socs- significantly suppressed tnf-induced ros levels and the subsequent apoptosis. such anti-apoptotic function of socs- was manifest not only by the suppression of jaks acting upstream of p mapk, but also protection of ptps which down-regulate the tnf alpha -induced jak / activities. we further show that tnf-alphainduced ptp inactivation can be prevented by socs which up-regulates thioredoxin levels. finally we present data that there is a molecular interaction between thioredoxin and ptp which is formed in response to ros-generating stimuli and sustained in socs- overexpressing cells. the results strongly suggest that socs- acts as an anti-oxidant and anti-apoptotic factor to sustain t cell homeostasis and survival under oxidative stress imposed upon inflammatory cytokines during infection or other immune scenarios. aim: inflammation is a cardinal host response to injury, tissue ischemia, autoimmune responses or infectious agents. the effects of inflammatory mediators on the glial function are of particular interest since astrocytes contribute to the local inflammatory responses in the cns by producing cytokines, chemokines, and maintain local homeostasis clearing released neurotransmitters. cxcl /sdf- a not only regulates cell growth and migration of hematopoietic stem cells but may also play a central role in brain development. moreover, it has been described that tnf-a mediates in cxcl effects on primary astrocytes, and it is clear that tnf-a participates in the pathogenesis of many neurological conditions. methods: we used the astrocytoma cell line u- , and sk-n-mc as neuroblastoma cells. detection of tnf-a mrna expression was carried out by rt-pcr. transcriptional activity was measured using luciferase reporter gene assays in transiently lipofectin transfected-cells. we performed cotransfection experiments of nfat promoter construct with a dominant negative version of nfat (dn-nfat). neuronal death was performed by mtt and tunel assays. nfat translocation was confirmed by western-blot. p and fas-l expression was carried out by western-blot. results: cxcl induced mrna-tnf-a and transcriptional activity of tnf-a promoter in human astrocytes. this cytokine by itself was not toxic when added directly to astrocytes, but when we investigated its effect on nb cultures, neuronal death increased in a direct and indirect way. surprisingly, tnf-a did not induce nf-kb activation in nb cells but it induced nfat activity. nfat translocation was confirmed by western-blot. neurotoxicity was absolutely reverted in the presence of ciclosporin. we discard p pathway as responsible for tnf-mediated toxicity since we did not find any alteration in p -ser levels in stimulated cells. in addition, we found increased fasl expression in neuroblastoma cells after h of tnf-a treatment. conclusions: cxcl induced-tnf-a promotes nfat activation in neuroblastoma cells and this event leads to increased apoptosis through an increase of fasl expression without alter p function/levels. s. y. demiroglu , r. dressel universitätsmedizin göttingen, zelluläre und molekulare immunologie, göttingen, germany objectives: intracellular hsp is part of the cellular stress response system and can inhibit specific apoptotic pathways. extracellular hsp on the other hand, is an immunological danger signal that can induce the antigen-specific activity of cytotoxic t lymphocytes (ctl). interestingly, hsp does not protect against cell death mediated by ctl. acute overexpression of hsp can even increase the susceptibility of target cells to ctl, which use the granule-exocytosis pathway to induce apoptosis (dressel et al. cancer res : ) . granzyme b is one of the main effector proteases of cytotoxic granules. therefore, we analyzed the effect of acute overexpression of hsp on granzyme b-induced apoptosis. methods: hsp was expressed in a human melanoma cell line under the control of a tetracycline-inducible promoter (ge-tet). the effect of hsp induction on granzyme b-mediated apoptosis was now analyzed after delivery of granzyme b into the cytosol of the target cells by the endosomolytic activity of adenovirus type . results: hsp did not protect the melanoma cells against granzyme b-mediated apoptosis when annexin v binding, loss of mitochondrial membrane potential, release of mitochondrial cytochrome c, and activation of caspase- were evaluated. instead, we observed a moderate but significant pro-apoptotic effect of hsp in ge-tet cells when late apoptosis was analyzed at the nuclear level by sub g peak measurements (p= . ). in contrast, a partial protection of ge-tet cells was observed after acute hsp overexpression when apoptosis was induced by staurosporine. conclusion: acute overexpression of hsp does not seem to protect tumor cells from granzyme b-induced apoptosis; it appears even to accelerate the progression of apoptosis to dna fragmentation and nuclear condensation. it is conceivable that this hsp effect is mediated by chaperoning pro-apoptotic molecules and improving their function as it has been reported for the caspase-activated dnase (liu et al., blood : ) . thus, granzyme b might be able to kill even those tumor cells that undergo an otherwise protective stress response. the work has been supported by the grants grk and dr / - . the authors thank prof. c. j. froelich (evanston, usa) for the granzyme b and dr. t. seidler (göttingen) for the adenoviruses. tumor necrosis factor (tnf) elicits its biological activities by stimulation of tnfr and tnfr , both belonging to the tnf receptor superfamily. tnfr -mediated signal transduction has been intensively studied and is well understood, especially with respect to activation of the classical nfkb pathway, cell death induction and map kinase signaling. in contrast tnfr -associated signal transduction is poorly defined. earlier findings demonstrated that only membrane tnf, but not soluble tnf, properly activates tnfr , resulting in depletion of traf from the cytoplasm. here we confirm that tnfr induced depletion of cytosolic traf by the use of tnfr -and tnfr -specific mutants of soluble and membrane tnf. corresponding with the known inhibitory role of traf in the alternative nfkb pathway, we show that tnfr induced activation of the alternative nfkb pathway. thus, we identified activation of the alternative nfkb pathway as a tnf signaling effect that can be specifically assigned to tnfr and membrane tnf. j. c. morales , d. ruiz-magaña , d. carranza , c. ruiz-ruiz universidad de granada, instituto de biopatologí a y medicina regenerativa. centro de investigación biomédica, armilla, spain different molecular mechanisms have been involved in the resistance of tumor cells to tumor necrosis factor-related apoptosis-inducing ligand (trail)-mediated apoptosis. epigenetic modifications commonly associated with tumor development, such as histone deacetylation, may influence the resistance of some tumor cells to trail by regulating gene transcription of trail receptors and other trail pathway related-genes. in the present study we have analyzed the effect of several histone deacetylase inhibitors (hdaci), belonging to different structural families, on trail-induced apoptosis in leukemic t cell lines. moreover, we have analyzed the activity of hdaci in normal t lymphocytes. we have found that, to a greater or lesser extent, all hdaci potentiate the induction of apoptosis in leukemic t cells by enhancing the signals triggered upon trail ligation, from the activation of the most apical caspase- . in contrast, hdaci do not sensitize primary resting or activated t lymphocytes to trail-mediated apoptosis. the analysis of the expression of several pro-and anti-apoptotic proteins involved in the trail-signalling pathway indicates that most hdaci regulate the expression of trail-r and c-flip in leukemic t cells, but not in normal cells, which may explain their selective pro-apoptotic effect on leukemic cells. zfp l is a zinc finger containing protein that is involved in post-transcriptional gene regulation. it can bind to mrnas containing adenine uridine rich (are) regions and subsequently mediate their degradation. we have previously reported a role for this protein in promotion of b cell apoptosis. one mechanism whereby zfp l may mediate cell apoptosis could be by degradation of cell survival gene mrnas. the bcl- protein is an important cell survival protein at different stages of b cell development. bcl- mrna also contains are regions that could possibly be targeted by zfp l protein. in the present study, we have tested the ability of zfp l protein to bind to a bcl- mrna are probe and to degrade bcl- mrna. recombinant bacterially expressed zfp l protein was shown to bind specifically to a bcl- are probe by rna electrophoretic shift assays (remsas). furthermore, remsas using cell lysates of ramos burkitt lymphoma b cells stimulated to express high levels of endogenous zfp l also provided evidence that endogenous zfp l in b cells could bind to the bcl- are. in order to examine whether zfp l binding to bcl- are resulted in bcl- mrna degradation, actinomycin d rna degradation assays were carried out on murine embryonic fibroblast (mefs) cells from zfp l knockout mice and wild-type mice using quantitative real-time pcr analysis. bcl- mrna was expressed in both wild-type and knockout mefs. the half-life of bcl- mrna was found to be extended in knockout mefs compared to wild-type mefs suggesting that zfp l does play a role in degradation of bcl- mrna. overall, our data are consistent with a role for zfp l in degradation of bcl- mrna which could be a mechanism for the reported role of this protein in induction of b cell apoptosis. the epstein-barr virus (ebv) is a common human herpes virus, which can predominantly infect two types of human cells: lymphoid cells and epithelial cells. its infection is associated with several human malignancies (hodgkin's lymphoma, burkitt's lymphoma, nasopharyngeal carcinoma), where it expresses limited subsets of latent proteins among which the latent membrane protein lmp . since lmp is able to transform numerous cell types, it is considered as the main oncogenic protein of ebv. the principal mechanism of lmp function is based on mimicry of activated member of the tnf receptor super family (tnfr), by its ability to bind a similar sets of adapters and to activate overlapping signalling pathways like nfkb, c-fos-jnk, pi -kinase...involved in the regulation of cellular processes. we previously generated two unique model, a monocytic (te ) and lymphocytic (nc ) immortalized by ebv and which expressing type ii latency program. here we developed original dominant negative (dn), by generating a fusion between gfp and tes or tes (transforming effectors site) derived from the c-terminal intracellular part of lmp , in inducible vectors. then, we generated cell lines conditionally expressing these dns. we showed these dns not only inhibit survival processes resulting to the impairment of nfkb and akt pathway but increase apoptosis in this cell lines. we demonstrated that this pro-apoptotic effect is due to i) the depletion of lmp 's specific adapters and ii) the recruitment of theses adapters by dns interestingly allowed generation of apoptotic complex involved tradd, fadd and caspase- . using this nc tumorigenic model in scid mice, we showed that induction of the dn lmp -tes prevent development of tumours and mouse death. these dominant negative derived from lmp could be used to develop therapeutic approaches in malignant diseases associated with epstein-barr virus, but also in inflammatory pathologies. recent studies indicate that suppressors of cytokine signaling (socs) proteins play, in addition to their action as cytokine signaling inhibitors in the immuneinflammatory response, multiple roles in cell survival, differentiation, and apoptosis in diverse cell systems. since tumor cells often exhibit aberrant expression of socs genes, which may be involved in determining resistance to anti-tumor therapies, we have investigated the role of socs isoforms during dna damageinduced apoptotic response and cell cycle changes in various tumor cell types. by using tumor cell lines transduced for over-expression or knock-down of distinct socs isoforms, it is found that socs and socs differentially affect apoptosis and cell cycle changes induced by dna damaging agents in a cell type-specific manner. in t lymphocytic leukemia cell line jurkat, socs exhibited anti-apoptotic effect in response to ionizing radiation, hydrogen peroxide, and etoposide by inducing suppression of p mapk activities, while socs promoted apoptosis with an increase in p activities. in contrast, both socs and socs display proapoptotic effect in rko colon cancer cell lines upon exposure to gamma radiation or ros-generating agents. notably, effects of socs proteins on cell cycle changes induced by dna damaging agents were rather similar in that over-expression of either socs or socs induced a slight decrease in g or s phase cells and a prominent increase in g /m cells, regardless of their distinct effects on apoptosis. the analysis of cell cycle regulator proteins, however, revealed that different mechanisms are operating to regulate cell cycle via distinct cyclins and cdk inhibitors affecting g /s transition and g /m arrest induced by socs or socs . socs promoted dna damage-induced p induction and g /m cyclin b expression, while socs induced decrease in g cyclin e expression. the results suggest that socs isoforms potentially modulate growth of tumor cells exposed to dna damage via complex network involving apoptotic response and cell cycle regulation in a cell type-specific manner. the heat shock protein (hsp ) is a highly conserved a widely expressed molecular chaperone. it is known to regulate the activity of several protein kinases or the proper folding of client proteins. hsp has also been identified as an important regulator of cellular survival. besides these intracellular functions, extracellular hsp can initiate cross presentation or immune responses. apoptotic cell death occurs permanently in multicellular organisms, without initiation of an immune response. however the mechanisms which prevent an inflammatory response to apoptotic cells are not understood to date. hsp is released during necrotic cell death and proinflammatory effects of extracellular hsp have been observed. thus, we asked whether apoptozing cells cleave hsp during apoptosis or how hsp is disposed by these cells. we induced apoptosis either in activated or resting primary human cells and analyzed the hsp protein content. we observed that hsp is degraded during apoptosis resulting in the formation of a fragment of about kda. this fragment was to be observed exclusively in activated cells, while it was not detected if resting cells were induced to undergo apoptosis. analyzing the isoforms of hsp (hsp alpha and hsp beta) we could show that the kda fragment is formed after degradation of the alpha isoform of hsp . further, we were able to show, that hsp cleavage is dependent on caspase activity and most probably mediated by calpain. analyzing the cytokine response of monocyte derived phagocytes to apoptotic cells in presence or absence of exogenous hsp and caspase inhibitors. we observed a rather proinflammatory cytokine profile, if cleavage of hsp was inhibited or if exogenous hsp was added. these results demonstrate that cleavage of hsp represents a mechanism preventing the release of proinflammatory molecules from apoptozing cells. activity. mifc integrates the advantages of flow cytometry and fluorescence microscopy in one system, the imagestream. upon induction of autophagy, cytosolic lc -i is processed to lc -ii, which then remains associated with the autophagosome until its degradation upon fusion with the lysosome. an increase in steadystate levels of autophagosomes can be due to enhanced autophagy or decreased lysosomal activity. mcf- gfp-lc cells were therefore incubated in starvation medium for hours, +/-bafilomycin, which potently inhibits lysosomal activity. classical gating strategies allowed the detection of cell populations of interest, which were further analyzed on single cell levels. we conclude that imagestream-based analysis provides an improved method in terms of objectivity, sensitivity and significance, to quantify autophagic activity. our results clearly show the need for discrimination between "steady-state" levels of autophagosomes and "current flux" of fully functional autophagy, i. e. quantification of autophagic flux. jnk seems to mediate the bcl- /beclin- control of autophagy. recently, jnk was shown to be necessary for beclin- upregulation, and jnk-mediated phosphorylation of bcl- is associated with both, starvation-and ceramide-induced autophagy. the nfkappab pathway mediates critical survival signals during starvation, which have been linked to the inhibition of autophagy. we report here the novel findings that under conditions of starvation, pharmacological inhibition of nfkappab decreased the autophagic flux in mcf- cells, while jnk inhibition shows an enhancing effect on autophagy induction. ingenol -angelate (pep ), a novel activator of protein kinase c (pkc), has been shown to induce apoptosis in acute myeloid leukemia cells. we show here, that in contrast to leukemic cells, pep provides a strong survival signal to resting and activated t cells. this anti-apoptotic effect was dependent upon the activation of pkcv, a pkc isoform restricted to t cells and myocytes. expression of pkcv in the acute myeloid leukemia cell line nb turned their response to pep from an increased to decreased rate of apoptosis. furthermore, our data show that pep inhibited t cell apoptosis through the activation of nfxb downstream of pkcv, leading to increased expression of the anti-apoptotic proteins mcl- and bcl-xl. we conclude that pkcv expression determines whether pkc activation leads to an anti-or pro-apoptotic outcome in the cell types analyzed. this finding may be of considerable importance for the development of pkc -targeting antileukemic therapies. the neuronal growth factors, neurotrophins, and their receptors are widely expressed in a variety of non-neuronal tissues including the immune system. several reports indicate that survival and activation of normal b lymphocytes are regulated by nerve growth factor (ngf) and brain-derived neurotrophic factor (bdnf) autocrine circuits. however, the production and the role of neurotrophins were not evaluated in b lymphoma cells. diffuse large b-cell lymphoma (dlbcl) is a common and often fatal malignancy. despite major advance in the treatment (r-chop protocol) which improves the clinical outcome of patients, a subset of patients does not respond or relapses after the initial treatment; the exact mechanism of such resistance is not entirely clear. we hypothesized that autocrine neurotrophin survival circuits could contribute to the chemoresistance of dlbcl tumor cells. this hypothesis was investigated with dlbcl cell lines (su-dhl). thus, we evaluated the ability of su-dhl cells to produce neurotrophins (ngf, bdnf) and to express their receptors (p , trka and trkb) in different cell culture conditions. our preliminary data show for the first time the production of neurotrophins by dlbcl tumoral cells whose level decreased in apoptotic conditions, in association with bad dephosphorylation suggesting its pro-apoptotic role. furthermore our results suggest that up-regulation of autocrine circuits (expression of trka known to be involved in survival signaling pathways) may contribute to cell survival and thus drug resistances of tumoral b cells. objectives: ataxia telangiectasia (a-t) is a rare disorder caused by mutations in the ataxia telangiectasia mutated (atm) gene. this gene encodes atm, a protein kinase which has a major role in dna double strand break response. a-t patients suffer from a variety of immune system defects including lymphopenia, immunoglobulin deficiencies and impaired class switch recombination, they also have an increased incidence of cancer especially leukaemia and lymphoma. the susceptibility to lymphoid tumours and immunodeficiency could be partly due to failure of extrinsic apoptotic processes involved in regulation of the immune system. although atm is known to have a central role in the induction of apoptosis in response to unrepaired dna double strand breaks its role in extrinsic apoptotic pathways is unclear. this study aimed to investigate if atm has a role in fas induced apoptosis. a bank of lymphoblastoid cell lines (lcls) derived from a-t and normal individuals and tumour samples with wildtype or mutant atm were used in the study. apoptosis was induced by incubating cells with the fas activating antibody ch and analysed by flow cytometry. expression of the caspase inhibitor cflip which inhibits fas induced apoptosis was detected by western blot. results: there was no significant difference in the susceptibility to fas induced apoptosis or cflip protein expression between atm mutant and control groups. however cells expressing high levels of cflip protein do show greater resistance to fas induced apoptosis than those with lower expression. whilst the lcls expressed both long and short forms of cflip, the tumour cells expressed only the long form. conclusion: atm mutations do not affect susceptibility to fas induced apoptosis or alter cflip protein expression in lcls or tumour cells. cflip protein levels and fas susceptibility vary greatly between individuals but this is independent of atm status. high expression of cflip protein correlates with reduced apoptosis in response to ch treatment but there is no clear difference in cflip expression between atm wildtype and mutant cells. labdane diterpenoids have a broad spectrum of biological activities including antibacterial, antiviral, and anti-inflammatory properties. however, little is known about their possible role in the apoptotic cell death machinery. we report that labdane diterpenoids induce apoptosis in different tumor cell lines by activating caspase in the extrinsic death receptor pathway, with subsequent participation of mitochondrial signaling. activation of caspase by diterpenoids was followed by a decrease in mitochondrial membrane potential, the release of apoptotic factors from mitochondria to the cytosol, and subsequent activation of caspases and . diterpenoids also led to time-dependent cleavage of bid. inhibition of caspase- abrogated these processes, suggesting that the death receptor pathway plays a critical role in the apoptotic events induced by labdane diterpenoids. in addition, pretreating cells with neutralizing antibodies to fas ligand, tumor necrosis factor receptor (tnf-r ), and tumor necrosis factor (tnf)-a receptor (trail) inhibited diterpenoid-induced apoptosis, revealing it to be dependent on these death receptors. diterpenoid treatment also induced a significant increase in the generation of reactive oxygen species (ros). however, increased ros production was not directly involved in diterpenoid-triggered apoptosis. these results demonstrate that labdane diterpenoids induce apoptosis through activation of the death receptor pathway. conclusion: cell proliferation and differentiation are tightly regulated networks and it is believed that in cell differentiation, even in cancer form, cells precluded from proliferation. whether these changes affect the level of differentiation or the change of survivin expression can affect the proliferation and differentiation pathways are the hypotheses that need further investigation. synthetic alkyl-lysophospholipids (alps) are a group of unnatural lipids with promising anticancer capability. a prototypic member is the ether lipid -o-octadecyl- -o-methyl-rac-glycero- -phosphocholine (et- -och ; edelfosine), which induces selective apoptosis in tumor cells through activation of fas/cd independent of its ligand fasl/cd l. fas/cd is activated by edelfosine via its translocation in lipid rafts. in this study we showed that edelfosine promotes cell death in multiple myeloma and various solid tumor cell lines in a death receptor-independent manner. edelfosine-treated cells could not be protected against cell death after inhibition of caspases by zvad-fmk while fasl-stimulated cells stayed mostly alive. furthermore cells could not be rescued by addition of zvad-fmk in combination with necrostatin- , an inhibitor of death receptor-induced necrosis. fas resistant solid tumor cells overexpressing members of the anti-apoptotic bcl -family as well as cells overexpressing the cellular regulatory protein flip went in contrast to fas stimulation to apoptosis after treatment with edelfosine. therefore we suggest that edelfosine induces a death receptor-independent cell death pathway in a wide range of tumor cells. apoptosis represents a cellular "suicide" mechanism which allows the control of cell number from tissues and elimination of cells that present dna mutations or having an abberant cell cycle, those cells being predisposed to malignant transformation. thus, elucidating the mechanisms of programmed cell death process seems to be of great importance for malignant transformation, tumour evasion and therefore for anti-cancer therapy. many anti-cancer drugs act during physiological pathways of apoptosis, leading to tumour cell destruction. the present study focused on the potential influence of oncolitical treatment ( -fluorouracyl) associated with natural compounds (curcumin, genistein, quercitin) on the dynamics of the cell cycle and levels of apoptosis in colon cancer cell lines (e. g. colo , sw , lovo, caco- , ht- ) . in addition, expression of antigens involved in tumour proliferation and apoptosis (ki- , pcna, p , bcl- ) was compared with gene expression in the presence or absence of stimuli treatment. percentages of apoptotic cells were detected by using annexin v/fitc and propidium iodide double staining, while progression through cell cycle phases was evaluated by using pi staining. correlation analyses between the individual profile of the stimuli modulated gene expression with the coded protein expression were performed by using data from rt-pcr with specific primers, and indirect immunofluorescence followed by flow cytometry, respectively. stimuli treatment of colon cancer cell lines differentially induced higher levels of apoptosis as compared to untreated tumour cells, while cell cycle distribution of dna changed. data obtained showed a various expression and functional behaviour of the markers under study associated to colon cancer cells, suggesting their possible involvement in regulating the interactions between tumour cells and host immune system. the results obtained might further lead to the establishment of an experimental pattern for the corroboration of cell and molecular mechanisms involved in the tumour progression and the treatment resistance of colon tumors using cell lines. the effect of modulatoy agents on proliferation and apoptosis could be used in clinical departments in order to elaborate new therapeutical approaches and act as useful instruments in elaboration of individualized treatment schemes. extensive tissue trauma and malnutrition results in disorders of programmed cell death influencing the patients susceptibility to infections. the purpose of our study was to assess the effect of pancreatic cancer surgery and immunonutrition on the apoptotic signaling pathways. the randomized studies were performed in patients after pancreatic cancer resection with preoperative standard or enteral immunonutrition. lymphocytes expressions of bcl- , bax, caspase , , , nfkb, parp / kda, tnfr /cd a and cd /fas were assessed by western-blot and flow cytometry. results: before and after surgery the expression of bcl- , bax, nfkb, parp was significantly lower and expression of caspases, tnfr as well as percentage of cd cells significantly higher as compared with control group. caspase expression was significantly higher as compared with nfkb, parp and tnfr . in comparison to the standard nutrition preoperative immunonutrition increased bcl- and nfkb expressions and decreased caspases and parp expressions. in addition, we found a significant down-regulation of bcl- expression after surgery, but insignificant in patients with preoperative immunonutrition. conclusion: preoperative enteral immunonutrition has an modulative effect on apoptotic signaling pathways after pancreas resection and possesses antiapoptotic properties. this modulatory effect of glutamine and omega- fatty acids has no influence on patients outcome. the capacity of medicinal herbs to modulate cellular and humoral immune response could have useful applications in some immune-mediated disorders, infections and cancers. in this study the immunomodulatory effects of salvia mirzayanii a native plant that is widely distributed to iran was investigated. s. mirzayanii is used for the treatment of infectious and inflammatory diseases and as a tonic in folk medicine. study of the effect of this plant on the activated human peripheral blood lymphocytes showed stimulatory effects at lower concentrations and inhibitory effects at higher ones (p x . ). in flow cytometry analysis, accumulation of apoptotic cells in the sub-g phase of cell cycle of the mitogen-treated lymphocytes exposed to the inhibitory doses of the extract was observed. dna fragmentation analysis of these cells showed a typical dna laddering. immunization of the extract-treated mice with the antigen decreased delayed hypersensitivity skin reaction as well as the antibody titer at higher concentrations (p x . ). these results indicated the presence of immunomodulatory compounds in the extract of s. mirzayanii and suggest that the induction of apoptosis in lymphocytes might be the mechanism responsible for the inhibitory effect of the extract observed at higher concentrations. a new randomized, double-blind, placebo-controlled clinical trial was conducted with healthy volunteers receiving either la ( cfu/day) or placebo, during days prior to uv ( × . med). blister roofs, liquid and skin biopsies were collected , and days after uv exposure from non-irradiated and irradiated skin areas and used for identification of cells involved in uv-induced immune response, quantification of inflammatory cytokines, a dna damage marker (p ). while a similar decrease of lc for both groups was observed on day after uv exposure compared to placebo, la- group presented a faster increase of a new subset of epidermal dendritic cells (dc), namely early lc precursors (cd a low cd -) associated with a minor recruitment of monocytes. concomitantly, inhibition of il- and a tendency to inhibit il- was observed in la- group compared to placebo. on day , la- group presented a greater recruitment of early lc precursors and a trend to increase cd a low cd + lc precursors compared to placebo. additionally, a faster reduction of inflammatory and immunosuppressive cytokines (il- , tnfa, il- , and il- ) was observed in la group compared to placebo. we show that la limits uv-induced immune-suppression and skin inflammation. this contributes to the recovery of the skin immune homeostasis, confirming the previously observed benefits of la supplementation for photoprotection at a lower dose ( log). the thymus is one of the primary lymphoid organs and plays a central role in the immune system. it provides the essential microenvironment for proper t cell development. in the thymus, the maturation of t cells depends on many interactions between t cells and different stromal cell types, mainly composed of epithelial cells (tecs). foxn is a winged-helix/forkhead transcription factor, which is crucially required for proper epithelial cell differentiation in the thymus. foxn appears to be expressed in all epithelial cells of the early thymic rudiment starting around e . . previously, we have used a lineage-tracing system to confirm the existence of a bi-potent epithelial progenitor cell. using the cre-loxp system, we showed that a single epithelial cell, when reverted to express foxn in a nude (foxn -deficient) background, can give rise to a functionally competent thymic microenvironment. hence, we hypothesize that the epithelial progenitor cell expresses foxn . if true, it should be possible to target this cell type by use of foxn -promoter driven transgenes. conditional targeted cell ablation is a powerful method to elucidate the physiological function of cell populations and their regenerative capabilities. currently, we are using three different strategies of conditional targeted cell ablation in order to examine functional characteristics of epithelial bi-potent progenitor cells within the thymus. intracellular accumulation of poly glutamines is known to cause neurodegenerative disorders, such as huntington's disease. considering this, we are expressing transgenic egfp variants containing either or glutamine residues under the control of foxn promoter, leading to different degrees of tec degeneration. furthermore, also under the foxn promoter, we are using the transgenic expression of human diphtheria toxin receptor and the transgenic expression of the bacterial nitroreductase enzyme that converts the pro-drug metronidatole (mtz) into a cytotoxic cross-linking agent for conditional cell ablation. preliminary results describing the phenotypes of these mice will be presented. t. kamei , , y. toriumi , k. kimura european university viadrina, frankfurt (oder), germany, shimane institute of health science, izumo, japan, shimane university faculty of medicine, department of pediatrics, izumo, japan, japan yoga niketan, yonago, japan as a method in relieving stress, yoga is popular today. many reports of physical changes describing how yoga improves respiratory, circulatory, endocrine, and metabolic functions by yogic practice have been reported until now. we examined changes of electroencephalograph (eeg) and cellular immunity before, during, and after yoga exercises, in an endeavor to detect the correlation between them. the subjects consisted of eight yoga instructors who had been practicing yoga for several years. a -minute-rest period, followed by a -minute yoga exercise called asana, a -minute respiratory exercise called pranayama (various specialized respiration methods continuously performed with the eyes closed), and a -minute meditation were performed. throughout rest and yoga, brain rhythms were continuously recorded via two disc electrodes placed on each forehead (fp ). blood samples were drawn before and after each exercise. nk activity and percentages of t-cell and b-cell subsets were measured. during the pranayama period, both a positive correlation between the change in abundance of the activated alpha waves and the ratio of changes in nk activity (r= . , p x . ), and a positive correlation between the change in abundance of the activated alpha waves and the ratio of changes in the number of t lymphocytes (r= . , p x . ) were observed. furthermore, a positive correlation was also observed between the change in amplitude of the activated alpha waves and the ratio of change in the number of cd (r= . , p= . ). these findings suggest that yoga creates a stress-free and mentally concentrative state which activates the functions of nk cells and t lymphocytes, mainly of cd , within a short period of time. we conclude from these results that yogic respiratory exercise may be able to activate cellular immunity and to help recover the mental and physical harmony of human. yoga is considered to have an effect of some re-activation of a latent ability of harmonization in which humans naturally possess. t regulatory cells play a central role in the suppression of immune responses thus serving to induce tolerance and to control persistent immune responses that can lead to autoimmunity. several recent studies suggest also that diverse populations of regulatory t cell play an important role in regulating t-helper response to allergens, maintaining functional tolerance and preventing allergy. here we demonstrate that cd + cd + t regulatory (t reg ) cells are critical in controlling the immediate hypersensitivity response of bone marrow mast cells (mcs) without affecting cytokine release. this effect is shown to require a cell-cell contact and depends on interaction between ox l expressed on mcs and the constitutive expression of ox (members of the tumor necrosis factor [tnf] and tnf receptor family, respectively) on t reg cells. this interaction does not alter the activation of plc-g, syk and lat in ige/ag stimulated mcs upon co-incubation with t reg cells, whereas it induces a decrease in the phosphorylation levels of akt. moreover, we find that upon co-incubation with t reg cells, mcs show increased levels of camp, which is known to inhibit mcs function, as a result of ox l signal. antagonism of camp in mcs reverses the inhibitory effects of t reg cells restoring normal ca + responses and degranulation. the cross-talk between t reg cells and mcs through ox -ox l interaction defines a previously unrecognized mechanism controlling mcs degranulation. loss of this interaction may contribute to the severity of allergic responses or inflammatory disease. active regulation has emerged as a very essential mechanism for both inducing and maintaining peripheral tolerance to non-pathogenic environmental antigens. a healthy immune system responds to antigens with a combination of polarized th or th effector cells and the induction of antigen specific foxp + regulatory t cells (treg). it is believed that the dominant subset determines the quality of the eventual immune response. in allergic asthma there is a clear dominance of th cell responses to non-pathogenic environmental antigens. recently it was shown that the specific transcription factors that characterize the th and treg subset, gata and foxp respectively, counter regulate each other (mantel y et al., ) . we hypothesize that children with allergic asthma will respond to allergens with low induction of foxp + tregs and high gata + th cells. in order to prove this hypothesis pbmc of children with allergic asthma and non-sensitized healthy controls are stimulated with allergens, tetanus toxoid, and lps. almost million allergic patients are sensitized to the major birch pollen allergen bet v , which cross-reacts with major allergens of fagales (e.g., alder, hazel, hornbeam, oak) pollen and plant food allergens. the epitopes of bet v recognized by allergic patients' ige antibodies belong to the conformational type and therefore have not been characterized in detail. here we used antibodies raised against peptides spanning the bet v molecule in ige competition experiments to search for sequences which are involved in ige recognition. the strongest inhibition (i.e., g %) of patients' ige binding to bet v was obtained with polyclonal and monoclonal antibodies specific for peptides comprising aa - (p ) and aa - (p ) of bet v . cross-reactive ige epitopes between bet v and related pollen allergens and plant food allergens involved primarily p . p and p are not adjacent peptides in the bet v sequence but define a surface-exposed patch on the three-dimensional structure of bet v . as determined by surface plasmon resonance, monoclonal antibody mab specific for p and mab specific for p showed high affinity, i. e., dissociation constants, k(d) = . e- m and k(d) = . e- m, respectively. interestingly, peptide-specific mabs inhibited allergic patients' ige antibodies equally well as peptide-specific polyclonal rabbit antibodies but only the latter inhibited strongly allergen-induced basophil degranulation. this finding indicates that the surface patch defined with anti-p and anti-p antibodies contains several ige epitopes. in summary, we have defined a surface-exposed patch on the bet v allergen which seems to harbor the majority of the ige epitopes and may be used for the rational design of active and passive immunotherapy strategies against birch pollen and related allergies. background: antigen-specific th cells as well as tc cells, induced by biolistic gene transfer using plasmid dna encoding the model allergen b-galactosidase (bgal) under control of the fascin promoter (pfascin-bgal), inhibited the elicitation of systemic th immune responses and suppressed ige production in an experimental mouse model. moreover, protective biolistic dna vaccination with pfascin-bgal prevented th -mediated lung pathology (eosinophilia) in sensitized mice locally challenged with bgal protein, but led to the recruitment of th /tc cells into the lung, associated with substantial neutrophilic infiltration and the induction of airway hyperresponsiveness (ahr). objective: to analyze the modalities of ahr induction in mice biolistically vaccinated with pfascin-bgal. methods: balb/c mice were immunized with pfascin-bgal using the gene gun. subsequently, mice were challenged by consecutive intranasal application of bgal protein. cd + and cd + t cells, respectively, were depleted before and during the provocation phase by intraperitoneal injection of anti-cd (gk . ) or anti-cd ( . . ) monoclonal antibodies. neutrophilic granulocytes were depleted by treatment of animals with either anti-gr- monoclonal antibody rb - c or monoclonal antibody nimp-r . one day after the last challenge airway reactivity was assessed by whole body plethysmography, bronchoalveolar lavage (bal) was performed and the frequency of ifn-g-producing cd + effector t cells in the lung was determined. results: whereas neutrophilia in the lung of immunized and challenged mice was considerably alleviated by depletion of cd + t cells, ahr was not significantly affected, implicating that the elicitation of ahr by cd + t cells is dissociated from the activity of neutrophils. this notion was verified by elimination of neutrophils during the provocation phase, likewise leading to unaltered ahr. in contrast to cd + t cells, cd + t cells induced strong neutrophilic infiltration and ahr. transfer experiments with cd + or cd + t cell, separated from the airways of vaccinated and challenged mice, will probably reveal details of the effector mechanisms of th and tc cells operative in the elicitation of airway inflammation. conclusions: robust type immune responses, although highly effective in the counter-regulation of local th -mediated pathology, might as well trigger inflammatory reactions in the lung and provoke the induction of ahr. respiratory epithelial cells function as physical barrier and have shown to be active participants within the process of defense against pathogens and recognition of allergens. upon activation they release inflammatory mediators thereby creating a micro environment in which recruited immunocompetent cells induce a local immune response. house dust mite (hdm) extract as a source of allergens has been shown to induce a broad panel of genes upon stimulation of epithelial cell line nci-h . the proteolytic activity of these hdm allergens has been proposed to be involved in the activation process. the aim of this study was to compare the influence of hdm extract on respiratory epithelial cells with grass pollen allergen-induced activation of these cells with regard to the mechanism of activation, gene expression level, and the level of induced cytokine and chemokine release. in contrast to the hdm major allergen der p , we were able to show that the major allergen of phleum pratense, phl p , although sharing molecular similarities with der p , does not display any enzymatic activity under physiological conditions. therefore, in this study respiratory epithelial cells were stimulated with grass pollen extract and purified phl p . chemokine and cytokine release was determined by multiplex enzyme-linked immunosorbent assay and mrna was used for cdna-microarray analysis. first data show that both, hdm extract and grass pollen allergens, induce the release of il- and il- from nci-h cells. furthermore, stimulation with hdm extract leads to the release of tnf-a, gm-csf and ifn-g. interestingly none of these mediators was induced after stimulation with grass pollen extract or purified phl p . in contrast to hdm extract grass pollen allergens induce the release of mcp- from respiratory epithelial cells, as well as moderate levels of il- . detailed characterization of the response on gene expression level might give new insights into the pathophysiology of grass pollen allergy and a comparison with hdm induced expression profiles will be helpful towards understanding the allergic response in general. (supported by dfg sfb tr ) results: collectively, responses to blg, but not to bsa, were observed in all groups analyzed, included healthy controls. nevertheless, distinct profiles of response were obtained: children with ige mediated cma had a significant increased level of proliferation (mean±sd of stimulation index(si): . ± . ) and of il- (mean±sd: ± pg/ml), and reduced il- (mean±sd of il- -spot forming units/ x cells (sfu): ± ), compared to healthy subjects ( . ± . si, p x . ; ± pg/ml, p x . ; ± sfu, for proliferation, il- and il- , respectively); children with non-ige mediated cma had a significant reduction of il- ( ± pg/ml), compared to ige patients (p x . ) and an increased, although not statistically significant, production of ifn-g ( . ± . sfu) compared to control ( . ± . sfu) and to ige-allergic patients ( . ± . sfu). finally, tolerant patients showed reduced il- ( ± pg/ ml, p x . ) and proliferation ( . ± . si), compared to acute ige-cma children. interestingly, the high level of il- observed in all groups might have a counter-regulatory effect, since its neutralization resulted in an increase of proliferation to blg; by contrast, il- was undetectable in all patients even blocking the il -receptor. conclusion: blg-specific, immune responses can be recalled in peripheral blood of cma patients, as well as of normal and tolerant children. a th -like response with il- and proliferation is dominant in ige-mediated cma patients; by contrast a th -skewed response with ifn-g is present in non-ige-mediated allergic and in those children who outgrew ige-allergy. y. f. tang , b. chua , f.c. lew , a. ho , k. wong , k.l. wong , d. m. kemeny national university of singapore, immunology programme, yong loo lin school of medicine, singapore, singapore allergic inflammation of the airways causes changes in the lung wall that can lead to chronic inflammatory disease such as asthma. using a mouse model, this response can be divided into an induction phase, in which cd th t cells specific for airborne allergens are produced, and an effector phase, during which they are recruited to the lung. in the lung, recruited th cells orchestrate the inflammatory response marked by eosinophilia, mucus hyper secretion and increased airway hyperresponsiveness (ahr). previously we, and others, have shown that transfer of cd t cells inhibits the induction of the th response. here we have investigated the effect of cd t cells on the effector phase of the inflammatory lung response. in vitro activated ot-i cd t cells were transferred to ovalbumin (ova)/ alum immunized mice one day before the first of airway challenges with ova. eosinophil infiltration was inhibited by transfer of cd + t cells ( . %± . % to . %± . %). when ifn-gamma -/-ot-i cd t cells were transferred, we found that the inhibitory effect on eosinophilia was reduced ( . %± . %), suggesting an important role for cd t cell ifn-gamma. cd c + cd + cd blung dcs from cd transferred mice secreted higher levels ( pg/ml) of il- p following ex-vivo stimulation as compared with animals that were not given cd t cells. these data show that, in addition to regulating the induction of the allergic immune response, cd t cells can subsequently divert the local lung environment to one that favors th immunity. the chain terminator drug abacavir triggers a serious hypersensitivity reaction in % of patients with hiv infection. this reaction is strongly associated with hla-b* and appears to be mediated by cd + t cells producing inflammatory cytokines. we show that cd +t cell responses can be primed in vitro, in normal blood donors who are hla-b* +, but not in non-b* + donors. cd t cells, but not cd t cells, are expanded by abacavir pulsed autologous apc over a -day culture, producing ifn-gamma and tnf-alpha upon re-stimulation with apc expressing hla-b* . similar responses were detected in abacavirhypersensitive hlab* + patients. responses were not detected using mutant apc deficient in tap or tapasin, or when normal apc were aldehyde fixed before loading with abacavir, indicating a reliance on the conventional mhc-i ag presentation. responses were exquisitely restricted to hla-b* since they were undetectable using apc expressing closely related hla-b or b allotypes. responses to apc expressing mutants of hla-b* demonstrated a crucial role for residue . isolation of peptide fractions from abacavir-loaded cells has led to the identification of specific fractions recognised by an abacavir-specific t cell line. our data suggests that abacavir forms a conjugate with an endogenous peptide that is presented by hla-b* triggering cd t cells. we speculate that this form of altered self is highly immunogenic, behaving like a form of allogeneic mhc-i, contributing to the responses observed in abacavir naï ve individuals. the molecular mechanisms underlying altered hla-b* may be relevant to the role of other disease-associated class i allotypes such as hla-b and b . a. jenckel , s. bulfone-paus , n. föger research center borstel, immunobiology, borstel, germany mast cells play a crucial role in acute inflammatory and allergic reactions. upon activation, mast cells secrete a vast array of preformed and newly synthesized inflammatory mediators. recent work has begun to appreciate an important role of the actin cytoskeleton in mast cell activation. the actin-associated protein coro-nin a (coro a), a coronin family protein preferentially expressed in hematopoietic cells, is critically involved in various actin-mediated cellular functions of leukocytes. recent data of our group also indicate a regulatory role of coro a in mast cell function. coronin proteins have been described to be differentially phosphorylated in vivo. however the molecular mechanisms by which coro a is regulated in response to physiological stimuli are still poorly characterized. here we investigated the modalities of coro a phosphorylation during the activation of mast cells. immunoprecipitation studies combined with phospho-specific western blotting techniques revealed a transient phosphorylation of coro a on serine residues upon antigen-specific engagement of fc-epsilon-receptors. as the phosphorylation status of coro a can influence its association with the actin cytoskeleton, we analyzed the subcellular localization of coro a during mast cell activation. cell fractionation experiments demonstrated that the association of coro a with the actin cytoskeleton significantly decreases in response to mast cell stimulation, concomitant with the increase in coro a phosphorylation. a functional correlation between coro a phosphorylation and its association with the actin cytoskeleton in mast cells was further indicated by structure function experiments employing specific phosphorylation mutants of coro a. thus, coro a is a downstream effector molecule of fc-epsilon-receptor signaling and likely is involved in the dynamic reorganization of the actin cytoskeleton during mast cell activation. allergen-specific t and b lymphocytes play an important role for the pathogenesis of asthma. t cells orchestrate the infiltration of the lung tissue with eosinophils and neutrophils and provide help for allergen-specific immunoglobulin production. recently, we have shown in a mouse model for allergic airway inflammation that b cells directly interact with t cells in the inflamed tissue and locally produce ige. to analyse t/b-interaction in the inflamed tissue in more detail, we developed a novel adoptive transfer system using ovalbumin-specific t cells and nitrophenolspecific b cells. recipient mice are then challenged intranasally with an np-ova conjugate. this system allows to track single allergen-specific t and b cells in all stages of the immune reaction using flow cytometry and immunohistology. in addition, cells can be re-isolated by flow-sorting for in-depth analysis. using this system we could define several phases of the inflammatory reaction. t and b cells first become activated in the lung-associated lymph nodes. granulocytes can be found very early in the lung tissue and also activated t cells very rapidly emigrate to the site of inflammation. however, clusters of allergen-specific t and b cells can only be found in later stages of the reaction. as another focus, we used our in vivo system to define the role of t cell costimulatory molecules for airway inflammation. costimulatory receptors are key regulators of t cell activation and differentiation and therefore promising targets for therapeutic intervention. of special interest is the t cell-specific icos molecule which is important for t/b cooperation as well as the regulation of chronic inflammatory reactions. using icos knock-out mice we were able to delineate the specific role of icos for the different stages of airway inflammation. in particular, we analysed the impact on t cell subset differentiation, cytokine production and allergen-specific immunoglobulin production. the integrity of the actin cytoskeletal network is critcal for a large variety of cellular functions. coronins constitute a family of evolutionary highly conserved wdrepeat containing proteins that have been implicated in the regulation of actin cytoskeletal dynamics. in mammalians seven coronin family members have been described. the high degree of sequence conservation amongst coronin family proteins suggests common features and functions. however, individual family members may also have developed additional selective and specific functions. our recent studies on coronin a (coro a) deficient mice have demonstrated that coro a exhibits an inhibitory function on the cellular steady-state f-actin content, is required for chemokine-mediated functions in t cells and is involved in the maintenance of t cell homeostasis. coronin b (coro b) is a closely related homolog of coro a and the two genes are co-expressed in hematopoietic cells. to address the question of functional redundancy in vivo, we have generated coro b deficient mice and crossed them with coro a deficient mice to obtain coro a/coro b double deficient mice. analysis of t lymphocytes from coro a/coro b double deficient mice revealed defective chemotactic responses and a severe peripheral t cell lymphopenia in double-deficient mice, which was significantly exacerbated as compared to the respective single knock-outs. an analysis of coronin deficient mast cells also revealed an involvement of coro a/coro b in the regulation of actin cytoskeletal dynamics and the function of mast cells. however, in contrast to the inhibitory effects of coro a/ b deficiency on t cell function, mast cell degranulation and migration was enhanced in coro a/ b double deficient mast cells. thus, depending on cell type specific requirements, coronin proteins can either exhibit positive or negative regulatory functions. additional studies will investigate molecular and regulatory mechanisms by which coronin proteins control actin cytoskeletal organization and function of immune cells. together, our studies here reinforce and expand our appreciation of the importance of actin-cytoskeleton regulatory proteins for immune cell function. initially found by serial analysis of gene expression, murine samsn (also known as hacs or sly ) is a putative adaptor and scaffold protein with a sterile-alphamotif (sam), a src homology (sh ) domain and a predicted bipartite nuclear localization signal. the samsn gene is located on mouse chromosome and encodes a well conserved protein with amino acids, which is predominantly expressed in hematopoietic tissues. initial overexpression studies suggest a contribution of samsn in b cell activation and differentiation, however its physiological function is yet unknown. to investigate samsn expression in lymphatic and myeloid cell types in greater detail we employed the sensitive method of quantitative real-time pcr. our data revealed an expression of samsn in all tested hematopoietic cell types. the highest expression level of samsn mrna was seen in mast cells compared to lower levels in macrophages, dendritic cells, cd + and cd + t cells and b cells. the other two members of the sly family of adaptor proteins -namely sly (hacs ) and sly (sash ) -were expressed only at a very low level in mast cells. the high level of samsn mrna expression in mast cells, together with minimal expression of other sly family proteins in these cells, implicates an important role of this adaptor protein for mast cells. to address the potential role of samsn in mast cell differentiation and function we are analyzing bone marrow derived mast cells from samsn deficient mice. initial in vitro experiments indicate normal proliferation and differentiation of samsn deficient mast cells. in additional studies we are now investigating the effects of samsn deficiency on mast cell activation processes, such as degranulation, cytokine production and the signal transduction cascade. analyzing the role of samsn in mast cells will help to define the biological function of this novel class of adaptor proteins. introduction: we showed previously that the ability of murine igg antibodies to mediate anaphylactic reaction is directly dependent on the amount of sialic acid residues attached to the carbohydrate chain n-linked to the antibody fc region (silva et al; j.immunol., ). then, we hypothesize that differences in the glycan composition mainly the sialylation grade observed between the anaphylactic and non-anaphylactic igg abs may be resultant of the differential expression of the glycosyltransferase, essentially sialyltransferase, coding genes during its synthesis in b cells. objective and methods: to prove this hypothesis it was analyzed the expression of st siai-v; st galnac i-iv, st gal ii -v genes quantitatively by real time-pcr in the hybridomas producer of these two types of igg abs. results: we observed that the expression of st gal i, iii and v coding genes was similar in both hybridomas, while the st gal ii and iv genes were less expressed in the hybridoma producer of non-anaphylactic igg . in addiction, the expression levels of st sia and st galnac genes in the hybridoma producer of anaphylactic igg were significantly higher when compared to those observed in the hybridoma producing of non-anaphylactic igg . conclusion: these data suggest a direct correlation between the sialylation grade observed in the carbohydrate chain attached to the igg abs and the expression of sialyltransferase enzymes in the hybridomas producer of these molecules. financial support: cnpq, capes, fapesp. basophils are innate immune cells endowed with important effector functions during allergic inflammation and parasite infection. their activation in terms of histamine and cytokine production is mediated through immunoglobulin-dependent and -independent mechanisms, raising the question whether stimulation of tolllike receptors (tlrs), which have been described in basophils, has a similar effect. we found that, in contrast to other tlr agonists tested, only the doublestranded rna poly(a:u) induced the typical t h cytokine and histamine production in vitro. this compound was also fully active when administered in vivo since it activated basophils and promoted their recruitment to the periphery. we took advantage of a murine model of allergic asthma to establish the pathophysiological relevance of this finding. using both adoptive transfer and depletion of basophils, we established not only that these cells contribute directly to the severity of asthma symptoms, but also that a mimic of viral infection can aggravate the disease through their activation. this is the first evidence for a mechanism of exacerbation of allergic asthma induced by a mimic of viral infections, mediated through basophils. ishes the airway hyperresponsiveness and airway inflammation in experiment murine asthma models. to investigate the effect of activation of nkt cells at different allergic asthma progression, we administered balb/c mice with a-galactosylceramide (a-galcer), a stimulator for nkt cell activation, before or after ova immunization and measured the airway inflammation of that mice after times of intranasal ova challenge. in our results, the total numbers of bronchoalveolar lavage (bal) cells were higher in mice administered with a-galcer before ova immunization compared to that of mice administered with a-galcer after ova immunization. moreover, significant increased percentage and cell numbers of eosinophils in bal of mice administered with a-galcer before ova immunization was noted. il- and eotaxin are the most potent cytokine/chemokines for the recruitment of eosinophils. il- and eotaxin levels in the bal fluid were higher in mice administered with a-galcer before ova immunization compared to that of mice administered with a-galcer after ova immunization. these data demonstrate that activation of nkt cells at different allergic asthma progression dictates the different outcome of asthma. in addition, the activation of nkt cells in naïve mice induces airway inflammatory responses. the potential risks of treatment with nkt cell activation on human diseases should be considered. objective: bronchial asthma is a complex disease of the lung and is characterized by a variety of symptoms such as airway hyperresponsiveness, reversible airway obstruction, high serum levels of ige and inflammation. histologically, there are infiltrations of eosinophils, degranulated mast cells and hyperplasia of airway globlet cells in addition to lymphocytes. the transcription factor irf mediates the differentiation into th cells by activating multiple genes which are independently crucial for the development of naive t cells into th cells. because irf is expressed in many different tissues, it can be considered as a master switch factor for th cell differentiation. methods: here, we tested mice deficient in irf in the murine acute asthma model to evaluate its importance in this th cell-mediated disease. the protocol setup was the following: sensitizations s. c. with ova, followed by challenges via ova inhalation and adoptively transferred wildtype cd + t cells prior to initial sensitization. in our experiments, we could demonstrate that only after priming of irf deficient mice with the help of adoptively transferred cd + t cells, asthma symptoms in these mice were more severe than in wildtype controls. as an example, eosinophil infiltration into the lung was increased by . fold. likewise, ovaspecific antibodies and numbers of goblet cells (fig. ) were also significantly higher in irf deficient mice. conclusion: interferon regulatory factor plays a role in the severity of the development of asthma. in its absence, proinflammatory parameters in the lung are increased significantly. this effect is only visible in the presence of wildtype cd + t cells. mechanistically, a potential counterregulation of asthma by th cells is not available in irf knockout mice. together with our previous report that irf represents a susceptibility gene for allergy in the human, our data highlight irf as key in regulating the severity of asthma. the sensory neuropeptide substance p (sp) acts as an important stress mediator with its own stress axis in the skin modulating mast cell as well as antigenpresenting cell (apc) activity. here we postulate that stress-dependent communication between nerve fibers and immune-competent cells can also occur in spleen and affects the course of inflammatory disease. to address this question, atopic dermatitis-like allergic dermatitis (ad) was induced in c bl/ mice by intraperitoneal sensitization and intradermal challenge using chicken egg ovalbumin. animals were additionally exposed to noise stress for hrs prior to challenge. in this model, stress lead to a relative hyperinnervation of the immune-competent areas of the spleen. at the same time, an increased number of apc could be observed in these areas and contacts between nerve fibers and apc were found. under the same conditions, we were able to show increased nk -receptor and ppt mrna levels. accordingly, sp had the capacity to raise the number of antigen presenting cells in spleen and altered the profile of cd c expressing apc substets characterized by cd and cd expression in vitro. in vivo we found a stress dependent shift of cytokine mrna levels towards a th- cytokine profile and increased levels of il- mrna. further the number of cd + t-regulatory cells was increased in vitro. additional analysis of the quality and function of neuro-immune interactions in the spleen will reveal the role of the observed stress-induced alterations in allergic inflammation. proton pump inhibitors (ppis) that are the cornerstone of gastroesophageal reflux disease therapy have been reported to improve asthma and eosinophilic esophagitis (ee) in patients with associated symptoms. the most accepted explanation for these findings is based on the belief that pathologic acidic reflux can act as a triggering factor for these diseases through proximal extent and laryngopharyngeal reflux in asthma and impairment of the epithelial barrier in ee. under these considerations, acid suppression is believed that could prevent these pathogenic mechanisms. nonetheless, a number of evidences suggested the possibility that ppis could have a direct effect in molecular pathways involved in asthma and ee: ) the inhibitory mechanism of ppis implies alkylation of cysteine residues in gastric atpase , ) asthma and ee are prototypic th diseases in which the cytokines il- and il- play a principal role through the activation of the transcription factor stat , and ) we have recently demonstrated that some chloromethyl ketones can downregulate stat by mechanisms involving cysteine alkylation. on the theoretical basis that cysteine reactivity of ppis may affect the regulation of stat , we analyzed its effect in the activation of stat by il- and il- . we found that treatment of cells with ppis inhibited the ability of il- and il- to signal stat activation in a dose-dependent manner in multiple cell types from different origin. given the important role of these mechanisms in asthma and related diseases, our findings show a novel mechanism to understand the effect of omeprazole in these diseases. in argentina more than three million people suffer from asthma, and the number is rising. asthma is defined as a disorder characterized by chronic airways inflammation that results in high mucus production and airways hyperresponsiveness. a th mediated immune response prevails in these patients. in the asthmatic exacerbation period, crisis (cr), triggered by viral infections or other factors, there is a high prevalence of bacterial overinfection. our objective is to compare immunological parameters and in vitro response of lymphocytes to bacterial antigens in the same patient, at that moment and at a time of stability between episodes (i). we studied asthmatic patients both at cr and i. we evaluated eosinophils, basophils and ige expressing b lymphocytes; as well as t regulatory cells (by expression of cd and cd high (treg)), that might inhibit the development of a th response, together with gdt cells, which function in asthma is not completely understood, but could have a role in the increased airway responsiveness. to evaluate the t cell response, mononuclear cells were cultured for hours in the presence of m. tuberculosis (m) or s. pneumoniae (spn), or absence of them (c). then the percentage of activated cells was determined (expression of cd or cd at hours). all the parameters were evaluated in peripheral blood by flow cytometry. discussion: even though the pathophysiological characteristics of asthmatic patients in periods of cr and i are different, no significant differences were observed in the parameters (cell populations and cell response to bacterial antigens) evaluated when compared for the same patient at cr and i. we might be able to detect differences if we studied cells from the lungs, the target organ. we demonstrate that murine and human lc expressed the h r. the level of intracellular ccl production in human lc was reduced after stimulation with h r agonists and basal production could be restored when h r was blocked with the specific antagonist jnj . moreover histamine and a h r specific agonist augmented the migration of lc from the epidermis as shown in ex-vivo migration experiments using human skin and in-vivo migration experiments in mice. in conclusion, the h r is expressed on murine and human lc and influences the immunomodulatory function and migration of these cells. these findings underline the relevance of the h r in allergic skin diseases and encourage further exploration of the h r as a therapeutic target in allergic skin diseases. expression data of the non-coding rna gene, prins (psoriasis susceptibility-related rna gene induced by stress) identified and characterized by our workgroup, suggests a role for prins in psoriasis susceptibility and in cellular stress response. in order to asses the function of prins, we aimed to identify genes regulated by prins and intracellular molecules interacting with this stress-induced non-coding rna. to identify prins regulated genes, we carried out a cdna microarray chip experiment on hela cells where the expression of prins was silenced. this experiment identified g p , an interferon-inducible anti-apoptotic gene that was down-regulated by prins silencing. g p was strongly expressed in proliferating keratinocytes and markedly upregulated in involved psoriatic epidermis compared to healthy epidermis. to detect prins interacting proteins we applied ribonucleoprotein (rnp) purification in hacat cells. with the help of matrix-assisted laser-desorption ionization time-of-flight (maldi-tof) method we identified nucleophosmin, a protein that physically interacts with prins rna. nucleophosmin is a ubiquitously expressed nucleolar phosphoprotein which shuttles continuously between the nucleus and the cytoplasm. immunohistochemical experiments revealed that the expression of nucleophosmin was significantly elevated in psoriatic involved epidermis, localized to the dividing cells of the basal layer. our data indicate that the non-coding prins rna forms a molecular complex with nucleophosmin that regulates stress-induced cellular processes. we suppose that the abnormal functioning of this complex may result in the altered regulation of genes among them the anti-apoptotic g p which can contribute to the pathogenesis of psoriasis. atopic dermatitis (ad) is a chronic inflammatory skin disorder based on a genetic predisposition and triggered by environmental factors characterized by eczematous skin lesions, pruritus, and typical histopathological features. rituximab is a monoclonal anti-cd antibody therapy that targets pre-b cells and mature b cells, but not plasma cells. ad is generally considered as a biphasic, with switch to initial th to chronic th -predominant disease, in which rituximab may have multiple effects. objectives: to report three patients with severe ad refractory to conventional treatments and to anti-ige monoclonal treatment (omalizumab). materials and methods: three patients with severe refractory ad with high levels of serum ige that received weekly intravenous infusions of rituximab at a dose mg/m body surface each. subsets of lymphocytes were analyzed with multiparametric-flow cytometry (facscalibur, bd) at baseline and at specific intervals after treatment. serum immunoglobulins levels were quantified by nephelometry. results: at baseline, all patients had highly elevated levels of total ige ( g , ; g , ; g , mg/dl, respectively). all patients underwent prior treatment with omalizumab for months, with only partial response. then, we started rituximab therapy, resulting in a clear and complete improvement of ad eczema area and severity of skin lesions in all patients. remission of pruritus was observed from the nd week after initiation of rituximab therapy up to year. whereas allergen-specific ige levels were not altered, we observed a large reduction in total serum ige concentrations after initiation of therapy with rituximab. in the first treated patient (follow-up year), ige levels decreased from , to , mg/dl. the other two patients are in the and -months of the follow-up period. importantly, during follow-up no other therapies were required for ad control. conclusions: treatment with an anti-cd antibody led to a dramatical improvement in our series of patients with severe refractory ad. this study support further evidence on the efficacy and safety of rituximab in severe ad. we have previously demonstrated that chronic topical exposure of mice to the contact allergen dncb or to the respiratory sensitiser trimellitic anhydride (tma) preferentially activates t helper (h) and th cells, respectively. in addition, a single application results in divergent cutaneous cytokine production and the migration of langerhans' cells (lc) with different tempos. to explore events occurring after allergen application, balb/c strain mice were exposed to a single topical dose of either %dncb, %tma or to vehicle alone for . - h. measurement of cytokine production from skin exposed to the allergens was performed by cytokine bead array. exposure to dncb provoked rapid production of il- (mean= pg/ml, n= , p x . ), il- (mean= pg/ml, n= , p x . ) and il- a ( pg/ml, n= , p x . ) in skin compared with tma-or aoo-treated mice. in subsequent experiments, mice received an intradermal injection of ng/ear of murine recombinant il- or of the known regulator of lc migration; il- b. interleukin- b induced a significant loss of epidermal lc numbers, measured as a function of reduced frequency of mhc class ii positive cells within epidermal sheets, after ( %) and h ( %) (n= , p x . ). in contrast, il- or control injections were without effect. however, il- administration caused an increase in cutaneous il- production ( pg/ml, n= , p x . ) compared with control injection and naï ve tissue ( and pg/ml, n= , ns). in addition, systemic treatment with anti-il- antibody failed to impact on lc migration provoked by dncb ( % reduction; n= , p x . ). in parallel experiments dncb-induced lc migration was blocked by treatment with anti-tumour necrosis factor (tnf)-a antibody, another cytokine known to regulate lc migration. however, dncb-induced cutaneous il- a ( pg/ml, n= , p x . ) and il- ( pg/ml, n= , p x . ) expression was reduced to baseline levels by anti-il- treatment. these data demonstrate that il- is not involved in the regulation of lc migration, unlike il- b and tnf-a. however, il- is involved in the regulation of the production of other cutaneous cytokines provoked by dncb. therefore it is hypothesised that il- may influence lc and dermal dc maturation, via the expression of il- a and il- . allergic contact dermatitis (acd) caused by nickel ions (ni) represents the most common form of human contact hypersensitivity. along with other allergies its incidence is increasing in the us and worldwide (nhanes iii survey ), but the majority of molecular events underlying this kind of t-cell mediated disease are still widely unknown. to elucidate initial molecular mechanisms (sensitization phase) taking place at the primary allergen contact site in human skin a differential proteomic approach was chosen. by applying dige technology (differential gel electrophoresis), software analysis and mass spectrometric protein identification to cell lysates of allergen stimulated human keratinocytes, seventeen proteins were identified that are specifically regulated by metal allergen ni. in the attempt to further characterize the role of a certain down regulated p -mapk-pathway related protein (p prp) in acd, we analysed its regulation, differential distribution of phosphorylated isoforms as well as its subcellular localization. our results strongly support an involvement of p mapk pathway in allergenspecific signaling responses. it is expected that identification of differentially allergen-regulated proteins and detailed analysis of acd-associated signaling events in primary keratinocytes will lead to a better biomolecular understanding of the initiation of human contact hypersensitivity. (work supported by eu-project novel testing strategies for in-vitro-assessment of allergens, lshb-ct- - , www.sens-it-iv.eu.) objectives: schnitzler's syndrome is a rare disease characterised by chronic urticaria, monoclonal gammopathy, fever, and arthralgia/arthritis with marked elevation of acute phase reactants. in the long term, % of patients develop a lymphoproliferative disorder. schnitzler's pathogenesis is unclear; immunosuppressive treatment is ineffective and high dose steroids are usually required. the recent finding that treatment with il- receptor antagonist (il- ra; kineret) is extremely effective has raised the issue of the role of the inflammatory cytokine il- b and of il- -like cytokines in the pathogenesis of the disease. methods: two patients with recently diagnosed schnitzler's syndrome were treated with kineret, obtaining rapid disappearance of fever and urticaria and normalisation of acute phase reactants in one month. blood samples were collected before and after initiation of therapy. serum cytokine levels were measured by elisa, and expression of il- -related genes by real-time pcr on mrna from blood cd + monocytes. results: compared to normal controls, schniztler's monocytes had similar expression of il- , il- bp, and caspase- , both before and after therapy with kineret. il- b expression was similar to controls before therapy, and was decreased five-fold after therapy. at the serum level, neither inflammatory (il- b, tnfa, il- ) nor anti-inflammatory cytokines (il- , tgfb) were detected. as expected, il- ra was only detectable after therapy. il- was detectable in schnitzler's sera at higher levels than in controls ( . vs. . pm) and decreased after therapy ( . pm). the circulating il- inhibitor il- bp was lower than in controls and not affected by therapy. thus, free il- levels were increased in schnitzler's patients as compared to controls ( . pm vs. . pm in controls) and decreased after therapy ( . pm). conclusions: schnitzler's syndrome is not associated to enhanced expression of il- -related cytokines (il- b, il- ), nor of the il- /il- -converting enzyme caspase- in blood monocytes. however, the high circulating levels of il- suggest an increased activity of caspase- , as in the case of autoinflammatory diseases. experiments are in progress to test this possibility. atopic dermatitis (ad) is a chronic relapsing allergic skin disease with a high and growing prevalence. currently around % of the children in industrialized countries are affected. in most cases patients exhibit increased systemic ige-levels (so-called extrinsic form) accompanied by sensitization to allergens. while ad is frequently cleared until adulthood, many patients develop allergic rhinitis and asthma. most ad-patients show topical colonization with staphylococcus aureus indicating a defective innate immune response. as a class of pattern recognition receptors, toll-like receptors (tlrs) are essential for pathogen recognition and critical for the induction of an effective adaptive immunity. all known tlrs except tlr signal via myd to induce nfxb-dependent gene transcription. tlrs are also known to be involved in the pathogenesis of autoimmune diseases. as chronic ad also has an autoimmune component, the study of myd signaling in ad might provide new insights into the function of tlrs. to investigate the role of tlrs in the immunopathology of allergic reactions and skin infections, we induced ad-like symptoms in c bl/ myd -deficient mice by repeatedly sensitizing the mice to ovalbumin (ova) after mechanical disruption of the skin barrier by tape stripping. first results show that myd -/-mice display reduced inflammation of the treated skin area compared to wildtype mice. immunostainings of skin biopsies reveal reduced acanthosis and infiltration of inflammatory cells into the dermis compared to wildtype. skin-draining lymph nodes are less enlarged in the ova-treated knockout mice compared to wildtype and differ in cellular composition. serum antibody levels determined by elisa show reduced systemic total and ova-specific igg -titers in ova-treated myd -/-mice compared to wildtype mice, although the nacl-treated myd -/-control group has higher total antibody titers than the wildtype nacl control group. total ige levels are increased in the knockout mice compared to wildtype mice under both conditions. to further investigate the role of staphylococcus aureus during ad development, we will include topical application of the superantigen staphylococcal enterotoxin b (seb) or living bacteria into our analyses. following this approach, we anticipate to obtain new insights into the role of the innate immune system in allergic reactions of the skin. introdution: the skin of vertebrates is the target for over , species of hematophagous arthropods. among these are ticks, which are long-term feeders and interact with host defenses for days to weeks. little is known about specialized strategies for eliminating ectoparasites, but ticks can induce immune responses in hosts. bovines present variable and heritable levels of resistance to the tick rhipicephalus microplus and are the only model in which distinct outcomes of infestation can be examined in the same species of host. in order to obtain some of the immune correlates of these outcomes, we examined expression of candidate genes and quantified populations of leukocytes and subpopulations of lymphocytes present in the inflammatory infiltrates elicited by tick bites in skin of genetically resistant and susceptible bovine breeds, respectively, nelore (bos taurus indicus) and holstein (b. t. taurus). methods: skin biopsies ( mm punch) were taken at the feeding sites of ticks from susceptible and resistant cattle (each phenotype n = ) or from non-infested contra-lateral sites. expression of mip- a, igf- , mcp- and ip- genes was quantified with realtime rt-pcr. sections of paraffin-embedded skin were stained with may grünwald-giemsa for differential cell counts. lymphocytes in sections of frozen skin were phenotyped with specific antibodies using immunoperoxidase technique; in infested skin, histological sections were limited to the area of the tick's cement cone. results: as expected, hosts recruit cutaneous inflammatory infiltrates around the tick's mouthparts. however the composition of infiltrates presented with significant differences that varied according to the phenotype of infestation. inflammation of nelores contained significantly more basophils, eosinophils and mononuclear cells expressing cd , cd , cd , cd , mhc class ii, and p than that of holsteins. lymphocytes expressing wc and cd antigens were significantly diminished in infested skin of holsteins when compared with control skin (p x . ). infested skin of nelores contained significantly more message for mip- a, igf- , mcp- and ip- than that of holsteins. conclusions: although ticks secrete molecules that inhibit cell adhesion and chemokines, resistance correlates with the capacity to recruit and maintain populations of leukocytes that generate effector immune responses. supported by cnpq, capes, fapesp, and icttd. in the last decade it has become clear that keratinocytes play an important role in the skin immune system. upon stimulation, keratinocytes produce high amounts of proinflammatory chemokines and cytokines and express receptors which are involved in immunoregulation. in a number of inflammatory skin diseases such as eczema or psoriasis infiltrating lymphocytes are found in close vicinity to keratinocytes, enabling interaction of these two cell types. it has been proposed (goodman et al.) that keratinocytes rather support a th response by interacting lymphocytes. we examined this hypothesis with autologous cultures of keratinocytes derived from the outer root sheet of the hair follicle co-cultured with cd + t cells from the same donor. during the coculture either seb or antigen were added. in all experimental approaches the addition of keratinocytes resulted in higher production of ifng by t cells. furthermore, we set up an experimental approach were autologous antigen-pulsed monocytes were also added. again, the induction of ifng by the presence of keratinocytes resulted in a marked and significant increase of ifng production by t cells. we were able to show that il- plays a crucial role in the induction of ifng in t cells keratinocyte interaction. in addition blocking of lfa- in the co-cultures resulted in significantly reduced ifng production by t-cells underlining that icam- /lfa- binding is also crucially important for ifng induction. we conclude from our study that keratinocytes rather support a th than a th local response pattern by virtue of il- secretion and icam- /lfa- interaction. this property of keratinocytes may account for the observed cytokine switch in allergic eczematous skin from a th like micromilieu in acute towards a th dominated milieu in chronic lesions. the genotyping of ccl l gene in patients with psoriasis could allow describing subcategories of patients based in clinical parameters and disease severity. therefore, it could be also used as a clinical diagnostic tool, potentially modulating the efficacy of new treatments, or even to be used as a therapeutical target of psoriasis. this work was supported by project grants of merck-serono and instituto de salud carlos iii (pi / ). psoriasis is an inflammatory dermatosis with % prevalence among caucasians. hla-cw allele is the gene that confers susceptibility to psoriasis and it is placed near to tnf loci with several snp in promoter region. the most common polymorphisms are two g to a transitions in - and - positions. strong association was found between polymorphisms in the - region with psoriasis. in several diseases, the association with hla and clinical manifestation is different between genders, for example in spondyloarthropathy and hla-b , and this is a question of increasing interest. the objective of this study was to identify clinical and molecular differences between male and female in brazilian psoriatic patients. sixty-nine individuals assisted at the dermatology outpatient clinic of the teaching hospital, university of campinas, with diagnosis of psoriasis of early-onset (up to years of age) were selected. hla-a -b -c -dr -dq alleles and tnf- and - snp were differentiated by pcr/ssp. analyzing the total group, patients ( . %) were male, ( . %) were female. in the male group, the mean age at disease onset was , years. severe forms were seen in this group (psoriatic arthritis in cases and erythroderma in ). seven patients ( , %) had a favorable evolution of the disease, but ( , %) developed extensive psoriasis, covering over % of body surface requiring systemic treatment. the main molecular risk factor for the disease, cw* allele was positive in cases ( , %), tnf g/a genotype was found in ( , %) and tnf g/a in ( , %). in the female group, the mean age at disease onset was , years, one case of psoriatic arthritis and one of erythroderma. twenty-nine ( , %) had a favorable evolution of the disease and ( , %) an unfavorable evolution. cw* allele was positive in cases ( , %), tnf g/a genotype was presented in ( , %) and tnf g/a in ( , %). severe disease was seen in male patients. there was no difference in frequency of cw* allele between male and female groups, but there was a tendency of significant difference in tnf g/a genotype. we found that c bl/ mice were more susceptible than balb/c and dba/ mice. higher susceptibility was reflected by higher footpad swelling and transient systemic dissemination. analysis of serum cytokine level revealed differences in production of proinflammatory cytokines, such as il- and mrp / , among different inbred strains of mice. furthermore, we identified the cells which are involved in this cytokine production. as expected, histopathological analysis showed that s. aureus infection induces an influx of monocytes and granulocytes. our study shows that not only bacteria-but also host-specific differences are associated with different courses of s. aureus skin infection. aims: to investigate the cause and to study the clinical symptoms and the laboratory findings of the anaphylactic reactions in the pediatric population of our country, considering that these are very often dangerous situations which demand direct treatment and increased alertness. methods: cases, which were studied retrospectively, included children ( boys and girls), aged - years, who had an anaphylactic reaction, out of the that were examined in total. the statistical analysis of the data was held with the spss program. the commonest causes were proved to be food ( %-particularly sea food and dried fruit), drugs ( %-usually antibiotics and non-steroidal antiinflammatory drugs), as well as insect bites ( %-mainly caused by hymenoptera). the symptoms included mainly the presence of pruritic pomphus with erythema ( %), and gastrointestinal symptoms ( %), while there were quite many cases with dyspnea, nasal congestion, but also angioedema. total ige g was found in out of the severest cases ( , %), in which the adequate control was held, while in their vast majority ( out of ) there was no previous anaphylactic reaction. on the other hand, it was proved in total, that in , % ( cases) there was a hereditary family history of atopy, while in children ( %) there was also a personal history of asthma. finally, at a great percentage ( %) eosinophilia was found, while a statistically significant seasonal distribution during spring and summer was registered. conclusions: it has, therefore, been shown that )the anaphylaxis is quite often in the pediatric population, with the commonest causes to be food and drugs, which are often thoughtlessly used. ) in particular, in many cases it is proved that there is a personal but also a family history of atopy. ) increased attention should be, thus, given in these cases -especially during spring and summer-for their early diagnosis as well as for their effective treatment (adrenaline, antihistamines and corticosteroids) particularly for the severest cases, where the hospitalization of the patient is also necessary. allergic contact dermatitis (acd) is an adaptive inflammatory response of the skin triggered upon exposures to certain chemicals or metal ions. as classical type iv delayed hypersensitivity reaction this response is mediated by t-cells. since many ingredients in consumer products might exert allergenic potency, there is a need for an appropriate screening and characterization of the chemicals used according to this toxicological endpoint. up to now the identification of potential allergens completely relies on animal testing, like buehler assay or guinea pig maximization test (gpmt). due to economical and ethical reasons, as well as driven by the enforcement of certain governmental regulations (i. e., cosmetics directive), the development of an in vitro test system for identification of potential sensitizers is mandatory. since dendritic cells (dcs) play a pivotal role in the initiation of contact dermatitis we chose dcs to characterize known sensitizers in their ability to activate these cells and subsequently examined the molecular interplay between dcs primed by allergens and t-cells in the test tube. the known allergens nickel, dinitrochlorobenzene (dncb) and cinnamic aldehyde were tested for their ability to alter the expression of several immunomodulating surface molecules on dcs derived from monocytes that display a langerhans cell (lc) type-similar phenotype. we used multicolour flow cytometry to detect differences in expression patterns of surface molecules that have been associated with maturation. in addition to the upregulation of cd we could observe dose dependent upregulation of programmed death ligand (pdl- ) and downregulation of the dendritic cell immunoreceptor (dcir). furthermore we observed enhanced t-cell proliferation in mixed leukocyte reactions (mlrs) applying lcs stimulated with allergens ex ante. since changes in the expression of only single cell molecules are unlikely of being sufficient for reliable identification of possible contact allergens, we are aimed at analyzing a wide pattern of various surface molecules by multicolour facs and propose that this might be a reasonable approach to screen for contact sensitizing properties of chemicals. our findings are of particular interest for further development of new in vitro assays, using immune cells, to detect the sensitizing potential and quantify the sensitizing potency of chemicals. we want to present the case of a year old iraqui patient with arabic ancestors who had been suffering from psoriatic arthritis since years. in march a treatment with fumaric acid esters in combination with ibuprofen was introduced. this led to the complete healing of the skin lesions. for this reason the dose could be reduced to one tablet fumaric acid esters ( mg) every second day. in april the patient presented himself in the consult with multiple livid papules with a diameter of mm in the area of the auricle. the histological examination showed an hhv- positive kaposi sarcoma. the differential blood cell count demonstrated a lymphocytopenia. the hiv-serology was negative. the staging examinations (chest x-ray, gastroscopy, coloscopy, abdominal and lymph node sonography) showed no signs of visceral involvement. after the diagnosis the treatment with fumaric acid esters was discontinued. over the course the livid papules showed a spontaneous complete regression. a spontaneous regression is known from the iatrogenic ks caused by immunosuppressive therapy when the immunosuppression is terminated. as our patient also showed a spontaneous regression of the kaposi sarcoma after stopping the treatment with fumaric acid esters we propose a causative relation. sarcoidosis is a multisystemic granulomatous disease with unknown etiology. although the immunopathogenesis of sarcoidosis remains unknown there are some supportive evidence for the significant role of th type immune response. recently, suppressor of cytokine signaling (socs) proteins have been identified as regulators of cytokine signaling pathways. in this study we aimed to evaluate the roles of socs , socs and foxp in the immünopathogenesis of sarcoidosis and their association with responsiveness to treatment. peripheral blood (pb) and broncholaveolar lavage (bal) mononuclear (m) cells from sarcoidosis patients in remission following treatment (responders, n: ), the patients who showed recurrence or progression after treatment (non-responders, n: ) and stage i/ii sarcoidosis cases which were followed up without any treatment (untreated, n: ) were evaluated for socs , socs ve foxp mrna expressions by taqman pcr, and also flow cytometric analysis was performed for lymphocyte markers including cd , cd , cd , foxp , cd + cd high , cd + foxp + . expression of socs and foxp- mrna in pbmcs and balmcs from responders were found to be significantly higher in comparison to other two groups . socs was found significantly elevated in pbmcs of responders when compared with other two groups. it was also elevated in balmcs of responders when compared with with those of untreated cases. the proportions of cd , foxp , cd +cd high , cd + foxp + cells in pbmcs and balmcs of responders were found to be increased in comparison to nonresponders and untreated cases. our data demonstrates that socs , socs and t regulatory cells may have potential roles in the control of sarcoidosis. we think that if the roles of socs and socs molecules and t regulatory cells are well characterized, new therapeutic approaches targetting cytokine signal supressors, which can strenghten the regulatory responses, may be beneficial for the sarcoidosis cases resistant to conventional therapy. the inorganic dust, containing free crystalline silicon dioxide (fcs) is critical for the development of silicosis. several studies supported the view that fibrotic responses mainly depends on the regulation of the immune response to the fcs in affected individuals. the role of fcs in induction of a local and systemic inflammation and pulmonary fibrosis are still debates.we studied the changes of neopterin, as a marker for ifn-g dependent macrophage activation and circulating immune complexes (cic), as a marker of humoral immune response, in patients with silicosis and workers exposed to dust containing fcs.we survey a group of silicosis patients, with mild ( ), moderate ( ) and severe ( ) silicosis, coal workers, exposed to inorganic dust containing fcs (exposed), and healthy workers without exposure to dust aerosol (controls).the serum quantity of neopterin and cic, containing iga(igacic), igg(iggcic) and igm(igmcic) was detected by elisa. differences between investigated groups were detected by student's t-test and a p-value less than . was considered significant.neopterin level was significantly elevated in exposed ( , ± , ng/ml) compared to controls ( , ± , ng/ml; p= , ). moreover, the neopterin level in exposed was similar to silicosis patients ( , ± , ng/ml).the levels of iggcic was significantly elevated in the exposed compared to controls ( , ± , au vs , ± , au p= , ) and to silicosis patients ( , ± , au p= , ). in contrast, igmcic was significantly elevated in silicosis than in exposed ( , ± , au vs , ± , au; p= , ).in comparison with exposed, significantly higher igmcic was found only in mild, but no in moderate and severe silicosis. in contrast, the level of iggcic in mild and moderate silicosis was significantly lower compared to the exposed (p= , and , respectively).the obtained results showed that activation of alveolar macrophages mainly depends on the presence of fcs in the respirable dust fraction and precedes the clinical data for pulmonary fibrosis. the dynamics of cic suggest the involvement of fc-receptors mediated regulation of the immune response in the progression of pulmonary fibrosis, and could be useful marker for exposure to inorganic dust containing fcs. described pathologic similarities between sarcoidosis (sa) and tuberculosis (tbc) suggest m. tuberculosis antigens as caustaive agentes. it seems that in the genetically different predisposed hosts, the same antigens may cause the development of sarcoid or tuberculous inmune response. so different hla haplotypes have been described as a predisposing factor to develop sarcoidosis (hla a* , b* , drb * (these of good prognosis) * /* /* /* ) or tbc (drb * /* /* /* and associations with dqa * /* /* ) we describe two cases of two female patients from the same geographic region with mantoux and zhiel-neelsen negative tests and high levels of tnf diagnosed of tbc and sa respectively. both of them debuted with the same clinical manifestations: fever, abdominal pain, and asthenia and shared similarities in the images from the tc study (pulmonary nodules and mesenteric adenopaties). we found the same results for the flow citometry analysis of the non-caseificant granulomes as well as the same anatomopathologic characteristics. after being treated with anti-tbc drugs, the first one presented a good clinical improvement, so she was diagnosed as tbc. the second one did not improved, so she was treated with corticosteroid, with good results. therefore, she was diagnosed as sarcoidosis. after hla analysis, we noticed that the tbc patient was hla a* , b* and drb * (sarcoidosis good prognosis haplotype) and the patient diagnosed as sarcoidosis was hla a* , drb * . as the results show, could there not be a direct relationship between the hla system and the development of sa or tbc, or in contrast, was the first patient missdiagnosed of tbc being a good prognosis sa? objectives: experimental mouse models for acute asthma are well established, yet models for chronic asthma have several shortcomings. for example, current chronic models show decreased inflammation over time and only marginal effects on airway remodelling. experimental models for chronic asthma are essential for development of new therapeutics and must include changes that closely resemble clinical conditions. ovalbumin (ova), house-dust-mite (hdm) and cockroach (cra) proteins are commonly used to trigger an asthma like response in mice and, for this reason, were used in the present study. the objectives of our work were to compare the most frequently used mouse models of chronic asthma and to develop a mouse model of chronic asthma that clearly displays pivotal features of severe human asthma. methods: for the induction of asthma, mice were initially sensitised by intraperitoneal injection of ova, hdm, cra or a combination of all three, followed by repeated challenge by intratracheal application of ova, hdm or cra. inflammation in lung was measured by analysis of cell influx into the bronchoalveolar lavage (bal) and by determination of chemokine and cytokine levels in bal and lung tissue using elisa and multiplex technology. additionally, serum levels of ige and igg antibodies were measured. airway remodelling was assessed by histological staining for mucus production, immune cell influx, smooth muscle thickening and fibrosis. results: significant differences were measured in cell influx, chemokine/cytokine and total ige levels. compared to hdm and cra, ova induced an higher cell influx in the bal, hdm showed an increase of chemokines in bal and increased ige levels in serum. using a combination of all three proteins resulted in the most severe form of asthma. conclusions: to our knowledge, this is the first study that directly compares the most commonly used mouse models in regard to their potential to display a pathology specific of severe asthma. the most sustained and severe form of asthma was induced by the combination model. this model offers particular advantages for evaluating existing and novel therapeutic agents. furthermore, this model could contribute to understanding of the mechanisms underlying chronic asthma. the present study focused on peri-smi connective tissue capsule formation, the most frequent post-operative local complication in patients receiving smi. we investigated the local immune processes via the phenotypic and functional characterization of lymphocytes within the fibrotic tissue. to this end, intracapsular lymphoid cells and peripheral blood mononuclear cells (pbmcs) from the same patients were isolated and analyzed via facs, concentrating on t-effector cells (teff) and t-regulatory cells (tregs: cd + , cd ++ , foxp + ), cytokine profiles, t-cell receptor (tcr) repertoire and reactivity against human heat shock protein (hhsp ). intracapsular tregs were visualized by immunohistochemistry and functionally tested in suppression assays. the cellular composition of intracapsular mononuclear cells showed a preponderance of cd + t-helper cells and a significant subset of tcrg/d + cells, exceeding that observed in peripheral blood. il- , il- , il- , tgf-b and ifn-g production prevailed, pointing to a th /th weighted immune response. furthermore, intracapsular t-cells displayed a restricted tcr a/b repertoire (monoclonal/oligoclonal) as well as a preferential reaction with hhsp . importantly, numbers of intracapsular tregs were inversely proportional to the degree of fibrosis and showed less suppressive capacity as compared to peripheral tregs. our results suggest that silicone triggers a specific local immune response via activated th /th cells, promoting fibrosis due to the production of profibrotic cytokines. clonal restriction of the tcr repertoire is a further indication for a specific antigen driven immune response preceding capsular fibrosis. in this context, hsp might be a prominent candidate. taking into consideration that it is ubiquitously expressed, it might be the "missing link" between local and systemic side effects of smi. the inverse correlation between the degree of capsular fibrosis and the number of intracapsular tregs suggest that tregs may initially be able to inhibit the progress of capsular fibrosis. however, as numbers of tregs, as well as their suppressive capacity decreases over time, fibrosis develops. supported by the competence center medicine tyrol (kmt) and the lore-and-udo-saldow donation. objectives: recent findings have proven that silicone induces a local inflammatory response with subsequent fibrotic reactions. the present project deals with the standardization and further development of a modified elisa test system (silisa ® ) for the identification of patients with a risk for fibrotic side effects to silicone mammary implants (smis) based on the protein signature adhering on the surfaces of such devices ( ) . the current silisa ® is a test system for the simple detection of the adhesion pattern of proteins from patients' sera to silicone. the optimization of the silisa ® comprised inter-and intra-assay standardization, robustness, specificity and sensitivity. the essay was further developed with antibodies against annotated proteins that were not yet tested in the past. all experiments were carried out in a well plate format for high-throughput analysis. statistical analysis has been performed using spss. the extended essay has been successfully established in the system with antibodies against seven already tested proteins, including c-reactive protein, collagen-i, collagen-iii, fibronectin, igg, c -complement, myeloid related protein and two new proteins, integrin-ß and fibrinogen. data from more than patients have been obtained and exploited so far. the intra-and inter-assay variability of the test was reduced to less than % and %, respectively. patients with fibrotic reactions to silicone were successfully identified using a pattern of protein deposition to silicone. conclusion: applying the silisa ® , sera from five different groups were tested: silicone patients with and without fibrotic reactions, female and male individuals without any contact to silicone and hospital's medical staff with potential silicone contact. the distribution pattern of eight proteins showed differences in patients developing strong fibrotic reactions to silicone compared to controls. muscular lesion is a frequent matter in sportive medicine and myodegenerative diseases. necrosis of the damaged tissue and activation of inflammatory response characterize the initial phase of muscle repair. this work aimed to analyze the tissue repair after induction of lesion in skeletal muscle from mouse lineages with distinct cytokine secretion patterns. it was included at least mice per group with distinct cytokine pattern: th (c bl/ , c bl/ ) and th (balb/c). muscular injury was performed by injection of bupivacaine. both th -dominant strains presented more areas with regenerating myofibers and macrophages at dpi. regional lymph nodes showed significant increase of cellularity and relative numbers of cd bupivacaine-inoculated balb/c mice compared to non-inoculated matched mice at dpi. balb/c mice showed increased collagen expression and decrease of mmp- activity associated with more mrna for tgf-b . this study shows that the immune background of the mouse may affect the remodelling processes in skeletal muscles that occur in response to bupivacaine injection promoting muscle regeneration (th cytokines) or myonecrosis and collagen deposition (th cytokines). the severe, life-threatening heart failure in some of the patients with dilated cardiomyopathy (dcm) is imputed to the stimulatory autoantibodies against the second extracellular loop (ec ii ) of the ß -adrenergic receptor (anti-ß ec ii ). to analyze their pathogenic impact as a single causal factor we used a human-analogous lewis rat model of dcm, where monthly subcutaneous immunization of the rats with the ß ec ii peptide as a glutathione-s-transferase (gst) fusion protein induced production of anti-ß ec ii and eventually dilated cardiomyopathy. in this model we isolated a ß ec ii -specific rat monoclonal antibody (clone f ), and showed by elisa that it binds to the linearized ß ec ii peptide. additionally, we confirmed with flow cytometry that f also binds the ß ec ii in its native conformation, i. e. directly labeled circular ß ec ii (dyl -ß ec ii ) peptide. moreover, we demonstrated activation of the ß -adrenoreceptor by f using a fluorescence resonance energy transfer (fret) assay system in vitro. these data further corroborate the pathogenic role of anti-ß ec ii antibodies in mediating dcm in this animal model, thus rendering them a potential therapeutic target. therefore, we investigated a novel anti-ß ec ii -specific peptide-based therapy, by intravenously applying a circular ß ec ii peptide in the dcm lewis rat model to neutralize the anti-ß ec ii antibodies. while the peptide therapy strongly reduced the anti-ß ec ii titers in the serum by up to % and consecutively lead to clinical remission, elispot assays for the detection of ß ec ii -specific antibody-secreting cells (asc) indicated no difference in the number of long-lived plasma cells in treated animals. in contrast, elispot and flow cytometrical analyses revealed a decrease in the number of ß ec ii -specific memory b cells in the treated animals, indicating that this cellular compartment is most likely also targeted by the peptide therapy. our newly developed anti-ß ec ii -specific therapy, thus, not only neutralized the pathogenic autoantibodies, but also depleted antigen-specific memory b cells involved in the generation of these autoantibodies. these results provide the rationale for further development of this therapeutic strategy for eventual application in patients with autoimmune dilated cardiomyopathy. cardiovascular diseases like myocarditis and subsequent dilated cardiomyopathy (dcm), are a frequent cause of mortality in humans with dcm being the most common reason for heart failure in young adults. infections with coxsackievirus b or cytomegalovirus can lead to an acute inflammation of the heart muscle that is followed by an autoimmune response directed autoantigens in the heart, such as the alpha isoform of cardiac myosin (myhca). immunization with the well-characterized myhca - epitope elicits autoreactive cd + t cell responses that have been shown to be the major mediators of autoimmune myocarditis in balb/c mice. it is known that professional antigen presenting cells (apcs) such as dendritic cells are crucial for initiating and maintaining t helper (th) cell responses affecting the heart muscle. however, the detailed analysis of the interaction between these cells in the context of autoimmune myocarditis has been hampered by the lack of appropriate analytical tools. we therefore generated a tcr transgenic mouse harboring t cells that specifically recognize the myhca - peptide. in a first step, hybridoma cells were generated by fusing bw tcra -cd lymphoma cells with myhca - -specific th cells. tcr expression and antigen specificity was assessed by facs analysis and elispot assay. following subcloning, the variable regions of the expressed tcr were characterized by pcr-sequencing. the rearranged v(d)j regions were subcloned into tcr cassette vectors and linearized constructs were injected into the pronuclei of fertilized oocytes. using this novel tcr tg mouse we plan to investigate in detail the activation of myhca - -specific t cells during the process of autoimmune myocarditis. furthermore, this new tool will help to generate a high resolution analysis of the contribution of different apcs in the activation and differentiation of autoreactive th cells during inflammatory heart disease. m. relle , a. schwarting , p.r. galle university medical center of the johannes gutenberg university mainz, medical clinic i, mainz, germany several mouse or rat models have been established to explore the role of proteinase (pr ), in anca-associated glomerulonephritis, vasculitis or pulmonary inflammation but these studies have demonstrated that anca alone are not sufficient to induce these diseases directly. therefore, we assessed the expression, mobilization and enzymatic activity of pr in mouse bone marrow, kidney, spleen and peripheral blood by immunohistochemistry and immunoblots, as well as the proportion of pr -positive neutrophils in the peripheral blood of frequently used mouse strains. neutrophils were mobilized from the bone marrow by an intraperitoneal injection with human il- . pr -mrna from the murine cancer cell line wehi- was amplified by race-pcr and subsequently sequenced. sequence comparisons were done with dnasis software package and the blast tool of the ncbi. promoter analyses were performed with the genomatix software matinspector. we could demonstrate, that mouse bone marrow is a reservoir for functional neutrophils, which are rapidly mobilized after injection of furthermore, we identified an alternative pr -promoter in the second intron of the mouse pr gene. this promoter is active in the bone marrow, in embyros and in cancer cell lines, indicating that its expression is not restricted to myeloid cells. fine structural analyses of this alternative promoter revealed differences not only between the rat and the mouse promoter but also between different mouse inbred strains. taken together, we have shown that the maturation processes of mouse neutrophils differ from those of human granulocytes. the identification of an alternative pr transcript and its promoter indicates that the murine pr may have additional, as yet not described, functions in hematopoiesis and cancerogenesis. objective: recent studies show that in vivo administration of och, a synthetic lipid that specifically activates natural killer t (nkt) cells, results in suppression of th mediated immune responses in autoimmune diseases. nkt cell activation depends on lipid presentation via the mhc-i like molecule cd d on antigen presenting cells such as mature dendritic cells (mdcs) and upon activation by och nkt cells rapidly produce large amounts of th cytokines. the goal of this study was to investigate the effect of och and och-primed dendritic cells on atherogenesis. methods & results: ldl receptor deficient (ldlr -/-) mice were fed a western type diet and atherosclerosis was induced via collar placement around both carotid arteries. subsequently the mice were treated i. p. with och (n= ) or pbs (n= ) twice a week for seven weeks. the injections with och did not affect atherosclerotic lesion size. to improve the presentation of och to nkt cells in vivo, bone marrow-dendritic cells were maturated via tlr activation, in the presence/ absence of och. subsequently we transferred . x mdcs (n= ) or och-primed mdcs (n= ) ( times) to ldlr -/mice. afterwards the mice were put on a western type diet to induce atherosclerosis. vaccination with och-primed dcs resulted in a . % reduction in plaque size compared to mice treated with mdcs (p x . ). during the experiment no effect on serum cholesterol levels was observed, but at the end of the experiment there was a significant . % (p x . ) reduction in cholesterol levels in the mice treated with och-primed dcs. the number of nkt cells in blood and liver was monitored and a to -fold increase in these cells was detected days after the last treatment with och-primed dcs (p x . ). additionally, the nkt cells in the liver of mice treated with och-primed dcs produced more il- . discussion: we conclude that immunotherapy using och-primed dendritic cells efficiently activates nkt cells, resulting in a th phenotype of the nkt cells and this leads to an efficient protection against atherosclerosis. these data indicate that immunotherapy based on ligand specific primed dcs may be a novel way to treat atherosclerosis. systemic lupus erythematosus (sle) is characterized by high serum titers of igg anti-nuclear antibodies secreted by plasma cells. however, the characteristics of the igg+ plasma cell antibody repertoire in sle has never been determined on a single cell level and little is known about the role of germinal center (gc) reactions for the development of sle autoantibodies. the igg inhibitory fcgriib knock-out mouse on the c bl/ background is a strain specific lupus autoimmune model that is characterized by the spontaneous development of autoantibodies to nuclear antigens such as dsdna and chromatin. to characterize the igg+ plasma cell compartment under normal circumstances and in autoimmunity we have cloned and expressed igg antibodies from single isolated gc b cells and plasma cells derived from spleen, bone marrow and lymph nodes of wild-type c bl/ and fcgriib deficient mice. igh and igl chain gene sequence analyses revealed no major differences in the ig gene usage between wild-type and autoimmune mice, but gc b cells of fcgriib were enriched for antibodies with positively charged igh cdr regions and anti-nuclear specificity. the overall frequency of autoantibodies was similiar between wild-type and fcgriib deficient mice. however, strongly autoreactive antibodies to dsdna and murine igg c were isolated only from fcgriib deficient mice, but not from c bl/ control mice and somatic mutations contributed to their generation. in summary, our data suggest that the gc reaction plays an important role for the development of self-reactive antibodies in fcgriib deficient mice. the finding that the frequency of autoreactive antibodies is higher in gc b cells than in spleen or bone marrow plasma cells may indicate that autoreactive gc b cells are partly regulated even in the absence of fcgriib. autoantibodies against double-stranded dna (dsdna) and nucleosomes (ncs) represent a hallmark of systemic lupus erythematosus (sle). however, the factors leading to the autoimmune response against these nuclear autoantigens are not fully identified. high mobility group box protein (hmgb ), a nuclear dnabinding protein and an extracellular proinflammatory mediator gets tightly bound to modified chromatin during apoptosis. it is not released, since apoptotic cells are immediately engulfed by phagocytes. conversely, in conditions of clearance deficiency, which is observed in a subset of patients with sle, non-ingested apoptotic cells, may undergo secondary necrosis, thereby releasing ncs containing the "endogenous adjuvant" hmgb . we investigated if hmgb -containing ncs contribute to the breakdown of immunological tolerance against dsdna and ncs. we found that hmgb remains associated with ncs released from late apoptotic cells in vitro. hmgb -ncs complexes were detected also in the blood of patients with sle. hmgb containing ncs from apoptotic cells induced secretion of il-b, il- , il- , and tnfa as well as expression of co-stimulatory molecules on human and murine macrophages and dendritic cells (dc), respectively. cytokine release from murine macrophages was dependent on myd and toll-like receptor . neither hmgb -free ncs from living cells nor from apoptotic hmgb -or hmgb / -deficient cells induced marked cytokine production or dc activation. specific inhibition of hmgb activity by the antagonistic a box domain significantly reduced capacity of "apoptotic "ncs to induce tnfa and il- release by macrophages. immunizations with hmgb -containing ncs from apoptotic cells induced anti-dsdna and anti-histone igg responses in non-autoimmune mice in tlr -dependent manner. in conclusion, hmgb in complex with ncs activate antigen presenting cells thereby contributing to the loss of immunological tolerance against ncs/dsdna and, hence, to the immunopathogenesis of sle. objective: apoptotic cells are considered to be a major source for autoantigens in autoimmune diseases such as systemic lupus erythematosus (sle). in agreement with this, defective clearance of apoptotic cells has been shown to increase disease susceptibility. still, little is known about how apoptotic cell-derived self-antigens activate autoreactive b cells and where this takes place. methods: injections of fluorescently labelled syngeneic apoptotic cells were traced using immunofluorescence microscopy. binding studies were performed using apoptotic cells and cho cells transfected with class a scavenger receptors (sr). repeated injections of syngeneic apoptotic cells in sr deficient and wild type mice were conducted and antibody production by autoreactive b cells was measured. autoreactivity against sr was followed in two sle prone mice strains over the development of disease and in a cohort of sle patients. an antibody against the sr was injected together with several antigens to directly evaluate the possible role of autoantibodies against the receptors. results: in this study, we find that apoptotic cells are taken up by specific scavenger receptors expressed on macrophages in the splenic marginal zone and that mice deficient in these receptors have a lower threshold for autoantibody responses. autoantibodies against sr are found before the onset of clinical symptoms in sle-prone mice, and they are also found in diagnosed sle patients. furthermore, injections of an antibody binding sr enhance the antibody production by b cells when co injected with either apoptotic cells or tnp-ficoll. conclusion: our findings describe a novel mechanism where autoantibodies toward scavenger receptors can alter the response to apoptotic cells, affect tolerance, and thus promote disease progression. because the autoantibodies can be detected before onset of disease in mice, they could have predictive value as early indicators of sle. e. glasmacher , k.p. hoefig , e. kremmer , v. heissmeyer stretches and helps in the selection of the correct splicing borders. a allele of (r h) creates a strong binding site for a splicing enhancer protein srp according to bioinformatics. our findings indicate that, the putative branch point, r h snp and the t stretch located downstream of exon two, plays a role in the alternative splicing of bank . finally, we believe that bank delta protein work as a dominant negative isoform in b cell activation and antobodies production, and may antagonize the effect of the full-length protein. these properties of the delta protein may contribute to the observed reduction in sle susceptibility. s. beermann , r. seifert , d. neumann hannover medical school, pharmacology, hannover, germany the biological function of histamine is mediated by four different receptors, namely histamine h receptor (h r), h r, h r, and h r. during an immune reaction histamine acts as a local proinflammatory mediator and contributes to the polarisation of the adaptive immune reaction by modulating the activity of dendritic cells and t cells. in these cells, histamine may modulate the synthesis of characteristic t cell cytokines such as ifng, which plays a central role in a number of autoimmune diseases. the present study was initiated to analyze the involvement of histamine on the induced production of ifng by immune cells. mouse spleen cells were stimulated in vitro by either immobilized a-cd antibodies or cpg-oligonucleotides (cpg-odn) in the presence or absence of histamine or -methylhistamine, a h r-selective agonist. ifng production was evaluated by analysis of cell culture supernatants by elisa. both, histamine and -methylhistamine concentration-dependently reduced ifng production in splenocytes obtained from control c bl/ mice induced by either a-cd antibodies or cpg-odn. this histamine effect was completely inhibited by the h r-specific antagonist famotidine, while h r-, h r-, and h /h r-selective antagonists had no or only moderate effects. interestingly, the h r-selective reagent jnj , which serves as an antagonist on human cells, did not inhibit the histamine-mediated reduction of a-cd induced ifng synthesis, but in contrast it slightly enhanced the histamine effect. thus, at the murine h r, jnj may be a partial agonist. we conclude that histamine modulates the induced production of ifng by t cells via mainly the h r and, to a much lesser extend, the h r. using this assay system, cells obtained from control c bl/ mice will be compared to those from sle-prone mrl lpr/lpr mice and the respective wild type strain mrl +/+ . i objectives: resolvins are products of omega- fatty acids and they exert potent anti-inflammatory properties. in this study we examined their effects on cytokine release in healthy subjects and autoimmune patients. to test the in vitro effects of ng/ml resolvin e (rve ) on the release of tgfb, il- and il- in the culture of peripheral mononuclear cells ( x /ml) stimulated by phorbol ester (pma) ( nm), and the combination of pma and ionomycine ( mg/ml) for hours. methods: mononuclear cells were prepared by ficoll-uromiro gradient centrifugation from healthy subjects and from patients each with sle and sjögren's syndrome (ss). level of cytokines was measured by elisa method. results: in the patients with sle (p = . ) and sjögren's (p= . ) mononuclear cell stimulation by pma resulted in a reduced release of tgf b compared with controls. rve significantly reduced tgfb release from control mononuclear cells stimulated by either pma (p= . ) or pma+ionomycin (p= . ), however rve was ineffective at reducing tgfb release in the sle and ss patients. rve caused a non-significant decrease in il- release from control mononuclear cells, but was again ineffective in sle and ss patients. the production of il- was not significantly modified by rve in any of the groups tested. the release of tgfb by ng/ml of rve can be significantly reduced in healthy control subjects but not in subjects with sle or ss. at the single dose of rve tested, il- and il- release were not significantly affected in healthy or autoimmune patients. omega- fatty acid derived rve may affect inflammation in healthy patients by reducing tgfb production but its effects on inflammation in sle and ss patients may be expected to be smaller or non-existent. in addition, the tgfb release in the pma activated mononuclear cells of sle and sjögren's patients is less than that of healthy subjects. g. e. fragoulis , a.k. tsirogianni , m. herrmann , h. m. moutsopoulos , m.n. manoussakis university of athens, dpt pathophysiology, athens, greece, university of erlangen-numberg, institute for clinical immunology, erlangen-numberg, germany objectives: altered phagocytic capacity has been shown to characterize systemic lupus erythematosus (sle) that is thought to lead to impaired clearance of apoptotic remnants. herein, we assessed comparatively the phagocytic capacity in the peripheral blood of ss and sle patients and investigated the phagocytosis of apoptotic/necrotic cells in the salivary glands of ss patients. methods: patients studied included with primary ss (american-european criteria ) and with sle (acr criteria ). age-and sex-matched healthy blood donors to the ss and sle groups ( donors each) were also studied in all assays. the phagocytosis capacity (phagocytosis index) was assessed by flow cytometry, as previously (gaipl et al, j autoimmunity, ) using heparinized whole blood from individuals studied mixed with a commercially available preparation of fluorescent microbeads (mb-phagocytosis) or a preparation of propidium iodide-stained necrotic cell-derived material obtained from heat-treated normal pbmc (snec-phagocytosis). salivary gland biopsies of patients with ss with and without malt lymphoma ( patients each) were also assessed by confocal microscopy for the presence of apoptotic/necrotic material (tunel assay) and the presence of macrophages (cd -staining). results: in agreement to previous studies, mb-phagocytosis was found significantly decreased in granulocytes and monocytes of sle patients (both for p= . ). in ss patients, defective mb-phagocytosis involved only monocytes (p x . ) and significantly correlated with the presence of extraglandular manifestations (p= . ). compared to controls, snec-phagocytosis was significantly increased in the granulocytes of sle (p x . ) and of ss (p= . ). in the salivary gland biopsies of ss patients, the lymphoepithelial lesions and germinal center-like structures manifested significantly increased infiltrations by macrophages. these lesions were also characterized by notable accumulation of apoptotic/necrotic material that resided both inside and outside the phagocytes. these phenomena were significantly more intense in the salivary gland lesions that manifested malignant in-situ b-cell lymphoma. conclusion: in a manner similar to sle, ss patients appear to manifest altered phagocytic capacity. this may be associated with the observed accumulation of apoptotic/necrotic cells in the salivary glands that in turn, may participate in the chronic autoimmune reactions and/or the lymphoma-generating processes that characterize the disorder. the autoantibodies to various enzymes are often found out in sera of systemic lupus erythematosus (sle) patients, but clinical value of such antibodies often is not understood. the purpose of work was to study the of antibodies generation to the basic enzyme of purine metabolism -adenozine deaminase (ada) in sle and to reveal the relationship of studied antibodies with clinical and laboratory features of pathological process. methods: healthy persons have been included in our study and sle patients ( women and men) with various clinical signs ( persons had st degree of disease activity, persons - nd degree of pathological process activity). women had habitual noncarrying of pregnancy (hnp) in anamnesis. antibodies of igg class to ada (anti-ada) determined by technique of indirect elisa developed by us with the use of immobilized form of ada as an antigenic matrix. b glicoprotein-i-dependent antiphospholipids (aphl) of igg classes were determined using commercial "anti-phospholipid screen igg/igm" test set (orgentec diagnostica). results: at admission an anti-ada was revealed in , %, aphl of igg class -in , % sle patients. it has been noted that igg-aphl were found out in anti-adapositive patients more often and in higher antibody titer, than in anti-ada-negative sle patients (x = , ; p x , ). development of cytopenic syndrome was noted reliable more often in sle patients with associated presence of igg-aphl and an anti-ada in comparison with patients who has not the combinations of these antibodies in blood (x = , ; p x , ). the increased levels of anti-ada were revealed in of women with hnp, and the combination of anti-ada and aphl ( / ) was found out more often than isolated anti-ada ( / , x = , ; p x , ) or isolated aphl ( / , x = , ; p x , ). conclusion: taking into account the imbalance of immunoregulatory functions in sle, the further studying of autoantibodies to ada generation seems to be very promising. presence of hnp in anamnesis is the evidence of necessity of careful biochemical monitoring of aphl and anti-ada in women for the prevention of abortus fetus and administration of adequate therapy. objectives: sjögren's syndrome (ss) is a chronic inflammatory and lymphoproliferative autoimmune disease, characterized by dryness of the mouth (xerostomia) and the eyes (keratoconjunctivitis sicca). dendritic cells (dc) are the most potent antigen-presenting cells that play a crucial role in initiating and maintaining primary immune responses. two main subsets of dc have so far been identified in human peripheral blood: myeloid dc (mdc), which can be further divided into mdc and mdc , and plasmacytoid dc (pdc), also known as ifn-a/b producing cells. the pivotal role of dc in inducing and maintaining tolerance could be critical in ss as alterations among dc populations might contribute to autoimmunity. purpose of this study was to quantify mdc , mdc and pdc in peripheral blood from primary ss patients by flow cytometry and compare the results with gender-and age-matched healthy controls. methods: blood samples from pss patients fulfilling the american european consensus group criteria (aecc) and gender-and age-matched healthy controls were collected in heparin tubes. dc populations were stained with the blood dc enumeration kit, miltenyi, according to the manufacturer's manual. cells were analyzed on a facs canto ii, bd, and data analysis was performed with flowjo software, tree star. for the statistical analysis, a two-tailed mann-whitney u test was performed using prism, graphpad. results: pss patients have significantly reduced amounts of pdc (p= , ) and mdc (p x , ) in peripheral blood. conclusion: alterations in dc populations have been considered to play a role in autoimmune diseases such as systemic lupus erythematosus (sle) or diabetes. in ss patients, up-regulation of interferon-regulated genes has been shown previously. therefore, decreased pdc numbers in peripheral blood from pss patients might explain the fact that an increased ifn signature is found in salivary glands of pss patients, but no elevated levels of ifn-a are measured in serum. recently we reported that malignant cd + b cells from patients with b chronic lymphocytic leukemia (b-cll) produce granzyme b (grb) and are rapidly undergoing apoptosis in a granzyme b-dependent manner following interleukin (il- ) stimulation. several autoimmune diseases have been linked to both elevated frequencies of cd + b cells and increased il- levels. we therefore hypothesized that il- may have similar biological effects on cd + b cells in autoimmune diseases. here we demonstrate that the amount of il- in the serum of systemic lupus erythematosus (sle) patients but not of healthy subjects highly correlated with serum levels of grb. in contrast to b cells from healthy individuals, where no baseline grb expression was found, we demonstrate that up to % of cd + b cells in sle individuals expressed grb. in-vitro experiments revealed that il- was able to induce expression of grb in b cells from individuals with sle and other autoimmune diseases including psoriasis and rheumatoid arthritis. this effect was direct and was strongly enhanced by engagement of the b cell receptor or toll-like receptor . importantly, il- significantly decreased the cd +/cd -b cell ratio in both sle peripheral blood and healthy cord blood samples, suggesting a preferential induction of cd + b cell death. these results suggest that il- -induced grb may play a regulatory role for cd + b cells similar to what we described earlier in b-cll cells. this is the first report uncovering an interrelation between il- and grb levels in sle and showing that il- reduces the cd +/ cd -b cell ratio in b cells from sle peripheral blood and healthy cord blood. endogenous il- may therefore play a disease-modifying role and may explain elevated grb serum levels in autoimmune diseases. further studies should evaluate the therapeutic potential of il- in sle and other autoimmune diseases. r. de palma , e. d'aiuto , s. vettori , g. abbate , g. valentini second university of naples, clinical & experimental medicine, napoli, italy ssc is considered an autoimmune puzzling disease whose pathogenesis is unknown. in the last years, there have been increasing evidences that an interplay between activated t cells and fibroblasts could play a pivotal role in promoting matrix accumulation in systemic sclerosis (ssc). we have previously shown that peripheral t cells from ssc patients with early diffuse disease co-cultured with autologous fibroblasts expand the same t cell clonotypes found in the affected skin. here, using the same experimental approach, we found that the t cell clonotypes expanded in co-cultures are ab positive, hla-dr positive, and promote apoptosis of autologous ssc fibroblasts. we also found that, in these co-cultures, ssc fibroblasts up-regulated fas and underwent apoptosis that paired with the expression of fas ligand (fasl) on cd + t cells. finally, when we added a blocking anti-fas antibody to the co-cultures, we observed a marked reduction of fibroblast apoptosis, suggesting that engagement of fas/fasl had a critical role in mediating apoptosis in co-cultured fibroblasts. it has to be reminded that the absence of fasmediated apoptosis in vivo could be due to several reasons, as the increase of soluble fas in sera of patients affected by ssc. moreover, in the co-culture supernatants we found tgf-beta, il- beta, il- and il- , cytokines known to have a role in promoting fibrosis in systemic sclerosis. taken together, these data suggest that t cell response in ssc may represent an attempt of the immune system to kill fibroblasts, cells that are likely to be altered and expressing (auto)antigens. indeed, fibroblasts of ssc patients have been shown to display a persistently activated phenotype characterized by excessive production of collagen and other extracellular matrix proteins. however, the overall outcome of the t cell response triggered by fibroblasts in ssc, while unable to control the activity and the growth of fibroblasts, contribute to sustain inflammatory loops leading to fibrosis. these findings may lead to change our view about the pathogenesis of this disease and other autoimmune diseases. systemic lupus erythematosus (sle) is a chronic inflammatory autoimmune disease that is associated with a major breakdown in b cell self-tolerance as reflected by elevated serum igg levels of predominantly antinuclear antibodies (anas). serum antibody titers are maintained by antibody-secreting plasmablasts and longlived plasma cells, which reside in survival niches of the bone marrow. however, the antibody repertoire of bone marrow plasma cells, which may include cells expressing autoreactive and potentially pathogenic antibodies, has not been characterized in sle. to determine the frequency, specificity and immunoglobulin gene characteristics of autoantibodies in the long-lived plasma cell compartment in sle, we cloned and expressed igg antibodies from single facs purified cd +cd +cd +cd + bone marrow plasma cells of patients with sle and tested the recombinant monoclonal antibodies for self-reactivity. our preliminary data on the ig gene repertoire and reactivity profile of human igg+ sle bone marrow plasma cells in comparison to healthy controls will be discussed. z. amirghofran , e. moazemi godarzi , e. kamali sarvestani , e. aflaki shiraz university of medical sciences, shiraz, iran, islamic republic of interleukin (il- ) has been shown to be related to the pathogenesis of systemic lupus erythematosus (sle). two polymorphisms in the promoter region of il- gene at positions - g/c and - g/c have been described that are key regulators of il- gene. in the present study the relationship between these two polymorphic sites and disease susceptibility in a group of iranian patients with sle was investigated using polymerase chain reaction-restriction fragment length polymorphism method. the genotype distribution and allele frequencies of il- gene polymorphism at - position showed no significant difference between sle patients and controls. at this position the frequency of gg genotype as well as g allele was higher than c allele in both patients and control groups. in contrary, both allelic and genotypic frequencies at the - position significantly differed in sle patients and controls. at this position gg genotype was observed in . % of patients compared to . % in the control group (p x . ). the frequency of - g allele in patients ( . %) was also higher than in controls ( . %) (p= . ). the haplotype study showed no significant difference between patients and healthy subjects. the relationship between these polymorphisms and clinical manifestations and laboratory parameters were investigated. - polymorphism was associated with the presence of antinuclear antibodies in all patients and rash and hematuria in male patients (p x . ). at - polymorphism, a significant difference with regard to photosensitivity in male patients (p= . ) was found. in conclusion, results of this study showed that - polymorphism plays an important role in susceptibility to sle and that - polymorphism could influence the presence of antinuclear antibodies in the patients. the eukaryotic constitutive proteasome is the main protease expressed in most tissues. recently we have elucidated a functional importance of the second proteasome form, inducible immunoproteasomes, in regulating nf-kb activity during the intestinal inflammation. in comparison with healthy controls and patients with ulcerative colitis (uc), there was increased expression of immunoproteasomes in the inflamed mucosa of patients with crohn's disease (cd) at both mrna and protein levels. in our very recent work we have shown that the proteasome subunit pattern might be suitable for diagnostic differentiation between cd and uc patients. since ifn-g has been shown to be the main inducer of immunoproteasomes in various murine and human cell lines and the ifn-g levels are highly elevated in inflamed intestine of cd patients, induction of immunoproteasomes in cd might be mediated by this cytokine. our data with human leukemic t cell lines and primary macrophages show a significant increase in the nf-kb controlled production of proinflammatory cytokines after the ifn-g-mediated induction of immunoproteasomes in these cells. in the dss-induced colitis model we have observed a diminished colonic inflammation in the absence of the proteasomal immunosubunits. therefore we here suppose that immunoprteasome are involved in the complex inflammatory response during the chronic intestinal inflammation by increasing nf-kb activity in the epithelial and immune cells. however, it remains to be determined whether these results have an important implication for the treatment of chronic gut inflamation in humans. objectives: inflammatory bowel diseases (ibd) including crohn's disease (cd) and ulcerative colitis (uc) are characterized by unknown etiology and chronic intestinal inflammation. noninvasive serological tests to differentiate cd from uc have been searched for a long time. testing for panca together with ascas has good predictive values to identify patients with ibd.the aim of this study was to find evidences for diversity of ascas and anti-mycobacterium paratuberculosis antibodies (anti-mpt) by elisa method. in addition, to examine whether combination of these elisas is useful for distinguishing cd from uc. methods: the study population contained patients with ibd ( with cd, with uc, with gluten sensitive enteropathy, gse) and healthy control subjects. serum asca igg, asca iga and anti-mpt antibody levels were measured by solid phase enzyme immunoassay. adsorption of asca positive sera was performed by baker's yeast suspension. results: elevated level of asca igg, iga and anti-mpt was shown in cd and gse but not in uc compared to healthy controls. serum levels of asca igg, iga showed a significant positive correlation with anti-mpt antibody levels in cd. repeated adsorptions with yeast removed asca igg and iga from sera of patients, but did not change levels of anti-mpt. these results indicate the diversity of asca igg, iga and anti-mpt (accordingly their antigens) and suggest that combination of these elisa can have a role in the differential diagnostics of ibd. it is now well recognised that the majority of lymphocytes may be located within tissues, not in blood, and yet these tissue-resident lymphocytes are relatively understudied, especially in humans. we have extracted cells from human gut biopsies (both normal and inflamed gut) in order to characterise the immune cell populations that exist therein and which molecules may be of paramount importance to their function. we show that distinct populations of t cells exist within the gut and that the ratio of these populations changes down the length of the gut, with the so-called 'unconventional' double negative t cell population (ie tcrab+ve, cd -ve cd b-ve) predominating in the healthy colon whereas these cells are overwhelmed by infiltrating cd (+) cells in inflammatory bowel disease (ibd). having previously shown in mice that gut-resident t cells express high levels of the regulator of g protein signalling- (rgs- ) protein, we have now found substantial over-expression ( - fold) of rgs- in human gut-derived t cells, particularly in this unconventional t cell population. furthermore, levels appear even higher in t cells derived from inflamed gut. transfection of rgs- decreases primary t cell responses to cxcl and ccl , strongly implying that it may regulate t cell localisation. thus, rgs- may be a novel target for modulating t cells in ibd, consistent with which snps in rgs- have been associated with both coeliac disease and type diabetes. mechanisms involved in the induction of oral tolerance (ot) or systemic immunization through the oral rout are still poorly understood. in our previous studies we have shown that when normal mice eat peanuts they become tolerant, with no gut alterations. conversely, if they are immunized with peanut proteins prior to a challenge diet (cd) containing peanuts they develop chronic inflammation of the gut. our aim is to evaluate the consequences of the introduction of a novel protein in the diet of animals presenting antigen specific gut inflammation. adult, female c bl/ j mice were divided in control (c) and experimental (e) groups. c -c received peanut protein immunization, animals of the control groups c were sham immunized, and control group c received ovalbumin (ova) immunization. the experimental group was immunized with peanut protein extract. before initial exposure to a day peanut containing cd, the experimental group was divided into groups (e -e ). ova feeding began days prior cd (e ) on day (e ), (e ), (e ) and (e ) during cd. our results show that oral exposure to a novel protein (ova) in the absence of gut inflammation (e ) leads to low levels of systemic antibody titers, comparable to tolerant animals. conversely, as off initial induction of inflammation, groups submitted to ova (ot) protocol develop increasingly higher systemic antibody (ab) titers similar to animals of the immune control group. in conclusion our protocol indicates that timing is more important than the antigenicity when a novel protein is offered, in the diet. nanoparticles of various types are increasingly used as constituents of food supplements and so called nanofood. since nanoparticles induce inflammatory reactions in the lung, there is an urgent need to also study the toxicological potential of nanoparticles in the intestine. therefore, we assessed the effect of particles on dendritic cells (dc) as key players in the manifestation of intestinal immunity.in in vitro studies we could show that ultrafine tio as well as ultrafine silica led to a mature phenotype of the cells when particles were added to cultures of immature bone-marrow-derived dc. this effect appears to result from enhanced cell death in immature dc but also from direct stimulation of the cells.to analyse the mechanisms underlying this effect we looked for apoptosis as well as for induction of the inflammasome since it has been shown that crystalline silica leads to activation of caspase and secretion of bioactive il -b.in our hands certain nanoparticles induced apoptosis of immature dc, as well as enhanced secretion of active il -b. we therefore hypothesize that particles can induce the inflammasome which leads to the activation of dc.to study the impact of nanoparticles on intestinal inflammatory processes in vivo, we induced colitis by applying % destrane sulphate sodium (dss) in the drinking water for days to wildtype mice. when ultrafine nanoparticles were administered on day and by gavage feeding, we observed an amelioration of disease symptoms when scoring the degree of epithelial disruption and inflammation. in future experiments we will also analyse the effect of different particles in the il -/model of colitis to assess the contribution of particles to the induction and pathogenesis of disease. m. schmohl , n. schneiderhan-marra , m. blum , g. stein , m. schmolz , t. joos nmi-natural and medical sciences institute at the university of tübingen, biochemistry, reutlingen, germany, edi gmbh, reutlingen, germany the human immune system represents a highly complex system that protects the organism against diseases. there is an impressive network of immunoregulatory signals within the immune system as well as between the different healthy and diseased organs. epithelial layers function as a barrier against pathogens. as the gastrointestinal tract, which is occupied with a large variety of microorganisms, represents the outside of the body, the immune system has to establish and maintain a strong presence at the mucosal boundary. the ability to discriminate between pathogens while remaining relatively unresponsive to food antigens and the commensal microflora is achieved by a plethora of largely unknown regulatory mechanisms. this ability appears to be breaking down with chronic inflammatory bowel diseases (ibd) like crohn's disease and ulcerative colitis [ ] . to date treatment options are restricted to controlling symptoms, putting and keeping the dis-eases in remission and preventing relapse. therefore, there is an urgent need for a more detailed understanding of the inflammatory events taking place during the disease. for this purpose a human organo-typic (hot) co-culture model is used, which allows analyzing the collaborative regulation between the immune system and the gut epithelial cells. the human caco- cell line, as a model for the gut-epithelium cells, are cultivated on the top side of special culture vessels, fitting as inserts into carrier wells of -well culture plates, containing whole-blood. this co-culture set up mimics the physiological barrier to perorally applied biologicals/drugs and allows measuring their effect on the immune system. as a read out miniaturized and parallelized sandwich immunoassays will be used to detect alterations in the intracellular mapk and rtk-signalling of the epithelial cells as well as in the extracellular communication via cytokines and chemokines at the interface of the two organs. this approach will provide new insight into the inter-and intracellular signalling of gut epithelium and the immune system, which will finally result in a better understanding of the etiology of inflammatory bowel diseases. inflammatory bowel disease (ibd), including crohn's disease (cd) and ulcerative colitis (uc), is characterized by an upregulation of pro-inflammatory cytokines that play an important role in pathogenesis. osteopontin (opn) is a cytokine implicated in several immunological diseases and, although expressed constitutively in normal intestine, is upregulated in intestinal mucosa and in the plasma of ibd patients. opn has been shown to be either pro-inflammatory or anti-inflammatory for experimental uc, indicating a controversy in this field, while its role in experimental cd remains unknown. in our study we investigated the role of opn in experimental colitis using two mouse models: trinitrobenzene sulphonic acid (tnbs) colitis, a t h -associated model that resembles cd, and dextran sulphate sodium (dss) colitis, a t h -like-associated model for uc. deficiency of opn (either by antibody-mediated neutralization or use of opn -/mice) resulted in suppression of disease phenotype in both colitis models, revealing that opn, and especially, the secreted isoform of opn (opn-s) is important for the initiation of acute intestinal inflammation. importantly, we discovered that opn drives il- production and t h polarization and decreases recruitment of cd + cd + foxp + t regulatory (treg) cells in mesenteric lymph nodes (mlns) of mice with colitis. also, there was an effect of opn on recruitment of cd c + dcs, which were significantly elevated in mlns of opn -/or anti-opn-treated, as compared to opn +/+ or ig-treated control mice. this finding implies that opn deficiency results in enhanced recruitment of regulatory cd c + dcs which may mediate treg induction and protect from colitis. overall, our findings indicate that opn is proinflammatory in both types of colitis, by promoting pathogenic t h and attenuating treg cell recruitment, implying also common mechanisms in the pathogenesis of cd and uc. c. shen , , g. van assche , p. rutgeerts , a. liston , j. l. ceuppens k.u. leuven, autoimmune & genetics lab, vib, leuven, belgium, k. u. leuven, experimental immunology lab, leuven, belgium, k. u. leuven, department of pathophysiology, gastroenterology section, leuven, belgium background: haptoglobin (hp) is one of the acute phase proteins synthesized during inflammation. hp- allele is associated with the disease behavior in crohn's disease but not in ulcerative colitis. however its role in inflammatory bowel disease has not been defined. aim: to determine whether hp modulates the immune responses in experimental colitis. methods: we induced types of colitis dss (th /th ), tnbs (th ) and oxazolone (th ) in hp ko mice. neutralizing anti-il- mab was injected into dss and tnbs hp ko mice. severity of colitis was evaluated by body weight, colon length and histology. th /th cells were analyzed by flow cytometry. cytokines were measured by elisa or rt-pcr. ) compared to the wt mice, hp ko mice developed much severer dss and oxa induced colitis. dss induced lethal colitis in hp ko but not in wt mice; ) in dss but not in oxa colitis mice, il- , ifn-g, tgf-b and il- were significantly increased (p x . , dss vs control) in lamina propria and mesenteric lymph nodes (mln), and this is much evident in hp ko mice compared to those in the wt (p x . , ko vs wt). in tnbs colitis, we found elevated il- and ifn-g (p x . , tnbs vs control). although not significant, il- was also somewhat upregulated; ) in dss colitis we observed that il- enhanced differentiated th cells in vitro, this effect could be abrogated by coculture with serum from wt but not hpko mice. furthermore, in vitro in the presence of tgf-b, il- and il- , more mln-t cells from hpko mice differentiated into th cells; ) anti-il- mab improved dss and tnbs colitis, and partially rescued hp ko mice from lethal dss colitis. in line with this, mice treated with anti-il- showed reduced il- , il- and ifn-g in both mln and lp (p x . , anti-il- vs control). our results reveal that hp has a protective role in the development of mucosal inflammation. in dss and tnbs colitis hp may exert its beneficial effect partially through inhibiting production of il- , supporting further pre-clinical and clinical application of hp for treatment of crohn's disease. p. engelmann , g. talabér , g. süt" o , p. németh , t. berki university of pécs, clinical center, department of immunology and biotechnology, pécs, hungary, university of pécs, clinical center, department of immunology and rheumatology, pécs, hungary objectives: inflammatory bowel disease (ibd) resembles as an autoimmune-like disease. ibd is most common in developed countries: it is calculated that . million people in europe suffer from ibd. several hypotheses are raised in the pathogenesis of inflammatory bowel disease. one of the most favored is the dysregulation of the immune response due to failure of regulatory t cells. the most well known regulatory t cells are the cd +cd hi+ t (treg) cells. furthermore, other immune-regulatory cells are known such as invariant natural killer t (inkt) cells producing both th and th cytokines rapidly upon antigen (lipid) stimulation. methods: based on this hypothesis we aimed to investigate the role of various immune-regulatory t cells in human ibd. we attempt to measure the proportions of inkt cells, treg cells in peripheral blood of patients with crohn's disease (cd) and ulcerative colitis (uc) compared to normal controls. blood samples were collected from normal controls and ibd patients; then lymphocytes were labeled for inkt and treg markers with specific monoclonal antibodies and measured with flow cytometry. results: according to our results a decline in the total inkt cells of ibd patients was observed, interestingly the proportions of cd + and double negative (dn) inkt subgroups showed a characteristic shift among the study groups. percentages of dn and cd + inkt subpopulations were assessed after gating of total inkt populations. in controls we observed high percentage of dn inkt cells ( . ± . %, mean ± sem), while cd + inkt cells ratio was moderate ( . ± . %). in uc and cd patients we found a reduced proportion of dn inkt cells (uc: . ± . %; cd: . ± . %, mean ± sem), while the percentage of cd + inkt cells was elevated (uc: . ± . %; cd: . ± . %, mean ± sem) in both disease groups. proportions of foxp + treg cells also showed a decline in ibd patients comparing to normal controls. conclusion: this study can provide useful data about the pathogenesis of ibd and can lead to identify and characterize new cellular and molecular targets with possible therapeutic use in human autoimmune disorders. objectives: the aim of this project is to explore whether exosomes from tgf-b gene modified bone marrow-derived immature dendritic cells (md-imdc) have the function of systemic immune inhibition and protective effect on the development of inflammatory bowel disease (ibd) in mice, the underlying mechanism was also investigated. methods: exosomes were isolated from supernatant of md-imdc transfected with tgf-b adenovirus (tgf-b -exo). the t cell inhibitory function of tgf-b -exo was determined by mixed lymphocyte reaction (mlr) in vitro. to evaluate the protective effect of tgf-b -exo in the development of ibd, dextran sulfate sodium(dss) induced murine ibd was established and mice were treated with tgf-b -exo. the main symptoms of ibd were observed. the inflammatory degree of colon was also evaluated by histological examination. the relative cd + foxp + treg cell numbers from spleens and mesentery lymph nodes (mlns) were analyzed by facs. results: it was demonstrated that tgf-b -exo could inhibit the proliferation of t cells in mlr in vitro. in murine ibd model, after treated with tgf-b -exo, the main symptoms of ibd such as weight loss, diarrhea and grume sanguinopurulent stool were all alleviated and the inflammatory degree of colon was also reduced. analysis of cd + foxp + regulatory t cells (treg) revealed that the relative numbers of cd + foxp + treg increased in lymphocytes from mesentery lymph nodes (mlns) of inflammatory site but not from spleens. conclusions: these results demonstrate that immunosuppressive exosomes obtained from tgf-b gene modified md-imdc can delay the development of ibd. this protective effect is mediated by the induction of cd + foxp + treg. tgf-b -exo might provide a novel strategy for the therapy of ibd. results: hcv-specific cytokine expression by cd + t-cells was similar in the four vaccinees as observed by ifng, il- production-profiles. however, the killing capacity of expanded cd + t-cells was distinct as observed by the competence to kill ns -peptide presenting transfectants in vitro. as depicted in figure , cd + t-cells cells from both vac (cleared ) and vac (chronic) produced il- and ifng after stimulation with ns -peptide . however, specific killing of the peptide loaded transfectants was only observed in vac , who was able to clear its hcv infection, and this was not observed not in any of the other chimpanzees, who became chronic carriers. [ figure ] killing of ns peptide presenting cells was restricted to the vaccinee that was able to clear hcv infection. these results suggest that controlling hcv replication as initiated by this dna-prime mva-boost vaccine-protocol was partly mediated by antigen specific cd + t-cells. hence, the effector mechanisms induced were distinct between the animals and clearance of the infection was correlated with induction of killing competent cd t-cells. objectives: infection by hepatitis c virus (hcv) is characterized by its high tendency to chronicity, which is usually associated with a low or absent t-cell response against viral antigens. immune response specific for non-structural protein ns from hcv was associated with viral clearance. we have demonstrated that fusion of an antigen to the extra domain a from fibronectin (eda) targets the antigen to tlr -expressing dendritic cells and improves its immunogenicity. thus, we tested if covalent linkage between eda and ns might constitute an alternative for vaccination against hcv infection. methods: recombinant plasmids expressing a secretable version of ns or eda-ns under the control of cmv promoter were prepared. recombinant ns and the fusion protein eda-ns were produced in e. coli. the recombinant proteins were tested in vitro on their capacity to activate maturation of bone marrow derived dendritic cells and to favour antigen presentation. hhd transgenic mice (expressing the human hla-a molecule) were immunized with the recombinant plasmids or with the recombinant proteins, in the absence or presence of poly(i:c) and anti-cd agonistic antibodies. elispot and chromium release assays were carried out to measure the immunogenicity of the different vaccination strategies. intrahepatic expression of hcv-ns rna was measured after a hydrodynamic injection with a plasmid encoding hcv ns . results: immunization of mice with the plasmids expressing eda-ns , but not ns alone, induced strong t cell responses against the main hla-a restricted cytotoxic t cell determinants from ns . the recombinant eda-ns fusion protein, but not ns , was able to activate in vitro maturation of bone marrow derived dendritic cells as well as the production of tnf-a by the thp- monocyte cell line. immunization of hhd mice with eda-ns fusion protein induced both cd + and cd + t cell responses against ns and, when immunized with poly(i:c) and anti-cd antibodies, was able to down-regulate the intrahepatic expression of hcv-ns rna. the recombinant eda-ns fusion protein may be considered for the development of prophylactic or therapeutic vaccines against hcv infection. vaccination is the most efficient strategy to prevent from microbial infections and to control epidemics but are still not available in the case of hiv infection even years after virus detection. therein we propose the intra-dermal inoculation of dna vaccine that present a plasmid vector exploiting the binding capacity of the bovine papillomavirus e protein encoding an artificial multi-component hiv antigen. this inoculation is followed by electroporation in order to increase dna uptake. we used skin as site for vaccination because, being the first line in host defence, it is populated with various cells of immune system. among them, langerhans cells (cd +cd a+), located in the epidermis, are dendritic cell subset capable to elucidate specific cd + responses. the present work emphasizes molecular and cellular biodistribution of the dna vaccine in the skin after intra-dermal vaccination in macaques, as one of the most relevant animal models in hiv studies. technical approach considers an intra-dermal injection of dna followed by topical electroporation of the injection sites. skin and draining ln biopsies were collected at different time points. these biopsies were used for ihc fluorescent staining in order to establish biodistribution dna-encoded antigens and co-localisation with different cell types. kinetic of antigen expression was studied by bioluminescence in vivo imaging. t cell responses were measured by ifn-g elispot assays up to years after dna vaccination. we show that a dna vaccine delivery method combining intra-dermal injection and electroporation dramatically increased the expression of the vaccine antigen selectively in the epidermis, increased the frequency of cd a+ cells in the draining ln in association with the antigen expression, and increased the cellular response persistence, at high levels, for more than two years after the last vaccine boost. our data suggest that electroporation after intradermal injection of dna vaccine involves langerhans cells from the epidermis that elucidate qualitative anti-hiv immune responses. this new approach that comprise new dna vaccine followed by non-invasive electroporation, induce long-lasting cellular response that could be crucial in prophylactic / therapeutic vaccine design. presenting cells was developed. murine coronavirus-based virus-like particles encoding epitopes from the lymphocytic choriomeningitis virus glycoprotein or human melan-a, in combination with the immunostimulatory cytokine gm-csf, selective targeted dcs in vitro and in vivo resulting in vector-mediated antigen expression, and efficient maturation of dcs. in mice, a single application of only low doses elicited strong and long-lasting cytotoxic t-cell responses which provided protective antiviral and antitumor immunity. furthermore, the efficient activation of human tumor-specific cd + t cells by mature dcs transduced with melan-a-recombinant human coronavirus e indicates that this novel vaccine platform mediates the delivery of antigens and immunostimulatory cytokines to those cellular components of the immune system that initiate and maintain protective immunity. as the application of gm-csf already enhanced immunogenicity, we are now trying to further modulate the coronavirus vector-induced immune response with the reverse genetic setup of recombinant coronavirus-based vectors expressing different immunostimulatory cytokines. thereby cytokines will be acting on t cell and dc level. to enhance t cell response interleukin (il ) and interleukin (il ) will be involved, and fms-like tyrosin kinase ligand (flt l) will be expressed to modulate dendritic cells. il is known to enhance early t cell expansion and limits t cell overshoot, whereaes il guarantees survival of high affinity t cells during memory phase. on the other hand flt l enhances dc proliferation and accumulation. with these approaches modulation of the immune response generated by this novel vaccine platform will be examined in viral and tumour models to get insight on the antigen specific ctl response, synergistic effects of the cytokines and protective as well as prophylactic vaccination approaches. f. demircik , ag waisman uniklinik mainz, . med, mainz, germany in murine cytomegalovirus (mcmv) infection, cytotoxic cd t cells and nk cells play a critical role. previously it was shown that mice deficient for b cells are more susceptible to mcmv-related disease, caused by virus reactivation. to better understand the role of b cells and antibodies in the response to mcmv, we made use of different mouse strains that lack b cells, secreted antibodies or il- production by the b cells. we found that for the initial t cell response to the virus b cells are important, but antibodies do not play an important role. this implicates b cells as potential important antigen presenting cells (apcs) in the activation of the virus-specific t cells. the reduced t cell response to the virus was observed whether the mice were b cell deficient from birth or if they were depleted later in life. six month after infection mice were tested for the memory cd t cell response. interestingly, we found that in mice that lack antibodies (mice that lack b cells all together and mice that have b cells but no secreted antibodies) maintain a rather high t cell response to viral peptides, in a level similar to the acute response days after infection. we conclude that antibodies probably remove residual viruses from the body and therefore prevent the continuous activation of t cells. finally, we tested the role of il- produced by b cells by conditional deletion of the il- gene in these cells. we found that b cell secreted il- has a suppressive effect on the t cell response to mcmv, as this response is elevated in these mice. we conclude that b cells are important for an efficient acute response to mcmv and that antibodies play a role in eliminating residual viral particles, thus implicating a dual role for b cells in the efficient acute and memory response to mcmv. this work is supported by the deutsche forschungsgemeinschaft grant sfb to aw. objectives: ebv infection leads to life-long viral persistence. although ebv infection can result in chronic disease and malignant transformation most carriers remain disease-free due to an effective control of the virus by t cells. ebv-specific ifng-producing t cells could be demonstrated in acute and chronic infection by many researchers. recent studies in hiv and leishmania provide, however, evidence that assessing ifng alone is insufficient to assess the quantity and quality of a memory t cell response and support the crucial role of multifunctional t cells in disease control. in this study we therefore analyzed ebv-specific t cell responses in peripheral blood (pb) and bone marrow (bm). methods: paired pb and bm samples were obtained from healthy virus carriers who underwent total hip arthroplasty. t cells were expanded for days in the presence of il- and il- with exposure to overlapping peptide pools of latent ebna- and lytic bzlf- antigens. ebv-specific immune responses were assessed exvivo and after expansion by multiparameter flow cytometry staining for live/dead discrimination marker, cd , cd , cd , ccr , cd ra, il- , tnfa, ifng and cd a. the majority of ex vivo ebv-reactive cd + t cells as well as ebna- -reactive cd + t cells were il- and tnfa-producing memory cells, the later being more frequent in bone marrow (cd +, median, ebna- : bm . %;pb . %; bzlf- : bm . %;pb . %, p= . ). after in vitro expansion a major subset of ebv-specific cd + and cd +t cells displayed a differentiated effector ifng/tnfa phenotype. a comparable number of ebv-specific cd + and cd + t cells retained, however, a tnfa single, tnfa/il- or triple producer phenotype resembling early differentiated or multifunctional memory t cells, respectively. interestingly, both cd + and cd + t cells generated from bm revealed significantly higher cytotoxic potential. sorting of ccr /cd ra differentiation subsets, revealed that ebv-specific t cells were predominantly expandable from the central memory compartment. conclusion: our data shows that multicolor assessment of ifng, tnfa and il- delineates various subsets of ebv-specific memory t cells, which reflect the profile of a protective immune response. human adenovirus (hadv) can cause serious morbidity and mortality in immunocompromised patients after allogeneic stem cell transplantation (allosct). reconstitution of hadv-specific t cells has been reported to be associated with sustained protection from hadv disease, but epitope specificity of these responses has not been further characterized. furthermore, the relative contribution of hadv-specific cd + and cd + t cells in the protection from hadv disease after allosct remains to be elucidated. in this study, we demonstrate, by sensitive measurement using intracellular cytokine staining combined with cd or peptide-mhc tetramer staining, that clearance of hadv was associated with a combined hadv hexon specific cd + and cd + t cell response in both pediatric and adult allosct recipients. based on this observation, we developed a clinical grade method for the rapid generation of t cell lines with high and defined specificity for hadv hexon epitopes for adoptive immunotherapy. activation of hadv hexon-specific cd + and cd + t cells in peripheral blood with a hexon protein-spanning pool of synthetic -mer peptides followed by ifng-based isolation allowed rapid expansion of highly specific t cell lines from healthy adults, including donors without detectable frequencies of hadv hexon-specific t cells. the frequency of hadv-specific t cells was increased to - % in the t cell lines and the absolute numbers of both hexon-specific cd + and cd + t cells were to log increased compared to the starting material. detailed analysis showed that hadv-specific t cell lines recognized multiple mhc class i and ii restricted epitopes, including known and novel epitopes, and showed specific and efficient lysis of hadv infected target cells. this strategy may be used for adoptive transfer of donor-derived hadv hexon-specific cd + and cd + t cells for treatment of disseminated hadv infection after allosct. several studies showed that hbv persistance correlates with a failure of an efficient virus-specific t-cell response. induction of hbv-specific t cells by vaccination may be an innovative approach to overcome virus persistance. dna prime-recombinant adenovirus serotype (ad ) boost strategy proved to be effective in stimulating t cell responses and control of viral infections. woodchuck hepatitis virus (whv) and its host the woodchuck are a useful peclinical model for investigating the new therapeutic approaches. the efficacy of plasmid dna and ad vaccine vectors expressing whv core protein was first examinated in c bl mice. groups of mice were immunized with a dna prime-ad boost regimen or with dna and ad alone. ad was injected i. m. or s. c. t cell response was evaluated by intracellular ifng staining of splenocytes stimulated in vitro with whc-derived peptide pools. anti-whc antibodies were detected by elisa. we detected cd + t cell responses against peptide pools and in spleens of dna and dna-ad immunized mice. however, in prime-boost group the percentage of of detected ifng+ cd + t cells was lower in comparison to dna group. in splenocytes of animals vaccinated with ad very weak cd + t cell response was observed. in dna vaccinated animals we determined high level of anti-whc already after second immunization. after boosting with ad level of antibodies did not change. those antibodies were only igg a subclass what indicates th t helper type of response. ad -immunized mice had over -fold lower level of anti-whc: both igg a and igg subtypes were detected. the weak response induced by ad may be due to the low expression of whcag. in ongoing expreriments we improved the protein expression level by insertion of an intron. we currenly investigate the new construct in mice. the new peptide construct containing four m e-peptide sequences coupled to t helper epitopes from the plasmodium falciparum cs protein and the hepatitis b virus antigen was administered together with adjuvants intranasally and subcutaneously as described (mozdzanowska et al., virology journal ) into various mouse strains. in contrast to its predecessor peptide, we found that vaccination induced much higher anti-m e serum ab titers against peptide and native m e. this correlated with a large number of m -specific ab-secreting cells in lungs and bone marrow. moreover, the serum of vaccinated mice was also crossreactive against the influenza virus subtype a/fm (h n ), which contains a variant m e-sequence different in amino acid positions. importantly, this new peptide vaccine regimen showed significant protection against viral challenge with influenza a strains x (h n ) and the highly pathogenic pr/ (h n ) with remarkably reduced viral titers in lungs and noses of mice. in conclusion, our studies show promising results towards the further development of vaccination with m e as a potential "universal" influenza vaccine. this research is supported by a nih t fellowship ca - , the nih grant ai and a grant from the commonwealth of pa. l. yu zhejiang university, zhangzhou, china interleukin- (il- ) is a cytokine produced by stimulated mononuclear macrophage system. in this report, -day-old chicken embryos were vaccined with the plasmid dna (pci-chil- ) encoding chicken interleukin- and the copy numbers of chil- in peripheral blood, spleen and bursa of fabricius at different time points post-embryonic-vaccination were detected by real-time fluorescent quantitative pcr. the polyprotein of infectious bursal disease virus (ibdv) was prepared into dna vaccine, and the dna vaccine was co-administrated with pci-chil- in -day-old chicken embryos, then boosted after two weeks, and challenged with virulent ibdv four weeks later. the results indicated that allantoic cavity vaccinated with pci-chil- could accelerate high concentrations of chil- in nonage peripheral blood, accelerate high expression of chil- in nonage spleen and bursa of fabricius and promote the body early immune response capacity. embryo vaccination with chil- could significantly enhance the nonage proliferation responses of t lymphocytes from spleen and b lymphocytes from bursa. meanwhile, it could raise the nonage neutralization antibody level and inhance the protection against virulent ibdv induced by dna vaccine. the results indicated that the nonage immune responsing to ibdv dna vaccine was highly enhanced by embryonic coadministration with chil- (p x . ). due to the unique role of the hair follicle in percutaneous penetration, drug delivery systems, which target active compounds to the hair follicle, may result in a better penetration and a higher efficiency of hair and skin therapy ("follicular targeting"). applications in immunotherapy, e. g. transcutaneous vaccination, are of particular interest, because skin antigen-presenting cells (apcs) can be found at particularly high densities in hair follicle-bearing skin, where they are concentrated around the upper portion of the hair follicles. in in vitro studies on human skin explants, we demonstrated that nanoparticles, due to their ability to aggregate in the hair follicle openings and to penetrate along the follicular duct, are promising carrier systems for transfollicular drug delivery. transcutaneously applied nanoparticles in the size range of nm, were capable of penetrating the epithelium and entered into human epidermal lcs, suggesting that such particles may be used to transcutaneously deliver active vaccine compounds, via the hair follicle. the use of the skin as target organ for vaccine has been spurred by recent implication of epithelial dendritic cells (dc) in cd cell cross-priming and suggests that vaccination via the transcutaneous (tc) route may be relevant in the induction of cellular immune responses. advanced studies in vivo using functional vaccines are, however, essential to further assess the potential of particle-based vaccines in transcutaneous vaccination. for this purpose, we developed a standard operating procedure (sop) for transcutaneous vaccine delivery on human skin based on our current knowledge on follicular penetration. in a pilot study on volunteers and a phase i study on volunteers vaccinated with an influenza vaccine, we found that this newly developed sop is safe and efficient at inducing a significant increase in cellular immune responses mostly composed of antigen-specific cd cells. induction of t cell responses has become one of the major goals in therapeutic vaccination against viral diseases and cancer. this study proposes new perspectives for the development of vaccination strategies that triggers t cell immune responses in humans. objectives: all anti-hiv- neutralizing antibodies are directed toward the viral envelope glycoproteins (gp) and the transmembrane protein gp . two sites on gp and gp are attractive targets for vaccine design: the epitope in the third hypervariable region (v ) is recognized by the human monoclonal antibody - d and the epitopes in membrane proximal external region (mper) were recognized by the human monoclonal antibodies e and f . in order to elicit anti-hiv- neutralizing antibodies we have designed virus like particles (vlps) displaying either the gp -v region or the gp -mper. the vlps are based on the acyltransferase component (e chain) of the pyruvate dehydrogenase complex of geobacillus stearothermophilus. the e chain self-assembles into a nm protein scaffold resembling a vlp and that contains copies of e . efficient display and refolding of the v and mper regions in e vlps are obtained by using engineered plasmid which allows insertion of exogenous oligonucleotides at the ' of the gene coding for e . the priming and boosting with a combination of vlps and specific hiv- envelope dna were used to immunize mice and rabbits. results: the v -e and mper-e vlps were purified as stable mers from e. coli cells after refolding in vitro from inclusion bodies followed by gel filtration chromatography. binding of - d, e and f antibodies to hiv-e monomers was confirmed by western blot. we obtained high titers of hiv- gp -specific antibodies in mice immunized with a combination of vlps plus dna (hiv- sf gp ). these antibodies generated a low ( %- %) level of neutralization. moreover immunizations were also performed in rabbits, a better model for induction of neutralizing antibodies. three doses of e vlps plus dna elicited a low titer of hiv- gp specific antibodies. additional rounds of immunizations in rabbits will be performed, in combination with gp plasmid dna, to enahance the responses to envelope and to induce neutralizing activity against these key epitopes. our results demonstrate that e vlps are able to display antigenic determinants of hiv and to induce high titers of hiv- -specific antibodies. the e vlps represent a promising tool for a vaccine design. now a day we paid for vaccination of previous generations. as a result morbidity sharply increases, but we haven't well-tried scheme of immunity renewal yet. every clinic, every center do it in there own way, while vaccination is continued, even when it's not necessary, for example, grip, nobody know strain exactly. the most unpleasantly think is that most of physicians don't know what immunity mean specifically, general they think about vitamins, that isn't fit for forming immunity because of many reasons. we offer a way of immunity according to the world scientific theory and practice. the method is based on biochemical, electrophysiology, and biology way of correction physical status. at first we normalize and activate current settings that are going to the diseased organ, vascular system, gastrointestinal tract, spleen. all of it attends indemnity necessary microelements that were extracted from wild officinal herbals. we don't concentrate only on the one or two types of immunity, fist of all we take into account structure and dynamic of immunogeneration system. in our clinic we use this method; immunity is restored very quickly and kept during long time even if organism gets any complications, which can worsen the situation. that's why when we secure new physical statement in the cns program we forming new nearest and distant men health. we tell local state mechanism of disturbances from disturbances, that develop in blood, lymphatic system, tissues and hypothalamus, when pathological process exist long time. it's completely different disturbances of physician state, which should have different therapeutic approach. the threat of an influenza pandemic has become evident in recent years, emphasizing the requirement for influenza vaccines that are broadly cross-reactive against different subtypes with pandemic potential. we have previously shown that baxter's vero cell-derived h n whole virus candidate vaccines are highly immunogenic both in animal models and in human clinical studies, and cross-protective in mice and ferrets. more recently, it was reported that cross-reactive heterosubtype immune responses against highly pathogenic h n influenza virus could also be achieved by immunizing subjects with a trivalent seasonal influenza vaccine; however the induction of cross-subtype protection could not be addressed in this study with human subjects [ ] . the study reported here evaluated whether the seasonal influenza vaccine, when used either as a monotherapy or in combination with a h n whole virus wild-type vaccine, could induce an immune response and protect mice against h n influenza virus infection. a trivalent seasonal influenza vaccine was shown to elicit anti-h n antibody and t cell responses and partially protected mice against a lethal challenge with wild-type h n virus. the protective efficacy of the trivalent vaccine derived mainly from the h n component. moreover, passively transferred serum of mice immunised with seasonal influenza vaccine protected naïve mice from infection with h n virus, suggesting that antibodies are the main contributor to protection. h n specific serum did not inhibit neuraminidase activity of h n virus suggesting that protection was not mediated by neuraminidase n -specific antibodies. next, we investigated the combination of the trivalent seasonal influenza vaccine and the h n whole virus wild-type vaccine. a prime with the seasonal influenza vaccine followed by immunisation with the h n vaccine enhanced anti-h n antibody response, cellular immunity and protection compared to a single immunization with an equivalent sub-optimal dose of the h n vaccine. hence, hetero-subtype immunity can be achieved by immunization with a trivalent seasonal influenza vaccine, which can be further boosted with a h n candidate vaccine. [ ] gioia c et al. aims: to register the compliance of the population to the old and new vaccines of the national vaccination program for the children up to years old, and to investigate the possible causes of the potential shortages, in order to approach even more successfully the further goal of this whole attempt, which undoubtedly is the future control of important generalized infections. methods: in the study we checked the vaccination history of children in the first grade of primary school in the area of central and west macedonia. there were greek and foreign children. as fully vaccinated were considered those who had already undergone at least one dose of hib, meningococcus and pneumococcus, two doses of hav, as well as four doses of dtp-sabin, while in the cases of a lack of vaccination, the causes were investigated and the adequate recommendations and information were given. in all the cases, except for the nationality, the sex, and the educational and social level of the parents were registered. results: the percentages of the compliance found, are presented in the following ) it should be underlined that, as shown in the table, the percentages of the obligatory-free of charge vaccines were close to %. ) high percentages were noted also for meningococcus, either because it is an old vaccine (it has been available for seven years), or because the bacteria is considered quite dangerous (it has been emphasized through the media). ) on the contrary, as far as the hepatitis a and the pneumococcus vaccines are concerned, low percentages were found, either because of the lack of adequate information-fact that was also shown in our study-or even because of their cost. ) finally, a statistically significant difference was found relating the response to the vaccination coverage, between greeks and foreigners, but also between the greeks themselves, in relation to their educational and socioeconomic level. objective: over the past three decades, the incidence of type diabetes has dramatically increased in europe and north america, inversely correlated to the decrease of infections. according to the hygiene hypothesis, pathogens may prevent the onset of the disease. om- , a bacterial extract of both gram positive and gram negative bacteria already used as an immunomodulatory treatment in children, has been shown to protect non obese diabetic (nod) mice from diabetes development. we aimed here at understanding the mechanism underlying this protection. methods: nod mice and nod-cd -/mice, which are devoid of natural regulatory t cells (tregs), were treated with om- . cytokine secretion, activation and proliferation of b cells and foxp + tregs were monitored. as toll-like receptors (tlr) recognise microbial molecules and trigger innate and adaptive immunological response, cells from mice deficient for tlr , tlr or the myd adaptor protein were used to further address the mechanisms driving the immunomodulatory activity of om- . two synthetic tlr agonists used as adjuvant in human (om- -dp and om- -mp-ac) were also tested for their capacity to protect nod mice from diabetes. the om- -induced protection of diabetes required natural tregs, as nod-cd -/mice were not protected. remarkably, om- activated b cells and not t cells, promoting their proliferation and il- secretion, two phenomena that were tlr -and myd -dependent. om- -dp and om- -mp-ac two synthetic murine tlr agonists effectively prevented diabetes onset in nod mice, promoted the expansion of cd + cd + foxp + t cells and the proliferation of il- secreting b cells in a dose-dependent manner. conclusion: our results argue for the involvement of tlr signaling in the protective effect of om- on development of diabetes and show that two other tlr agonists induce proliferation of b cells and their secretion of il- as well as stimulation of regulatory cd + cd + foxp + t cells. activation of the innate immunity by tlr-stimulation using those products already used in clinics, may prevent the onset of diabetes in those at risk of developing the disease. d. de wit , a. legat , s. thomas , m. van mechelen , p. hermand , m. goldman institute for medical immunology/université libre de bruxelles, gosselies, belgium, glaxosmithkline biologicals, rixensart, belgium aminoalkyl glucosaminide -phosphates (agp) are lipid a mimetics which are considered as interesting candidates for the development of synthetic vaccine adjuvants targeting toll-like receptor (tlr ). since natural lipid a from bacterial lipopolysaccharide (lps) depends on membrane-bound or soluble cd (scd ) for its tlr ligand activity, we investigated the involvement of both forms of cd in the responses elicited by crx- , a prototypical agp. first, we found that crx- efficiently induces nf-kb and irf activation in hek cells transfected with tlr and md- genes, whereas the responses to lps required co-transfection of the gene encoding membrane-bound cd . likewise, crx- efficiently induces the synthesis of nf-kb and irf- dependent cytokines in whole blood of a patient with paroxysmal nocturnal hemoglobinuria, a disease in which a defect in membrane-bound cd prevents lps responses. we then observed that monocyte-derived dendritic cells (dc) which are devoid of membrane-bound cd respond to crx- but not to lps in serum-free medium. the addition of the soluble form of cd did not modify the levels of il and tnf produced by crx- stimulated dc but increased the levels of interferon-b (ifn-b). when scd was added to hek cells expressing tlr /md- , nf-kb activity was not modified but irf activity was increased in a dose-dependent manner in response to crx- . we will further compare the responses induced by crx- in wild-type and cd deficient mice. we previously showed that the transcriptional transactivator (tat) of human immunodeficiency virus possesses the unusual ability to raise a humoral immune response in the absence of adjuvant. these observations prompted us to examine whether such a property can be used to boost the immune response raised against poorly immunogenic peptides. as we previously observed that the autoadjuvant property is controlled by a determinant located within the core-and cysteine-rich regions of the protein, we decided to investigate whether the grafting or the co-injection of a peptide partially containing this determinant (ptat) can raise a humoral immune response against two model peptides. these two peptides, which originate from diphtheria toxin (pdt) and from toxin alpha (pt), both contain an i-ad restricted t-cell epitope but are nonetheless non-immunogenic in balb/c mice of the h- d haplotype when injected with alum. the ptat, pdt, pt, ptatpt and ptatpdt constructs were prepared by chemical synthesis, purified by reverse phase hplc and characterized by mass-spectrometry. pdt+ptat, pt+ptat, ptatpt and ptatpdt were respectively injected twice at two weeks interval in balb/c mice and animals were bled and days after the second immunisation. the sera were subsequently incubated in microtiter elisa plates previously coated with pt and pdt peptides respectively in order to assess the humoral immune response. we observed a lack of antibody response for the immunizations made with the mixture of peptides (pdt+ptat and pt+ptat) but an anti-pdt and anti-pt response for the immunizations made with the two hybrid constructs (ptatpt and ptatpdt). our results indicate that a humoral immune response can be raised towards non-immunogenic peptides using a determinant involved in the autoadjuvant property of tat, that the phenomenon requires the covalent coupling to the peptide antigen and that it is therefore not related to a bystander effect. interleukin- gene polymorph isms (c t, g a, c a and a t) and susceptibility to brucellosis in iranian patients russian federation some epidemiological and observational data suggest that farm and pets exposure [ ] in early childhood may be conducive to reduced atopy. currently, there is a lack of consensus regarding underlying immunological mechanisms, especially in prenatal period. as we previously reported the decreasing of intracellular ifn-g production by cbmc statistical analysis was performed using the kruskal-wallis and mann-whitney tests. results: we revealed that newborns from rural mothers (n= ) have higher amount of both nonactivated (subtype infg+/cd -, p= . ) and activated (subtype infg+/cd +, p= . ) cbmc, producing ifn-g, as compared with newborns from urban mothers (n= ) exposure to pets and the risk of allergic symptoms during the first years of life intracellular interferon-g production by cord blood mononuclear cells as predictor of atopic dermatitis forming in infants: a one-year prospective birth cohort study pc / to what extent t-spot.tb could be used in the diagnosis of tuberculosis in children exposed to tb infection? s. a tb) in children, especially in bcg-vaccinated is difficult for diagnosis because of the low percentage of smear positivity ( - %) and clinical futures only in severe forms of disease. the purpose of the present study was to evaluate the diagnostic value of t-spot.tb (oxford immunotec, oxford, uk) compared to tuberculin skin test (tst) in children exposed to tb contact in the family. forty three children with a history for bcg vaccination/revaccination, treated in the university clinic for lung diseases in children sofia, bulgaria were enrolled in the study. the patients were divided according to age in the following groups: months - years (n= ), - years (n= ) and - (n= ) tb has the highest diagnostic value in children n years of age in early childhood the diagnostic value of t-spot.tb and tst does not differ cfp- antigen is more sensitive for detection of tb-specific t cells compared to esat- antigen. . in children with tst - mm t-spot.tb has a high diagnostic value objectives: the goal of this study is to determine the role of tlr and tlr in the development of spontaneous lupus disease by creating tlr or tlr deficient c bl/ lpr/lpr mice. methods: tlr and tlr deficient lupus prone mice have been generated by crossing c /bl -tlr -/-or c /bl -tlr -/-mice with c /bl lpr/lpr mice which develop a moderate type of lupus related to fas deficiency. we analysed the phenotype of the disease, autoantibody production and renal injury. statistical comparisons were performed using the mann-whitney u-test. results: these mice developed a less severe disease and few immunological alterations. indeed, in tlr or tlr deficient lpr mice, glomerular igg deposits and mesangial cell proliferation were dramatically decreased and anti-nuclear, anti-dsdna and anti-cardiolipin autoantibody titers were significantly reduced. however, the response against nucleosome remained unaffected, indicating a role of tlr or tlr in the production of autoantibodies directed against certain slerelated autoantigens. analysis of b cell phenotype showed a significant reduction of mz b cells, particularly in tlr deficient mice suggesting an important role of tlr in the sustained activation of these cells likely involved in autoantibody production. interestingly, the lack of tlr also affected the production of cytokines involved in the development of lupus disease. conclusion: our data show that deficiency in tlr pc / expression of full length mcl- and its splice variant in juvenile systemic lupus erythematosus (jsle) neutrophils: differential modulation by gm-csf granulocytemacrophage colony-stimulating factor (gm-csf) can prolong neutrophil survival by increasing mcl- , an anti-apoptotic protein. a splice variant of mcl- arises by removal of exon and induces cell death rather than preventing it. here we investigate the expression of both the full length mcl- (mcl- l) and its splice variant (mcl- s) in jsle neutrophils compared to controls and investigate whether the addition of gm-csf changes the expression of both isoforms of mcl- . method: neutrophils were isolated from children (diagnosed x years) with jsle (n= ) and non-inflammatory conditions (control, n= ) and incubated with control serum, jsle serum alone or with jsle serum plus pg/ml gm-csf. quantitative real time pcr was used to assess mcl- l and mcl- s mrna expression (mean ± sem) following incubation in the above conditions and immediately following neutrophil isolation the ratio of mcl- s to mcl- l was also higher in jsle patients compared to controls (p x . ). the addition of gm-csf to jsle serum was associated with an increase in mcl- l ( . ± . ) and a decrease in mcl- s ( . ± . ) mrna expression the addition of gm-csf to jsle serum can abrogate the increased neutrophil apoptosis. alternative splicing is recognised to play a significant role in the regulation of proteins involved in cell death. our results suggest that jsle neutrophils may be more apoptotic due to differential expression of mcl- compared to controls, with jsle neutrophils having greater expression of the pro-apoptotic isoform mcl- s, and less anti-apoptotic full length mcl- cyld is a tumor suppressor gene known to play an important role in the nf-kb pathway. to analyze the function of cyld in vivo we used the cyld ex / mouse strain, which is characterized by loss of the full-length transcript and overexpression of a short splice variant of the cyld gene (scyld) to further investigate the connection between scyld overexpression in t cells and colonic inflammation, we used an adoptive transfer model of colitis. therefore naive cd + cyld ex / t cells were transferred into rag -/-mice which were analysed by mini-endoscopy weekly after cell transfer. here we could demonstrate that cyld ex / cd + t cells exhibit less capacity to induce colitis compared to control cells. consequently we investigated if regulatory t cells (t regs ) of cyld ex / mice are capable to control inflammatory responses. for this purpose cd + cd + cells were co-transferred with naïve wt cd + t cells into rag -/-recipients. interestingly, rag -/-recipients of cyld ex / t regs displayed strong features of colitis compared to control recipients showing that these cells were unable to inhibit inflammatory responses. our findings demonstrate that overexpression of scyld leads to a hyperresponsive t cell phenotype and higher production of inflammatory cytokines by t cells pc / the role of hla complex in inflammatory bowel disease: crohn's disease and ulcerative colitis de investigación biomédica en red de enfermedades hepáticas y digestivas (ciberehd). university hospital virgen arrixaca the allele frequencies of hla class i in cd and uc patients were not different to those observed in controls, although we found an increased frequency of a* in cd vs uc. haplotype frequencies of hla class i and ii in cd and uc were also not different to those observed in controls. however, we found increased frequencies of drb * , * and * alleles, and a decreased allele frequency of drb * in cd vs uc patients and controls. these data are in concordance with other previous studies suggesting that, in patients with isolated colonic cd, drb * is associated with the development of severe disease and positive association of cd with drb * and drb * . indeed, drb * was negatively associated with cd. this allele appears to confer protection against all subgroups of cd, in all ethnic groups including japanese. however, hla-drb * frequency allele, associated in unselected patients with cd in other studies, was not different in our cd and uc patients, and controls. additionally, an increased frequency in hla-drb * in cd was not found in our patients in a different manner to other reported studies. on the other hand, in our uc patients, allele frequencies of drb * were strongly increased with respect to cd and controls. however, the frequency of drb * was decreased in uc with respect to cd and controls. in this sense, our data are agreed with other reports showing that hla-drb * is associated with uc in european, north american, japanese and korean populations methods: a total of children were studied, ( boys and girls), up to years of age, with symptoms suspicious for epstein-barr virus infection. the elisa method was used to look for specific antibodies against the capsid of the virus vcaigg and against the nuclear antigen ebv-igm, while taking into consideration the possible increase of the vcaigg title between two serum samples. results: totally, positive cases of children were found ( %) with active infection : boys ( x years of age, - years of age and g years of age) and girls ( x years of age, - years of age and g years of age). pharyngitis was present in children ( , %), had fever ( , %) and had lymphadenitis ( %). the lab tests revealed leukocytosis up to . leukocytes in cases ( , %) and leukocytosis g . in cases ( , %). the most frequent complication documented was streptococcal superinfection in children ( , %) and thrombocytopenia in children ( , %). a past infection (negative ebv-igm values and positive vcaigg values) was virus infection is common among children and teenagers serum negative are mainly the children of little age and ) there is no statistically important difference between the two sexes, while on the contrary there is a seasonal distribution of the infection, with winter and summer outbreaks general hospital of rethymno, rethymno, greece shows that the il- ra , previously believed to be a decoy for il- only, is able to transmit a signal via il- . our results support this and may suggest that il- / il- ra signalling causes disease in oxazolone-induced colitis. currently we are dissecting the role of single cell populations expressing il- ra to establish which cells play a role in regulating the immune response to oxazolone-induced colitis. together this data can define a role for il- or il- ra and identify specific cell populations methods: splenic apcs exposed to enteroantigen (eag) +/-probiotics were used to stimulate cultured cd + cd -t cells to which titrated numbers of tregs were added. neutralizing antibodies against il- and il- b and elisa-based cytokine analyses were used to monitor the effect of cytokines secreted in the t cell cultures. results: exposure of apcs to eag and probiotics did not influence eag-specific cd + cd -t cell proliferation. however, exposure to three of the six probiotics tested (b. bifidum bi- , l. acidophilus ncfm tm and b. bifidum bi- ) consistently reduced regulatory activity of tregs in a cell-dose dependent manner. the tregreducing activity of probiotics was analyzed using fractionated components of the b. bifidum bi- strain. data indicated that bacterial cell-wall components were responsible for reducing treg activity and not components of nucleus or cytoplasm. the probiotic-induced down-regulation of treg activity was not mediated by increased intra-culture secretion of inflammatory cytokines such as il- or il- b. conclusion: we conclude that certain probiotic strains can modify apcs to cause reduced treg activity in an eag-specific t cell proliferation assay. this effect apparently depends on a direct apc-to-treg cell contact and not secreted cytokines. the apc/probiotics-mediated inhibitory effect on tregs may oppose antiinflammatory activities desired from probiotic therapy palmieri 'la sapienza dysregulated innate and adaptive immune responses against commensal flora lead to crohn disease (cd) and ulcerative colitis (uc), two different forms of inflammatory bowel disease (ibd), a lifelong inflammatory condition of the gastrointestinal tract methods: we analyzed pediatric cd patients ( active, remission), pediatric uc patients ( active, remission), and age-matched non-ibd controls. nkg d/ligand expression was evaluated by immunostaining and multiparametric facs analysis (on pbmc subsets), and by immunohistochemistry and twocolour immunofluorescence (on intestinal biopsies). differences between groups were analyzed with non-parametric and parametric tests; a level of p x . was considered significant. results: nkg d expression is selectively upregulated on circulating "innate-like" t cell populations (g/d and cd +cd + nkt cells), in active, but not in quiescent ibd patients; receptor upregulation correlates with disease type (observed in uc, but not in cd patients). in the same patient groups, the appearance of nkg d ligands on circulating monocytes is also observed. the dramatic increase of nkg d+ lymphocytes, and the strong upregulation of nkg d ligands on both epithelial and immune components, are observed in active ibd lesions. conclusions: our observations document the dysregulated expression pattern of nkg d/ligands on selected innate immunity populations in pediatric ibd patients, both at mucosal and systemic level pc / peripheral and intestinal regulatory t cell dynamics in pediatric ibd patients is a chronic inflammatory condition of the gastrointestinal tract characterized by dysregulated innate and adaptive responses against commensal flora. regulatory t cells (t reg) represent an important mechanism to suppress uncontrolled immune responses to bacterial flora. aims: to evaluate the frequency of regulatory t cells in the peripheral blood, and in inflamed and non inflamed mucosae of pediatric ibd and non mucosal regulatory t cells were identified by immunohistochemistry; circulating regulatory t cells were analysed by immunofluorescence and facs analysis. differences were analyzed with parametric and non-parametric testsconsidered significant. results: foxp + t reg were significantly increased in the intestinal lesions of active ibd patients (cd or uc), and returned to normal levels in post-therapy remission phase. at variance, circulating cd + t reg frequency was elevated in patients affected by both forms of ibd, independently of disease activity, as it persisted in the remission phase. a selective imbalance in the frequency of t and nk subsets characterized the abundant inflammatory infiltrate present in active intestinal lesions, and the normal immunological profile was only partially restored in mucosal samples of quiescent ibd patients. conclusions: regulatory t cells dynamics are differently regulated in mucosal tissues and at the systemic level, during the distinct phases of disease; t reg dynamics in pediatric ibd patients only partially matches previous data obtained in the adults; quiescent ibd is characterized by the imbalance of selected lymphocyte subsets, both in the mucosa and systemically the increased expression of immunoproteasomes in the inflamed mucosa of ibd patients was shown to contribute to this pathology by enhancing nf-kb activation. due to the relation between nf-kb and the immunoproteasome we have investigated whether specific inhibition of immunoproteasomes is suitable for therapeutic intervention in ibd. lmp knock-out mice are deficient in the essential catalytic immunoproteasome-subunit ß i and therefore are devoid of immunoproteasomes. to test our hypothesis, we employed the dss colitis model. in contrast to wild-type mice, colitis was attenuated in lmp knock-out mice characterized by reduced weight loss and less infiltration of lymphocytes in the mucosa confirmed by histology. in addition, lmp knock-out mice had lower levels of proinflammatory cytokines and chemokines compared to wild-type mice validated by rt-pcr and elisa. especially nf-kb regulated genes show enhanced induction in wild-type mice unlike lmp knock-out mice synaptic systems gmbh, braunschweig, germany objectives: although more than million people worldwide are chronically infected with hepatitis c virus (hcv) no prophylactic or therapeutic vaccines do exist to prevent or cure hcv infections. our major objective is to develop a dendritic cell (dc)-based immunotherapy enhancing virus-specific cellular immune response for treatment of hcv infections based on this approach we aim at generating adec- antibodies conjugated with immunodominant hcv proteins to induce hcv-specific protective immunity. methods: recombinant hcv proteins are expressed using "expression-ready-clones" containing n-terminally his-tagged hcv-core (aa - ) or hcv-ns (aa - ) sequences. protein purification is performed by metal-affinity chromatography on ni-nta-agarose hcv-specific t cell responses are monitored at different time points after immunization by facs and in vitro t cell proliferation assays. results: to obtain high amounts of recombinant ns and core we successfully optimized culture and protein purification conditions. briefly, ns was purified natively using pbs-based buffers with ph-gradient. in contrast, purification of core was performed under denaturing conditions in presence of guhcl and urea and a ph-gradient elution. moreover, optimized conditions allowing conjugation of adec- to recombinant hcv proteins were established with respect to duration of conjugation and buffer requirements needed to avoid protein precipitation. efficient conjugation was verified by western blot analysis. after successful generation of adec- / hcv-protein conjugates we are currently establishing optimized vaccination conditions to induce hcv-specific immune responses pd / mva-nef vaccination induces polyfunctional cd t-cells and increases the proliferative capacity of cd t-cells in hiv- infected individuals under haart several vaccination trials have made use of the modified vaccinia virus ankara (mva) as delivery vector. in a therapeutic vaccination trial, we demonstrated that mva expressing the hiv- protein nef (mva-nef) was safe in hiv- infected individuals under haart and immunogenic in regard to the elicitation of ifn-g mediated cd t-cell responses. recent advancements in polychromatic flow-cytometry technology revealed that the sole evaluation of ifn-g provides limited information on the quality of antigen-specific t-cell responses. the evaluation of several functions is essential, as simultaneous production of multiple cytokines by t-cells is associated with superior control of viral replication. methods: in a retrospective setting, we simultaneously assessed the production of ifn-g, il- and mip- b, the expression of the activation marker cd and the differentiation marker cd ra in nef-specific cd and cd t-cell populations during the course of the vaccination trial. furthermore we applied a multi-colour cfse based proliferation assay investigating the proliferative capacity and the simultaneous expression of ifn-g, il- and mip- b. results: following mva-nef vaccination, we observed a significant increase of the total nef-specific cd t-cell response and a significant increase of polyfunctional nef-specific cd t-cells, simultaneously expressing ifn-g, il- and cd . using the standard ics no increase of nef-specific cd t-cell responses was observed. however, by the cfse based proliferation assay, we could show a clear expansion and a generally enhanced proliferative capacity of nef-specific cd t-cells following mva-nef vaccination. notebly, we observed a correlation between the increase of ifn-g, il- and cd expressing cd t-cells and the increase of proliferating cd t-cells suggesting the possibility of a causal link between the two functions. conclusions: the mva-nef vaccine is able to change the quality and quantity of the nef-specific cd t-cell immune response and has the potential to increase the proliferative capacity of nef-specific cd t-cells in hiv- infected subjects under haart this preferential binding to the complex was evident in classical immunochemistry assays, as well as in surface plasmon resonance (spr) tests. this ab inhibited hiv- mediated membrane fusion and p -detected replication. db was found to nicely recapitulate the characteristics of the unconventional, protective immune response, which is taking place in naturally resistant esn individuals. further characterization of the antibody and of its binding epitope is ongoing following intradermal vaccination with mg dna and electroporation of balb/c mice, splenocytes have been incubated with peptides representing class i and ii epitopes, and specific t cell-responses were examined by elispot-assays. the specific antibody responses have been measured by sandwich eli-sas, and neutralizing antibodies have been investigated by hi-assays. results: the vaccibody constructs have been found to be expressed and correctly folded in vitro. the in vivo experiments further demonstrate the presence of neutralizing antibodies as well as the strong induction of antigen specific cd + and cd + t cells. conclusion: antibody and cellular immune responses against influenza hemagglutinin are enhanced when targeted to apcs. methods: the hcv recombinant proteins rns ( - aa) and rns a ( - aa) were conjugated with immunomax using the heterobifunctional reagent sulfo-smcc. balb/c and dba/ j mice were immunized intraperitoneally times at a month interval with different doses ( . - mg/mouse) of the proteins without adjuvants, as conjugates with immunomax, or with complete freund's adjuvant (cfa) the other combinations were not immunogenic at given doses. it should be noted that only conjugates stimulated production of antibodies that bound not only to recombinant protein but also to peptides imitating epitopes of ns protein. immunization with rns a-immunomax conjugate and rns in cfa ( . mg/mouse) induced a similar antibody activity, but a different t-cell responses. the conjugate induced splenic accumulation of t cells specifically reacting in vitro with ns a recombinant proteins of various genotypes, with peptides and with phages by cell proliferation and/or cytokine secretion. immunization with rns a in cfa induced cells proliferating in vitro after stimulation only with peptides; none of the antigens stimulated cytokine secretion. conclusion: covalent conjugates of hcv nonstructural proteins with immunomax effectively induce humoral and cell immune responses pd / degree of cross-genotype reactivity of hcv-specific cd t cells directed against ns the existence of multiple hcv genotypes characterized by marked sequence differences is a challenge for immune control. the aim of this study was to compare the antiviral cd t cell response targeting hcv genotype (gt ) and genotype (gt ) as the most predominant genotypes in germany and to determine the extent of cross-genotype reactivity of specific t cells. we analyzed a cohort of patients with past or ongoing intravenous drug use (ivdu) hypothesizing that multiple exposures to different genotypes may occur. methods: subjects ( with gt , with gt and anti-hcv-pos/hcv-rna-neg) were analyzed. hcv-specific t cells were expanded from pbmc in the presence of peptide pools covering ns from gt or gt . individual reactive peptides and the degree of cross-reactivity between the gt and gt variants were determined by ics. complete ns is sequenced from all viremic patients pd / anti-retroviral effects of type i interferon subtypes in vivo ifna subtypes , , or suppressed fvreplication in vitro, but differed greatly in their antiviral efficacy in vivo. treatment of fv-infected mice with the ifna subtypes , or , but not led to a significant reduction in viral loads. decreased splenic viral load after ifna treatment correlated with an expansion of activated fv-specific cd + t cells and nk cells in the spleen, whereas in ifna -and ifna -treated mice it exclusively correlated with the activation of nk cells. other ifna subtypes like ifna , and are under investigation pd / elimination of immunodominant epitopes from multispecific dna-based vaccines allows induction of cd t cells that have a striking anti-viral potential immunodominance limits the tcr diversity of specific, anti-viral cd t cell responses elicited by vaccination or infection. to prime multispecific t cell responses, we constructed dna vaccines that coexpress chimeric, multidomain antigens (with cd t cell-defined epitopes of the hepatitis b virus (hbv) surface (s), core (c) and polymerase (pol) proteins, and/or the ovalbumin (ova) antigen as stress protein-capturing fusion proteins. priming of mono-or multispecific, hla-a* -or k b -restricted cd t cell responses by these dna vaccines differed. k b /ova - -and k b /s - -specific cd t cell responses did although chronic infections remain asymptomatic in most cases, immunocompromised patients can suffer from severe and life-threatening ebv-associated diseases, such as posttransplant lymphoproliferative disorders (ptld). thus, immunotherapeutic strategies using adoptively transferred ebv-specific t cells are promising. one option is the generation and expansion of cd + and cd + t lymphocytes by using ebv-specific synthetic peptides for the stimulation of pre-existing memory t cells. aim of our study was to identify a set of mhc class-ii peptides for each antigen promiscuitive peptides with high syfpeithi scores were tested for immunogenicity using an ifn-g-elispot. pbmcs of at least healthy, randomly chosen blood donors were cultured for days in the presence of each candidate peptide. functional and phenotypic analysis of t cells of several donors was performed by multicolor flow cytometry. out of tested peptides could be identified as t-cell epitopes. two of them were defined as immunodominant, as more than % of tested blood donors showed peptide-specific t cell responses. so far, eight of the tested peptides could be identified as mhc class-ii epitopes. furthermore, a highly immunodominant class ii peptide mix consisting of peptides was selected. in conclusion, we could identify several new ebv-specific mhc class-ii epitopes which can be used for united kingdom, hospital de clínicas during persistent hbv infections, patients usually develop poor or no protective immune responses against viral antigens, which not only leads to the chronicity but also the unresponsiveness to conventional treatments.in order to overcome the unresponsiveness and to generate an effective therapeutic strategy for treatment for chronic hbv infections a chimeric tcr against hbsag, which aims to increase the percentage and quality of antigen-specific cd + t cells, was developed moreover, we pre-conditioned the liver microenvironment by injection of cpg oligodeoxynucleotides (odn) to optimize the recruitment of transferred cd + t cells to the liver and to overcome the tolerogenic microenvironment of the liver. we found that the il- -exposed cd + t cells showed at least five-fold increase of survival rate in vivo than il- -exposed cd + t cells did treatment of the recipients with cpg-odn could increase the percentage and also the total amount of transferred cd + t cells mainly in the liver. by in vivo brdu incorporation, we demonstrated that the higher in vivo survival rate of il- -exposed cd + t cells and the effect of cpg-odn were due to the up-regulation of the proliferation of those cells. to sum up, the cocktail therapeutic strategy could not only increase the survival rate of transferred cells but also direct the antigen-specific cd + t cells to the liver to exhibit their effector functions. the detailed mechanisms responsible for the il- and cpg-odn effects on the regulation hyper igm (him) and wiskott-aldrich syndrome (was) than those of corresponding controls (p x . ) . there was a significant elevation of t ada and ada activities in iga deficient patients as compared to healthy individuals (p x . ) . our results hypothesized that altered ada activity may be associated with altered immunity. therefore, serum ada level could be used as an indicator along with other parameters pd / hiv- sequence evolution after dendritic cell-based immune therapy in a phase i/ii clinical trial hiv rna was extracted from plasma samples collected before the startof haart and early after vaccination when haart was terminated. rna was amplified by rt-pcr and sequenced using standard protocols. sequences of the vaccine genes tat, rev and nef as well as control genes vif, vpr, vpu and parts of env were analyzed for variation between pre-and post vaccination time points. hiv sequences spanning known and predicted epitopes of the relevant hla alleles from each participant were analyzed in detail. results and conclusion: immune therapy was well-tolerated and no severe adverse effects occurred. after haart termination, plasma viral load became detectable in all patients after - weeks. follwing the viral rebound a set point was reached, that was lower than the viral load before start of haart. using various methods we evidenced newly induced or enhanced immunity after immune therapy (see abstract b. de keersmaecker et al.). for studying sequence evolution, complete sets of both pre-haart and post-vaccination sequences were obtained in out of patients. with one exception, variation in sequences of vaccine and control genes of pre-haart samples compared to samples taken early after vaccination was limited. this indicates that there was no significant impact of the immune response on virus evolution at this stage. more focussed analysis on viral sequences spanning specific hla islamic republic of newcastle disease (nd) is regarded throughout the world as one of the most important diseases of poultry, not only due to the serious and high flock mortality, but also through the economic impacts. the purpose of this study was to be informed from the possible influence of infectious bronchitis virus on immune response of chickens to nd live vaccine. one hundred and twenty, -day-old ross broiler chickens divided randomly into groups, experimental and a group as control one. the first experimental group vaccinated by a monovalent nd live vaccine with cl/ strain, and the second experimental group vaccinated by a bivalent newcastle disease and infectious bronchitis live vaccine with cl/ and h strains, via the drinking water at days of age at the same time, and the control group received no nd vaccine. the antibody response to vaccination was assessed using the hemagglutination inhibition (hi) test by taking blood samples three times, first the day before and the next, & days post vaccination. results indicated that, although the strain of studied nd live vaccines were the same united kingdom t cell-based ifng release assays from blood are an important advance for diagnosing tuberculosis infection but do not permit reliable treatment monitoring or distinction of active tb from successfully treated disease or latent infection. t-cell cytokine profiles vary with in vivo antigen load in viral infections cd t cells from patients with active tb and patients with successfully treated tb were analysed for simultaneous expression of ifng and il at the single cell level using multi-colour flow-cytometry after hours stimulation with ppd. moreover, cells were stimulated with esat- and cfp- receiver operator characteristics analysis revealed that a percentage of ifng /il dual positive cells x % served as an accurate marker for active tb patients (specificity %, sensitivity %), while frequencies g % were observed in treated as well as active tb patients. in conclusion, quantitation of antigen-specific t cells based on the analysis of ifng only does not allow distinction of patients with active and successfully treated disease pd / necessity of postpone bcg vaccination -lesson from primary immunodeficiencies v. thon methods and results: the czech national database of primary immunodeficiencies (pid) was established in and is connected with the european database of primary immunodeficiencies (esid). the prevalence of pid in the czech republic (approximately inhabitants) is . to . among these patients there are children diagnosed with severe combined immunodeficiency (scid) and chronic granulomatous disease (cgd) too. according to the czech national database of pid, out of children with later proved scid were immunised with bcg vaccine in the first days of life. nine of them developed disseminated and generalized bcg infections. five children with scid died. moreover, reactivation of bcg was also seen in healthy children after admission of combined vaccines with hepatitis b given at the age of twelve weeks. on the other hand, this was not the case in thousands of children of hbsag positive mothers who were vaccinated against hepatitis b after delivery in the first place and later immunized with bcg vaccine. systematic vigilance against tuberculosis (tb) and vaccination significantly lower the prevalence and risks of tb. in the czech republic, the prevalence of tb is currently . to inhabitants. unfortunately, temporary interruption of bcg vaccination in three large districts in the period of to led into higher incidence of tb and appearance of new cases of aviary mycobacteriosis. these complications were not observed in vaccinated children. conclusion: we recommend a change of current practice of bcg vaccination considering new immunization schedule with hexavalent vaccine pd / novel analogues of thalidomide inhibit cd expression and production of tnf-a, il- , ifn-g, cxcl- this work describes the synthesis and characterization novel thalidomide analogues, prepared in good yields using simple methodology. our results suggest that anti-inflammatory and immunomodulatory activity of these diamine compounds is potentially applicable in treating enl and other diseases. supported by: cnpq, fapemig and capes, brazil. of the b cell follicle. cta -dd augmented gc-formations, specific antibody responses and cell-mediated immunity to the t cell-dependent antigen np-cgg, but failed to do so when used together with t cell independent antigens, such as np-ficoll or np-dextran. this effect required adp-ribosyltransferase activity, as mutant cta r k-dd failed to exert an adjuvant effect. the adjuvant function appeared to correlate with the fdc-localization and turned out to require complement and/or complement receptors (cr) chitosan formulations varying in molecular weight, counterion and structure (i. e. soluble v/s particulate) were used in assays to examine expression of maturation markers via flow cytometry and cytokine production by elisa. we found that, in contrast to alum, plg and ps particles, chitosan induced bmdc maturation on its own, as determined by the expression of cd and cd . these effects were most prevalent with soluble chitosan chloride formulations but were also notable with soluble chitosan glutamate chitosan. the effect of chitosan on cytokine production was investigated using a panel of different tlr agonists in combination with chitosan particles. results show an increase in the secretion levels of il- a and il- b, while il- levels were not affected. finally we studied the role of inflammasome activation in the enhancement of il- b production. using bmdc from nlrp -/-mice we examined il- b production in response to different tlr and chitosan combinations. results show that the ability of chitosan to enhance il- b production is dependent on nlrp . collectively our data indicate that upregulation of maturation markers and enhancement in proinflammatory cytokine secretion mediated by chitosan severe sepsis, induced in mice by cecal ligation and puncture (clp), led to ho- expression in infiltrating peritoneal leukocytes, kidney and liver. mortality rate of clp increased from % in wild type (hmox +/+ ) mice to % in ho deficient (hmox -/-) mice. hmox -/-but not hmox +/+ mice developed end-stage multiorgan failure. mortality of hmox -/-mice was associated with increased peritoneal leukocyte infiltration, but not with increased pro-inflammatory cytokine secretion or bacterial load in peritoneum, blood or organs. clp induced a significant increase in cell-free hemoglobin free heme was found to sensitize primary hepatocytes to tnf, anti-fas antibody, h o or peroxynitrite mediated apoptosis. this cell death was associated with outward nuclear translocation and extra-cellular accumulation of the late-stage pro-inflammatory cytokine hmgb . similarly, circulating and cytoplasmic hmgb was increased in hmox -/-relative to hmox +/+ mice following clp. in conclusion, these data suggest that free hemoglobin and heme, released during severe sepsis, are important factors in the organ failure and death associated with severe and b- , -linked mannose residues elicit inhibition effect. it was found that inhibition activity of oligosaccharides increases with chain length. immunization with mannan-hsa conjugate allowed for the maturation of immune response generating specific antibodies with high avidity/affinity, whereas immunization with mannan alone elicited only low-affinity antibodies. in the future, an effective antifungal subcellular vaccine would be constructed using selected mannooligosaccharidic epitope and the appropriate carrier protein as inductor of immunological memory. acknowledgements: this work was supported by the grant agency of slovak academy of sciences all subjects received dtwp vaccine at - years of age (booster vaccination), following the national vaccination schedule of iran. blood samples were collected before and - weeks after the vaccination. immunogenicity of the vaccines was assessed by elisa using commercial kits. results: the geometric mean titers (gmt) of the antibodies induced against diphtheria and tetanus by dtwp-local were . and . iu/ml and those of dtwp-pasteur were . and . iu/ml, respectively. there was no significant difference between the immunogenicity of the two vaccines against diphtheria and tetanus. the gmts of antibodies produced against pertussis were . eu/ml for dtwp-local and . eu/ml for dtwp-pasteur vaccines, respectively (p x . ). no significant differences were observed in the antibody titers against diphtheria, tetanus and pertussis between the two vaccines before vaccination. conclusion: immunogenicity against diphtheria and tetanus was similar for the two vaccines pd / united kingdom haemorrhagic septicaemia (hs) is an acute disease of cattle and buffaloes in tropical countries, caused by pasteurella multocida serotype b: , a gram-negative coccobacillus. jrmt , an aroa mutant of pasteurella multocida, constructed previously in our laboratory, attenuated for virulence in the mouse and protects mice from challenge with the virulent strain. in this work, the immune response of calves was tested after intramuscular vaccination with single dose of cfu of jrmt . a possible contributory role of cellular immunity against hs was investigated in vaccinated and in control calves after challenge. a lymphocyte stimulation assay was used to assess the effects of a cell-free extract (cfe) of p. multocida on peripheral blood mononuclear cells (pbmcs) isolated from calves at different times after challenge. the results were indicative of a possible immunosuppressive effect of challenge with p. multocida b: on calf pbmcs. the suppressive effect was further investigated by in vitro experiments. calf pbmcs obtained from normal calves were treated with cfe for h before adding concanavalin-a (cona) pd -vaccination and immunotherapy against parasitic diseases pd / evaluation of simian adenoviral vector adch expressing msp- as a candidate blood-stage malaria vaccine this successful regime incorporated a human adenovirus serotype (adhu ) prime, boosted eight weeks later with a modified vaccinia virus ankara (mva) vector. adenoviral vectors have generated great scientific interest in recent years and appear to be superior viral vectors with great potential in vaccine regimes. their potential use in humans, however, is limited by natural anti-vector immunity to human adenoviruses, but this problem could be largely circumvented by the use of simian adenoviral vaccine vectors. recent clinical trials have suggested that the simian adenoviral vector adch is a promising clinical candidate. we have developed vectors (of human and simian origin) and mva encoding a novel construct based on p. falciparum msp- and have undertaken comparative immunogenicity studies in mice. the antigen, termed 'pfm while asymptomatic per se, the heterozygous sickle cell trait confers a survival advantage against malaria, the disease caused by plasmointo carbon monoxide (co), iron and biliverdin. when infected by plasmodium hb sad mice are protected against experimental cerebral malaria (ecm), a lethal neuroinflammatory syndrome that in many aspects recapitulates human cerebral malaria. ho- expression and activity are strictly required to suppress ecm in hb sad mice, as demonstrated by functional deletion of the hmox locus or pharmacologic inhibition of its enzymatic activity. the protective effect of ho- is mediated by co, which inhibits the accumulation of protein-free heme in plasma following plasmodium infection conclusion: topical treatment of cutaneous leishmaniasis with gsno accelerated healing and reduced local parasitism in the mouse suggesting that it may be ben gp expression was confirmed by sds-page and elisa using monoclonal antibody against gp . discussion: today researchers attempt to find a suitable vaccine for leishmaniasis. although some researchers have reported proper vaccines of interest, a - recp is recognized only by sera collected from resistant bovines infested with all stages of r. microplus, but not by sera from similarly infested, susceptible hosts. furthermore, this recognition was specific since sera from resistant non-infested bovines (naï ve animals) did not react with a - recp. our results show that reverse immunogenomics can be useful for discovery of new antigens for development of an anti-tick vaccine. supported by cnpq and fapesp. for the maintenance of ab-mediated vaccine-induced protection after re-challenge with the pathogen or the vaccine antigen. memory b-cell elispot together with ab titres might therefore prove useful as independent marker for duration of protection. objective: this study focused on establishing experimental conditions and optimizing the performance of the memory b-cell elispot assay by detection of specific memory b-cells against anti-tetanus vaccine and naturally acquired toxoplasma gondii infection as a model. methodology: twelve healthy subjects who had received the tetanus vaccine at least month previously were enrolled. peripheral blood mononuclear cells (pbmcs) were isolated from each donor using cell preparation tubes (cpt). plasma was obtained after centrifugation of cpt and stored at - °c until used for elisa. specific igg-secreting b-cells were determined by elispot assay, using tetanus toxoid (tt) and t. gondii surface antigen as model antigens. results: to optimize our assay, conditions were changed and compared to the previously established protocol. we detected low frequencies of total igg memory b-cells and tt-specific memory b-cells in all donors four seropositive and seronegative donors had positive responses in elispot. no correlations were found with serum antibody titers and frequencies of memory b cells (r= . , p= . ) or with t. gondii-specific b-cells conclusions: following optimization of several assay parameters, we demonstrated that the memory b cell elispot could be reliably used to determine low numbers of antigen-specific memory b-cells in individuals naturally exposed to infection or following vaccination our previous work demonstrated that il- also affects the cells of erythroid lineage, by stimulating development of early erythroid progenitors, bfu-e, but inhibiting the growth of late stage erythroid progenitors, cfu-e, from normal murine bone marrow. we also provided in vitro evidence that at least part of its effect on cfu-e is mediated by nitric oxide (no) generation. in the present study we demonstrated that the in vivo reducing effect of il- on bone marrow cfu-e was prevented by co-treatment with the no synthase (nos) inhibitor, l-name, implying that this effect is mediated through nos activation. the data obtained in cultured bone marrow cells showed the ability of il- to upregulate the expression of mrna for both the inducible (i)nos and the constitutive, endothelial (e)nos isoform. both the nos-inducing effect of il- and il- -related inhibition of cfu-e growth were dependent on p mapk activity, since the p mapk inhibitor, sb , markedly downregulated il- -induced activation of nos and reversed the growth inhibitory effects of il- on cfu-e. the in vivo stimulating effect of il- on bfu-e colony growth in the bone marrow was not affected by co-treatment with the nos inhibitor, pointing to different mechanisms for il- effects on bfu-e and cfu-e. however, the in vivo exposure of the mice to l-name, increased the number of various hematopoietic progenitor cells in the bone marrow, indicating that no itself is important regulator of hematopoietic progenitor cell activity. overall, the data presented gave an insight into the mechanisms by which il- acts on bone marrow cells and also revealed a link between the il- , no and hematopoiesis. further studies on il- -mediated induction of both inos and enos methods: a total of blood donor samples were tested for hbsag and anti-hbc with the immunoenzymic method elisa, while simultaneously, molecular blood test (nat) was applied. the positive samples for anti-hbc were also tested for anti-hbs and anti-hbc igm. results: a total of samples ( , %) were found anti-hbc positive conclusions: it is proven, therefore, that in some cases the levels of hbsag, following an infection from the hepatitis b virus, are probable to remain low, so that it is not possible to detect them using elisa method. in these cases anti-hbc can be the only serological marker of the infection. consequently, patients with positive anti-hbc and levels of anti-hbs x iu/l are possibly not immune enough, so that they can become blood donors. that was the reason why some blood donation centers in our country, until recent years when there was no capability for nat testing of blood donors, had iu/l as a limit for anti-hbs levels. however, in present days that nat testing of blood donors is used in our country, it has offered great safety and it is possible that anti-hbc testing will not be necessary, despite the fact that many blood donor centers have preserved the safety limit of iu/l anti-hbs in all the blood units, which also goes for our study pd / neonatal allo immune thrombocytopenia and igg glycosylation patterns michaelsen , national institute of public health in milder cases it can cause petechia and in more severe cases it can cause intracranial hemorrhage and death. the reason behind the variation in clinical symptoms is not fully understood, but is probably not due to differences in immunoglobulin isotypes or antibody affinity. recently influence of glycosylation patterns of igg on the biological activity has been realized. variation in carbohydrate structures attached to asparagine can cause differences in the interaction with fc-receptors, and hence a difference in thrombocyte elimination capacity of the igg molecule. patient sera from norway and the netherlands with different levels of antibody titres and severity of symptoms have been used to affinity isolate igg antibodies against the hpa- a alloantigen and analyze the glycopeptides using mass spectrometry. the glycosylation patterns have been analyzed for a possible link between severity of symptoms and variation in the glycosylation patterns. so far patients with serious symptoms seem to have increased galactosylation and sialylation and a high level of non core-fucosylated n-glycans on their anti-hpa- a iggs we monitored children ( boys and girls) in ages from . to . years with average age of . years. in of them all was diagnosed for the first time. subject had the second relapse of all. one patient received maintenance chemotherapy, all the rest ( subjects) induction chemotherapy. methods: leukocyte count and hemiluminescent analysis of whole blood were performed for all the patients during infectious complications twice: on neutropenia background and after the recovery of neutrophil number. hemiluminescent analysis for whole blood allows to estimate the functional activity of phagocytes, namely their bactericidal power and phagocytosis completeness. we valuated spontaneous and zymosan induced hemiluminescence. we used onsonised zymosan as the inductor of "respiratory paroxysm mice with a homozygous mutation in the rc h gene, that encodes the zinc finger and ring finger containing protein roquin, develop severe autoimmune disease. the observed lupus-like phenotype involves follicular helper t cells, which express higher levels of icos. these cells provide inappropriate t cell help to b cells, leading to the production of autoantibodies (vinuesa et al. , nature , - ). it has been shown that the half-life of icos mrna is shortened when roquin is over-expressed. such repression requires the 'utr of icos, in which a bp sequence, containing a possible mir- binding site, was sufficient (yu et al. , nature, , - ). mnab is the paralogue of roquin, and has been shown to bind to nucleic acids (siess et al. , j biol chem , - ). we demonstrate that in primary mouse t cells and embryonic fibroblasts roquin, but not mnab, inhibits translation of icos. we map critical domains in the roquin protein to icos repression using deletion-and point-mutants of roquin, as well as chimaeras that swap sequences from roquin to mnab and vice versa. addressing the mechanism of roquin mediated icos repression; we demonstrate binding of roquin to icos mrna in primary mouse t cells and in cotransfection experiments. our current work dissects the requirement of cellular rnai, the stress response pathway or p-body function by testing roquin repression of icos mrna in dicer-, tia- -and ago -deficient mef cells and in knockdown approaches. acknowledments: j m m-v is a recipient of a harvard real colegio complutense (rcch) grant. work in dr tsokos' lab is supported by grant phs nih r ai . we have recently reported that -hydroxyl- -methylindole- -acetonitrile ( -hma) isolated from brassica rappa inhibit nuclear factor-kappa b (nf-xb) activity in raw . macrophages. in this report, we investigated the effect of -hma on dextran sulfate sodium (dss)-induced colitis model in mice. methods: we induced colitis with dss in mice and evaluated disease activity index (dai), including body weight, stool consistency and gross bleeding, and tissue myeloperoxidase (mpo) accumulation. through h&e staining, histological change was observed. the expression of inducible nitric oxide synthase (inos), inhibitory kappa b-a (ixba) and nf-xb were detected by western blot and immunohistochemical staining. in-vitro system, the expressions of interleukin- (il- ), monocyte chemotactic protein- (mcp- ) in ht- human colon epithelial cells were measured by rt-pcr. results: in dss colitis model, the dai score and detection of mpo accumulation brevealed -hma significantly inhibited loss of body weight, suppression of diarrhea and bleeding, and infiltration of macrophages, leukocytes. moreover, h&e staining also indicated -hma suppressed the thickness of muscle layer, edema, mucosal damages by dss. these results were related to the regulation of nf-xb activation. -hma attenuated the dss-induced phosphorylation and translocation of nf-xb subunit p . in addition, this effect was accompanied with parallel blocking degradation of ixba. moreover, pretreatment of -hma significantly reduced the mrna levels of il- and mcp- stimulated by tumor necrosis factor-a (tnf-a) in the ht- cells. pretreatment of -hma also significantly blocked the ixba degradation and nf-xb p nuclear translocation stimulated by tnf-a in the ht- cells. these results were concurred with the effect on nf-xb activation in dssinduced colitis model. conclusions: these results for the first time demonstrated that alleviation of -hma mediated by regulation of nf-xb activation and suppression of chemokines in vitro and in vivo. therefore, -hma could be new potential therapeutic agent for inflammatory bowel disease.cd serves as receptor of hiv and is a self-antigen. we have previously characterized the anti-cd igg immune response in hiv- -exposed, seronegative (esn) subjects and we know that there is a peculiar specificity of these antibodies for epitopes induced by gp -binding and that there is an epitope specificity distinct from that seen in hiv-infected patients (second cd domain preferred). to generate antibodies able to inhibit the infection of hiv virus trying to learn from what happen in nature in esn we used a particular immunization procedure. we immunized mice with autologous cells expressing gp , reacted with the external domains of soluble human cd , in the absence of the target cells expressing the co-receptor ccr . the latter is the membrane molecule, which allows the complete reshuffling of the epitopic make-up of the cd -gp complex and trigger the membrane fusion between effector (gp expressing) cells and target (ccr expressing) cells. thus, in the absence of ccr we specifically enriched our immunogens with "frozen" conformational intermediates, that are presumably transiently exposed on the cell membrane during hiv- infections. a conventional protocol for the generation of monoclonal antibodies was used. db- (igg , x), one of the anti-cd antibodies obtained, recognized preferentially cd complexed to gp , as compared to cd alone, not competed for the gp binding site on cd and was specific for the second extracellular domain of cd . g. röder , l. geironson , a. darabi , m. harndahl , c. schafer-nielsen , k. skjödt , s. buus , k. paulsson copenhagen university, institute of international health, immunology and microbiology, department of experimental immunology, copenhagen, denmark, lund university, immunology bmc d , lund, sweden, lund university, rausing laboratory, division of neurosurgery, department of clinical sciences, lund, sweden, schafer-n, copenhagen, denmark, department of immunology & microbiology, university of southern denmark, odense, denmarkcytotoxic t-lymphocytes become activated by binding to mhc-i molecules presenting antigenic peptides. the loading of peptides onto mhc-i takes place in the er and involves different chaperones and enzymes. tapasin binds mhc-i molecules, integrates them into peptide-loading complexes, and assures that only 'optimal peptides' are bound to surface exported mhc-i molecules. how tapasin exerts this quality control, and the criteria for being an optimal peptide, are still unknown. here, we have generated the first n-terminal amino acids of human tapasin, tpn , and shown that this fragment of tapasin facilitates peptide dependent folding of hla-a* . to further investigate the properties of tpn and tapasin, we generated multiple mouse monoclonal antibodies towards tpn and wildtype human tapasin. one clone, atpn - / , was found to be specific for natural human tapasin and stained cellular er localized tapasin. using peptide chip technology, the epitope of atpn / was demonstrated to be located on tapasin [ ] [ ] [ ] [ ] [ ] , which recently was shown to be a surface-exposed loop of the tapasin structure. together, these results demonstrate that, the first n-terminal amino acids of tapasin are able to facilitate peptide-binding to mhc-i, and as well, this fragment can be recombinantly expressed in e.coli and fold into a structure, which at least partially, resembles that of wild-type human tapasin. we speculate that this region of tapasin might support empty, open and receptive mhc-i peptide-binding clefts effectively allowing an otherwise inherently unstable molecule to exchange peptide; i. e. this tapasin region might be essential for enabling peptide editing. a objectives: antigen processing and presentation through hla class i molecules is critical for an effective destruction of infected or transformed cells by cd + t lymphocytes. different intracellular routes governing the processing of endogenous and exogenous antigens have been described. we show here a strategy to introduce epitopes inside the cells for a productive cross-presentation to ctls. methods: to produce genetic in-frame tat fusion proteins, dna sequence encoding for the amino acid region - of the influenza a virus nucleoprotein (np) was inserted into the expression vector ptat-ha. starting from tatnpflu recombinant protein we produce hybrid proteins, in which the hla-b* -restricted np-flu epitope (aa - ) was replaced by hla-b or hla-a -restricted epitopes of ebv and hcv, respectively. cross-presentation was evaluated according to the standard cr release assay and through the ifn-g production. results: using hla-b or hla-a restricted viral epitopes we show that the two molecules cross-present the epitopes following two different pathways of processing: the hla-b molecules follow a proteasome-independent pathway which is active in different cell types, whereas the hla-a molecules present the epitopes in a classical proteasome-dependent pathway performed by dcs. furthermore, different hla-a restricted epitopes can be inserted in tandem and presented to the specific ctls without interfering each other. the data reported here offer new insights on how a same construct containing multiple epitopes from different viral or oncogenic proteins could be designed for vaccinal strategies. these findings also enlighten hla-b as a remarkable hla-class i molecule that, differently from hla-a , can present peptides through additional, unconventional antigen presenting routes. this could concur to an imbalance of the immunological properties of the hla-b molecules leading to a more effective response towards viral as well as self -antigens. objectives: although cytotoxic t cells (ctl) in human immunodeficiency virus (hiv- )-infected individuals can potentially target multiple virus epitopes, the same few are repeatedly recognized. ctl play a key role in limiting viral replication in infections caused by e. g. epstein-barr virus, cytomegalovirus, hepatitis c virus and hiv . consistent patterns of immunodominant and subdominant ctl-responses have been found between individuals with the same hla-alleles in both acute and chronic infection. as the ctl-response frequency in a population closely correlates with its relative magnitude in an infected individual, the terms immunodominance/subdominance have been used in both contexts. however, the factors determining these ctl-response hierarchies are largely unknown. while structural differences between peptide-hla class i complexes may be important for tcr-repertoire selection and clonal expansion, it is less obvious how they impact ctl-response hierarchy formation and timing. other factors may also contribute, e. g. epitope abundance at the cell surface. methods: antigen processing efficiency of ctl epitopes from the p -gag and p region was determined in vitro. mer peptides were digested with i s and c proteasomes and the fragments identified by mass spectrometry. for epitope precursor peptides generated by the proteasome, we then determined tap affinity, trimming by eraap and hla-binding affinities and analyzed patient responses by elispot. results: we show that ctl-immunodominance in regions of hiv- p -and p -gag correlates with epitope abundance, which is influenced strongly by proteasomal digestion profiles, transporter-associated-with-antigen (tap) affinity and endoplasmatic reticulum aminopeptidase (eraap)-mediated trimming, and moderately by hla affinity. proteasomal cleavage-preferences were affected by flanking and intra-epitope ctl-escape mutations and could modulate the number and length of peptide-epitopes, thereby affecting t cell response avidity and clonality. conclusion: our analyses reveal that antigen processing plays a pivotal role in determining ctl-response hierarchies, that viral evolution may modify cleavage patterns, and suggest strategies for in vitro optimization of ctl-epitope-based vaccines. t. f. gregers , g. koster , o. landsverk , f. skjeldal , o. bakke university of oslo, molecular biosciences, oslo, norway mhc ii is synthesized and assembles in the er together with invariant chain (ii). ii facilitates mhc ii assembly followed by transport to the mhc ii loading compartment (miic) where peptide loading occurs. miic is multivesicular late endosomal compartments resembling conventional multivesicular bodies (mvbs) found in all cells. it is not known whether the biogenesis of miics is regulated by the same mechanisms as formation of mvbs. expression of ii induces the formation of enlarged endosomes and we have previously shown that ii modulates antigen processing and presentation. we have suggested that ii itself can act as a tethering factor involved in fusion of ii containing endosomes, and our main question is whether ii can regulate the formation of an endosomal pathway dedicated for antigen processing and mhc ii loading.in order to investigate this we use cell lines expressing ii controlled by an inducible promoter, thus being able to control the ii expression level and thereby the endosomal size. live imaging and high through put microscopy of ii expressing cells treated with inhibitors and/or specific sirnas have revealed that ii induced endosomal fusion is independent on type iii pi kinases and thus ptdins( )p. this is in contrast to conventional endosomal fusion and mvb formation. thus other factors might be important for miic biogenesis. by using small rnai libraries targeting proteins known to be involved in endosomal pathways and microscope based screening we aim to identify factors that are able to knock out the formation of enlarged endosomes in ii expressing cells, and thus potentially identify molecules defining an antigen presenting cell. m. bouvier , l. visvabharathy , j. fu university of illinois at chicago, microbiology and immunology, chicago, united statesobjectives: adenoviruses (ads) cause persistent infections. the e - k protein from ad targets class i mhc molecules for retention in the endoplasmic reticulum (er), thereby preventing the cell-surface presentation of viral peptides. this escape from immune surveillance allows ads to freely replicate in host cells. the molecular mechanism of e - k-mediated class i retention is mostly undefined. it is clear that further characterization of this mechanism is important to understand the susceptibility of the class i antigen presentation pathway to immunomodulatory proteins and to elucidate the molecular basis of ad pathogenicity. we used biophysical and cell-based approaches to examine interaction between ad type e - k and class i molecules.results: we showed that e - k associates with immature (peptide-deficient) and mature (peptide-filled) hla-a molecules, with the mature form being more tightly associated. we also provided evidence that e - k does not compete with the class i assembly proteins for binding onto class i molecules. importantly, immature class i molecules sequestered by e - k can still bind peptides. together, these results suggest that ads have evolved to interfere with the early and late stages of the class i antigen presentation pathway. evidence was also provided that e - k displays an allele-and locus-specificity towards class i molecules with high-density lipoprotein (hdl) reduces the risk for atherosclerotic cardiovascular disease by promotion of cholesterol efflux from macrophage foam cells and by antioxidative as well as anti-inflammatory properties. recent data indicate that qualitative changes of hdl including oxidative modifications and alterations of the protein cargo of hdl may alter its biological activity. here we analyzed the anti-inflammatory potential of hdl and compared it with hdl obtained from patients with end-stage renal disease (esrd), which are characterized by a proinflammatory state and an associated significantly increased cardiovascular mortality. we demonstrate that freshly isolated, but not oxidized hdl from healthy individuals exerts profound anti-inflammatory properties on professional antigenpresenting cells (apc) such as monocytes and dendritic cells, which are regarded as the most potent apc. production of typical proinflammatory cytokines (il- , il- , tnf-a) were significantly suppressed by hdl after stimulation of monocytes or dendritic cells with toll-receptor ligands and , but also with the t-celldependent stimulus cd l (cd ) indicating an immunomodulatory effect independent of agonist neutralization by hdl. moreover, surface expression of crucial activation and costimulatory molecules like cd , cd , and cd was inhibited by freshly isolated, but not oxidized hdl. the negative regulatory effect of hdl on cytokines and surface receptors occurred at the transcription level, while hdl did not modulate the activity of the major inflammatory transcription factor nf-kb or the map kinases p and erk- / . strikingly, hdl from esrd patients not only failed to block, but rather promoted proinflammatory cytokine production and apc activation. these data identify hdl as a novel potent anti-inflammatory regulator of professional apc, which may help to dampen excessive inflammatory responses of the innate immune system. conversely, qualitative changes of hdl leading to a loss of its anti-inflammatory function might contribute to a proinflammatory state that is linked with excessive cardiovascular mortality in esrd patients. objectives: cd + t cell abnormalities may play a role in the autoimmune pathogenesis of churg strauss syndrome (css). on one side, th (il- +) cells may sustain autoantibody formation and eosinophilia, which are hallmarks of css. on the other, th (ifn-g+) cells could participate in vessel wall damage and granuloma formation. in order to define this th / th balance and to identify potential t cell target antigens (ags), we analyzed circulating cd + t cell responses to polyclonal stimuli and to myeloperoxidase (mpo) in css and healthy subjects. methods: ifn-g and il- expression in peripheral blood cd +cd + lymphocytes were measured in ccs patients and healthy subjects (hs) upon polyclonal stimulation, both by intracellular staining and by elisa. mpo-driven il- /ifn-g production was assessed by elispot on t cells co-coltured with autologous dendritic cells, stimulated either with heat-inactivated mpo, negative control protein or hexavalent vaccine (positive control recall ags). results: upon polyclonal stimulation, higher il- and lower ifn-g intracellular expression were detected in cd + t cells from css patients, as compared to hs (il- : . ± . % vs. . ± . %, p x . ; ifn-g: . ± . % vs. . ± . %, p x . ). similar results were obtained by elisa (il- : . ± . vs. . ± . pg/ml, p x . ; ifn-g: . ± . vs. . ± . pg/ml, p x . ). elispot counts of hexavalent vaccine-stimulated cd + cells were positive for il- in / ( %) css patients and in / ( %) hs, and for ifn-g in / ( %) css patients and / ( %) hs. mpo stimulation determined significant ifn-g release in / ( %) css patients, but not in hs ( / ) no il- response to mpo in both groups was observed. conclusion: polyclonally or recall ag-stimulated cd + cells from css patients show a th -polarized cytokine profile. mpo is here first identified as a css-related ag targeted by cd + t cells, and responses towards it are instead th -polarized. these data unfold one molecular target and possible pathogenic mechanisms of cd + t cells in css. a. voigt , e. opitz , k. savvatis , k. klingel , k. stangl , u. kuckelkorn , p.-m. kloetzel charité -universitätsmedizin berlin campus mitte, berlin, germany, charite -campus benjamin franklin, berlin, germany, universität tübingen, tübingen, germanymurine models of coxsackievirus b (cvb )-induced myocarditis mimic the divergent human disease course of cardiotropic viral infection. immunoproteasomes (ip) are crucial in the modulation of adaptive immune responses, in the maintenance of protein homeostasis and in the preservation of cell viability under stress conditions. our previous work has established that ip expression in the infected myocardium is linked to a strong enhancement of viral epitope generation.here, we investigated the impact of ip function in enterovirus myocarditis. mice, which are deficient in immunosubunit lmp of the stress-induced ip, were infected with x e pfu cvb nancy strain. in concurrence to wt littermates, we observed a pronounced up-regulation of cardiac ip subunit lmp as early as day p. i. in lmp -deficient mice. however, lmp -deficiency was linked to less severe myocarditis at day p. i. (he stain of cardiac tissue sections: wt . ± . vs. lmp -deficiency . ± . (grade of myocarditis; scale - ; p x . ). whereas the cardiac output (co) was reduced in wt littermates in enterovirusmyocarditis (p x . ), there was no difference in lmp -deficient mice in comparison to sham-treated mice. maximal left ventricular pressure and dpdt max were impaired in acute myocarditis in wt littermates. in contrast, systolic function was not affected by cvb infection in lmp -deficient mice. likewise, diastolic function was preserved in lmp -deficient mice upon enterovirus infection. our findings of less severe myocarditis in lmp -deficient mice were associated with tremendously reduced viral load in the myocardium of this strain.in conclusion, this study suggests an impact of lmp -immunosubunit function in regulatory processes of viral replication. absence of lmp confers host protection in enterovirus myocarditis. h. w. liao , j. xu , j.q. huang sun yat-sen university, guangzhou, chinathe characteristic of the dengue hemorrhagic fever/dengue shock syndrome (dhf/dss) is hematologic abnormality, which results from multiple factors including thrombocytopenia, coagulopathy and vasculopathy. the pathogenesis of endothelial dysfunction associted with vascular leakage syndrome however remains unknown. in this work, we showed that dengue virus serotype (den- ) strain induced apoptosis in human umbilical vein endothelial cells (huvecs). additionally, fas expression was increased on infected huvecs. trailr and tnfr - were constantly very low whereas trailr - decreased after den- infection. fasl was expressed at similar levels on huvecs throughout den- infection. the apoptotic rates in huvecs were decreased upon addition of caspase family inhibitors and activated caspase , caspase were also observed by western blot after by den- infection. there were no significant changes of no in our study. we thus proposed that the fas/fasl pathway might be involved in apoptosis induced by dengue virus in vascular endothelial cells in vitro. dermatolymphangioadenitis is a common complication of interruption of afferent lymphatics by cancer surgery combined with partial lymphadenectomy. it seems that skin microbes normally penetrating epidermis during hand work or walking are retained in the skin and subcutis because of lack of lymph drainage and evoke host reaction. aim. to study lymph node cellular reaction to bacterial antigens before and after ligation of afferent lymphatics. materials & methods. group i. s. epidermidis was injected daily for days into wis rat paw web tissue in saline containing . x cells. group ii. s.epidermis was injected as in group after ligation of lymphatics below the popliteal lymph node. nodes were isolated on day .they were weighed, the cell number was counted and cells were stained with mabs for immunohistochemical analysis. immunohistochemical pictures were analyzed by microimage program. results. group i. skin contained some mhcii cells. the popliteal lymph nodes became enlarged on the bacteria injected side. there was an increase in lymph node weight and cell concentration per g of tissue, compared to controls by factors , and , respectively (p x . ). immunohistochemical pictures showed increase in percentage of ox (migrating dendritic cell), mhc ii, his (granulocytes), ox (stem cells) and cd (icam i) subsets in the subcapsular , follicle, paracortex and medullary areas. group ii. after ligation of afferent lymphatics the weight of nodes was not significantly increased. skin showed presence of multiple mhc ii, ed (macrophages) and ox cells. popliteal lymph nodes contained evidently less of ox , his and mhcii cells than in group i (p x . ). summary & conclusions. afferent lymphatics transport microbial cells and/or microbes phagocytized by dendritic cells and macrophages to the regional node. local skin reaction is limited, whereas lymph nodes reveal acute reaction with mobilization of granulocytes from blood perfusing nodes. interruption of lymphatics saves nodes but skin reaction is strong and long-lasting. these observations seem to explain why damage to lymphatics during mastectomy or groin dissection is followed by recurrent attacks of skin inflammation. omega- fatty acids, and in particular docosapentaenoic acid (dha) and eicosapentaenoic acid (epa) from fish origin, have recently emerged as nutrients capable of modulating the expression of genes involved in inflammation and atherosclerosis and thus reduce the risk for cardiovascular events. our presentation focuses on the role of omega- fatty acids in the prevention and treatment of cardiovascular disease. it is based on reviewing and processing data obtained by search of scientific and medical databases. search terms used were: atheroma, atherosclerosis, cardiovascular disease (cad) ,coronary disease, antiinflammatory drugs, omega- fatty acids, epa, dha. we also searched epidemiological research web sites and screened the results of numerous controlled clinical trials which monitored the effects of omega - fatty acids consumption. the results indicate that omega- fatty acids supplementation is associated with a significant cardioprotection effect on both healthy individuals and patients with an established cardiovascular disease. omega- fatty acids appear to work by decreasing endothelial responsiveness to pro-inflammatory and pro-atherogenic stimuli, affecting molecular events not targeted by other drugs thus allowing their use as complementary treatments for the already implemented pharmacological treatments in inflammatory diseases. combined therapy with omega- fatty acids and statins shows a synergistic effect. methods: on ultracentrifugation of serum at density . and , g, top % layer contained lipoproteins only and - % layer contained lipoproteins as well as immunoglobulins. the bottom layer was shown to contain immune complexes (ic) by binding to coated anti apo(a) and detection with peroxidase labelled anti human immunoglobulins.both these forms of lp(a) were western blotted and probed with jacalin-hrp, anti-gal-hrp and anti apo(a)-hrp. anti-gal was prepared by affinity chromatography on guar galactomannan and complexed with lp(a) in vitro. ic formation by lp(a) was measured in terms of reduction in response in a new elisa for lp(a) involving addition of lp(a) sample to plate-coated jacalin, followed by anti-apo(a)-hrp detection. ic formation was also shown by migration of lp(a) from free lipid layer to ic layer below in ultracentrifugation. results: anti-gal and lp(a) could be liberated from precipitated ic using specific sugar. immune complexed lp(a) in serum was found to be more o-glycosylated, larger in size and binding more anti-gal than lp(a) in free form in western blots. while ic formations within homologous free anti-gal-free lp(a) pairs were few, those within heterologous pairs were more rampant. conclusions: lp(a) is a risk factor in vascular disorders including atherosclerosis, aneurysm, stroke and peripheral vascular diseases and is a component of atherosclerotic plaques, though mechanism of its uptake remains unclear. anti-gal comprising % of serum igg is rich in igg capable of complement fixation and macrophage mobilisation. present results offer a viable mechanism of lp(a)-mediated immune injury to vessel walls leading to vascular damage. even though no receptors have been detected for lp(a), unlike for ldl, the present results may explain the internalization of lp(a) in the form of lp(a) immune complexes by macrophages since the latter can phagocytose ic. extended specificity of the a-galactoside-specific anti-gal for t-antigen in lp(a) is akin to that observed in jacalin, pea nut agglutinin and galectin- . objective: the aim of this study was to determine if the combination of two genetic alterations, one affecting cell cycle regulation, such as the e f mutation, and other affecting b cell apoptosis control, such as bcl- over-expression, can induce the development of ais. methods: mice: mice with both genetic abnormalities were generated in a non-susceptible c bl/ (b ) genetic background. e f -/-bcl- tg were obtained backcrossing e f -/-and e f +/+hbcl- tg mice. e f -/-bcl- tg, e f -/-, e f +/+hbcl- tg and control mice (e f +/+) were followed up to mo-old. serologic studies: serum samples obtained at , and month of age were test for igg and iga ana and anti-dsdna by elisa. histopathologic studies: kidney paraffin sections of , and mo-old mice were stained with hematoxylin-eosin (h&e) and masson's trichrome to identify histological changes. immunecomplex deposits were studied by direct immunofluorescence on kidney using fluoresceinated goat anti-mouse igg, igm and iga. to evaluate b cell homeostasis, absolute number of b cell in blood, primary and secondary lymph organs were assessed by flow cytometry. in vitro proliferation was measured with [h ]-thymidine and brdu was used to assess in vivo proliferation capacity of immature b cells. results: overexpression of hbcl- tg in b lymphocytes of e f -/-mice induced the production of high titres of igg and iga ana and anti-dsdna, together with the development of a glomerulonephritis characterized by a moderated mesangial proliferation, mesangial immunecomplex deposits, mainly of the iga isotype, and the presence of tubular casts and lymphoid infiltrates with the presence of glomerular deposits. e f -/-bcl- tg mice showed an altered b cell homeostasis as demonstrated in proliferation and apoptosis studies. e f -/-mice showed neither autoantibodies nor nephropathy. this study demonstrates that the isolated deficiency of e f or the overexpression of a bcl- tg in the b genetic background do not induce an ais. when combined both genetic alterations, involving deregulation of cellular proliferation and survival affect lymphocyte homeostasis, induce a mild ais with overproduction of iga autoantibodies. an alteration in the b cell compartment, but not in the t cell compartment, seems to be underlying the syndrome described in the present work. in different mouse models of the autoimmune disease systemic lupus erythematosus (sle) loss of toll-like receptor (tlr ) abolishes the generation of antinucleosome igg a and igg b autoantibodies but exacerbates lupus disease. however, the tlr -dependent tolerance mechanism is unknown. here we show that loss of tlr in b cells of lupus prone mice prevents the generation of protective t cell-dependent self-reactive igm and thereby enhances the development of th and th t cells. transfer of a synthesized monoclonal polyreactive igm to tlr deficient lupus prone mice inhibits t cell activation and abolishes development of lupus disease. thus, these results document a protective tlr -dependent tolerance mechanism in b cells that induces the generation of self-reactive igm to prevent autoimmunity. cloning and production of polyreactive or antigen-specific igm might therefore be a powerful tool to treat autoimmunity. objectives: to investigate cytokine and autoantibody levels in serum from patients with primary sjögren's syndrome (pss), and to determine possible associations with focal mononuclear cell infiltrates, lymphoid organization, and age at the time of biopsy. methods: minor salivary gland tissue was obtained from a group of patients fulfilling the revised eu-us criteria for pss (n= ) (vitali et al. ) . ninety-seven of ( %) patients had focal mononuclear cell infiltrates corresponding to focus score (fs) g (fs+), while biopsies from / ( %) patients lacked characteristic focal mononuclear cell infiltrates (fs-). germinal center (gc)-like lesions were determined in / ( %) minor salivary gland biopsies. serum samples were used for cytokine and autoantibody evaluations. the mean level of unstimulated whole saliva was significantly lower in the fs+ patients compared with the fs-patients, and in the gc+ patients compared with the gc-patients (p x . ). interleukin (il) , il- ra, il- beta, il- p , il- , macrophage inflammatory protein (mip) alpha, mip- beta, eotaxin, interferon (ifn) alpha, and il- levels were significantly increased in the gc+ patients (n= ) compared with the gc-patients (n= ). in addition, minor differences in cytokine levels were found when comparing age groups. degenerative changes such as atrophy/fibrosis and fatty cell infiltration observed in the minor salivary glands of patients with pss may represent "burned out" inflammation. no significant differences were found in autoantibody levels in either of the groups, nor when comparing cytokine levels in the fs-and fs+ subgroups. the reduced salivary flow observed in gc+ patients may be influenced by the elevated levels of il- found in these patients (gao et al. ) . increased titers of th -associated cytokines, il- , il- beta and the il- subunit il- p , may indicate a higher activity of these cells in gc+ patients (nguyen et al. ) . differences in cytokine levels may be utilized when sub-grouping the ss patients into disease phases and may consequently have implications for treatment. objectives: c-reactive protein (crp) is an acute phase protein, produced by hepatocytes in response to the pro-inflammatory cytokine il- . the rapid increase of crp during inflammation makes it an excellent inflammatory marker, but for unknown reasons, blood levels of crp typically remain low in disease flares of systemic lupus erythematosus (sle), a systemic autoimmune disease. another feature of sle is the so called 'interferon (ifn) signature' which implies high levels of ifn-alpha and/or up-regulation of ifn-alpha related genes. ifn-alpha has a wide spectrum of immunomodulatory functions but is mainly known for its antiviral and anti-tumour effects. since high levels of ifn-alpha coexist with a muted crp response in sle disease flares and in viral infections, we hypothesized that ifnalpha inhibits crp synthesis. methods: crp promoter activity was studied in a crp-promoter and luciferase reporter transfected human hepatoma cell-line, hepg . production of the acute phase protein serum amyloid a (saa) and the negative acute phase protein transferrin were analysed by elisa as reference. results: the crp-promoter activity was inhibited by all ifn-alpha subtypes. mixes of type i ifns that were induced by sle-like immune complexes or virus also inhibited the crp-promoter activity. virus-induced purified leukocyte ifn-alpha had the most prominent inhibitory effect ( g %) on crp promoter activity. saa synthesis was inhibited by ifn-alpha in a similar fashion as for crp promoter activity, whereas transferrin was unaffected. conclusion: our data indicates that ifn-alpha is an inhibitor of crp-promoter activity. we suggest that this could explain the muted crp response seen in sle disease exacerbations. further, i may contribute to differences in crp response between viral and bacterial infections. background: b cell activating factor of the tnf family (baff) is an essential b cell survival and maturation cytokine. mice overexpressing baff (baff tg mice) develop lupuslike autoimmunity, b cell hyperplasia, and lymphomas. autoimmunity in these mice involves proinflammatory autoantibodies driving nephritis and sialadenitis, and was previously found to be t cell-independent (ti) and myd -dependent. this suggested the involvement of transmembrane activator and caml interactor (taci), which is a receptor for baff that is essential for ti immune responses and is upregulated by myd -dependent tlr activation. we assessed the role of taci in baff-driven ti autoimmunity. methods: we tested the importance of taci in ti autoimmunity by generating baff tg bone marrow (bm) chimeras reconstituted with taci -/or taci +/+ bm then comparing their disease severity by flow cytometry, autoantibody elisa, immunofluorescence microscopy for ig deposition. results: as expected, baff tg chimeras reconstituted with taci +/+ bm produced high levels of circulating proinflammatory autoantibody isotypes and rheumatoid factors (rhf), and ig deposition in the kidneys and salivary glands was observed. by contrast, baff tg chimeras reconstituted with taci -/-bm had greatly ameliorated levels of circulating proinflammatory autoantibodies, rhf, and ig deposition. b cell hyperplasia was greater in taci -/- baff tg chimeras. defects in the regulation of apoptosis contribute to the pathogenesis of human systemic lupus erythematodes (sle). autoantigens not being properly removed and thus exposed to the immune systeme might lead to the emergence of autoantibodies. physiologically apoptotic cells are removed without initiation of an inflammatory immune response and myeloid dendritic cells are believed to actively tolerize t-cells after phagocytosis of apoptotic material. these processes of silent apoptotic cell clearance seem to be disturbed in sle patients. a characteristic of apoptotic cell death is the shedding of membrane coated vesicles from the cellular surface (apoptotic cell blebbing). these microparticles have been recognized as mediators of intercellular communication. therefore, we were interested whether apoptotic cell derived microparticles can influence the function of monocyte-derived dendritic cells and whether those interactions might play a role in the pathogenesis of human sle. we observed an engulfment of microparticles by monocyte-derived dendritic cells. further, apoptotic cell-derived microparticles stimulated differentiation of immature dendritic towards a mature phenotype. however, microparticles caused a remarkable downregulation of mhc class ii molecules. further, we observed only a minor release of proinflammatory cytokines from monocyte-derived dendritic cells pulsed by membrane microparticles when compared to lps stimulated dendritic cells. finally, these dendritic cells pulsed by membrane microparticles did not cause a significant t-cell expansion. interestingly, dendritic cells obtained from sle patients showed significant variations in phenotype and cytokine secretion compared to normal healthy donor cells with absence of the mhc class ii downregulation and a higher constitutive secretion of il- . objectives: increased levels of il- , an innate and inflammatory cytokine of the il- family, can be detected in serum and organs of human autoimmune pathologies, as well as in autoimmune animal models. here, expression of il- and other genes of the il- /il- r families was examined in human systemic lupus erythematosus (sle) and in mrl lpr/lpr mice, which develop a chronic progressive lupus-like syndrome. methods: serum, urine, and monocytes were collected from patients and healthy controls. lymphoid (lymph-nodes, spleen, thymus, peyer's patches) and non-lymphoid organs (kidney, lung, liver, salivary and lacrimal glands) were collected from mrl +/+ and lpr/lpr mice of different ages. il- and il- bp were measured by elisa. gene expression was assessed by real-time pcr and expressed relative to b-actin. results: in sle, serum and urine levels of total and free il- are higher than in controls. serum il- correlates with disease activity and decreases upon remission. monocyte expression of the receptor il- rb is increased and correlates with disease severity, while expression of tir /sigirr (a down-regulatory receptor of the il- r/il- r family) is reduced. in mrl lpr/lpr mice, expression of il- , caspase- and il- rb genes precedes disease onset in lymph-nodes. in other organs, changes in il- -related genes (il- and tigirr- up-regulation, tir /sigirr down-regulation) occur after disease onset. free il- levels are abnormally high in lpr/lpr lymph-nodes before disease onset, while in other organs the increase occurs with disease. conclusions: free il- levels correlate with autoimmune lupus both in mice and humans. free il- may be pathogenic in murine lymphadenopathy, while is a disease correlate in lpr/lpr and a severity correlate in sle. both in human and mouse syndromes, upregulation of il- rb is a marker of pathology, suggesting increased il- -dependent activation. both in mouse organs and human monocytes, tir /sigirr expression decreases with disease, suggesting impaired control of il- r activation. thus, il- may be involved in autoimmune lupus pathology, and il- -related molecules can be both original diagnostic markers and novel therapeutic targets in autoimmunity. in this study we compared the epitope specificity of anti-topo i autoantibodies present in sera of dcssc, lcssc and sle patients. we have constructed an antigen fragment library displayed on bacteriophage lambda and screened this library with igg purified from patients' sera. regions of topo i selected from the library were expressed as recombinant fusion proteins and were tested with elisa and western blot. we unexpectedly found that antibodies against a fragment of topo i (fragment f (amino acid (aa) - ) could be detected in sera of healthy individuals and patients with inflammatory rheumatic diseases other than ssc and sle. using sera of dcssc, lcssc and sle patients we showed that the pattern of recognized epitopes is different between these patient groups. fragment f was recognized by all patients. fragment f (aa - ) was recognized by of dcssc patients. fragment f (aa - ) was recognized by of sle patients. analysis of clinical data revealed a significant difference between the f negative and f positive groups of ssc patients in age and in the duration of the disease. according to our results the newly identified fragments f and f could represent characteristic epitopes for dcssc and sle, respectively. background: previous studies demonstrated that depletion of regulatory t cells (tregs) results in autoimmunity in mice while their adoptive transfer prevents autoimmune diseases. studies performed by us and others showed that in human connective-tissue diseases a reduced number of tregs exists and this abnormality seems to be correlated with autoantibodies production and disease activity. objectives: based on these observations and the fact that rapamycin (rapa) has the ability to expand tregs and to induce anergy, we proposed to study the possibility to restore peripheral tolerance of cd + t cells isolated from systemic lupus erythemaosus (sle) patients by ex vivo expansion of tregs. methods: pbmcs or peripheral cd + t cells from sle patients were cultured in the presence of specific stimulation with or without rapa and ril- . by facs the initial percent of tregs and after expansion protocol were determined. in order to verify the suppressive capacity of expanded tregs, cd + t cells enriched in tregs were co-cultured with activated cd + effector t cells (teff) stained with cfse, after one week teff cells proliferation was measured by facs. additionally, cytokine and igg release in cell culture media were analyzed by multiplex and elisa, respectively. expanded cd + t cells anergy was also evaluated based on cbl-b, grail and foxp mrna by realtime rt-pcr. results: in vitro expansion of tregs was more efficient when the starting cells were cd + t cells. the presence of rapa during expansion protocol significantly increased the number of tregs. sle tregs cells expanded in vitro in the presence of rapa had the capacity to suppress proliferation of both sle and hd teff cells. rapa inhibits igg secretion in the pbmcs culture, inhibition dependent on tregs level. rapa during tregs expansion protocol stimulated some type of cytokines while suppressed others. rapa had the capacity to re-establish sle cd + t cells anergy by induction of anergy genes, grail and cbl-b. conclusions: our data show that the above described protocol permits ex vivo tregs expansion and that suppressive capacity of the expanded tregs depends on the source of both tregs and teff cells. in this study, we look for a more specific approach to remove b- cells through targeting p d by shrnas strategy. methods: we used the drugs, ly and wortmannin, pan-specific inhibitors against pi ks. then we designed shrnas carried by the lentiviral system and validated that several segments of them can sufficiently knock down the expression of p d. we then introduced either pan-specific inhibitors against all pi ks or p d-targeting shrnas into an sle-prone animal model, nzb/w f mice, for therapeutic purposes. the results suggested that pi ks are not only important for the development of b- cells but also remain essential to maintain their population after birth. shrnas carried on lentiviral systems were designed to knock down the expression of p d. either pan-specific inhibitors against pi ks or p d-targeting shrnas were introduced into the sle-prone animal model, nzb/w f mice. one inhibitor, ly , and shrnas delivered by low dose of lentivirus exhibited certain potential to retard the rising of anti-dna auto-antibodies and prolonged the life span. conclusions: our findings are promising for developing treatments for sle. moreover, knowing pi ks are critical for the maintenance of b- cell populations might shed light on future treating other diseases associated with b- cells, such as certain melanoma, lymphoma, or leukemia. a. m. zaghlool , m. alarcón-riquelme , s. kozyrev institution of genetic and pathology, uppsala university, uppsala, swedenrecently, we discovered that the bank gene, which plays a role in b cells activation pathway, is associated with systemic lupus erythematosus through a nonsynonymous substitution g/a (rs , r h). we identified that bank gene expresses two alternatively spliced isoforms, a full-length, and a shorter isoform that lacks exon (delta ). the two isoforms were detected differently in susceptible lupus patients depending on the presence of a risk haplotype. to address the question of how bank is spliced and what are the signals governing the expression of each isoform, minigenes with different genetic variants were constructed and the expression of the bank isoforms were tested in vitro. qpcr analysis revealed that, another t/c snp (rs ), which is in complete ld with r h snp and located in the putative branch point, has a strong affect on the isoforms expression levels. deletion of a polypyrimidine (py) stretch downstream of the skipped exon produced a dramatic decrease in the full-length expression levels, probably due to the loss of the binding site for protein tia , which bind to t objectives: cerebral ischemia is the most common presentation of antiphospholipid syndrome (aps), but several other neuropsychiatric features, including chronic headache, dementia, cognitive dysfunction, psychosis, depression, transverse myelitis, multiple sclerosis-like disease, chorea, and seizures have been associated with the presence of antiphospholipid antibodies (apl). we report the case of a subject with atypical movement disorder related to aps successfully treated with oral anticoagulation agents. case report: a -year-old woman with a previous history of recurrent foetal losses was admitted to our hospital due to cognitive dysfunction and headache. she presented involuntary movements that were characterized as mioclonic seizures and tonic spasms lasting from few minutes to several hours, followed by bilateral arrhythmic rapid purposeless jerks of the legs. mild executive dysfunction was observed. her deep tendon reflexes were symmetric and normal. pathological reflexes were absent. biochemical analysis, renal, hepatic and thyroid functions were preserved, prothrombin time and partial thromboplastin time were all normal. the immunoglobulin g (igg) isotope of anticardiolipin antibody (acl) was elevated, whereas igm isotype and anti- gpi antibodies were undetectable. the lupus anticoagulant (la) was negative such as antinuclear antibodies (ana). no evidence of epilepsy was revealed from electroencephalogram or signs of denervation from electromyographic studies. brain magnetic resonance imaging (mri) showed multifocal encephalomalacia probably linked to previous cerebrovascular accidents. she was diagnosed as having an atypical neurologic manifestation probably linked to aps. she was thus discharged with a low-molecular-weight heparin therapy subsequently changed to mild oral anticoagulation . the therapy leads to a late, gradual improvement of symptoms that persisted at the last year follow-up evaluation. conclusions: antiphospholipid syndrome may constitute a rare but treatable cause of atypical neurologic manifestation such as myoclonic movements. due to the possibility of an effective treatment, it is important to rule out this diagnosis, moreover in women with other associated features of aps (foetal losses, livedo reticularis, thrombosis). a.-s. korganow cnrs , strasbourg, france b lymphocytes from patients with systemic lupus erythematosus are hyperactive and produce autoantibodies. several b cell phenotypic characteristics have been reported, as the expansion of activated populations, and of a newly investigated memory compartment. a few genes have been suggested to be implicated. one of the thing that makes these results difficult to interpret is the heterogeneity of the lupic disease, and sometimes the analysis all together of quiescent, paucisymptomatic and highly symptomatic patients, treated with immunosuppressors or untreated.we made the postulat that "intrinsic" abnormalities of b cells could be a common point in very quiescent patients. we choosed patients, with minor clinical and/or biological manifestations of their disease, for at least monthes. known of them received immunosuppressive drugs since this period. the mean sledai score was below . b cell surface markers expression was determined by flow cytometry. we analysed most of the already described and phenotypically distinctive b cell populations. we confirm the presence of activated b cells even in quiescent patients. we do not confirm the significant increase of a specific memory b cell compartment. above all, we described a decreased expression of the cd surface protein for all patients. this cd lower expression is associated with cd lower levels. it is not associated with an evident gene expression alteration and in vitro stimulation restores a control phenotype. these findings suggest some mechanisms in lupus genesis. objectives: tgf-beta is a pleiotropic cytokine with wide ranging effects in proliferation, differentiation, immune suppression and apoptosis. recent work from our group has shown that tgf-beta signalling in t-cells is protective in a mouse model of colitis associated cancer. smad ubiquitin regulating factors (smurf) are ubiquitin ligases that are involved in the regulation of tgf-beta signalling. the aim of this study was to determine the function of smurf expression in t-cells on the pathogenesis of experimental colitis associated colon cancer. methods: we could isolate a known splice variant of smurf lacking an exon in the c -domain. to analyse whether this form has a regulatory role in colon associated cancer we generated a transgenic mouse strain that overexpresses smurf in t-cells. smurf expression were analysed by qpcr. wild type (wt) and transgenic (tg) mice were treated once with the mutagenic agent azoxymethan (aom) followed by three cycles dextran sodiumsulfate (dss). after each cycle, the inflammation of the gut and the tumor growth and size of every mouse were monitored by colonoscopy. results: smurf expression was upregulated by tgf-beta stimulation in t-cells and smurf was markedly upregulated in tumor infiltrating cd + lymphocytes in aom/dss treated mice. whereas wt mice suffered from severe colitis resulting in colon tumors beginning at day , smurf transgenic mice had less colitis and were significantly protected from tumor development. interestingly, t-lymphocytes overexpressing smurf showed an upregulation of the tgfbrii and an activation of smad and as compared to wild-type t-lymphocytes, which were previously described as typical smurf targets for degradation. in addition the transfection of smurf and a caga-luc plasmid into cos-cells for smad -promotor studies yielded the same effect as shown by upregulation of the smad activity. conclusion: although, wt-smurf has been described as a negative regulator of the tgf-beta signalling pathway, our data show surprisingly that a smurf splice variant upregulates the tgf-beta receptor expression and increases tgf-beta signalling effects. due to immunosuppressive effects on t-cells smurf has beneficial effects on mucosal inflammation and tumor development. smurf thus emerges as an attractive target for modulation of chronic intestinal inflammation and colitis associated carcinogenesis. the transcription factor stat has important functions in cytokine signalling in a variety of tissues. however, the role of stat in the intestinal epithelium is not well understood. we demonstrate that development of colonic inflammation is associated with the induction of stat activity in intestinal epithelial cells (iec) both in humans and in mice. studies in genetically engineered mice showed that epithelial stat activation in dss colitis is dependent on il- rather than il- . il- was secreted by colonic cd c+ cells in response to toll-like receptor stimulation. conditional knockout mice with an iec specific deletion of stat activity were highly susceptible to experimental colitis, indicating that epithelial stat regulates gut homeostasis. stat iec-ko mice, upon induction of colitis, showed a striking defect of epithelial restitution. gene chip analysis indicated that stat regulates the cellular stress response, apoptosis and pathways associated with wound healing in iec. consistently, il- and epithelial stat was found to be important in wound-healing experiments both in vivo and in cell culture experiments in vitro. in summary, our data suggest that intestinal epithelial stat activation regulates immune homeostasis in the gut by promoting il- -dependent mucosal wound healing. stat seems dispensable for gut homeostasis under steady state conditions, but is activated upon challenge to drive tissue regeneration and protection in situations of increased demand, as during colitis and injury. map and ma infection induced an increase in both cd and tlr expression at day and day after infection. mycobacterial infection did not result in differential tlr expression as compared to uninfected cells. cd is involved in stimulating th pro-inflammatory responses, although map may interfere with cd signalling ( ) . tlr signalling elicits anti-inflammatory responses, which can contribute to bacterial replication ( ) .in conclusion, monocyte-derived macrophages from crohn's disease patients show an increase in cd and tlr receptor expression in response to both map and ma infection. as ma is a known human pathogen of immunocompromised hosts, this findings further support a role for map in the immunopathology of crohn's disease. objectives: for our understanding of the pathogenesis of human ibd, animal models of intestinal inflammation are indispensable. most of them are based on a compromised intestinal barrier, and a deregulated immune response against components of the flora is considered to be critically involved in the development of ibd. the occurrence of extraintestinal manifestations suggests that cross-reactions against hitherto undefined auto-antigens could be responsible for the activation of the adaptive immune system. to further dissect the pathophysiological mechanisms responsible for initiation and progression of ibd and associated extraintestinal manifestations, we established a new antigen-specific model, in which the local activation of cd t cells by exogenous antigen leads to colitis. methods: eight million naïve cd + ot-i cells, transgenic for a t-cell receptor specific for an ova-derived peptide (siinfekl) in the context of h -kb, were transferred i. v. into b mice. at day and , mice were treated intra-rectally (i. r.) with % ethanol. thirty minutes later, ovalbumin (ova) or bovine serum albumine (bsa) were applicated i. r. proliferation of cfse-labelled cells was measured at day after the injection of ot-i cells. the phenotype of effector cells was evaluated at day by measuring ifng production and by in vivo cytotoxicity assay. based on histology and immunhistochemistry for cd , the severity of colitis was scored. results: local application of the exogenous antigen ova but not of bsa led to antigen-specific activation and proliferation of adoptively transferred naïve ot-i cd + t cells. these cells differentiated into fully activated effector t cells with the capacity to secrete ifng upon re-stimulation ex vivo and possessed in vivo cytotoxicity to siinfekl-loaded target spleen cells. furthermore ova treated mice displayed an inflammatory infiltrate in the colonic lamina propria with strongly elevated numbers of cd + t cells. our study demonstrates that the local activation of antigen-specific cd t cells by exogenous antigen in the colon leads to fully activated effector t cells with the capability to promote local intestinal inflammation in non-immune-compromised b mice. aims: to determine the immune system response of the greek population against helicobacter pylori (hp), given the fact that hp infection is a frequent causal factor of gastroduodenal ulcer and gastritis, and to study the distribution by age and sex, as well as the possible correlation with anemia markers (hematocrit, hemoglobin, iron, ferritin etc). the results of express qualitative detection method for igg and iga antibodies were studied of patients, ( male and female), with age average , years of age. patients who received antibiotics and excretory medicine in the last year were excluded. anemia laboratory tests were performed (hematocrit, hemoglobin, iron, ferritin), which were followed by statistical processing, using spss, x and t-test programmes. results: in patients ( , %,with age average , years of age) no antibodies were detected. on the contrary, in the remaining , male and female, ( , %, with age average , years of age), antibodies were detected. out of them, in cases the results were strong positive ( male and female) and weak positive in cases ( male and female). the statistical analysis that followed, showed no statistically important correlation with any of the anemia markers who were determined (hematocrit, hemoglobin, iron, ferritin, mcv and rdw). conclusions: it is proven, therefore, that: ) helicobacter pylori infection is relatively common in the general population ( , %). ) there is a statistically important correlation, as far as age (increased in elderly patients) and gender is concerned (clearly greater in women). ) there seems to be no correlation with anemia. it is evident, that the method is very useful, especially in elderly patients with dyspeptic complaints, (who frequently cannot undergo invasive procedures), and should not be neglected, given the fact that there is a great risk of helicobacter pylori infection in our country. abstract withdrawn by author m. durilova , t. ulmannova , k. stechova , k. tesarova-flajsmanova , v. stavikova , j. nevoral charles university, pediatrics, prague, czech republicobjective: was to analyze composition of cytokines in breast milk of mothers whose infants were diagnosed with allergic colitis and compare it to cytokine composition in breast milk of healthy controls. methods: breast milk of mothers whose infants were diagnosed with allergic colitis and mothers of healthy infants and no history of allergic disease was analyzed for presence of cytokines. breast milk samples were collected at the time of diagnosis of allergic colitis ( - weeks, average . weeks of infant's age) or at the age of weeks in control group. concentrations of the following cytokines were analyzed using elisa method: il- , il- , il- , il- , il- , ifn-gamma, tgf-beta , egf and eotaxin. man-whitney u test was used for statistical analysis, p x . was considered statistically significant.results: il- as the only cytokine was not detected in any of the tested samples in both groups. significant difference was seen in concentration of ifn-gamma, which was higher (p x . ) in breast milk of mothers whose infants were suffering from allergic colitis (range - . pg/ml, mean . pg/ml) than in control group (range - . pg/ml, mean . pg/ml). higher concentrations of il- and lower concentration of tgf-beta were observed in breast milk received by infants with allergic colitis but the difference was not statistically significant. conclusion: immunologic factors including cytokines present in breast milk passively and actively influence the developing immune system of the newborn. although their role is not exactly known, they are important in regulation of immunologic reactions and might be responsible for protective effects of breast milk from many diseases. inter-individual differences in cytokine composition of breast milk were previously found in many studies and their presence is influenced by various factors. the results of our study indicate that there might be a risk cytokine pattern in breast milk of mothers whose infants are suffering from allergic colitis. supported by national project no. - . background: ulcerative colitis is associated with excessive neutrophil infiltration into the lamina propria and intestinal crypts leading to the formation of crypt abscesses. the chemokine il- (murine homologs kc and mip- ) and its receptor cxcr are involved in neutrophil recruitment, thus blocking this engagement offers a new therapeutic strategy for inflammatory bowel disease. this study aimed to develop and characterize a pre-clinical in vivo model to test potential therapeutics targeting neutrophil migration. methods: peritoneal exudate neutrophils from transgenic b-actin-luciferase mice were isolated h post intraperitoneal injection of thioglycollate and phenotypically (facs analysis) and functionally characterized in an in vitro chemotaxis assay. four million exudate cells were injected intravenously into recipients with dextran sulphate sodium (dss) colitis followed by bioluminescence imaging of whole body and ex vivo organs at , , and h post-transfer. anti-kc antibody or its isotype control was administered at mg/mouse one hour before transfer followed by whole body and organ imaging hours post-transfer. results: facs analysis revealed % neutrophil purity, % of which were cxcr + . in vitro, the cells migrated towards kc and this was inhibited by anti-kc. in the bioluminescent imaging model, trafficked neutrophils were evident in whole body and ex vivo organ images of dss recipients at all time points. neutrophil recruitment to the colon was detected only in dss recipients and was inhibited by anti-kc, h post cell transfer. this study describes a novel in vivo model of neutrophil trafficking that can be used for pre-clinical studies to evaluate potential inhibitors of neutrophil recruitment. the human gut contains more than bacteria (known as the commensal microbiota) that are essential for normal function of our digestive and intestinal immunologic systems. the barrier function of the mucosal epithelium is reinforced by innate defense mechanisms and by immune exclusion mediated by secretory (s)iga and sigm. sigs are generated via epithelial polymeric ig receptor (pigr)-mediated transfer of iga and igm from the lamina propria to the intestinal lumen. to assess the role of sigs in colitis development, we constructed pigr knockout (ko) mice and tested them in the dextran sodium sulfate (dss) colitis model ( . % dss in drinking water for week, followed by pure drinking water for week). pigr ko mice suffered increased morbidity and mortality compared with wild type mice, but colitis was cured by depletion of intestinal commensals suggesting that one role of sigs is to prevent pathology induced by commensal microbiota. in contrast, % dss was lethal to all commensal-depleted mice, but these mice became anemic rather than suffering from bloody diarrhea. as previously documented by medzhitov and co-workers (rakoff-nahoum et al, cell ), treatment of commensal-depleted mice with the tlr ligand lps in drinking water protected against the lethality of % dss. thus, the commensal microbiota serve two distinct roles in the dss colitis model. at dss concentration of . % they may become pathogenic and drive an intestinal inflammation. at % dss commensals protect against the toxic effect of the chemical via their tlr ligands. in mice lacking sigs, due to deleted pigr, the severity of colitis induced by . % dss was greatly enhanced suggesting that one role of sigs is to prevent commensal microbiota from becoming pathogenic. ulcerative colitis (uc) is a human inflammatory bowel disease associated with chronic inflammation of the gastrointestinal tract. although uc is associated with a type immune response, current treatment strategies use broad anti-inflammatory drugs which are aspecific for the disease. in a mouse model resembling uc, oxazolone induces il- production which is an important pathological factor. neutralizing il- or il- prevents or ameliorates disease significantly. as many aspects of the mechanisms involving these th cytokines in colitis remain undefined, we used mice deficient in il- /il- or the key receptor through which they signal, il- ra, to further dissect their role in oxazolone-induced colitis. disease was exacerbated in il- ra -/mice with increased weight loss, mortality, inflammation and immunopathological symptoms. this was in contrast to il- /il- double deficient mice which were protected from colitis. removing il- production from il- ra -/mice, by using il- ra/il- double deficient mice, reversed the susceptible phenotype to protection. together these data strongly suggest that il- mediates susceptibility in an il- ra independent manor. recent evidence pc / introduction: the activation of cd + t-cells in the lamina propria play an major role in the pathogenesis of inflammatory bowel disease (ibd). whereas cd is associated with increased production of th -like cytokines, the cytokines profile in chronic uc is characterized by the increased production of several th cytokines, such as il- ,- and il- . however, the functional role of t cell transcription factors such as nuclear factor of activated t cells (nfat) in ibd is poorly understood. the aim of this study was to further analyze the role of this signal transduction pathway and its pathogenic significance in uc. cryosesctions of uc and cd patients were analysed by immunohistochemically methods. a significantly higher expression of nfatc was found in uc and cd colonic tissue compared to control specimen. transmitted to the th -mediated oxazolone-induced colitis model, nfatc -production is significantly increased in both diseases, too. nfatc deficient mice were analyzed in colitis model and are significantly protected against the development of intestinal inflammation compared to control mice, documented by loss of weight, histological score and miniendoscopy. interestingly, cyrosections of inflamed colonic tissue displayed a higher apoptotic rate in nfatc deficient mice compared to control mice, which can be observed by tunel assays, caspase and annexin v staining, as well as in lamina propria t cells. contrary, anti-apoptotic proteins, like bcl- and bcl-xl were downregulated for induction of apoptosis. this observation was associated with a reduced production of il- , ifn-gamma, il- and il- by mucosal t lymphocytes, tested by elisa assays. further studies with the oxazoloneinduced colitis model showed that nfatc regulates il- /il- in an indirect way. last, administration of il- blocked the protective effects of the nfatc deficiency in experimental colitis, suggesting that nfatc through il- signal transduction plays a direct pathogenic role in vivo. conclusion: our data define a unique regulatory role of nfatc in colitis by controlling mucosal t cell activation in an il- dependent manner. the examination of this signal transduction pathway emerges as a potentially new therapeutic target for inflammatory bowel diseases. the pivotal role of micrornas in the regulation of gene expression, in particular genes involved in the immune response, indicates that they may play an important role in the pathogenesis of inflammatory bowel disease (ibd) as well. the study of the expression of micrornas in ibd will unravel their role in this disease. in addition, micrornas by their mechanism of action, are promising new therapeutic agents or targets. a possible therapeutic application of micrornas is the introduction of novel, artificial micrornas or microrna mimics to regulate specific genes. because ibd is a heterogeneous disease in human we decided to define microrna expression in a well defined model of experimental colitis. as a result of this study we found a number of micrornas involved in different phases of experimental colitis. to study the role of mirnas in experimental colitis in mice we have used a well defined colitis model that resembles human ibd. this colitis is mediated by cd cd rb high t cells that are injected i. p. in scid mice. in control mice in addition to the cd cd rb high t cells also regulatory cd cd rb low t cells are transferred and no colitis develops. to study mirna expression we collected colonic tissue from the mice at different time points during colitis progression. after weeks a chronic progressive colitis developed characterized by a progressing wasting disease that was terminated at weeks. microrna was isolated from colons of mice in different stages of colitis progression ( , and weeks) and control mice that do not induce colitis (n= for each timepoint). from all mice we also processed a part of the colon for immunohistochemistry to determine disease progression at the various time point after induction of colitis.the rna isolation as well as the microarray analysis has been outsourced to miltenyi biotec gmbh, bergisch galdbach, germany. we used the mirxplore tm microarrays for microrna expression profiling. from micrornas that demonstrated an induction during the development of disease we selected micrornas for in situ hybridization and for a proof of principle of the efficacy in the cd cd rb high transfer model. objective: the purpose of this clinical trial (id: nct of www.clinicaltrials.gov) is to investigate whether the expansion of the thymus in adults can restore specific immune responses by administration of growth hormone (gh). methods: successfully highly active antiretroviral therapy (haart) treated hiv infected patients that failed to elicit a humoral response to tetanus toxoid (tt), or to hepatitis a (hva) or to hepatitis b (hvb) virus have been selected for the trial. growth hormone was given for months with the hope that they will reactivate thymic input and restore their specific responses to these vaccine antigens. patients have been randomized in groups: group a (n= ) receiving haart+ gh (for months) + tt+hva/b vaccines (at month post gh adminsitration); group b (n= ) receiving haart+gh but not vaccines; and group c (hiv control group, n= ) with haart+vaccines (at month ) but without gh. all patients are followed up months further. results: preliminary results show that an increase in thymic size was observed in gh recipients and not in controls. furthernore after weeks of administaring hormone the absolute numbers of cd incresase from ± to ± cells per mm (mean and sem; p x . ). in contrast, pacients who have not received the hormone but have been vaccinated showed a significant decay of the cd absolute numbers from ± to ± cells per mm (p x . ). viral load remained undetectable in all patients. despite the increase in cd counts the percentage of recent thymic emigrants (as assessed by the expression of cd ) as well as the proportion of naï ve and memory cells remained constant throught the trial in all patients. finally, specific responses to hepatitis a virus seem to be restored in a major proportion of patients treated with gh (group a) than in the other groups. conclusions: although the clinical trial is ongoing, the preliminary results seem to indicate an increase in the thymic size and some immmune restoration in patients treated with growth hormone before vaccination. a major problem of current vaccines is the requirement for cold chains to maintain vaccine potency. in the course of the eradication of small-pox, freeze-dried vaccinia virus which proved to be extremely stable was used to overcome this limitation (dryvax ® ). before usage, dryvax has to be reconstituted before vaccination using a bifurcated needle reflecting another drawback of classical vaccination -transmission of blood-borne pathogens. an alarming report by the who has estimated that as many as one-third of immunization injections are unsafe in four of its six geographical regions. each year, an overwhelming number of infections with hiv ( , - , ), hepatitis c virus (hcv; . - . million) and hepatitis b virus (hbv; - million) are thought to originate from the reuse of needles and syringes by health-care providers. in this report, we took advantage of the stability of freeze-dried vaccinia virus mva and directly applied it into the nostrils of mice without prior reconstitution. this direct mucosal application induced systemic antibody and t cell responses comparable to those achieved by intramuscular administration. importantly, mucosal application of lyophilized mva conferred protective immunity against a lethal vaccinia virus challenge. additionally, recombinant mva expressing the model antigen ovalbumin induced long-term and protective immunity against listeria monocytogenes-ova challenge. the data clearly demonstrate the potency of a simple needle-free vaccination, combining the advantages of mucosal application with the stability and efficiency of lyophilized mva. methods and results: seventeen haart-treated asymptomatic hiv- infected patients with g cd + t-cell counts and plasma hiv-rna levels of x . log copies/ml were treated with a dc-based vaccine. the vaccine consisted of autologous mature dc electroporated seperately with either sig-tat-dc-lamp, sig-rev-dc-lamp or sig-nef-dc-lamp mrna and were each administered at a distinct anatomical site. after four monthly vaccinations haart treatment was interrupted. pbmc from timepoints, before during and after vaccination, were analysed for ifn-g production (elispot), proliferation (cfse assay) and lytic capacity (fatt-ctl) in response to the antigens used in the vaccine. pbmc were screened upon ex vivo stimulation with pools of overlapping tat, rev, nef and gag peptides and ifn-g secretion was analysed using elispot ('peptide elispot'). elispot was also performed on re-stimulated t-cells with electroporated dc ('dc elispot') in vitro. responses were considered positive when the number of spot forming units per million t-cells was g and when the responses were g times the standard deviation above the mean of replicate negative controls (mock electroporated dc). / patients were screened with both peptide and dc elispot. an increase of responses to the vaccine-antigens after vaccination was found in both assays. based on the dc elispot data, we observed immune reactivity against tat in / patients before and / patients after vaccination. for rev, / patients showed a pre-existing rev specific response and / patients responded to rev after vaccination. nef was the most immunogenic antigen used in this vaccine with already / patients responding before and / patients responding after vaccination. for our control antigen gag, we observed an anti-gag response in out of patients before vaccination and / patients after vaccination. the results of the other assays correspond to the dc elispot results be it less pronounced. conclusion: therapeutic vaccination of hiv-infected patients with dc electroporated with tat, rev and nef induces and/or enhances antigen-specific t-cell responses, especially when monitored with the dc elispot. background: enterovirus (ev ) is an etiologic agent responsible for seasonal epidemics of hand-foot-and-mouth disease and causes significant mortality among young children. no effective vaccine for ev is available yet. polysaccharide purified from ganoderma lucidum (ps-g) has been known to be a strong immunopotentiator, therefore, we studied the immune enhancing effect of ps-g as the possible adjuvant with ev inactivated virus. methods: mice were immunized intraperitoneally with mg inactivated virus /mouse with one of the following samples: pbs, cfa/ifa, and ps-g. each mouse received the same dose of boosters after , , and weeks. blood samples were collected at , , , and days. the total serum anti-ev igg level was determine by elisa, and the neutralization assay was also done. to evaluate the cellular immune responses, spleens were harvested from all mice for splenocyte proliferation assay. cytokines assay regarding ifn-g and il- from splenocytes was also measured. results: immunization with ev /ps-g showed that the anti-ev igg levels were significantly increased compared with ev alone or ev /cfa/ifa in balb/c mice. neutralization assays demonstrated an effective protection of ev /ps-g group compared to ev alone. the splenocyte proliferation test showed that production of ifn-g significantly increased in ev /ps-g-immunized mice compared to those of ev or ev /cfa/ifa-immunized mice, indicating a th cells response elicited by heat-inactivated ev vaccine with ps-g adjuvant. conclusions: vaccine design is important for the development of immune response for pathogen, and adjuvants play the very important role to enhance the effect of vaccine. the results here suggested that ps-g can be used as a novel, safe and natural vaccine adjuvant. objectives: the search for a vaccine against hepatitis c virus is hampered due to the lack of an animal model to study vaccine-efficacy other than chimpanzees. here we describe the differential modulation of cd + t-cell responses induced by a dna prime mva boost vaccine regimen in four individual chimpanzees and their association with viral clearance. methods: an ex vivo expansion protocol was used to map peptide specific cd + t-cell responses against hcv-ns , studying induction of il- , ifng and tnfa cytokine production as well as killing capacity. to assess the killing capacity and mhc restriction of the peptides, a non-radioactive killing assay was designed. peptides that induced both il- and ifng production by cd + t-cells were tested for their competence to induce killing of transfected target cells that expressed chimpanzee mhc class i molecules. introduction: high levels of hiv- -recognizing cd + cytotoxic t lymphocytes (ctl) with a widespread specificity, especially against conserved epitopes, are considered to play an important role in the control of hiv- replication, and for the prolonged survival of infected individuals. a potential immunotherapeutic strategy would be the adoptive transfer of t cells, which are reprogrammed by introduction of an hiv-specific t cell receptor (tcr). up to now, such ctl were generated by retroviral transfer of tcr-encoding genes, which harbors several challenges (i. e., activation/inactivation of genes, life-long autoimmunity). methods: therefore, we investigated the transfer of tcr-rna into cd + t cells by electroporation, and chose tcrs which were able to recognize the hla-a restricted hivpol-peptide iv , or the hivgag-peptide sl . results: t cells, reprogrammed with these receptors, released the pro-inflammatory cytokines il- , tnf, and ifng simultaneously, and showed up-regulation of the activation marker cd , after stimulation with peptide-loaded target cells or target cells (i. e. ebv-transformed b cell and cd + t cells) presenting the naturally processed epitope. furthermore, these cells maintained their ability to proliferate after stimulation. more importantly, killing assays demonstrated that the tcrreprogrammed cd + t cells were capable to specifically lyse target cells (for at least three days) loaded with the cognate peptide, or presenting the naturally processed epitope. a comparison of our reprogrammed t cells with the parental ctl showed that the transfected t cells were only one order of magnitude lower in avidity than the parental ctl. also, the parental clone's recognition pattern of mutant peptides was preserved in tcr-rna-transfected t cells. the transfection of tcr-encoding rna into cd + t cells, may represent a simple, secure, and more flexible alternative to retroviral transduction, but has the benefit that a better evaluation of the transferred tcrs is possible. background: dendritic cells (dcs) are able to capture, internalize, and process antigens leading to potent activation of antigen-specific cellular immunity. the aim of this study was to investigate the capacity of splenic dcs that ingest antigen coated magnetic beads to induce hcv cellular immune responses. methods: splenocytes of flt l pretreated balb/c mice were incubated for hrs with hcv ns -coated magnetic beads. the cells were harvested and cells that contained beads were purified by passage over a magnetic column. the isolated population contained g % dcs and was used for immunization. dc expression of the maturation markers cd , cd and cd was determined before and after ingestion of ns -coated beads, showing a significant increase of all maturation markers induced by phagocytosis. cellular immunity was assessed using a conventional ctl assay, a cfse-t-cell proliferation assay, intracellular cytokine staining and tumor challenge (with stably ns expressing sp / cells). results: in immunized animals, the ctl activity was increased -fold compared to mock immunized mice. accordingly, tumor challenge with ns expressing tumor cells showed a significant reduction in tumor growth. the number of cd + ifn-g + cells was increased g -fold and the number of cd + il- + increased g fold in the dc-ns -bead immunization group. these results paralleled the proliferative response of splenocytes to ns protein obtained from immunized animals with the most significant response in the cd + population of dc-ns -bead immunized animals. the use of ns coated beads combines three important aspects of dendritic cell based immunization in a single step: targeting of the antigen, enrichment and maturation of dendritic cells. the induction of a strong th biased t cell response makes this approach a promising new tool in therapeutic immunization in chronically hcv infected patients. the success of anti-dec- antibody as a stimulator of strong inflammatory immune responses depends on the coadministration of non-specific dendritic cell maturation factors. in their absence, anti-dec- induces antigen-specific tolerance rather than immunity. we hypothesize that regulatory t-cell epitopes contained in anti-dec- promote a tolerogenic reaction that is only overcome through the co-administration of non-specific immuno-stimulators. this hypothesis is based on our recent discovery of a set of natural regulatory t-cell epitopes derived from human immunoglobulins that induce tolerance by stimulating regulatory t cells. we have verified experimentally that these epitopes generate an expansion of regulatory t cells and suppress inflammatory immune responses. here, we embarked on a proof-of-principle demonstration that a pro-inflammatory and non-tolerogenic anti-dec- antibody can be developed. we screened the anti-dec- sequence computationally for putative hla dr -restricted, regulatory t-cell epitopes as targets for mutations that will reduce epitope binding affinity for hla. amino acid substitutions predicted to interfere with hla binding were identified and are being incorporated into an array of anti-dec- antibody variants recombinantly fused to a test antigen, hiv gag. variant antibodies that do not interfere with dendritic-cell targeting will be evaluated for reduced tolerogenicity, as well as for enhanced gag immunogenicity, in terms of cellular and humoral responses. we predict that the modification of regulatory t-cell epitopes will significantly diminish tolerogenicity, enabling the use of modified anti-dec- as a hiv antigen-delivery system that obviates the dangers associated with non-specific activation of the immune system. supported by nih r ai a live oral vaccine based on human adenovirus (ad) has proved safe and effective in us military recruits for nearly years. in these experiments, we have investigated whether replication-deficient ad can be an efficient potential vaccine carrier for oral vaccination. ad vectors were used throughout to provide a benchmark for efficacy. we generated novel ad and ad vector systems based on dna recombineering to facilitate manipulation of the vector backbone and high throughput transgene insertion (http://adz.cf.ac.uk). egfp was inserted with a hcmv ie promoter as a model transgene. preliminary in vitro studies on bloodderived human and murine cells demonstrated that primary lymphocytes are less susceptible to transduction with ad than ad . ad routinely infected and provided transgene expression in˚ % of human cd + and cd + t cells. stimulation of the hcmv ie promoter post infection increased detection of egfp to - % of cd + t cells present, showing that ad infected a surprisingly large proportion of t cells. in comparison, ad provided egfp expression in x % of either cell type, even after t cell activation. in contrast, infection rates and transgene expression in dendritic cells of both human and murine origin with ad and ad vectors were comparable. preferential infection of dcs is likely to be beneficial in the context of a vaccine. in vivo, we observed that following oral delivery both ad and ad induced restricted yet strong expression in the intestine. the vectors were rapidly taken up into the peyers patches, with optimal expression detected day after dosing, and transgene expression being reduced below detectable levels by day . interestingly, when delivered together ad and ad vectors targeted the same subset of cells. together, these data show that ad is a viable alternative to ad -based vaccines which may also avoid unwanted infection of activated t cells. aims: monoclonal antibodies (mabs) represent some of the most promising agents for anti-cancer and anti-viral immunotherapies ( recently commercialized; g in clinical trials). to date, their therapeutic antiviral efficiency has mainly been measured by their direct effects on viral spread. however, their indirect effects on long-term immune control of viral replication through their immunoregulatory properties upon interaction with other components of the immune system has hardly been assessed. as induction of long-term protective antiviral immune responses still remains a paramount challenge for treating chronic viral infections, we asked whether neutralizing mabs, in addition to blunt viral propagation, may also modulate the endogenous immune response. methods: we have developed a preclinical mouse model of fatal leukemia induced by the frcas e murine retrovirus. mice were infected with frcas e and treated, or not, with a neutralizing mab (the mab). viral propagation, survival and development of immune responses were followed up for several months. results: using this model, we have shown that -treated/infected mice develop a long-lasting protective humoral immune response as well as a strong and sustained cellular immune response with high cytolytic activity which are not observed in leukemic non-treated/infected mice. these results show that neutralizing mabs act as immunomodulatory agents capable of inducing long-term protective immunity ( g months) with both humoral and cellular contributions, despite the fact that they were administered over a short period of time (gros et al, ; gros et al, ; michaud et al. submitted) . although the initiation and maintenance of this long-term immunity is multi-factorial, we have demonstrated the crucial importance of the uptake of antibody-coated, infected cells by dendritic cells in the development of enhanced primary and memory antiviral t-cell responses. conclusions: our results show that infected-cells/antibody immune complexes play an important role in the induction and maintenance of protective antiviral immunity through enhancement of primary and memory antiviral t-cell responses. our observation might have important consequences on the design of antiviral mab-based immunotherapies. objectives: we have analyzed the potential of virus-like particles (vlps) from rabbit hemorrhagic disease virus (rhdv) as a delivery system for foreign t-cell epitopes. to accomplish this goal, we generated chimeric rhdv vlps incorporating a cd + t-cell epitope (siinfekl) derived from chicken ovalbumin (ova). the ova epitope was inserted in the capsid protein (vp ) of rhdv at two different locations: ) the n-terminus, predicted to be facing to the inner core of the vlps (rhdv-vlp- ), and ) a novel insertion site predicted to be located within an exposed loop (rhdv-vlp- ). both constructions correctly assembled into vlps and we analyzed the immunogenic potential of both chimeric rhdv vlps in vitro and in vivo. in vitro, dendritic cells (dcs) were able to process and present siinfekl peptide in the context of mhc class i from chimeric rhdv vlps for cd + specific recognition in a dose-and insert position-dependent manner. moreover, chimeric rhdv vlps activated dcs for tnf-alpha secretion.in vivo, mice immunized with the chimeric rhdv vlps without adjuvant were able to induce specific cellular responses mediated by cytotoxic (ctl) and memory t cells. although both chimeric rhdv vlps were able to induce specific ifn-g-producing cell priming, insertion of the siinfekl peptide at the amino-terminal position (rhdv-vlp- ) was more immunogenic than insertion at position for induction of ctls and anti-viral immunity.more importantly, immunization of mice with chimeric rhdv vlps at the highest dose tested was able to control an infection by a recombinant vaccinia virus expressing ova in target organs. in addition, immunization with chimeric rhdv-vlp- at the highest dose tested was able to resolve vv-ova infection. conclusion: our data demonstrated that immunization with chimeric rhdv vlps was able to protect mice from a viral challenge, suggesting the potential suitability of these constructions for new vaccine development against animal and human viral infections. objectives: a major issue pertaining to use of dna vaccines is that despite successful proof of principle results in small animal models, low efficacies have been reported in human clinical trials and large doses are required to induce protective immune responses. in this study, we describe the targeting of antigen-encoded dna directly to dendritic cells (dcs) through a pathway that results in internalisation and transfection using a cationic lipopeptide containing arginine residues and the lipid dipalmitoyl-s-glyceryl cysteine, a known tlr- ligand. methods: agarose gel electrophoresis was used to confirm the electrostatic binding of lipopeptide to dna encoding for green fluorescent protein (gfp), influenza hemagglutinin (ha) or nucleoprotein (np). transfection efficiencies of dcs using these complexes were determined by flow cytometry using specific antibodies for each encoded protein. stimulation of t cells by np-transfected dcs was also investigated by measuring their ability to induce in vitro cytokine secretion by influenza virus-specific cd + t cells. subsequently, vaccine immunogenicity was ascertained by immunisation of mice via the intra-nasal route. results: electrostatic binding of the lipopeptide to dna plasmids was confirmed by gel electrophoresis where the positively charged amino acids of the lipopeptide were able to neutralise the negative charged phosphate groups within the dna backbone and retard its ability to migrate towards the anode. high levels of gfp, ha or np were detected in murine spleen-derived cultured dcs following transfection with these complexes concomitant with the upregulation of surface mhc class ii molecules compared to when dna alone was used. the ability of transfected dcs to stimulate cd + t cells from influenza virus-infected mice was also investigated. subsequently, vaccination by lipopeptide-dna complexes resulted in the induction of higher numbers of ifn-g producing np - specific cd + t cells in the spleen and lymph nodes of mice compared to those that received dna alone. conclusion: altogether these results demonstrate that the use of a tlr- targeting lipopeptide-based system that can facilitate the delivery of dna by directly targeting and concurrently activating antigen-presenting cells, such as the dc, could prove to be advantageous in enhancing cellular responses induced by dna vaccination. the level of interferon in blood serum of non-infected mice was determined by elisa kit and by the cytopathic test in the l cell culture after aplication of ridostine and ridostine ointment. the effect of the preparation on phagocytic activity of macrophages was evaluated in the monolayer peritoneal cell culture. the statistical processing of the data was carried out by the student t-criterion. ridostine was administered to patients once or twice in the case of high temperature. the clinical signs were recorded (temperature, rhinitis, headache etc). for prophylactic and treatment purposes the ridostine ointment was intranasally applied twice per day during days. the effectiveness of the preparation was evaluated by clinical sings and the level of cd +, cd +, cd +, cd + t -lymphocytes. results: ridostine significantly decreased accumulation of the virus in lungs of infected mice at the initial stage of influenza. ridostine and ridostine ointment stimulated synthesis of interferon and phagocytic activity. ridostine in clinical practice decreased the duration of influenza, attenuated clinical signs and was more effective at an early stage of the infection. prophylactic intranasal application of ridostine ointment by healthy volunteers (nurses and doctors) resulted in a high degree of protection during the whole epidemic season and an activation of t-cell immunity. application of ointment at an early stage of disease markedly activated t-cell immunity, reduced the duration of the disease and the intoxication syndrome by , - -fold. conclusion: interferon inducer based on natural dsrna stimulates some reactions of innate immunity and resistance to influenza virus. the drugs based on dsrna show promise for treatment of influenza. objectives: the creation of gene engineering vaccines against hepatitis c virus (hcv) based on recombinant proteins is one of relevant approach. since the immunogenicity of these proteins is low as a rule, the choice of adjuvant is very important. the aim of this work was to evaluate immunogenicity of covalent conjugates between nonstructural ns and ns a hcv proteins and immunomax ® , an acid peptidoglycan of plant origin, which displays immunomodulating activity. objectives: ifn-g takes part in the development of an anti-infectious reaction of the organism, which is connected with the peculiarities of its immunomodulating action. a/h n influenza virus inhibits the ifn-g synthesis (mibayashi et al., ) and causes a decrease in cd + and cd + t-lymphocytes content in lung and lymphoid tissues associated with an impairment of this cytokine production (tumpley t. m. et al., ) . thus, ifn-g is a promising drug for prophylaxis and treatment of avian influenza under conditions of monotherapy or complex therapy. an essential defect of this cytokine is its fast degradation in blood. the goal of this work was to study immunomodulating properties of an ifn-g structural analog with increased proteolytic resistance when it was used alone or in combination with double-stranded ifn inducers. methods: a recombinant human ifn-g analog deltaferon is distinguished by a amino acid deletion at the c-end of the molecule and substitutions of amino acids in positions - (tat'kov c. i. et al., ) . deltaferon was i. p. administered to male non-inbred mice in doses of - * iu once or twice at an interval of hours, alone or in combination with double-stranded yeast rna preparation ( mg/kg). the content of ifn-a and ifn-g in mouse blood serum was determined by the immunoenzyme method, proliferative activity of splenocytes -by mtt-test (mosmann t., ) . results: when deltaferon was administered in doses of - * iu alone it did not influence the content ifn-a in blood, but caused a transient increase in the level of ifn-g. the injection of the preparation in a dose of * iu led to a an increase in both spontaneous and conconavalin a-induced proliferation of splenocytes. the two-fold administration of deltaferon in the maximal dose increased a level of ifn-g and inhibited cell proliferative activity. the combined administration of deltaferon ( * iu) and dsrna markedly increased the level of ifn-a and enhanced splenocyte proliferation. the recombinant human ifn-g analog is able to enhance ifn-g synthesis, splenocyte proliferation and to modulate the effect of ifn inducer. these data suggest that the studies of the preparation as an antiviral agent during influenza are perspective. by using a combined approach of routes of immunization and vaccine delivery systems such as poly-lactic acid (pla) biodegradable nanoparticles, we have dissected the intensity and quality of both cellular and humoral immune responses in mice. we showed that the amplitude and quality of the immune response (humoral, cellular) at systemic and mucosal sites (blood, vagina) could be largely influenced by the choice of a pertinent cutaneous route of vaccination (intradermal (id), transcutaneous (tc), subcutaneous (sc)). while id and tc route remain mostly efficient for the induction of antigen-specific cd responses (tetramer+ hiv- gag p cells), the quality of humoral responses (igg, iga) remained distinct between the two routes. in addition, sc route is less efficient than id and tc routes for the induction of cd responses after pla-p immunization. we have also shown that a lower antigenic charge of pla particles was needed when pla-p were injected using id and tc routes of immunization. currently, we are dissecting innate cellular mechanisms that gave rise to distinct quality of immune responses. this unique possibility to modulate the quality of the immune response according to the skin route of immunization paves the way for new vaccine design strategies and highlights the capacity of nanoparticles-based vaccine delivery system. b. dí az-freitas , c. prego , s. vicente , m.j. alonso , a. gonzález-fernández university of vigo. area of immunology, department of biochemistry, genetics and immunology, vigo, spain, university of santiago de compostela, nanobiofar group, department of pharmacy and pharmaceutical technology, santiago de compostela, spainobjectives: the design of effective vaccine delivery nanovehicles is opening up new possibilities for making immunization more equitable, safe and efficient. in this work, we purpose polysaccharidic-based nanocarriers as delivery structures for virus-like particle antigens, using recombinant hepatitis b surface antigen (rhbsag) as a model. our aim was to evaluate in a murine model if these nanocarriers induce an immune response after intramuscular and intranasal administration. materials and methods: loaded chitosan-based nanocarriers were prepared by cross-linking the polysaccharide chitosan, in the presence of the stabilizer poloxamer , with a counter ion, tripolyphosphate, containing the free rhbsag. the immunogenicity of these polysaccharidic-based nanocarriers with or immunizations to balb/c female mice ( weeks old) was assessed following intranasal or intramuscular immunizations. blood samples from the mouse maxillary vein were collected at different intervals (from day to post-primary immunization). specific igg antibodies levels directed against rhbsag in serum were measured by indirect elisa in miu/ml. results: chitosan-based nanoparticles with particle size in nanometric range, positive zeta potential and an important rhbsag loading were prepared. the results of the specific igg levels achieved following intramuscular administration of the antigen-loaded nanocarriers, and also of the alum-adsorbed vaccine showed the significant adjuvant effect of the nanocarriers. the response elicited was delayed respect to the alum based vaccine, and very interestingly, igg concentration was much higher using antigen-loaded nanocarriers than with the conventional vaccine. after intranasal administration, chitosan-based nanoparticles generated a lower immune response compared with the intramuscular route, but increasing over the time, showing an interesting slow release of the antigen. the igg titers elicited were enough to induce full seroprotection against the disease ( miu/ml). polysaccharidic-based nanocarriers with interesting properties for improving vaccination with complex antigens were designed and in vivo behavior of these nanocarriers suggests their potential utility as controlled release vehicles for reducing the number of intramuscular doses administered. more studies are currently underway to fully validate the potential of these nanocarrier prototypes and to optimize alternative routes of immunization such as the intranasal route. the success of immunotherapeutical approaches strongly relies on specific antigen targeting to dendritic cells (dcs) in an environment that provides optimal immunostimulatory signals. in our research group a bio-safe coronavirus-based vaccine vector platform that delivers multiple antigens to professional antigen- background: infection with human immunodeficiency virus type (hiv- ) is characterized by dysfunction of hiv- -specific t cells. to control the virus, antigenloaded dendritic cells (dcs) might be useful to boost and broaden hiv-specific t-cell responses. poly electrolyte microcapsules are potent protein delivery vehicles which can be tailored with ligands to stimulate maturation of dendritic cells. we investigated the immune stimulatory capacity of dendritic cells loaded with these microcapsules, containing both p antigen from hiv- and the tlr ligand poly i:c as a maturation factor. methods: monocyte-derived dc (mddc) from healthy subjects were cultivated with polyelectrolyte microcapsules containing, poly i:c. potential maturation of dc was evaluated by flow cytometry. mddc from hiv-infected patients under highly active anti-retroviral therapy (haart) were similarly pre-incubated with p microcapsules containing p and poly i:c. these antigen loaded mddc were used to directly stimulate autologous peripheral blood lymphocytes (pbl) in elis-pot. they were also co-cultivated for days with autologous pbl to evaluate the immunogenic capacity. potential expansion of specific t cells was measured by comparing elispot responses of pbl before and after coculture, using a pool of overlapping p peptides. intracellular staining of interferon-gamma (ifn-g), interleukin- (il- ) and cd a after p stimulation was also performed. results: mddc from hiv(-) subjects, incubated for hour microcapsules alone did not induce maturation of dc, but when poly i:c was present the dc did mature. mddc from haart treated hiv-infected individuals, cultivated with p containing microcapsules with poly i:c, were able to efficiently expand and broaden autologous effector memory t cells which contain and secrete ifn-g and il- , upon p peptide restimulation. objectives: we aimed at investigating whether and how the distance of a cytokine from the vlp surface impacts on its function and whether the relative distance towards a co-presented antigen is critical for its biological activity. methods: we inserted one, two or four ig-like domains of hcd b between our model cytokine il- and the minimal gpi-anchor acceptor sequence. subsequently, we compared particle production by western blotting for p gag, targeting of cytokines to lipid rafts and and vlp upon isopycnic membrane separation and biological activity in il- dependent proliferation assays of il- variants. results: murine il- attached to either of the four fusion partners was biologically active in vitro as shown by induction of proliferation of the il- dependent cell line ht- . we found that the membrane anchors comprising one and four ig-like domains (il- :: iggpi and il- :: iggpi) resulted in severely reduced vlp production by producer cells and despite of an increased targeting of il- :: iggpi to vlp, a reduced stimulatory capacity of producer cell crude supernatant, when compared to il- fused to the minimal gpi-anchor acceptor sequence of hcd b (il- ::gpi). il- :: iggpi, however, showed comparable particle production and biological activity in vitro when compared to il- ::gpi. furthermore, il- fused to ig::gpi showed an increased capacity to co-stimulate primary p tcr transgenic t-cells specific for lcmv-gp - in the context of h -d b . conclustions: besides the minimal gpi-anchor acceptor sequence we have identified one additional membrane anchor, which displays superior capacity to target cytokines to vlp. ig::gpi has a biological activity in vitro, which is comparable to the minimal gpi anchor. moreover, il- ::gpi displays increased co-stimulatory potential in the context of specific mhcp complexes. this work was supported by grants sfb f -b of the austrian science foundation, the austrian research promotion agency (forschungsförderungsgesellschaft) bridge grant & biomay ag, and the christian doppler laboratory for immunomodulation. a. roemhild , interdisciplinary transplant laboratory charite berlin, insitute of nephrology and medical immunology, berlin, germany immunosuppressive treatments, e. g. after transplantation are often followed by an impaired or dysfunctional immune system. missing viral immunity, particularly against ebv, is an essential key player in the development of severe infections and posttranplant lymphoproliferative disorders (ptld). ptld affects - % of solid organ transplant recipients, depending on the organ transplanted. healthy individuals control ebv infection by ebv-specific cytotoxic t lymphocytes (ctls), but some patients under immunosuppression are unable to do so. in these cases, immunotherapy is increasingly used as a new approach for re-establishing a functional immune response by retransferring in-vitro expanded autologous virusspecific t cells into the patient. currently these t cells are generated by repetitive stimulations with ebv-infected autologous lymphoblastoid cells (lcls). due to a generation time of - months, many patients suffering from missing viral immunity and subsequent severe viral disease are excluded from therapeutic benefit. therefore, shortening the generation time would be an important step to make adoptive immunotherapy available for more patients. t cell lines were generated with two different protocols. in the first protocol t cells are generated by repetitive stimulation with ebv-infected autologous lcls. the second protocol is based on stimulation with different overlapping ebv peptide-pools and immunomagnetic cell isolation. expanded t cells were analysed using multicolour flow cytometry. cells were stained for diverse surface markers and intracellular cytokine production. cytotoxic capacity and specificity was determined by a calcein release assay. our group developed a new protocol for the production of ebv specific t cells, thereby shortening the generation time from , month to days. t cell lines are composed of cd and cd cells with a mainly effector memory like phenotyp. after restimulation the cells produce more tnfa than ifng. depending on the generation protocol t cells specifically recognized and lysed autologous lcls alone or loaded with ebv-peptides. the detailed characterization of ebv-specific t cell lines should help to further improve the adoptive immunotherapy and its outcome. the novel, short time generation protocol did not affect phenotyp and cytokine production of the t cells. nevertheless their therapeutic potential in vivo has to be tested in further experiments. s. s. schmucker , m. assenmacher , a. richter miltenyi biotec gmbh, r&d cell biology, bergisch gladbach, germanyadoptive transfer of virus-specific t cells provides a promising treatment of infection in immunocompromized patients. as expansion of virus-specific t cells from antigen-experienced donors is feasible, no reliable protocols for generation of antigen-specific t cells from naive hosts exist. in this study we established a cell culture system for priming of highly rare naive cmv pp -specific cd + and cd + t cells from cmv-seronegative donors in vitro.magnetically isolated naïve (cd ro -cd -) cd + and cd + t cells from pbmc of cmv-seronegative donors were co-cultured with autologous mature monocytederived dc loaded with cmv pp peptide pool and cd -depleted autologous pbmc as feeder cells in the presence of il- , il- , and il- . already - days after primary activation pp - /a -tetramer + cd + t cells were detectable for hla-a + blood donors. to analyze cd + t cells having other specificities than for the peptide pp - as well as probably primed cd + t cells, we looked for the production of cytokines after a second stimulation. we found ifn-g secretion in up to . % of the cd + t cells and up to . % of the cd + t cells after restimulation with pp peptide pool, but not with either irrelevant ie- peptide pool or without antigen, in each of eight donors tested. for generation of t cell lines, we magnetically enriched the primed t cells according to their ifn-g secretion. subsequent cultivation for days led to a - fold expansion of pp -specific t cells, defined by their sustained capability to produce ifn-g. evaluation of the antigen-specificity of the expanded t cells also showed upregulation of the activation markers cd and cd only if restimulated with the pp peptide pool. further cytokine analysis of the cells revealed co-production of ifn-g, tnf-a, and il- , indicating the functionality of the in vitro primed and expanded t cells.in conclusion, we established a cell culture system, which enables the in vitro priming and expansion of cmv-specific cd + and cd + t cells derived from the naive compartment. this should extend the application of adoptive t cell therapy to patients for whom immune donors are not available. a. i. wolf , k. mozdzanowska , l. otvos , j. erikson the wistar institute, philadelphia, united states, temple university, philadelphia, united statesthe influenza virus a matrix protein ectodomain (m e) sequence has remained highly conserved among various human influenza a strains and is therefore a promising target for a protective vaccine. based on previous work using a synthetic m e-based multi-antigenic peptide vaccine (mozdzanowska at al., vaccine ; virology journal ), we generated a novel peptide and investigated its efficacy in inducing an anti-m e antibody (ab) response and its ability to confer protection against viral challenge.objectives: cytomegalovirus (cmv) causes significant morbidity and mortality in patients after haematopoietic stem cell transplantation (hsct). due to limitations of current antiviral therapies, alternative approaches, involving transfer of donor-derived cmv specific cd + t cells, have been considered. clinical data confirm a crucial role for antiviral cd + t cells inversely correlating with the incidence of cmv reactivation and disease. cmv specific cells have to reach protective levels in order to be effective. levels of such cells correlating with protection against cmv infection and disease have only been reported in patients expressing hla-a* and hla-b* previously. considering other frequent hla alleles cmv specific cd + t cells were monitored longitudinally in hsct patients in this study to establish the cell number thresholds at which patients are protected from cmv reactivation. methods: we have correlated the pattern of different ex vivo cmv peptide specific cd + t cell responses (frequency analysis using tetramer staining and interferon gamma elispot analysis) with the cmv viral load (dnaemia) and clinical status in patients. different response groups were compared using the mann-whitney-u test.results: our results demonstrate that the presence of different cmv specific cd + t cells inversely correlates with the ability to detect of cmv reactivation in patients at different cell number thresholds. we show that the cell number thresholds for hla-a* /pp ( - ) ( . x cells/l) and hla-b* /pp ( - ) ( . x cells/l) specific cd + t cells are significantly lower than those for hla-a* /pp ( - ) ( . x cells/l) and hla-a* /pp ( - ) ( . x cells/l) specific cd + t cells in hsct recipients post transplant. this difference is also evident in healthy cmv seropositive volunteers. conclusion: these findings suggest a differing efficiency of the responses restricted by the two sets of alleles. the data merit further studies using larger patient cohorts and are important for considerations regarding the epitope restriction and quantities of ag specific t cells to be monitored after therapeutic strategies for cmv in hsct patients. ( , - mcg) . no adverse effects were indicated during trials (up to month of observation).hiv-specific antibodies were induced by dose-dependent manner, the most prominent response was detected after th immunization with mcg of vichrepol.no differences were detected in cd + and cd + t cell counts and cd +/cd + ratio, so there was additional safety issue concerned to the possible sensitivity of vaccinees to hiv infection. the results of phase i clinical trials of vichrepol vaccine were approved by who authorized russian national control institution and transition to phase ii immunogenicity trials was recommended. objectives: to improve the vaccination efficiency of adenoviral vectors for anti-retroviral vaccination, we constructed adenoviral nanoparticles by fusion of the vaccine antigen to the adenovirus capsid protein pix. the adenoviral nanoparticle vaccine was evaluated in the friend virus (fv) mouse model and compared to conventional adenoviral vectors. methods: adenoviral nanoparticle vectors were constructed by deletion of pix from the adenoviral genome and insertion of the fusion protein encoding sequence as transgene. for vaccination against fv, that is a retrovirus complex of friend murine leukemia virus (f-mulv) and spleen focus forming virus, we constructed fusion proteins of pix and the f-mulv surface env protein gp or gag. to elucidate underlying mechanisms we produced displaying-only nanoparticles and plasmid dna encoding either pixgp or gp alone. conventional adenoviral vectors were used that express full-length f-mulv env and gag. the vaccines were tested in cb f hybrid mice that are highly susceptible to fv infection and develop viremia and splenomegaly after fv infection. results: vaccination of cb f mice with adenoviral nanoparticles expressing fusion proteins containing gp resulted in protection from viremia and splenic enlargement after fv challenge that was superior to vaccination with conventional vectors. immunological analyses showed that the adenoviral nanoparticle vaccine induced a significantly higher number of f-mulv env-specific cd + t cells and higher antibody titers than a conventional adenoviral vaccine expressing the vaccine antigen. we could show that for the beneficial effect it is necessary that the fusion protein is incorporated into the adenoviral particle and it also has to be expressed from the adenoviral vector in vivo. conclusion: adenoviral nanoparticles are a useful tool for the induction of antibody and cd + t cell responses that are superior to conventional adenoviral vectors. this new type of adenovirus-based vaccination vector combines genetic and protein vaccination and should make adenoviral vectors even more interesting for vaccination purposes. . antibody levels were monitored by elisa and hemagglutination inhibition assay, viral excretion in nasal washes was assessed by quantitative rt-pcr, and cellular production of ifn-gamma was measured via flow cytometry. results: we found that animals vaccinated with caf exhibited higher levels of serum igg and mucosal iga than the ones which received the vaccine alone, and that they excreted - % less virus. animals that received only vaxigrip were producing ifn-gamma after challenge, a sign of infection by low virulence influenza strains, whereas the animals that received also caf did not show any increase in their levels of ifn-gamma. conclusion: caf enhances the protection conferred by the commercial inactivated vaccine against strains matched by the vaccine. evaluation of the t-cell specific immune response is very important for global eradication of measles and rubella. peripheral blood lymphocytes (pbl) from children aged - years old ( boys and girls) -group , and children ( boys and girls) - years old -group were isolated on a gradient of density before vaccination ( or revaccination) with priorix, week, and months after and incubated with cfse. then million/ ml pbl were incubated in rpmi- supplemented with % fcs (the negative control), at presence of mcg/ml pha (the positive control) or at presence of the measles or rubella viruses antigens in a humidified atmosphere containing % cÎ at °c within day. intensity of a fluorescence estimated on fl by flow cytometr facscalibur (bd biosciences, usa). cytokines production was measured in the same cultures by bioplex technology (biorad, usa). in the negative control % pbl in both groups did not enter mitosis. in the positive control % of cells have passed one and more mitoses. in group measles or rubella antigens did not induced lymphocytes to enter mitosis, like in negative control, before the vaccination and in a week, however in months - % of lymphocytes demonstrated antigen-specific proliferation. in group , on the contrary, before the vaccination the most part of cells ( - %) has not entered division, but - % of cells have passed and more mitoses. in a week specific lymphocyte proliferation decreased and in months it was increased up to - %. production of the interleukin (il) , ifn-g, tnf-a, il- , il- was more informative than il- , il- , il- , il- . measles and rubella antigens induced cytokines production in pbl of immune children and did not influence on pbl of intact children. thus, it was shown, that both methods can be applied to revealing the specific cellular immune response to measles and rubella antigens. objectives: broadly neutralizing human monoclonal antibodies (mab) and patients' sera recognizing functionally conserved epitopes on hiv envelope (env), such as the gp cd -binding site (cd bs), appear to be uncommon. therefore, new approaches are needed to elicit the humoral response on these conserved epitopes. here we describe the generation of two anti-idiotype (ai) murine antibodies recognizing human anti-hiv- neutralizing polyclonal iggs directed against the cd bs. the mabs were shown to react with an anti-cd bs human neutralizing mab (b ), to elicit antibodies that recognize the gp molecule and an anti-hiv- neutralizing response in rabbits, confirming them as cd bs mimotopes. these mabs were also used as probe to detect the expression of clonally distinct anti-gp antibodies in sera of hiv-infected individuals. methods: broadly neutralizing sera were collected from long-term non-progressor patients. anti-cd bs iggs were purified and used to immunize mice for hybridoma generation. mabs reacting in elisa with the anti-cd bs igg fraction were used to immunize rabbits. rabbit sera were then tested for anti-gp titer and hiv neutralizing activity by pseudovirus-based neutralization assay. sera from hiv-infected individuals at various clinical stage of infection were studied to validate an immunoenzymatic assay able to detect the reactivity to the ais. serial dilution of b in sera from healthy hiv-negative donors were used to determine elisa sensitivity. results: two clones (p and p ) reacted in elisa only with the cd bs-directed igg fraction. the clones were also recognized in elisa by b . p and p -immunized rabbit sera showed a strong anti-gp titer. in the pseudovirus assay the ais-immunized rabbits showed a neutralization activity against virions bearing hxb strain glycoproteins. in particular, / rabbits in the p group and / in the p group showed an % hiv neutralization at dilutions ranging from : to : . the immunoenzymatic assay used, allowed to detect a p and p reactivity in hiv-positive sera and was able to detect a b concentration equal to ng/ml. conclusions: these data demonstrate that immunogens designed on the idiotype of broadly neutralizing abs are feasible and could help in the design of effective anti-hiv vaccines or diagnostic assays. yellow fever vaccines ( d and dd) are well tolerated, with a very low rate of severe adverse events (yf-sae), such as serious allergic reactions, neurotropic (yf-and) and viscerotropic (yf-avd) diseases. viral and host factors have been postulated to explain the basis of yf-sae, especially those able to modify the host immune response to the yf vaccine. however, the mechanisms underlying the occurrence of yf-sae still remain unknown. in the present investigation, we present a detailed immunological analysis of a -year-old us citizen female patient, who developed yf-and characterized by encephalitis associated with pancreatitis and myositis following d- vaccination. our findings highlighted that yf-and exhibited decreased expression of fc-g-r in monocytes (cd , cd and cd ) along with increased levels of nkt-cells (cd + cd +/-cd +/-/cd + ratio) and activated t-cells (cd + and cd + ) and b-lymphocytes. enhanced levels of plasmatic cytokines (il- , il- , il- , il- and il- ) besides exacerbated ex vivo intracytoplasmic cytokine pattern, mainly observed within nk-cells (inf-g + , tnf-a + and il- + ), cd + t-cells (il- + and il- + ) and b-lymphocytes (tnf-a + , il- + and il- + ). the analysis of cd + t-cells revealed a complex profile with increased frequency of il- + and ifn-g + and decreased percentage of tnf-a + , il- + and il- + cells. depressed cytokine synthesis was observed in monocytes (tnf-a + ) following in vitro antigenic stimuli. these results support the hypothesis that a robust magnitude of the adaptive response and abnormalities in the innate immune system may be involved in the establishment of yf-sae. this is the first case report of yf-sae investigated by members of the international laboratory network for yellow fever vaccine associated adverse events. g. mester , h.-g. rammensee , s. stevanović eberhard-karls-universität tübingen, department of immunology, tübingen, germany adenovirus (adv) is a widespread pathogen in humans and can persist in its hosts after infection. persistent virus is an important cause for severe disease in immunocompromised individuals, e. g. bmt recipients, with high rates of mortality. however, the cellular immune response against adv is poorly characterised, and very few t cell epitopes have been published up to now. thus, our aim was to detect dominantly immunogenic adenoviral cd t cell epitopes by analysing the responses of healthy blood donors who have overcome infection. we have predicted possible cd t cell epitopes for the frequent mhc class i alleles a* , a* , and a* from the proteins pii (hexon), pviii, and e a of adv strains ad and ad by using the syfpeithi software developed by our group (www.syfpeithi.de). subsequently we performed a -day recall stimulation of pbmcs from at least healthy donors with synthetic peptide followed by ifn-g elispot screenings to identify naturally occurring t cell responses and assess their frequency in the population. tetramer and intracellular cytokine stainings were also carried out to confirm the presence of specific cd t cells. we could identify new peptides eliciting ifn-g responses, several of which were confirmed as novel cd t cell epitopes. amongst others we found at least one immunodominant epitope recognised by more than half of the healthy donors for each examined hla restriction as well as, to our knowledge, the first adenoviral epitope derived from a protein other than hexon. these findings will be helpful to identify frequently immunogenic and thus promising candidate peptides for in vitro t cell priming or expansion preceding adoptive transfer, which has been proven to be a valuable therapeutic approach in the treatment of persistent viruses in immunocompromised patients. methods: sle ( ara criteria) was diagnosed in a -year-old african female patient with hiv- (clade c) infection. good initial response occurred on hydroxychloroquine and steroids followed by disease flare and drop of cd t-cell count x cells/mm . initiation of mg mmf bid was associated with biological and clinical remission of sle and cd t-cell increase. no opportunistic infections or cancers were noted during a -year follow-up and the patient remained always naive to art. hiv- -specific cd and cd t-cell responses were analyzed after months of mmf by ifn-g elispot assay and polychromatic flow cytometry assessing ifn-g, tnf-a and il- production following stimulation with a panel of hiv- -derived optimal epitopes ( / -mers) covering various hiv regions and a pool of hiv- -derived peptides ( -mers overlapping by aminoacids) encompassing the entire gag protein. all peptides are derived from hiv- consensus strain iiib. results: highly polyfunctional hiv- specific cd and cd t-cell responses against gag were detected. epitope-specific cd t-cell responses were identified: except for one response restricted by hla a* and another one by hla cw* , all the others were restricted by hla-b alleles and mostly by b* (n = ). seven out of responses were strong enough to be further analysed with regard to their functional profile and shown to be highly polyfunctional (i. e. ifn-g+, tnf-a+ and il- +) regardless of the viral region and hla restriction. conclusion: strong, broad and polyfunctional hiv- specific cd and cd t-cell responses known to be associated with nonprogressive infection were detected during mmf treatment.we therefore suggest that mmf use in the context of sle-hiv is not detrimental to the establishment or preservation of protective hiv- t-cell immunity. the rabies virus was propagated in the vero cell line. virus was titrated by focus fluorescent units. virus preparations having a titer of dl /ml were inactivated with b-propiolactone. aluminium hydroxide gel or squalene, at different concentrations were adsorbed to the inactivated rabies virus. male mice of the strain cf- of - g and no less than four weeks age, were distributed in six groups for intraperitoneal immunization, group a was immunized with virussqualene, group b with virus-aluminium hydroxide, group c with the antigen alone, group d with saline buffer-squalene, group e with saline buffer-aluminium hydroxide and group e was inoculated with mock-infected cell culture supernatant. mice were boosted at the th day. all mice were properly bled to prepare preimmune sera and hyper-immune sera. at the end of the immunization protocol the igg raised against the rabies virus was tested by an indirect elisa. results: the highest titers of neutralizing antibodies were obtained with similar concentrations of either squalene-or aluminium hydroxide-based vaccine formultaions. there was a significant difference in the neutralizing antibody titers produced by mice immunized with the antigen (inactivated rabies virus) adsorbed to the adjuvant, as compared to those obtained from mice immunized with the antigen alone, as expected, no neutralizing antibodies were detected on mice inoculated with saline buffer or mock-infected vero cell supernatant. conclusions: the use of either squalene or aluminium hydroxide as adjuvant in the canine antirabic vaccine formulation increases immunogenicity, almost to the same extent. aluminum hydroxide adsorbed to the antigen seems to be a better option, since squalene is more expensive than aluminium hydroxide. supported by: concyt- , cofaa and cgpi- . . state of vaccine-induced measles immunity was determined by means of elisa in - , - and - years since revaccination with live measles vaccine (lmv) before and after tuberculosis chemoprophylaxis. statistic data were processed with t-, w-and u-criteria. results: during the first three years since lmv revaccination igg level was middle (children with negative and long-term positive mt) and high (children with conversion and hyperergic mt). in - years since lmv revaccination uninfected and long-term infected children showed a significantly decreased (p p . ) measles immunity and antibody level much lower (p p . ) than among children with mt conversion. in - years the comparison group kept decreased (p p . ) measles immunity, the majority ( ± . %) of persons had minimal protected igg level, but the observation groups were characterized by average immunity level, which was higher (p p . ) than in the comparison group. comparing measles immunity level before and after tuberculosis chemoprophylaxis demonstrated the following: measles igg level among long-time infected children on completion of chemoprophylaxis decreased (p p . ), the majority ( . ± . %) of persons lost protected antibody level; among children with mt conversion in - years since lmv revaccination immunity state didn't change, but in further periods antibody level decreased (p p . ) to low values; among children with hyperergic mt igg level decreased (p p . ) and reached low (in - years), minimal protected (in - years) and lower than protected (in - years) values. -at the early stage of tubercular infection process measles immunity was higher compared to uninfected with mycobacterium tuberculosis persons, which fact is connected with immunomodulatory action of low-molecular peptide of bacterial cell wall -muromildipeptide.-in remote periods since lmv revaccination and on completing preventive tuberculosis treatment decreased measles immunity was observed.-in countries with high tuberculosis morbidity chemoprophylaxis level among children with latent infection is high, which can indirectly influence population measles immunity. objectives: to evaluate the balance of ifn-gamma and il- producing cells in lungs during the immunotherapy of tuberculosis with the dna vaccine encoding the heat-shock protein (dnahsp ). methods: balb/c female mice were infected by intra-tracheal route with h rv mycobacterium tuberculosis. immunotherapy with endotoxin free dnahsp genetic vaccine was done at days , , and post-infection. each dose consisted of micrograms of dna vaccine in the quadriceps. intracellular cytokine staining of cd +, cd + and gamma-delta t cells from lungs were determined and days after the end of the therapy. bacilli loads, histopathological and morphometric analysis of lungs were also evaluated. differences of p x . were considered significant (t test). results: at day after the end of the immunotherapy, dnahsp treated mice exhibit increased numbers of absolute cd + and gamma-delta t cells when compared to non-treated animals. the percentage of ifn-gamma and il- producing gamma-delta t cells were the same between treated and non-treated animals. in contrast, dnahsp treated mice showed more ifn-gamma producing cells in both cd + and cd + cell populations. at day after the end of the therapy, the main observation in mice which received dnahsp treatment was the augment of all three populations producing ifn-gamma. although non-treated animals also increased the frequency of cd + and gamma-delta t cells positive for ifn-gamma, they did not increase the numbers of ifn-gamma cd + cells, together with a more frequency of gamma-delta t cells producing il- . finally, the immunotherapeutic effects of dnahsp vaccination also included the diminution of bacilli loads in lungs, spleen and liver and the reduction of inflammation in lungs as determined by the histopathological and morphometric analysis. the results presented here indicates that cd + cells producing ifn-gamma and the reduction of the frequency of gamma-delta t cells secreting il- , are the main effects of dnahsp immunotherapy of murine tuberculosis. furthermore, these results have important implications since they indicate the importance of an appropriate balance of il- and ifn-gamma levels for the combat of the bacilli and the reduction of the immunopathologic damage in lungs. the detection of quantitative changes in mrna expression levels are currently being performed using either genome-wide (microarray) or single gene (real-time pcr) screening methods. because these techniques are technically challenging and too costly to be applied on a routine basis in resource poor settings, we have developed a reverse-transcriptase multiplex ligation-dependent probe amplification (rt-mlpa) method. rt-mlpa is a reliable, robust, low cost and user friendly technique permitting rapid mrna expression profiling of as many as loci in a single reaction. genes of interest can be selected on a tailor-made basis. the assay is highly reproducible, has an extensive dynamic range of - log depending on the genes of interest, and a pcr amplification step within the rt-mlpa ensures assay sensitivity, which is an essential prerequisite for the relative quantification of scarcely expressed genes. since this assay is relatively high throughput ( -well format), requires only ng rna per sample, and allows mrna profiling in direct ex vivo whole blood samples (from e. g. pax-gene tubes), it is an exceptionally suitable technique for performing semi-large scale gene expression analyses in human cohort studies. to illustrate this, we have been able to successfully implement this assay in different laboratories in sub-saharan africa. thus far we have applied rt-mlpa to characterize the human immune response to mycobacterium tuberculosis, with particular emphasis on the expression of genes associated with protective host cellular immunity and human disease susceptibility. a particularly useful application of the rt-mlpa is the identification and monitoring of host-biomarker profiles that predict (protection from) tuberculosis (tb) disease in latently infected household contacts or (in)adequate responsiveness to therapy in active tb patients. initial data sets already probe differences in immune reactivity in populations, yielding new candidate biomarkers associated with tb disease. these biomarkers may provide new and relevant information that can be applied in future tb studies for rapid, easy, semi-quantitative and reliable detection of host immune biomarker profiles. preclinical m. leprae infection is a major source for leprosy transmission. therefore, early detection of individuals infected with m. leprae is crucial. however, to date there are no diagnostic tests available that can identify preclinical leprosy. such tests will contribute to the prevention of leprosy disability and its further transmission by otherwise undiagnosed and untreated index cases.newly developed hla based bio-informatic tools combined with comparative genomics have created novel opportunities to help design improved tests for early detection of m. leprae infection.using this post genomic approach, we were able to identify candidate proteins and peptides unique to m. leprae containing predicted t cell epitopes restricted via several major hla-class i and ii alleles. since the selected genes were of unknown function, their expression in m. leprae bacilli was assessed.evaluation of the immunogenicity of these m. leprae proteins in pbmc from a brazilian population showed that candidate antigens induced significant ifn-g levels in m. leprae infected individuals but not in healthy controls from an endemic area.importantly, among exposed healthy controls % had no detectable igm antibodies to the m. leprae specific pgl-i, but instead responded to one or more m. leprae antigen(s). to further improve the diagnostic potential of these m. leprae sequences, synthetic peptides spanning all m. leprae proteins were analyzed similarly. determination of cumulative t cell responses towards of these peptides that activated pbmc of leprosy patients increased the sensitivity compared to single peptides to % in pb, % in rx and % in hhc, without compromising specificity.since diagnostic tools should be applicable in several populations regardless of the genetic background, these m. leprae antigens are also tested in populations on the african (ethiopia) and asian (nepal) continent.in addition, we have applied these antigens in a new user-friendly ucp-lf assay to detect different cytokines. this assay proved to be more sensitive than elisa for detection of ifn-g and can be easily applied in field sites. tuberculosis, an infectious disease caused by mycobacterium tuberculosis (mtb), affects millions of people. m. bovis bcg is the vaccine against tuberculosis but its efficiency is variable for the pulmonary form of the disease. paratuberculosis, an enzootic bacterial disease in ruminants, due to mycobacterium avium subsp. paratuberculosis (map), has a significant economic impact on livestock production, and moreover, map infection may be one of the microbial triggers of crohn's disease in humans. map vaccines can delay apparition of clinical symptoms, but they do not prevent infection and they have a confounding effect in the skin-test based bovine tuberculosis control programs. cd l, a co-stimulatory molecule preferentially expressed on activated cd + t cells, is the ligand of cd . cd -cd l interaction induces the production of il- and the initiation of a th -type immune response. several studies show that cd l is required for the activation of macrophages and the maturation of dcs. moreover, cd l enhances the capacity of cd + t cells to produce ifn-g and to lyse mtb-infected monocytes. in this study we attempt to improve existing tb and map vaccines with a recombinant bcg expressing cd l. we have constructed the recombinant bcg strain expressing cd l (rbcg ) by electroporation of bcg with pgfm /signalsequenceag b-cd lec and an another recombinant strain with empty vector pgfm (rbcg ) as a control. the expression of cd l has been evaluated by western blotting. balb/c mice were vaccinated with the recombinant bcg vaccines. bcg growth kinetics were compared by counting viable bacteria (cfu) in spleen and lungs. the immune response was evaluated by measurement of th type cytokine secretion of splenocytes after in vitro restimulation with immunodominant antigens and selected peptides. two months post vaccination, mice were challenged with mtb and map and protection was evaluated. preliminary results show normal persistence of the two recombinant bcgs. analysis of the immune response shows an effect of cd l weeks after vaccination but not at and weeks. rbcg seems to be more protective against paratuberculosis than rbcg , but not against tuberculosis. another vaccination experiment is required to confirm these results. the effects of bcg-cd l on cultured dcs in vitro will further be explored. objectives: tuberculosis is a major health problem globally and it is of critical importance to develop an effective vaccine to prevent further spread of the disease. iron is a key nutrient for both mycobacterial infection and for a successful protective immune response by the host. the regulation of iron availability within the host involves the intracellular iron-binding protein ferritin and it is proposed here that the regulation of ferritin is tightly controlled in the host immune response to tuberculosis. methods: using the guinea pig model of mycobacterium bovis bacillus calmette-guérin (bcg) vaccination, populations of immune cells were isolated and restimulated ex vivo over a time-course study using purified protein derivative (ppd) of mycobacterium tuberculosis or infected with bcg or m. tuberculosis. the expression of ferritin in co-ordination with key immuno-regulatory proteins, tnfa, ifng and il- a, was examined using real-time pcr. to determine whether immuno-regulatory proteins are involved in the regulation of ferritin, cytokine cascades were inhibited in the ppd re-stimulation studies by the addition of guinea pig specific tnfa and ifng antibodies. results: a typical pro-inflammatory immune response was observed with significant up-regulation (p x . ) of tnfa, ifng and il- a after re-stimulation with ppd and mycobacteria. of interest was a trend in ferritin down-regulation after re-stimulation with ppd and bcg and this was significant (p x . ) after restimulation with m. tuberculosis. the down-regulation of ferritin was also affected by the addition of tnfa antibody in the ppd re-stimulation study. conclusions: ferritin is important in the storage and management of intracellular iron and its regulation must be tightly controlled to restrict iron availability from invading mycobacteria from sequestering free iron. the data indicate that the regulation of ferritin is very subtle and is affected by cytokine cascades that involve tnfa. these results contribute to our understanding of the role of iron and intracellular ferritin in developing a protective immune response to mycobacteria in the guinea pig model of tuberculosis. this work is funded by health protection agency phd studentship award. methods: anti-cd mab and ag a were chemically treated with sata and sulfo-smcc respectively, in order to produce a stable crosslinker between both proteins. crosslinking was confirmed by western blotting and cd binding on cd transfected l fibroblasts. the conjugates were tested in vivo in wild type and cd + cell-depleted mice for the induction of specific anti-ag a serum antibodies. splenocytes were challenged ex vivo with ag a and were examined for their ability to produce th -related cytokines. elispot assays were performed to determine ifng production and flow cytometry was used to analyse intracellular cytokine staining for tnfa, ifng and il- . we developed a method to successfully crosslink anti-cd mab to ag a. serum antibodies against ag a were detected after immunisation with this conjugate vaccine in both wild type and cd + cell-depleted mice. t cells derived from mice immunised with conjugate vaccine, and stimulated ex vivo, showed an increase in ifng production (elispot), when compared to mice vaccinated with ag a alone. production of two other th -related cytokines, tnfa and il , was also increased in these t cells as shown by intracellular cytokine staining. conclusion: our results suggest that anti-cd conjugate vaccines could provide a new way to increase vaccine efficacy. this new conjugate vaccine may be able to by-pass the need for cd + t cell help in the production of specific antibodies, which would be a major benefit of any therapeutic vaccine to be used in immunocompromised patients. h. schäfer , r. burger robert koch-institute, cellular immunology, berlin, germany, robert koch-institute, infectious diseases, berlin, germanyobjective: immunity against mycobacterial infections is mediated by both cd -positive and cd -positive t-lymphocytes. cd -positve cells respond to peptides derived from cytosolic proteins and presented on mhc class i molecules of antigen presenting cells (apc) via the endogenous pathway. some apc however, are able to take up extracellular antigens and present peptides thereof on mhc class i molecules. this process has been termed cross-presentation and has been shown to be of importance in the immune response against intracellular bacteria. to define the contribution of cross-presentation to activation of cd + t cells in the response against mycobacterial antigens, we analyzed the secondary immune response in the guinea pig. methods: purified t lymphocytes from guinea pigs immunized with bcg or complete feund's adjuvant were labeled with the intracellular fluorescent dye cfse and incubated with ppd and/or apc for days. surface phenotype and proliferation of t-lymphocytes were analyzed by flow cytometry.results: up to % of lymph node t lymphocytes form immunized guinea pigs proliferated after in vitro restimulation with ppd-pulsed macrophages. no difference was observed between bcg-(living mycobacteria) and freund's adjuvant (heat killed mycobacteria)-immunized animals. the responses of both t cell subsets were equally strong, although the killed immunogen should primarily target the exogenous pathway of antigen presentation and therefore preferentially prime cd + t-lymphocytes in vivo. similarly, the cd -positive subpopulation should primarily respond to soluble antigens presented on mhc class ii molecules. proliferation of both the cd + and the cd + subpopulation depended on the presence of apc. stimulation oft cd + cells as a consequence of direct loading of peptides onto mhc class i molecules was ruled out by using mhc-class i-positive fibroblast cells instead of professional apc, which did not lead to proliferation of primed t-lymphocytes. conclusion: cross-presentation of soluble antigens to cd -positive t cells is a highly effective means to stimulate the response of cytotoxic t cells against mycobacterial antigens even without direct contact to infected cells. therefore cross-priming might represent an important mechanism for the induction of the cellular immune response against intracellular pathogens and should be useful for the rational design of vaccines against mycobacterial diseases. objectives: due to broad antigenic cross reactivity of purified protein derivatives (ppd) with bcg vaccine strains and environmental mycobacteria, results of currently used tuberculin skin test (tst) is not reliable to evaluate the specific anti-tuberculosis immune response. therefore, new tools are required to improve mycobacterim tuberculosis (tb) diagnosis and treatment, including enhanced ability to compare new treatment strategies. among different antigens early secretory antigenic target (esat- ) protein is highly specific for tb complex and elicit strong t-cell response in human. in order to monitor the immune response against the pathogen, iranian and afghan adults ( patients with sputum smear and culture positive tuberculosis, recovered patients during months after full course of chemical treatment and healthy individuals) were recruited to quantify the frequency of esat- and ppd specific t-cells in their peripheral blood by home made elispot ifn-gamma assay. results: considering cut off of spot forming unit ( g spots per million), we found detectable response to esat- in almost % of patients with active disease. this frequency among treated patients after disease recovery was not significantly different and % of these individuals had detectable esat- specific response even after six months completing treatment. neither of healthy individuals showed such response. t cell response against ppd was identified in %, % and % of healthy participants, active patients and healing individuals, respectively. conclusion: elispot ifn-gamma assay showed a sharp induction of th immune response, against esat- , in tb patients which persists after successful treatment and full recovery. these results may show potential application of tuberculosis-specific elispot testing as a proxy measure of tb diagnosis and treatment. bcg is the only available vaccine today to fight tuberculosis, but it has been reported to be variably efficacious in the field. both environmental and genetic host factors as well bcg strain variability and virulence of the intruding m. tuberculosis strain have been suggested to affect the efficacy of bcg vaccination. in mouse and bovine models it has been shown that pre-exposure to mycobacterium spp. negatively affected protective efficacy of bcg. we use non-human primates (nhp) for the evaluation of new tb vaccine candidates and possible identification of immune mechanisms of protection. however, in naive rhesus macaques a variable efficacy of bcg reminiscent of the clinical situation was revealed. by meta-analysis we compared immune response parameters measured after vaccination using bcg strain danish in the context of protection measured by gross pathology evaluation after experimental infection with a constant m. tuberculsosis strain erdman. although numbers of animals used are relatively low, data suggest that both breeding origin as well as the immune status of monkeys impact on the efficacy of bcg. most remarkably, while bcg induced levels of ifng secretion did not correlate with protection, kinetics of secretion monitored after in vitro stimulation of peripheral blood lymphocytes did correlate. our findings would suggest that, in accordance with mouse and bovine experimental data and epidemiologic observations, possible pre-exposure to mycobacterial antigens beyond the current sensitivity of tb diagnostics for nhp, negatively affects the protective efficacy of bcg. together, these results are relevant for evaluation and interpretation of tb vaccine tests in nhp and support further research into the identification of (mechanisms of) protective immunity in the primate host. p. s. nagpal , p.k. upadhyay national institute of immunology, pdc- , delhi, indiaobjective: study was aimed for the preparation of dry powder formulation containing live mycobacterium indicus pranii for pulmonary immunization against tuberculosis. pulmonary delivery evokes both systemic as well as mucosal immune response against the antigen. secondly, pulmonary delivery is a needle-free delivery system, long desired for vaccine delivery. dry powder aerosol one such method, in which vaccine can be directly delivered to lung without any kind of invasion and it has an edge over liquid formulation being feasibility of storage at room temperature, long term shelf stability and higher drug content per unit mass as compared to liquid one. method: sodium alginate solution with suspended mycobacterium indicus pranii was aerosolized using laboratory modified nebulizer assembly. aerosols so generated were entrapped in cacl solution with poly-vinyl alcohol (pva) as surfactant. particle so formed collected by centrifugation and lyophilized for dry powder formulation. pva and alginate concentration varies the size, surface and shape of the particles. formulation so prepared was delivered to c bl/ mice directly into lung by endotracheal intubations of mice. proliferation index (pi) of spleenocytes of immunized mice was measured after in-vitro stimulation with mycobacterium tuberculosis antigen. result: . % alginate, . % pva concentration gives particles with size of - micrometer as confirmed by particle size analyzer and scanning electron microscopy. viability of the mycobacterium indicus pranii was best achieved with % trehelose and . % pvp (poly vinyl pyrollidone). there was fold increase in proliferation index of spleenocytes and releases pico-gram of interferon gamma after week of immunization. formulation also induces the activation of dendritic cells after their in-vitro incubation as shown by % increase in cd and . % in ccr expression as compared to blank alginate formulation. bacterial exopolysaccharides (epss) are heterogeneous polymers containing a wide array of homo-or hetero-carbohydrates as well as organic and inorganic substituents. epss are produced by many bacteria and play a critical role in helping these microorganisms to cope with adverse environmental conditions. some epss contain sulphate groups as inorganic substituents. the presence of these groups contributes to the biological activity of epss, which have been shown to have anticoagulant, insulinotropic, antiviral, antitumoral and immunomodulatory properties, among others. b is a constitutively sulphated eps produced by halomonas maura, a recently discovered halophilic bacterium. in preliminary experiments we found that modification of its eps by adding sulphate groups to the native polymer (thus obtaining b s) resulted in vigorous antiproliferative activity in several haematopoietic tumour cell lines. at the same time we found that other epss produced by closely related strains had only a very limited antiproliferative effect on these same tumour cells. it was therefore of interest to determine whether the antiproliferative activity of b s was mediated by the induction of apoptosis, and if so, to dissect the pathway triggered by b s. by cell cycle analysis we determined that b s is able to induce apoptosis in up to % of jurkat and molt- t cell leukemias. the examination of a large panel of haematopoietic and nonhaematopoietic cell lines revealed that apoptosis induced by b s is restricted to cells of the haematopoietic lineage and that leukemic t cells are particularly sensitive to death induced by b s, but that untransformed cells are not. a time-course of caspases activation indicated that caspase is the first to be cleaved, followed by caspases and , thus suggesting that b s triggers apoptosis through the mitochondrial pathway. it is noteworthy that b s also induces vigorous apoptosis in primary leukemic t cells obtained from the peripheral blood of patients. therefore, b s may well provide a satisfactory therapeutic alternative to patients with acute t cell leukemias, since current antitumoral drugs are very inefficient in the treatment of these types of cancer. particulate antigen delivery tools have been shown to enhance the induction of immune responses by targeting dcs. polyelectrolyte microcapsules form a new class of microcapsules generated by the sequential adsorption of oppositely charged polyelectrolytes onto a sacrificial spherical template which is consequently dissolved, yielding a hollow microcapsule surrounded by a thin shell. this layer-by-layer approach allows an efficient incorporation of macromolecules under nondenaturing conditions. by using the biopolyelectrolytes dextran-sulphate and poly-l-arginine, biodegradable microcapsules can be obtained. in this study, we have chosen the lungs as a non-invasive route for vaccine delivery. as demonstrated by flow cytometry and confocal imaging, dextran-sulphate/poly-l-arginine microcapsules were readily taken up by local pulmonary apcs and transported to the mediastinal lymph nodes, making them excellent tools for antigen targeting towards apcs. microcapsule instillation also affected the pulmonary apc activation status, indicated by the emergence of an apc population expressing increased levels of mhcii, the co-stimulatory ligands cd , cd and cd , and of the inflammatory cytokines il- , il- and mcp- . using ovalbumin (ova) as a model antigen, we have analysed the adjuvant properties of these polyelectrolyte microcapsules. analysis of the alveolar infiltrate, cd t cell and antibody profiles revealed that polyelectrolyte microcapsules display different adjuvant properties than the standard th and th /th skewing adjuvants alum and complete freund's adjuvant (cfa). in response to ova aerosol exposure, microcapsule based vaccination resulted in an alveolar infiltrate dominated by monocytes, while alum and cfa respectively induced typical eosinophilic and neutrophilic inflammations. striking differences were also observed on the level of cd t cell responses. microcapsule based vaccination resulted in a marked induction of il- secreting th cells, without inducing strong th (cfa) or th (alum) responses. these differences were also reflected on the level of the humoral immune response, with microcapsules being the sole adjuvant producing antibodies of all isotypes tested (igg , igg c and ige).in conclusion, polyelectrolyte microcapsules allow an efficient targeting of antigens to lung apcs, and possess immune stimulating activities distinct from alum and cfa. due to their capacity to generate th responses, polyelectrolyte microcapsules may become interesting tools to combat fungal and bacterial infections. pneumolysin is an important virulence factor produced by virtually all clinical isolates of streptococcus pneumoniae. the protein binds to cholesterol in cell membranes and creates transmembrane pores, leading to cell lysis. published findings have proposed that, at sublytic concentrations, the toxin causes a range of effects including activation of host complement, activation and chemotaxis of cd + t cells and increased production of pro-inflammatory cytokines in immune cells. in this study we investigated the interaction of pneumolysin with murine dendritic cells (dc). we found that pneumolysin induced the activation of dc, reflected in the enhanced expression of the costimulatory molecules cd , cd and cd and mhc class ii molecules. the toxin alone was found to be a poor inducer of cytokine production by dc but it did enhance the secretion of tlr agonist-induced cytokines such as il- and tnf-a. previous published findings have shown that pneumolysin activates peritoneal macrophages in a tlr -dependent manner. however, we found that pneumolysin was capable of activating dc from both wildtype and tlr -defective c h/hej mice, by inducing cell maturation and synergising with tlr agonists to enhance cytokine secretion. importantly, we also found that pneumolysin is a strong inducer of il- b secretion by dc, through its effects on caspase- processing, which is also tlr -independent. the results suggest that pneumolysin is a potent stimulus for dendritic cell activation and that this does not require tlr signalling. objectives: transmission of immune competence from mothers to newborns during pregnancy and lactation is crucial for education of neonate immune system in order to develop optimal protection against early life infections. the objective of the present study was to assess whether maternal supplementation with probiotics may enhance neonatal responses to measles immunization. methods: pregnant balb/c mice were supplemented with placebo (maltodextrin) or probiotics (lactobacillus paracasei ncc (st ) or lactobacillus rhamnosus ncc (lpr), each at x cfu/day), suspended in the drinking water, throughout the gestation period and up to the weaning of pups. at weaning, pups were immunized with live attenuated measles vaccine (mv-s, aventis-pateur). weight evolution of pups was followed from week to week of life. fresh feces were collected at , and weeks of life for determination of iga levels (assessed by elisa). pups were bled and weeks after immunization for determination of measles-specific igg and igg a antibodies. analysis of microbiota composition (plating on semi-selective agar media) was performed on fresh feces collected one week after weaning. results: all newborns grew normally and no significant differences in the weight were observed between the groups all along the trial. fecal iga production increased progressively in all pups from weaning, reflecting a normal development status. nonetheless, feeding mothers during pregnancy and lactation did not significantly affect post-weaning s-iga production in pups. lpr supplementation of the mothers significantly potentiated post-weaning measles-specific antibody responses in pups in comparison to control group. interestingly, no significant effect was observed in the st -fed group. finally, a modification of the microbiota composition was observed in pups of supplemented mothers. particularly, there was a significant increase in lactobacilli in pups from the lpr group as compared to controls. conclusion: this study supports the benefit of perinatal intervention with probiotics during pregnancy and lactation on immune maturation in the offsprings. moreover, these first results seem indicate that the effects are strain specific. chitosan, ( - )- -amino- -deoxy-beta-d-glucan, is a deacetylated form of chitin, an abundant biodegradable, positively charged natural polysaccharide. chitosan (chi) is used for antigen delivery through mucosal barrier due to its ability to disrupt tight junctions. here we studied the ability of chitosan nanoparticles to form complexes with proteins of different size and charge. nanoparticles (chi-np) were prepared from kda chitosan by ionotropic gel formation. bovine serum albumin (bsa) and myoglobin, human immunoglobulin g and superoxidedismutase (sod), and chicken lysozyme were fitc labeled. chi-np were preincubated with proteins at : ration and washed times. after washing chi-np containing bound proteins were run by denaturating gel electrophoresis. all proteins were able to form complexes. most effective binding was shown for bsa, sod, and lysozyme. the stability of chi-np complexes with proteins was studied in vitro on macrophage cell line raw . by confocal microscopy. for this chi was labeled with rhodamine and nanoparticles were coincubated with fitc labeled proteins before addition to the cells. we showed co-localization of chi and fitc for all proteins studied. these results demonstrate that chi-np form stable complexes which are internalized by macrophages. the family euphorbiaceae consists of a large group of plants whose compounds have been documented to possess anti-inflammatory activities, however, their effects as modulators of innate or acquired immunity has not been described yet. in the present study, different aspects of the immunomodulatory activity of extracts from euphorbiaceaes on peripheral blood mononuclear cells (pbmc) from healthy individuals were evaluated. the pbmc were exposed to the extracts w/o phytohaemagglutinin a (pha), cycloheximide (chx) or lipopolysaccharide (lps) as agents that induce proliferation, apoptosis and cytokine production in pbmc. the lymphoproliferative activity of pbmc was evaluated by thymidine incorporation and cfse dilution assay using flow cytometry. the mitochondrial membrane depolarization (as an early apoptosis indicator) was measured using dioc /propidium iodide staining by flow cytometry and tnf-a secretion in the culture supernatans by elisa. we found that up to euphorbiaceae's extracts had the ability to modulate one or more of the immune parameters evaluated in this study. however, only the bark extract of croton spp. insoluble in hexane:diclorometane:methanol (hdm) and the latex extracts of euphorbia cotinifolia and euphorbia tirucalli induced strong proliferation, apoptosis and also tnf-a production in pbmc. these extracts were subfractioned by sephadex column chromatography obtaining three subfractions with enhanced activity in comparison to the crude extracts. additionally, we started with the characterization of the specific immune effects of these subfractions on pbmc. all three subfractions induced proliferation predominantly on cd + cells. these effect was also observed in isolated t cells indicating that accessory cells are not necessary for the subfractions'activity. the lymphoproliferative activity of these subfractions was also not inhibited by the carbohydrates d-(+) galactose or a-methyl-mannopyranoside. these results demonstrate the presence of immunomodulatory compounds in plants from the euphorbiaceae family and suggest an antigen-presenting cell-and carbohydrate-independent mechanism of the subfractions to exert their effects. we found significant increase on lymphocytes and eosinophils populations obtained from lps + p. acnes-treated group in relation to control group.on th day, we detected a significant negative correlation between eosinophils absolute number and fec. both il- and ige serum levels were increased on animals from group i when compared to control.the enhancement on th immune response pattern induced by lps and p. acnes treatment diminished drastically parasitic load. conclusion: our findings support the idea of the use of immunostimulant as a helminthiasis control strategy in sheep, which stimulate non-specific mechanisms of resistance and therefore can act against nematodes infections. vaccines based on partially purify populations from the organism or recombinant subunits proteins have been recently developed and are often not sufficiently immunogenic by themselves due to the lack of innate immune stimuli. indeed, current influenza a vaccines do not generate significant immunity against serological influenza a virus subtypes and would thus be ineffective in the face of a pandemic novel variant. hence adjuvant usually needs to be added to those types of vaccines. here we show that wittycell compounds significantly augment cellular and humoral immune responses to commercial seasonal influenza vaccines. experiments performed in mice showed induction of specific cd + ctl cells against conserved proteins that were accompanied by the induction of ifn-g producing cd +t cells, following single immunisation. in addition increased hi titres and higher levels of specific igg a and igg b antibodies were found even long periods after single vaccination with reduced doses of vaccines. consequently, protection from lethality was observed following challenge with homologous or heterogonous influenza viruses in vaccinated animals. this promising finding on the improvement of seasonal influenza vaccines by wittycell compounds in these preclinical studies strongly provides support for the careful evaluation in phase i clinical trials in humans. s. lindgren , , n. almqvist , a. lönnqvist , s. Östman , c. rask , e. telemo , a.e. wold university of göteborg, department of clinical bacteriology, göteborg, sweden, university of göteborg, department of rheumatology and inflammation research, göteborg, swedenobjective: dietary antigens normally evoke immunological tolerance. a prerequisite is their processing by intestinal epithelial cells, which leads to the appearance of a tolerogenic form in the serum of fed animals that confers antigen-specific tolerance when transferred to naï ve recipients. the gut microbiota may influence the handling of dietary antigens as atopic diseases have increased in western societies in parallel with reduced complexity of the infantile commensal microflora. we have observed that children neonatally colonized with s. aureus in the gut seem protected against development of food allergy. here we examine whether a s. aureus toxin affects tolerogenic processing by the intestinal epithelium. methods: mice were given s. aureus enterotoxin a (sea; . mg/ml) in the drinking water for days and, days later, mg ovalbumin per os. one hour postfeeding, serum was transferred to naï ve recipients, whose tolerance to ovalbumin was tested in a model of allergic airway inflammation (sensitization followed by intranasal challenge with ovalbumin). results: recipients of serum from sea pretreated ovalbumin-fed donors exhibited increased tolerance compared to recipients of serum from ovalbumin-fed donors not pretreated with sea. this was demonstrated as reduced ovalbumin-induced airway inflammation with diminished influx of eosinophils into the lungs and reduced antigen-induced production of interleukin- and interleukin- . examination of gut sections from sea treated donor mice revealed increased density of cd a + intraepithelial lymphocytes. our results show that sea promotes oral tolerance induction, possibly by facilitating tolerogenic processing of soluble antigens by the absorptive intestinal epithelium via activation of intraepithelial lymphocytes. abstract withdrawn by author to develop an efficient vaccine against cp pneumoniae we cloned chlamydial genes encoding proteins of the outer membrane like ompa, omcb, and pmp , proteins of the inclusion membrane like incc, secreted proteins like cpaf, and the heat shock protein groel. cpg-dna , a highly stimulatory oligonucleotide for apcs, was used as adjuvans. subcutaneous co-injection of ompa, omcb, pmp , or groel together with cpg-dna reduced the chlamdial burden in nasally infected mice. however, symptoms like substantial loss of body weight were not influenced. in contrast, a low dose infection with cp. pneumoniae almost completely prevented the loss of body weight upon challenge. to improve the efficacy of the vaccine we used poly-dl-lactide-co-glycolide microspheres loaded with the protein ompa or pmp together with cpg-dna . the microsphere based vaccine offers the advantage that antigen and adjuvans are delivered to the same apc. intranasal but not subcutaneous vaccination of mice with ompa or pmp microspheres efficiently lowered chlamydial burden upon challenge and prevented loss of body weight. pmp microspheres induced protective ifng-secreting cd + t-cells and raised pulmonary pmp -specific iga levels in vivo. also, pmp microspheres caused lower il- serum levels upon administration than the injection of pmp together with cpg-dna , indicating fewer side effects. objectives: staphylococcus (s.) aureus superantigens are highly potent t cell mitogens and the causative agents of toxic shock syndrome (tss) and food poisoning. most s. aureus have superantigens and patterns are highly variable. to date, the role of superantigens in bacteraemia is not well defined.to analyse whether superantigens play a role in bacteraemia, we investigated s. aureus strains and anti-superantigen antibody responses in cases of s. aureus bacteraemia in iv drug users and cases in nonaddicts. a rise in neutralising antibody titers indicates that superantigens are produced during infection. the study comprised iv drug users with positive s. aureus blood culture and an equal number of age-and sex-matched nonaddicts from the original fintrova and finlevo trials (ruotsalainen ).all s. aureus isolates were analysed by sequence-based genotyping (spa-typing), and multiplex-pcr was applied to determine the superantigen gene pattern. sera from patients were obtained at diagnosis (day ) and four weeks thereafter (day ). neutralising capacity of the sera was tested against the superantigen cocktail produced by the respective infecting strain as well as a panel of representative recombinant superantigens.results: genetic analysis confirmed our previous observation that most strains harboured superantigen genes, which were linked to staphylococcal lineages (holtfreter ) . there were no major differences in superantigen gene patterns in isolates from iv drug users and nonaddicts. interestingly, the staphylococcal lineage st (spa-type t , agr , and sea, seb, sek and seq) was much more prevalent among bacteraemia strains from iv drug users than from nonaddicts (p= . ).most iv drug users had neutralising antibodies against enterotoxins already at onset of bacteraemia, likely due to previous encounters with the infecting strain. we frequently observed a rise in antibody titers during infection. surprisingly patients with st strains did not show any elevations in neutralising antibody levels. conclusion: s. aureus bacteraemia induces an antibody formation against staphylococcal superantigens. this indicates that superantigens are produced during infection. however, the action of superantigens is frequently modulated by specific neutralising antibodies. this and the special behaviour of s. aureus st strains need further investigation. objectives: down syndrome (ds) is associated with recurrent infections, hematological malignancies and auto-immune diseases, suggesting immunological changes. to test for more severely disturbed specific antibody response we investigated the antibody response to the highly immunogenic protein antigen tetanus toxoid (tt), which is part of the dutch immunization program. methods: after booster vaccination at and years of age, quantitative (titer) and qualitative (avidity) tt responses were investigated in and ds children, respectively. samples were taken before and - weeks after vaccination. tt-specific igg and igg-subclass antibodies were measured in serum by quantitative enzyme linked immunosorbent assay (elisa), avidity of igg -anti-tt by an avidity elisa. the results were compared with reference values from the laboratory. results: at years, post-vaccination anti-tt-titers were decreased (geometric mean total igg, igg , igg and igg ). at years, ds children had lower postvaccination geometric mean igg anti-tt-titers only. post-vaccination igg -anti-tt avidity levels were decreased in / and / ds children at four years and nine years of age, respectively. the quantitative and qualitative anti-tt-responses in both ds groups are shifted downwards compared to the reference values. although the anti-ttresponse increases towards normal titers with increasing age, the avidity (qualitative response) is still abnormal at that age, showing that ds children have profound and lasting difficulties with specific anti-tt antibody formation. kda; , kda; , kda; , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] kda and , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] kda were expressed by a majority of examined strains independently of the associated diseases. we assume that these omps could be conservative proteins of h. pylori. conclusion: considering omps as potential targets in the search for disease-related biomarkers and potential vaccine antigens, the identification of h. pylori omps as well as the elucidation of their role in modifying the host immune responses seems to be very important research subjects. the increasing cases of severe diarrhoea and invasive lethal infections in children caused by salmonella typhimurium are a major public health problem in mexico. the rapid dissemination of multidrug-resistant s. typhimurium, and the lack of a licensed vaccine against non-typhoidal infections reduce the possibilities of an effective treatment. the objective of this study was to evaluate if the high incidence of non-typhoidal multidrug-resistant salmonella infections was associated with a reduced in anti-salmonella immunity. a cohort of families, from a mexican agricultural community with a high incidence of endemic salmonella infections, was followed prospectively for an -month period. sera were obtained from healthy subjects from the same community ( months to years of age). the highest incidence of salmonella-associated diarrhea, / , occurred in children under years of age. the lowest incidence, / , was observed in the population aged to . whereas serum from individuals ranging - years of age showed maximum igg , igg and iga anti-s. typhimurium titres, children less than years-old did not show detectable igg and igg titres and had weak igg , iga and igm antibody levels; only their igg levels were comparable to those detected in adults. moreover, the levels of igg and igg antibodies were lower in adults with a diarrheal-associated episode. interestingly, s. typhimurium yuhs - , a commonly isolated human strain from this endemic area, resisted the complement-fixing activity of antibodies although it was sensitive to opsonisation and to fc-mediated phagocytosis by human monocytes. these data contributes to define the protective immune response involved in anti-s. typhimurium immunity. diseases caused by the yeast of candida genus are a serious clinical and social problem. despite this fact, there is no effective prevention against these opportunistic pathogens yet. although c. albicans is the major cause of the mycoses ( %), the number of the multiresistant non-albicans isolates increases. c. dubliniensis, which was described only recently as serious human pathogen, belongs to the group of these resistant isolates. the surface mannan of candida cells is component of the cell wall mannoprotein complex and participates in an initial contact with its host and subsequently with the host defense mechanisms. because of complexicity of this homopolymer it is necessary to identify a subunit of the mannan that is most effectively recognized by the immune system and thus influences the specificity of the induced antibody response (immunodominant epitope). in our study we prepared oligosaccharides from acid-stable part of mannan c. dubliniensis by conventional acetolysis. this procedure specifically cleaves the a- , linked mannopyranose units of mannan backbone and releases the side oligomannosyl branches. obtained oligosaccharides were used in inhibition elisa and spr (surface plasmon resonance) measurements. the reason of these measurements was to quantify interactions of these oligosaccharides with anti-mannan antibodies present in rabbit serum after -fold immunization with mannan-hsa conjugate injections in week intervals as well with inactivated c. dubliniensis whole z. neščáková , s. bystrický institute of chemistry, slovak academy of sciences, bratislava, slovakiaour newest approach to sub-cellular vaccine against gram-negative bacterial pathogens exploits detoxified lipopolysacharide (detoxified lps) as the target antigen. this is achieved by conjugation of carbohydrate to a protein carrier which secures the t cell dependent immune response. the goal of the immunization with this conjugate is to generate the effective production of memory b cells. here we prepared subcellular conjugate with the detoxified lps from vibrio cholerae strain o using polymer carrier and a protein. the cell immunity induced by the vaccination with the conjugate was evaluated in mice, namely their peripheral blood and the spleen. activation and differentiation of b-cell populations in the time-dependent manner was determined by flow cytometry analysis of these samples. a single-platform approach based on flow cytometry and defined number of fluorospheres was used to count b cells. however in our hands this method, previously used in humans, had to be adapted for mouse blood samples first. the protocol allows quantifying cells simultaneously with cytometric immunophenotyping without cell loss or other cell preparation steps. like pan-b cell marker cd , expressed almost on all blood and tissue b cells, was used. here we investigate the characteristics and development of antibody (iso)types after secondary immunization with mencc or plain polysaccharide and the possible role of certain antibody responses in maintaining immunity after vaccination. methods: volunteers, age - years, were immunized with mencc or received a secondary immunization with mencc or plain menc ps. blood samples were obtained before and seven time-points after immunization. igg, iga, igm, igg , igg and avidity were assessed by a multiplex immunoassay. functional antibodies were determined by a serum bactericidal assay. results: high levels of antibodies were still present years after primary mencc immunization. secondary immunization resulted in increased igg and sba titers after to days. in primed individuals, igm was still present, and this only increased following a secondary immunization with plain ps. in addition, immunization with ps induced a higher igg response compared to mencc immunization. discussion: secondary immune responses are quiet slow. the composition of the ig (iso)type distribution is different between mencc and plain ps and might be of influence on functional titers. although this study indicates that immunological memory was previously induced by a single mencc vaccination, it highlights the importance to sustain protective antibody levels against a rapid invasive organism such as n. meningitidis. the immunological effectiveness of these two semi-synthetic immunogenic conjugates was established according to antigen-specific titers of igg, iga and igm isotypes and by phagocytic and respiratory metabolic activities of granulocytes. results: prime-boost immunization strategy resulted in enhanced production especially dlps-specific igg and igm isotype antibodies in both experimental groups (peak titers : ). igm-igg isotype switch was more pronounced with o-sp. peak values of dlps specific iga isotype were signicantly lower than igg and igm ones ( : vs. : ). flowcytometric simultaneous determination of phagocytosis and stimulated oxidative burst of granulocytes revealed conjugate induced enhancement, more evident with o-specific polysaccharide and ip final boost (stimulation index was . fold of normal control). subcutaneous immunization gave a weaker stimulation: . fold of normal control. the second de-oac conjugate exerted different pattern of stimulation, sc intervention was more effective. our results are indicative for immunological effectiveness of novel dlps derived glycoconjugates; thus promising further application in cholera subcellular vaccine. this work was supported by apvv - and vega / / grants of the slovak grant agencies. background: the yeast candida albicans is an opportunistic pathogen that causes infections in immunocompromised individuals with a high morbidity and mortality levels. a long-acting, effective and safe vaccine that protects against medically important candida species should significantly reduce the incidence of various forms of candidasis by these etiologic agents. mannan, polysaccharide component exposed at the most external layer of the fungal cell wall, contains a backbone consisting of a- , -linked d-mannopyranose units and many branches composed of a- , a- , and/or b- , -linked mannopyranose units that are connected to the backbone. investigation of oligosaccharides immunomodulatory functions could be considered as an important part of their protective immunity against fungal diseases. objective: in this study, for mice immunization, synthetically prepared oligosaccharide (heptamannoside) conjugated to protein carrier (bovine serum albumin, bsa) was used. methods: in order to study the immunogenicity of heptamannoside -bsa conjugate as inducer of hummoral and cell-mediated responses, balb/c mice were subcutaneously immunized without adjuvant ( mg oligosaccharide per one conjugate dose) two times in days intervals and then intraperitoneally or subcutaneously boosted. cell-mediated and humoral responses were analyzed on day after injections by flow cytometric immunophenotyping of peripheral blood leukocytes and by measuring the levels of mannan specific antibodies presented in serum using elisa. results: prepared conjugate was immunogenic and re-injection elicited increase of mannan specific serum antibodies levels. intraperitoneal boost elicited significantly higher igg and igm levels than subcutaneous boost. immunization also induced changes in proportions of major lymphocyte subpopulations in peripheral blood. introduction: bulgarian immunomodulator respivax enhances the natural resistance of organisms and specific immunity towards the most frequent respiratory pathogens. it is composed of killed bacterial bodies and lysates of six microbial species (streptococcus pneumoniae, branhamella catarrhalis, streptococcus pyogenes, haemophilus influenzae, staphylococcus aureus, klebsiella pneumoniae). the immune response in respiratory tract includes not only systemic immunity in lungs, but also balt as a part of common mucosal immune system. most animals develop balt after antigen stimulation and this tissue plays a central role in antigen uptake and local immune response regulation. therefore, immunostimulation of balt may contribute to more efficient mucosal immunity in respiratory tract. aim: to study balt development in different terms after oral application of respivax in guinea pigs. methods: male guinea pigs ( g- g) were treated orally with mg respivax five consecutive days. after the last application, on days , , , , and six animals on each term were sacrificed and lungs were removed. morphological changes were evaluated on mm thick serial sections, stained with hemalauneosin. the populations of cd , cd and b cells were identified on cryosections by using indirect peroxidase immunostaining. zio technique was used to detect intraepithelial dendritic cells (dc). results: balt was not identified in control animals. in the treated group on day subepithelial lymphocyte infiltrates and diffuse lymphocytes in lamina propria were found. the following two terms were presented by hyperplasia of lymph epithelium with massive complexes of intraepithelial lymphocytes. they were composed mainly of cd positive cells, which number reached maximum at the end of the second week. on day b cell lymphoid follicles with different size were found. lamina propria was presented by abundant lymphocyte infiltrates, composed of cd and cd positive cells. on days and the morphological reaction in the airways was reduced, characterized with small size lymphocyte accumulates. numerous intraepithelial dc's were detected in treated animals, comparing to controls in which only a few were identified. conclusion: oral administration of respivax in guinea pigs resulted in significant immunomorphological reaction in the airway mucosa presented by increased number of dc and balt development. g. gupta , s. majumdar bose institute, molecular medicine, kolkata, indiavisceral leishmaniasis (vl) caused by the protozoan parasite, leishmania donovani, is characterized by the loss of ability of the host to generate an effective immune response in the form of free radicals and proinflammatory cytokines. chemokines, particularly cc chemokines, have been shown to render protection against leishmania infection. there is no clear understanding about the immunoprotective role of cxc-chemokines in vl.in the present study, the comparative potential of cxc chemokines, interferon gamma inducible protein- (ip- ) and interleukin- (il- ) in restricting leishmania donovani infection via the release of nitric oxide (no) and proinflammatory cytokines was studied in an in vitro model. no, a crucial mediator for ip- mediated leishmanicidal activity, was found to be dependent on inducible nitric oxide synthase (inos ) expression and was linked to the mapk signaling pathway via antagonistic regulation of p mapk and erk / . further, ip- was also able to abrogate the survival of leishmania in an in vivo model of vl by restoration of th cytokines and no. thus this study strongly demonstrates that ip- , like cc chemokines, is involved in rendering a protective response in vl via upregulation of proinflammatory mediators. african trypanosomiaisis (at), known as sleeping sickness, is an orphan and extremely debilitating disease in human, cattle and domestic animals. at is caused by the protozoan trypanosoma brucei and at the present, there's no safe or efficient pharmacology intervention. the dna vaccines could be the answer for this disease by being able to induce production of igg antibodies and induce of th /th cytokines mediated by cd + t cells and activating cd + t helper cells. in this study, we shows that balb-c mice immunized intramuscularly with a single dose of plasmids encoding three antigenic candidate genes from trypanosoma brucei, named invariant surface glycoprotein (isg), trans-sialidase (tsa), and fosfolipase c (plc) are able to produce igg antibodies anti-trypanosoma. this immunization process was able to control the mortality level when mice were submitted to challenger assay with trypanosoma brucei brucei parasites. in mice co-infected with s. ratti and l. major (nl) neither clearance of l. major nor strongyloides infection was changed. mice co-infected with s. ratti and p. yoelii (nl) showed the same course of parasitaemia as single infected mice. these results suggest a strictly compartmentalized and successful immune response in both murine co-infection models, s. ratti and l. major or p. yoelii. if this compartmentalization is also observed in the antigen specific cytokine response of ex vivo prepared lymphocytes will be the topic of further investigations. in the present study ,we evaluated tsa -encoded dna vaccine against l.major in balb/c mice. igg and ifn-g values were markedly increased in the immunized group ,which were significantly higher than in the control groups (p x . ) following immunization and after challenge with leishmania major. il- values were increased in all groups, but there was no statistical difference between the groups(p g . ) following immunization and after challenge with leishmania major. the immunized mice with the dna vaccine presented a considerable reduction in diameter of lesion comparing to the control mice and indicated a significant difference was observed between the immunized and the control groups (p x . ) in this regard . the survival time of the immunized mice with the vaccine was significantly higher than the control groups (p x . ) after the challenge with leishmania major. the immunized mice had significantly lower parasite load comparing to the control mice(p x . ). the findings of this study indicated that the tsa -encoded dna vaccine increased the cellular response and induced protection against infection with leishmania in the mice. the tsa -encoded dna vaccine may be an excellent candidate for future vaccine developments against leishmania. there is a lot of evidence showing that bcg vaccination at mucosal site via intranasal, intragastric and intrarectal routes are effective in conferring protection against virulent mycobacterium and several non mycobacterial infectious diseases. in this study the protective effect of autoclaved leishmania major (alm) vaccine in combination of either rectal or subcutaneous bcg on susceptible balb/c mice was evaluated.one month after bcg vaccination, balb/c mice were immunized subcutaneously twice with alm+alum at week intervals. three weeks after booster injection, × stationary phase l. major promastigotes were inoculated subcutaneously in one footpad. immunological evaluation at before and post infectious challenge, showed strong proliferative responses in the spleen cells of the rectal immunized group after stimulating with parasite lysate. high level of interferon gamma was induced in the spleen and significant increase in the serum ratio of igg a/igg was observed only in rectal immunized group. rectal immunized mice showed comparable nitric oxide production and inos induction in peritoneal macrophages .the obtained results in rectal bcg vaccinated group showed no mortality but low parasite burden in the liver and spleen and suggested protective efficacy of intrarectal bcg immunization against leishmaniasis might be due to the long-lasting induction of type immunity. methods: two groups of balb/c mice were infected by l. tropica. one group was infected subcutaneously into the left footpad and the other group intradermally into the left ear dermis. mice were challenged by l. major in the right footpad after establishment of l. tropica infection. the immune response was evaluated at two intervals: one week and one month after challenge. single cell suspensions were prepared from draining lymph nodes of mice. cells were stimulated by phorbol myristate acetate (pma). cell surface markers and cytokine production were determined by intracellular cytokine assay using flow cytometry. the following parameters were assayed in the two experimental groups: lesion development, delayed type hypersensitivity (dth) to l. major challenge, production of gamma interferon (ifn-g) and interleukin (il- ), and cellular expression of cd and cd . results: infection through subcutaneous route in comparison to the intradermal route induces significantly higher levels of dth and ifn-g, lower levels of cd + lymphocytes, and higher protection against l. major challenges. conclusion: intradermal infection of l. tropica, in comparison to subcutaneous infection, induces significantly more protective immunity in balb/c mice. therefore, we propose the route of infection as an important variable in this experimental model. this factor should be considered for development of an appropriate experimental model for human l. tropica infections. objectives: many mammals exhibit a periparturient relaxation of immunity (ppri) to gastrointestinal nematode parasites culminating in increased worm burdens. it has been suggested that the extent of ppri may have a nutritional basis as this effect on host resistance is considerably augmented when protein supply is scarce. subsequent studies have shown that increased dietary protein intake can ameliorate this phenomenon. however, this effect is often confounded with increased food intake and thus increased energy levels. here, we aim to dissect the effects of protein and energy nutrition on the immune status and resistance to gastrointestinal nematodes in the periparturient host. the nippostrongylus brasiliensis lactating re-infected rat model was utilised as a well established model for mammalian ppri. lactating rats, re-infected with , infective n. brasiliensis larvae on day post parturition, were offered one of three levels of crude protein at one of two levels of metabolisable energy (me). parasite burdens were assessed by counting worms in the small intestine at day post secondary infection. histological counting of intestinal inflammatory cells, assessment of antibody levels and measurement of cytokine mrna levels in the mesenteric lymph nodes were carried out to assess the host immune status. results: increasing cp supply, but not increased me supply, reduced worm burdens. whilst feeding treatment did not affect eosinophil and goblet cell numbers, increased cp supply increased mucosal mast cell numbers and levels of n. brasiliensis specific antibody (total igg, ige, igg and igg a). this was independent of level of me supply. feeding regime did not affect levels of the type- cytokines il- and il- . conclusion: this study effectively demonstrates that increasing protein supply per se can decrease periparturient parasite burdens. this anti-parasitic effect correlates strongly with an upregulation of immune effector mechanisms, namely accumulation of mast cells and production of antibody. this data emphasises the role of immunonutrition in combating infectious disease. protein supplementation of periparturient mammals has considerable potential as a non-chemotherapeutic method of controlling gastrointestinal nematode parasites. background: gp is the major surface glycoprotein of leishmania that exhibits protease activity and has an important role in the biology of the parasite. the aim of this study was cloning and expression of gp of l.major strain mrho/ir/ /er. methods: l.major promastigotes were grown in rpmi supplemented with % fcs. l.major rna extraction and cdna synthesis were carried out. gp gene segment was amplified by specific primers and cloned into ptz r to construct ptz r/gp . the presence of gp into ptz r was confirmed by pcr. then, ptz r/gp was sent to determine the sequence of its nucleotides. after that the gp gene segment was sub-cloned into pet a (+) expression vector and transformed into e.coli bl (de ) plyss and gp protein was expressed in presence of mm iptg. objectives: the development of a vaccine against malaria caused by plasmodium falciparum is an urgent public health priority. influenza virosomes represent an innovative human-compatible antigen delivery system that has already proven its suitability for subunit vaccine design. at appropriate antigen doses, seroconversion rates of % were achieved against two synthetic malaria peptide-mimetics in malaria naï ve volunteers (genton et al., plos one, ) . the aim of this clinical trial is to proof that virosomes are a suitable delivery system for malaria peptide-mimetics in malaria semi-immune subjects. objectives include demonstration of safety and tolerability of virosome formulated malaria peptide-mimetics and determination of the humoral and cellular immune responses against these malaria peptide-mimetics. particularly, boosting of pre-existing naturally acquired anti-malaria immunity will be investigated. the study design was a single centre, randomized, controlled, double-blind, age deescalating trial including volunteers. male volunteers ( and years) for the adult group, and children of both sexes ( - years) were enrolled. subjects received virosomal formulations containing mg of ama -c (pev t), an apical membrane antigen- derived synthetic phospatidylethanolamine (pe)-peptide conjugate and ug of uk (pev t), a circumsporozoite protein derived synthetic pe-peptide conjugate. comparator groups received the influenza vaccine inflexal v. volunteers received two injections at study days , and . results: safety and tolerability defined as occurrence of local and systemic adverse events and incidence of clinically significant hematological and biochemical abnormalities are assessed. this vaccine showed a very good safety and tolerability profile in all study participants. curcumin dissolved in dmso when administered orally to p.berghei infected mice has been shown to have antimalarial activity, enabling % of the treated mice to survive till days after infection by which time all of the untreated mice had died. under such condition we found that bioavailability of curcumin was only . % of the amount fed and it remained in circulation in the blood only for minutes post feeding. we therefore prepared curcumin bound to chitosan nano particle to improve it's delivery and found that oral feeding of such particles not only increased its bioavailability to . % ( of the amount fed but it's circulation was sustained till hrs post feeding. under such conditions when mgm of curcumin bound to mgm of chitosan nano particles were fed one time daily for days post infection to plasmodium yoelii infected mice % of mice were cured and survived atleast for days without any infection and were resistant to reinfection with the same parasite. curcumin under such condition accumulated preferentially in infected erythrocytes, the quantity increasing with increase in parasitemia and fluorescence microscopy revealed that it was bound to the parasite. like chloroquine, curcumin inhibited hemozoin formation in vivo and heme polymerization in vitro in a dose dependent manner. we believe that it is one of the ways by which curcumin may be killing the parasite. among immune cells, nk and gamma-delta t cells are suspected to play a critical role in the early control of plasmodium falciparum parasitaemia and to influence malaria adaptive immunity. gamma-delta vgamma vdelta t cells, a non-conventional t cell subset specific of primates, are activated and expanded during primary p.falciparum infections in response to malaria non-peptidic phosphoantigens, and they are an important source of ifn gamma. furthermore these cells inhibit in vitro growth of p.falciparum blood stages by a granule exocytosis-dependent cytotoxic pathway and granulysin -an nk and t cell specific cytotoxic molecule has been incriminated. so far, the precise mechanism of the parasite inhibitory capacity of those cells, as well as the parasite blood stages involved remains unclear. to further investigate the anti-parasitic activity of gamma-delta t cells an rnai strategy based on a lentiviral vector approach was undertaken. we demonstrate that granulysin, but not perforin is essential for the anti-parasitic activity of gamma-delta t cells. concerning parasite blood stages, we show that both mature infected red blood cells and the free invasive form (merozoite) trigger gamma-delta degranulation and granulysin release, but noteworthy merozoites were the only stage affected by gamma-delta t cells. in addition, we also provide evidence that such a mechanism may occur in infected patients. altogether these data highlight a new mechanism by which gamma-delta t cells might directly contribute to malaria immunity opening new perspectives based on gamma-delta t cells to prevent or cure malaria. the immune system has a number of mechanisms to prevent self-destructive responses. amongst these, regulatory t cells (treg) have the ability to actively suppress effector responses. many questions surround the issue of antigen specificity of treg, since selective inhibition of only the pathogenic response, leaving the rest of the immune system intact, is the ideal therapeutic goal. the purpose of the project is to develop a model of robust, highly specific regulation operating in vivo that can be studied to understand the underlying mechanisms. such a model is provided by murine autoimmune hemolytic anemia (aiha) induced by immunisation with cross-reactive rat red blood cells (rbc). mice recover from disease due to the development of regulation with exquisite specificity, which suppresses only responses to self-epitopes whilst selectively allowing those to rat-specific determinants to be boosted. the re-establishment of tolerance is associated with the loss of t-cell proliferative responses, and emergence of il- responses, to epitopes on the dominant rbc autoantigen, anion exchanger- (ae- , or band ) protein, and protection can be transferred by injecting splenocytes from recovered mice into naï ve recipients. here we show that transfer of tolerance to naï ve recipients is dependent on ido mediated immunosuppression as mice receiving previously tolerised splenocytes under the cover of methyl tryptophan, an inhibitor of ido, were refractory to tolerance and developed hemolytic disease. induction of ido is therefore an important process in antigen-specific tolerance, and initiators of ido activity, including ctla- + regulatory t cells or soluble forms of ctla- , may also be crucial components of this regulatory pathway. consequently, this finding has important implications for our understanding of tolerance processes in autoimmune disease. objectives: it was shown alpha-fetoprotein (afp) induced immunosuppression of cell-mediated immunity in vivo. our previous work discovered afp-activated mice bone marrow hematopoietic stem cells (hscs) suppressed effector reactions of cell-mediated immunity in vitro. we investigated relationship existed between afp-induced hscs suppressor activity and immunosuppression of cell-mediated immunity during afp-produced teratocarcinoma development. methods: animal models, experimental oncology methods, immunomagnetic separation, cultural methods, cellular biology methods, flow cytometry, multiplex protein analysis, methods of molecular biology and rna interference (rnai) were used in this work. results: as a result, there was a negative correlation (r medium =- . ) between dynamics of hscs suppressor activity elevation in spleen and inhibition of nk cells, nkt cells and cd + t cells cytotoxic activities ex vivo during tumor growth. besides, the inhibition of spontaneous and induced cytokines productions such as ifng, tnf-a and tnf-b from these types of immunocompetent cells negatively correlated with increasing of suppressor factors expression such as tgf-b (r medium =- . ) and il- (r medium =- . ) in isolated splenic hscs ex vivo. analysis of effector cd + t cells in spleen showed decrease of t h cells quantity and simultaneous t h cells number increase during teratocarcinoma development. moreover, it were found elevated numbers of cd + cd + ctla- + -and cd + cd -il- + il- regulatory t cells in spleen as well as increasing suppressor activity of isolated regulatory t cells ex vivo. number boost kinetics of t h , t h and regulatory t cells were correlated (r th =- . , r th = . and r th = . and r tr = . ) with kinetics of hscs suppressor activity level. in addition, dynamics of regulatory t cells activity were linear (r th = . and r tr = . ) to hscs suppressor activity level in spleen during tumor growth. quantities of tgf-b -and il- -produced hscs in spleen were correlated (in some cases negatively but in other positively) with cell-mediated immunity effector reactions alteration during teratocarcinoma development also. however, inhibition of afp expression by rnai caused to inhibition as immunosuppression activity of hscs and their appearance in spleen as well as normalization of cell-mediated immunity effector reactions. conclusion: thus, hscs suppression activity is correlated with changes in cell-mediated immunity during endogenous afp productions by teratocarcinoma cells and may play a role in afp-mediated dysfunction of normal immunoregulation during afp-produced tumor development. syphacia obvelata, a murine pinworm gastrointestinal nematode, is common even in well-managed animal colonies. although often considered as irrelevant, pinworm infections were shown to alter hosts' immune responses and to interfere with the experimental settings. our studies showed that naturally aquired s.obvelata infection also influences the hosts' hematopoietic responses, inducing the increased production and release of the cells of granulocyte-macrophage, as well as of erythroid lineage from the bone marrow of the infected cba mice. while the enhanced myelopoiesis compensates the increased peripheral demand for a larger supply of tissue neutrophils and macrophages, the cause of stimulated erythropoiesis is less obvious, but as infection consequence clearly underscores the disturbed and altered hematopoiesis. beside cellular changes, we also evaluated the impact of the s.obvelata on mitogen-activated protein kinases (mapk) signaling in bone marrow cells and found that infection upregulated all three mapk families, p , jnk and erk. additionally, s.obvelata enhanced the expression of mrna for the inducible nitric oxide synthase (inos). to evaluate how this pinworm infection modifies hematopoietic cells' reactivity, we also examined the influence of interleukin- , t cell-derived cytokine implicated in the regulation of hematopoiesis and inflammation, on the bone marrow cells. bone marrow myeloid and erythroid progenitors from s.obvelata-infected mice displayed altered sensitivity to il- , as compared to non-infected controls. the infection also altered the effect of il- on mapk activation by preventing its stimulating effect on p mapk. moreover, in s.obvelata-infected animals il- markedly down-regulated the expression of both inos and constitutive, endothelial (e)nos, not affecting the low basal nitrite production, which was opposite to the effect previously observed in noninfected mice, i. e. il- induced no production through the activation of both inos and enos. besides highlighting the importance of working under pinwormfree conditions when using experimental murine models for immunohematopoietic investigations, the data obtained pointed to the multiple layers of modulatory ability of this pinworm parasite and confirmed that the overall orchestration of the host response to the parasites is a complex process still being unraveled at both the cellular and molecular level. .-validation of the method: in order to determine linearity, analytical range, and reproducibility, three different sera with previously identified mc were serially diluted from ⁄ to / with a normal serum pool. .-implementation as a standard method for analysis of the mc in patients with paraproteinemia. the method showed good linearity: r g . . the analytical range was from g/l to g/l. the coefficient of variation (cv) was x % for [mc] n g/dl, and x % for [mc] x g/dl. this procedure was successfully implemented to quantify the mc in serum samples between march and february, . among these samples, we have quantified light chains, heavy chains, igd and biclonal paraproteinemias. conclusions: . we have developed a simple, reproducible and low-cost method to quantify the mc using standard analyses of serum protein electrophoresis (spe), serum albumin, and densitometric quantification of mc and albumin regions. . the procedure allows monitorization of the mc in patients at diagnosis, after therapy, and evaluation of complete remission. objectives: direct influence of alpha-fetoprotein (afp) on immunosuppressor factors synthesis as well as immunosuppressor activity of bone marrow hematopoietic stem cells (hscs) was detected in our previous work in vitro. we investigated possible role of endogenous-produced afp in induction hscs immunosuppressor activity at tumor-bearing mice. methods: animal models, experimental oncology methods, immunomagnetic separation, cultural methods, cellular biology methods, flow cytometry, multiplex protein analysis, inhibition assay, colorimetric elisa, methods of molecular biology and rna interference (rnai) were used in this work. results: our results demonstrated afp endogenous synthesis adduced to elevation of immunosuppression activity of hscs in bone marrow. this afp effect becomes developed from day and reached plateau level after day of teratocarcinoma insertion. moreover, cd + cd cells showed in spleen and main lymph nodes from day and achieved plateau level after day of teratocarcinoma growth. however, immunosuppression activities of purified hscs from spleen and lymph nodes discovered at day and had a maximum pick at day of teratocarcinoma inoculation. besides, immunosuppression activity of hscs from spleen and lymph nodes was more than , times lower than in bone marrow in the same period of tumor development. isolated hscs from bone marrow, spleen or lymph nodes produced similar spectrum of suppressor factors such as tgf-b , il- , pge and no. inhibition of such suppressor factors lead to levelling of hscs immunosuppression activity ex vivo. kinetic of hscs quantity and activity had significant correlation (r cell number = . and r activity = . ) with afp level dynamics in blood serum. in addition, inhibition of afp expression by rnai caused to diminishing of hscs immunosuppression activity as well as hscs appearance in spleen and lymph nodes. conclusion: therefore, afp plays role not only specific inducer of hscs immunosuppression activity but also as a factor of activated hscs penetration into spleen and lymph nodes during afp-produced tumor development. cd -ligand molecules -that are powerful immunomodulators -are strongly expressed by activated platelets; membrane-associated cd l is cleaved to soluble (s)cd l. we sought to examine the levels of scd l in platelet concentrates (pcs) having led to an acute transfusion reactions (atr), and to test for its biological effect on b-lymphocytes. we recorded atr episodes that could lead to investigation of residual platelets in container. two fractions of aliquots from each pc (and controls) were prepared, one for assay of individual supernatant fractions and one of corresponding lysates of platelets; scd l -along with other products -were assayed by quantitative elisa. levels of il , cd p and pdgf-ab in pc supernatants and lysates from pcs associated with atr were similar to that in controls. supernatants of pcs associated with an atr contained higher scd l levels compared to controls, and -in a inversely correlative manner -corresponding platelet lysates contained lower levels of scd l . to examine if scd l was biologically active, we stimulated purified b cells recovered from healthy blood donors and exposed those normal b cells to supernatants and cell lysates of pcs implicated in atr, or control material, and we measured il- secretion. the il- concentration was consistently below - pg/ml in pc supernatants and lysates, and unstimulated b cells did not secrete detectable levels of il- . the addition of supernatant from atr-associated pc samples to purified b cells consistently resulted in sustained il- production over control (p x . ) at d after the onset of the culture, while -in a inversely correlative manner -corresponding platelet lysates contained lower levels of scd l (p x . ). pre-incubation of b cells with cd -blocking antibodies substantially abrogated il- secretion, unlike isotype-matched control. the partial blocking of cd binding on cd + b cells strongly suggests a potentially synergistic role in b cells for cytokines other than scd l (under investigation) and indicates a sustained role for pc-derived scd l. these data prompt us to investigate a larger series of events and controls to delineate on the one hand if certain factors can be responsible for an enhanced production of scd l by collected/stored cpa. objectives: there is accumulating evidence for a role of natural killer (nk) cells in the antitumor response against hematological malignances. nk cells exert their action by means of a large panel of structurally distinct activating receptors that recognize their ligands on target cells. analysis of activating receptor pathways in nk cells has revealed a dominant role for natural cytotoxic receptors (ncrs) and dnax-accessory molecule- (dnam- ) in the lysis of acute myeloid leukemia (aml) blasts. here, we investigate the expression of these activating receptors on nk cells from aml patients stratified by age. methods: we analyses by flow cytometry peripheral blood mononuclear cells (pbmcs) from aml patients before specific anti-leukemia therapy and healthy donors. all results were analyzed statistically using spss version . results: aml patients under years showed a significant reduction in the expression of dnam- , nkp and nkp compared with age-matched controls. both healthy individuals and aml patients older than years showed a reduction of these receptors compared with young donors. in contrast, we have found that nkp expression was increased in some patients of aml. on the other hand, the analysis of ligands for these activating receptors on leukemic cells showed a high variability that was not correlated with age or fab subtype. in addition, an inverse correlation in the expression of dnam- on nk cells and its ligand cd on aml blasts has been found in aml patients under years. to analyze if leukemia cell were involved in the modulation of these receptors we have performed in vitro cocultures of leukemic blast and healthy nk cells. the initial recognition of aml cells by nk cells may represent a crucial process to prevent tumor development. here we described for the first time, a decrease of dnam- expression on aml patients and confirm previous reports showing a significant decrease of nkp and nkp on aml-nk cells. altogether, these alterations of the major receptors involved in nk cell-mediated cytotoxicity of leukemic cells represent an important mechanism of immunoescape that may correlate with disease progression and patient survival. a. stelmaszczyk-emmel , e. gorska , u. demkow , m. wasik medical university of warsaw, department of laboratory diagnostics and clinical immunology of developmental age, warsaw, polandglucocorticosteroids are often used in leukemia treatment. their therapeutic use is limited due to several side effects. one of them is multidrug resistance phenomenon, which causes lack of patients response for treatment. dexamethasone is used in schedule of children's all-b treatment and the response on glucocorticosteroids therapy is very important. the aim of this study was to examine whether dexamethasone changes multidrug resistance of lymphoblasts in all-b and their tendency to begin apoptosis. the study involved children with all-b. bone marrow cells isolated by centrifugation on histopaque at the day of diagnosis were cultured for or hours with or without dexamethasone in concentration - m. analysis of: p-gp surface expression, p-gp function (rhodamine test), phi (carboxy-snarf) and apoptosis test (annexin-v and pi test) were performed with the use of flow cytometer coulter epics xl. for statistical analysis nonparametric wilcoxon test was used.the results showed that p-gp expression on lymphoblasts was , %± , . after hours of lymphoblasts incubation with or without dexamethasone any statistically significant changes were observed. average percentage of lymphoblasts with rhodamine efflux, which characterized p-gp activity, was , %± , . after hours of cells incubation with dexamethasone there was seen significantly higher percentage of cells able to eliminate rhodamine ( , %± , , p= , ). average phi in lymphoblasts was , ± , . acidification of cells incubated hours with dexamethasone was seen in - % percentage of cells ) . rest of lymphoblasts showed alkalization (phi - , ).the percentage of lymphoblasts in early stage of apoptosis after hours incubation with dexamethasone (annexin-v test) was higher than in control cells ( , % vs , %; p= , ). we concluded that dexamethasone does not influence surface p-gp expression on lymphoblasts of patients with all b but significantly increases activation of this protein. functional test should be performed to evaluate multidrug resistantace of leukemic cells, because surface expression of p-gp is not identical with its activity. moreover, dexamethasone alkalizes cytoplasm of lymphoblasts and induces early stage of their apoptosis. those effects may contribute to the treatment outcome. . however, small numbers of clonal pc can also be detected in the peripheral blood (pb) of the majority of patients and, in a minority of cases, mm is transformed into pc leukaemia (pcl). here, we describe that tumoral pc can express cd d/cd integrin with high or, sometimes, low affinity states, which is associated with their retention in or release from the bm, respectively. objectives: to evaluate the activation state of cd on malignant pc from bm and pb, as well as its regulation. methods: pc and active integrin expression on these cells were detected with anti-cd and anti-cd (clon huts ) moab, respectively, by flow cytometry. to study the integrin activation with divalent cations and pc index proliferation (brdu+ cells), we used short-term pc cultures ( hours). results: cd active form was expressed in the majority of normal and tumoral bm pc from healthy subjects ( . ± . , n= ) and mm patients in the early stages of the disease ( . ± . , n= ). in these cells, huts epitope was clearly upregulated by mn + . in contrast, circulating pc were almost all huts negative, and levels did not significantly augment when these cells were exposed to mn + . moreover, not only pb but also bm malignant cells from pcl patients were also huts negative and divalent cation refractory. it was also observed that pc from pcl patients showed an increased proliferative index ( . ± . brdu+ cells, n= ) in comparison to pc from mm patients in the first stages of disease ( . ± . brdu+ cells, n= ). these results suggest that the active form of cd must be expressed on pc to retain these cells within the bm environment. moreover, its downregulation is associated with increased numbers of circulating pc and disease progression. multipotent mesenchymal stem cells derived from (hucb) represent promising candidates for the development of future cellular therapy strategies. they are able to self-renew and they terminally differentiate into multiple lineages, including bone, cartilage, muscle, bone marrow, fat and other diverse connective tissues. in the first part of this study, we compared different protocols for the expansion of human mesenchymal stromal cells (hmsc) starting from diagnostic samples of bone marrow aspirates and the cord blood (cb). the protocols differed in the presence of either % fetal bovine serum (fbs) with and without fgf ,or % human platelet lysate (hpl), %hpl, ( % fbs + % hpl), ( % fbs + % hpl). we obtained a significantly better expansion with hpl, compared to cells with a selected batch of fbs and in fewer days.in the second part of this study, we focused on proteins that were differentially expressed during osteogenic, adipogenic and vascular muscular differentiation by western blotting. we compared the quality and the quantity of protein expression before and after differentiation (day ). two bm and two cb differentially expressed spots were observed between the two groups (before and after differentiation).we noted the low pourcentage of hmsc in cb samples: in ten samples, only two made msc colonies as in bm samples. we were also interested to the different coloration: osteogenic diffentiation was determined by alizarin red s staining, for adipogenic differentiation, the cells were stained with oil red o to visualize lipids droplets. background: inherited bone marrow failure syndromes (ibmfs) comprise a group of genetic disorders characterized by single or multiple cytopenias, as well as distinctive clinical features and varied molecular pathways. activation of p tumor suppressor pathway leads to cell cycle arrest and initiates apoptosis. we studied the presence of p dna (as a marker of cell cycle dysregulation); in bone marrow of children with fanconi anemia (fa) and those with acquired aplastic anemia (aaa). subjects and methods: this is a cross sectional study that involved: ) ten cases with fa diagnosed on the basis of dna breakage analysis, ) ten cases with aaa, and ) ten normal control cases. the presence of p dna was measured in both bone marrow and peripheral blood samples using a real-time quantitative pcr by taqman assay. results: p dna was demonstrated in bone marrow of % of children with fa, compared to % in children with aaa (p x . ), while, no p dna was seen in normal control. a positive correlation between dna breakage and presence of p dna was seen in bone marrow from fa (p x . , r . ). the presence of p tumor suppressor gene by real time pcr in bone marrow of fa may represent an early indicator of significant dna genetic alteration in those patients. key: cord- -osstpum authors: nan title: abstracts oral date: - - journal: am j transplant doi: . /j. - . . .x sha: doc_id: cord_uid: osstpum nan abstracts evl (target - ng/ml), aza or mmf with standard (sd) or reduced (rd) csa. data at months post-tx is presented here. results. the proportion of patients with cmv events, including serious adverse events and laboratory evidence of cmv infection, has remained low and broadly similar following introduction of cc administration of evl and use of concomitant rd-csa. cmv syndrome and cmv organ involvement occurred in < % of recipients receiving evl in each of the studies. the highest rates of all cmv parameters occurred with mmf + sd-csa or aza + sd-csa. conclusion. the low incidence of cmv infection and cmv-related events observed in heart tx patients receiving fd-evl and sd-csa as de novo immunosuppression has been maintained in newer regimens of cc-evl with rd-csa. cmv syndrome and cmv organ involvement are rare with evl-based immunosuppression after cardiac tx. the low rate of cmv infections in evl-treated patients may contribute to improved long-term outcome and a lower incidence of cav; confirmatory data are awaited. fibronectin-α β interactions enhance p mapk phosphorylation and metalloproteinase- expression in cold liver ischemia/reperfusion injury. sergio duarte, xiu-da shen, takashi hamada, constantino fondevila, ronald busuttil, ana j. coito. the dumont ucla transplant center. expression of endothelial fibronectin (fn) is an early event in liver ischemia/reperfusion (i/r) injury. we have recently shown that cs peptide facilitated blockade of fn-α β leukocyte interactions regulates metalloproteinase- (mmp- ) expression in steatotic orthotopic liver transplants (olt). this study tests the function of the cs peptide therapy upon mmp- , and further dissects putative mechanisms, in an alternate model of cold ischemia/reperfusion (i/r) injury. methods and results: cs peptides were administrated through the portal vein of sprage-dawley (sd) rat livers before and after h cold storage ( µg/rat). sd recipients of olts received an additional dose of cs peptides h post-olt. cs therapy significantly increased the d olt survival rate ( % vs. %, n= /gr, p< . ). cs peptides reduced sgot levels (u/l) at h ( ± vs. ± p< . ) and h ( ± vs. ± , p< . ) post-olt. cs treated olts showed good preservation of lobular architecture, contrasting with severe necrosis and sinusoidal congestion in controls. moreover, cs treated livers were characterized by a profound decrease in t ( ± vs. ± , p< . ), nk ( ± vs. ± , p< . ) and ed ( ± vs. ± , p< . ) cells as early as h after i/r. neutrophils, as indicated by mpo activity ( . ± . vs. . ± . , p< . ) were depressed by cs therapy. this correlated with decreased mrna expression of tnf-α ( . ± . vs. . ± . , p< . ) and cycloxygenase- ( . ± . vs. . ± . , p< . ) . leukocyte transmigration is dependent upon adhesive and focal matrix degradation mechanisms. mmp- , which is inducible and expressed by infiltrating leukocytes, was profoundly depressed in h cs- peptide treated olts, at both mrna ( . ± . vs. . ± . , p< . ) and protein ( . ± . vs. . ± . , p< . ) levels. moreover, mmp- activity evaluated by zymography was reduced by ∼ -fold in the cs- group. interestingly, phosphorylation of mitogen-activated protein (map) kinase p ( . ± . vs . ± . , p< . ) was selectively downregulated in the h cs- treated olts. in conclusion, this work supports a broad regulatory role for fn-α β interactions on mmp- expression by leukocytes, likely mediated through activation of p mapkinase, and matrix pathological breakdown associated with leukocyte infiltration. this data provides the rationale for the development of novel therapeutic approaches in cold liver i/r injury. ischemia reperfusion injury is the most common cause of acute kidney injury in both native and allograft kidneys. the pathogenic mechanisms of renal ischemia reperfusion injury include changes at the level of the microvasculature as well as the tubules. within the microvasculature, leukocyte-endothelial interactions likely play a role and contribute to the well-established microvasculature dysfunction (e.g., endothelial permeability) and alterations in endothelial-leukocyte interaction occur during ischemic acute kidney injury. our previous studies have demonstrated that t cells modulate renal ischemia reperfusion injury in a murine model, we hypothesized that t cells could mediate changes in renal vascular permeability during ischemia reperfusion injury. we performed a min bilateral renal ischemia followed by reperfusion in c bl wild type mice and in t cell deficient (nu/nu) mice with or without t-cell adoptive transfer from their wild type littermates and evaluated rvp by evans blue dye extravasations (ebde). the time course studies of rvp showed marked increases in renal ebde within the early - hrs after ischemia. cd positive pan t-cells but neither cd nor cd t cells were found to infiltrate into post ischemic kidney within hrs, and comparison was made with other leukocytes using immunohistochemistry technique. gene microarray analysis demonstrated that the gene for tnf-α (tnfaip ), a potent mediator of microvascular permeability as well as a t cell product, was increased in the kidney early after ischemia. tnf-α, ifn-γ and il- protein by an intracellular cytokine staining technique was found increased early in peripheral circulating t cells and later in renal t cells after renal ischemia. the rise in rvp was significantly attenuated in t cell deficient mice nu/nu at hrs compared to the wild type littermates and this attenuated rvp in t cell deficient mice was restored after adoptive transfer of splenic t cells from wild type littermates into these nude mice. these data demonstrate that t cells traffic early into post ischemic kidney, produce tnf-α and other cytokines that can increase rvp, and directly participate in the increased rvp during acute ischemia reperfusion injury. t cell-endothelial interactions are a likely mechanisms underlying the pathophysiologic role of t cells in renal ischemia reperfusion injury . background: ischemia/reperfusion injury (iri) is a major cause of organ dysfunction after intestinal injury and transplantation. the interaction between the innate and adaptive immune systems has become a major area of focus in this field. our preliminary work showed that mice undergoing intestinal iri experienced worse survival and tissue injury in conjunction with increased infiltration of pmn and cd + cells as compared to sham mice. the purpose of this study was to investigate the role of the t cell in intestinal iri through the use of genetically deficient mice. methods: under anesthesia, male c bl wild-type mice (wt) and cd knockout mice (cd ko) underwent min of warm intestinal iri by clamping of the sma. separate survival and analysis groups were performed. intestinal tissue was harvested at h and h. tissue was analyzed by histology, cd immunostaining, myeloperoxidase activity (mpo), and semi-quantitative pcr for several cytokines/chemokines. results: wt had significantly worse survival compared to cd ko ( % vs. %, p= . ), and worse histopathological injury mostly involving mucosal sloughing. wt had higher mpo activity than cd ko ( . ± . vs. . ± . at h, p= . , and . ± . vs. . ± . at h, p= . ) and increased cd + cell infiltration. there was also increased mrna production of cytokines/chemokines in wt vs. cd ko at both timepoints; specifically, the data with respect to b-actin at h was: il- ( . conclusion: this study demonstrates for the first time in the intestine that cd + t cells are important mediators of iri. in the absence of cd + t cells, better outcomes were associated with less pmn infiltration implying a link between the innate and adaptive immune systems. an alteration in chemotactic signaling potentially effected by cd + t cells may play a role in this process. these results confirm the important function of cd + t cells in intestinal iri and justify further investigation into their complex role in this process. messenger rna assessment in urine sediments from renal transplant patients is rapidly evolving as a non-invasive diagnostic tool. t cells, macrophages, and often b cells are present in rejecting kidney grafts. we questioned whether urinary mrna levels of markers representing these cell types ((regulatory) t cells: cd ε, foxp , cd , tgf-ß; macrophages: cd , s a ; b cells: cd ) are associated with rejection. materials in our institute urine samples a year from over renal transplant patients are being collected for mrna quantitation purposes. urine sediments are pelleted and stored in rna-preserving solution. we initially investigated the effect of incubation time of urine on mrna integrity. next, in a case-control study urine samples taken at time of biopsy-proven rejection were compared to samples taken during stable graft function. median time of sampling ( days versus days posttansplant) did not significantly differ between groups. rna ( . ± . µg) was extracted using rneasy spin columns. message of the markers mentioned above was quantified with q-pcr and normalized. results incubation of urine for h or longer at room temperature after acquisition resulted in a % decrease in s rrna signals (p< . ). however, storage of urine for up to h at ºc did not result in decreased mrna expression. in the case-control study, all markers tested showed the highest expression levels in urine rejection samples. when corrected for multiple comparisons, only tgf-ß ( -fold, p= . ) and foxp ( -fold, p= . ) expression was significantly increased in rejection samples compared to non-rejection samples. tgf-ß levels highly correlated with foxp levels (r = . , p< . ), suggesting a mechanistic relationship in vivo between the two molecules. conclusion rna integrity in urine samples stored at ºc is maintained for at least h. detection of increased tgf-ß and foxp message in urine from patients with a kidney transplant represents a means for indicating occurrence of graft rejection. this implies that mrna urinalysis renders a suitable molecular tool for non-invasive patient monitoring in clinical practice. the data furthermore suggest that rejection is associated with tgf-ß-mediated immune mechanisms in which regulatory t cells play a role. the significance of b cell and plasma cell infiltration in renal allografts remains controversial. we previously established that transcript sets associated with ifng effects or t cells reflect the inflammatory burden in renal allografts. in the present study, we identified b cell associated transcripts (bats) and immunoglobulin transcripts (igts), reflecting b cell and plasma cell infiltration, respectively. using microarrays, we analyzed bat and igt expression in relationship to histologic lesions, diagnosis, and renal function in renal allograft biopsies. immunostaining confirmed that bat and igt expression was associated with b cells and plasma cells in the graft. expression of bats and igts was increased in biopsies with rejection ( . ± . , . ± . ) compared to non-rejection ( . ± . , . ± . ), but was not different between t cell ( . ± . , . ± . ) and antibody mediated rejection ( . ± . , . ± . ) and also occurred in some non-rejecting biopsies (recurrent gn). bat and igt scores correlated strongly with time post transplant (fig ), which was best modeled as a dichotomous relationship: biopsies ≤ months did not express bats or igts above the level of control kidneys. other inflammatory markers were not time dependent. in biopsies ≥ months, bat and igt expression correlated with interstitial inflammation, tubular atrophy, and interstitial fibrosis. in a multiple regression analysis, only time post transplant and interstitial inflammation were independently related to bat and igt scores. when correcting for time post transplant, bat and igt scores did not correlate with renal function at the time of biopsy or future function months post biopsy. bats and particularly igts are a time dependent feature of injured and inflamed renal allografts. corrected for the effect of time, bats and igts do not correlate with outcomes, indicating that b cell and plasma cell infiltrates have no specific role for the mechanism of injury independent of the inflammatory burden. their accumulation in late allografts may indicate emergence of specialized lymphoid compartments in tissues with long standing low level inflammation. pearson correlation coefficient between pbts expression and renal function pbts egfr at %change in egfr from biopsy months after biopsy baseline to biopsy biopsy to months after qcats - . * - . * - . * - . grits - . ** - . ** - . ** - . irit_d - . - . - . . irit_d - . ** - . ** - . ** . * irit_d - . ** - . ** - . * - . kt . ** . . ** - . * p< . , ** p< . thus the pbts have prognostic value. surprisingly, the transcripts in the biopsy with greatest prognostic value for future gfr or recovery of gfr were not related to rejection (cytotoxic t cell or ifng associated) but to the degree of injury response. we propose that the common pathway linking various injuries (immune and non immune) to gfr is through the degree of injury response these events induce in the epithelium, particularly those in the irit_d set. early rejection from anamnestic response in offspring-to-mother or husband-to-wife kidney transplant. kwan tae park, song chul kim, duck jong han. surgery, asan medical center, seoul, korea. accelerated rejection can be developed in the immediate post-transplant period resulting from anamnestic response due to the exposure to fetal hla antigen during the previous pregnancy in case of offspring-to-mother or husband-to-wife. however, accelerated rejections in these groups have been rarely reported. cases of offspring-to-mother (offspring group) and cases of husband-to-wife (spouse group), who has been sensitized to spouse via their children, underwent kidney transplants from january of to august of at our institution and retrospectively reviewed. control group was female kidney recipients transplanted at the same period from living related donors other than offspring and from living unrelated donors other than husband. acute rejection (ar) rate within months after transplant were . % ( / ) in offspring group and . % ( / ) in spouse group. the ar rates were not different between the two groups, however they were significantly higher than control group in both groups, namely . % ( / ) for offspring control and . % ( / ) for spouse control. the mean onset of ar was . day ( - ) and it was significantly later than that of control groups ( . day, p< . ) and % of ar were accelerated rejections within days after transplant. proportion of acute humoral rejection was significantly higher in both groups than control group ( . % vs . % in offspring group, % vs % in spouse group). most of the ar was successfully reversed by steroid pulse and/ or plasmapheresis, ivig, rituximab. any risk factors for the ar such as number of pregnancy, preoperative cdc cross matching, pra, immunosuppressant and antibody induction couldn't be identified. serum creatinine level in ar patients were higher than ar free patients by postoperative month, but last follow up creatinine level didn't show any statistical difference ( . mg/dl in ar vs . mg/dl in ar free). year graft/patient survival rate were not different between ar and ar free groups and study and control groups. risk of higher rejection rate accompanied with higher accelerated and humoral rejection was identified in female kidney recipients from offspring or spouse donor in comparison with control group. considering the anamnestic response of this cohort of female recipients, more prudent preoperative screening and careful immediate postoperative immune monitoring are required for the avoidance of rejection and impaired graft survival. kaplan-meier survival curves showed that ∆upc > . were associated with decreased patient and allograft survival. these data show for the first time that regardless of the histopathology, ∆upc > . one month after rejection is associated with poor patient/allograft outcomes. from histology to microarrays the histopathological hallmark of t cell mediated rejection (tcmr), is interstitial infiltration. assessing infiltration by histology is arbitrary, limited in reproducibility, and has never been assessed against independent standards. we recently reported that a set of cytotoxic t cell-associated transcripts (qcats) could quantitatively assess the t cell burden in tissue. objective of the present study was to re-examine the current diagnostic criteria in relationship to the qcats. in renal allograft biopsies, we assessed how histology predicts the qcat burden. an independent diagnostic threshold for qcats was established in control samples. applying this qcat threshold revealed current diagnostic criteria for histology to be flawed; the threshold for interstitial infiltration is too high ( % specificity, % sensitivity), the types of infiltration to be considered (i.e. i-banff) are wrongly defined, and tubulitis is not increasing diagnostic accuracy. changing the criteria by lowering the histological threshold for cortical infiltration from % to %, taking into account all interstitial cellular infiltration (= i-total) and ignoring tubulitis increased sensitivity to % with a decreased specificity of %. but, this includes biopsies having interstitial infiltration without qcats, indicating that histology might not discriminate between 'active' and 'inactive' infiltration. both the refined histological and the qcat threshold had prognostic value in terms of future renal allograft function. we propose a refined histological scoring system that better predicts the active t cell burden and outcome than the current banff criteria for renal allograft rejection. there has been an increasing and well justified interest regarding the long term renal consequences of kidney donation. the collective evidence suggests, however, that kidney donors enjoy a normal life span and their lifetime risk of esrd may not be different from non-kidney donors. assessing kidney function utilizing serum creatinine is not without limitations. therefore, to better assess kidney function, yeas ago, we began a large effort to measure gfr using the plasma disappearance of iohexol and first void urinary albumin/creatinine ratio (acr) in randomly selected kidney donors who donated at our institution. results: we have performed over donor uninephrectomies since the inception of our program in . of these, donors underwent iohexol gfr at our general clinical research center. microalbuminuria is defined as acr between - mg/g and macroalbuminuria as acr> mg/g. the mean age was . ± . years, . ± . years have elapsed since donation, hemoglobin was . ± . g/l, systolic blood pressure (sbp) was . ± . mmhg and diastolic blood pressure (dbp) was . ± . mmhg. . % of donors had a gfr greater than ml/min/ . m and . % had a gfr between - ml/min/ . m . the detailed renal profile of these donors is shown in the table below. multivariate analysis that adjusted for age, gender, ethnicity, time from donation, sbp, dbp and body mass index (bmi) identified age at donation, female gender and bmi as independent predictors for gfr< ml/min/ . m and time from donation and systolic blood pressure as independent predictors for micro and macroalbuminuria. conclusion: this is the largest effort describing measured gfr in previous kidney donors. it is reassuring to find out that the majorities have a preserved gfr and only a minority has albuminuria. the risk factors for reduced gfr and albuminuria are analogous to what has been described in the general population. cystatin c levels and long-term donor health markers cystatin-c < (n= ) psychosocial and physical health of lkds following donation is of utmost importance. unfortunately, this issue has been neglected to a large extent. we sought to examine the current practices regarding psychosocial follow-up of lkds after surgery across transplant (tx) centers. we conducted a -question online survey regarding this practice and issues related to lkd. the survey was e-mailed via listservs of professional tx societies. several questions allowed for more than one response. characteristics of the tx centers that participated in the survey are found in table . only . % actively follow donors regarding mental health, substance abuse, or quality of life issues after donation. the professionals most often involved are social workers ( %) and nurse coordinators ( %). the majority of follow-up is conducted via phone ( . %); though appointments ( . %) and questionnaires ( . %) are also utilized. % initiate follow-up with donors - weeks post-operatively, whereas only % of programs maintain contact at both - months and months. . % offer post-operative psychosocial support; % only under certain circumstances. this support is provided by social workers ( %), psychologists ( %), and/or psychiatrists ( %). support is available indefinitely in % of programs. . % assume the costs of post-operative medical and psychosocial care indefinitely; . % for a specified period of time. . % bill the recipient's insurance and . % bill the donor's insurance. . % of centers accept donors without health insurance and only . % purchase insurance on behalf of donors to cover post-operative health care needs. the results of this survey clearly demonstrate that post-operative psychosocial follow-up of lkds is uncommon and that current practices are widely variable. without routine follow-up of donors, tx centers are less likely to capture post-operative psychosocial issues that may result from organ donation. standardized post-operative follow-up of lkds should become a mandatory part of their care, which will require increased support from health care policy makers. the role of hepatitis c and race in patient and graft survival in combined kidney and liver transplantation. dilip moonka, ravi k. parasuraman, kim a. brown, alissa kapke, dean y. kim. gastroenterology, henry ford health systems, detroit, mi; nephrology, henry ford health systems, detroit, mi; biostatistics, henry ford health systems, detroit, mi; transplant institute, henry ford health systems, detroit, mi. the influence of hepatitis c (hcv) and race are not well understood in combined kidney-liver transplant (klt). hcv has a negative impact on patient and graft survival in liver recipients whereas african-american (aa) race is a negative prognostic factor in kidney recipients. aim: to determine the influence of hcv and aa race on patient and graft survival in klt. methods: the unos public use database was used to identify patients undergoing klt who had known hcv ab status. % were aa and % were non-hispanic white (nhw). . % were hcv ab positive. groups were assessed for patient and graft survival. results: there is a significant gradient in patient survival and both kidney and liver survival from nhw patients without hcv to aa patients with hcv (table) . the patient survival at five years drops from % to % along this gradient. there is also a % drop in liver and an % drop in kidney graft survival. on pairwise testing, the difference in patient survival at yr between all patients without hcv and those hcv ab positive drops from % to % (p= . ). the difference in yr survival between all nhw and aa patients (regardless of hcv status) drops from % to % (p= . ). on multivariate analysis, hcv and the combination of hcv and aa race remains associated with diminished survival but race alone does not. conclusions: patients with hcv who undergo a klt transplant are at increased risk for poor overall survival and poor survival of kidney and liver grafts. this effect appears even more pronounced in aa patients. this knowledge is critical to individual centers in assessing risk and benefit from klt in these groups and these groups represent an opportunity for improved interventions. kidney: pediatrics background: pediatric renal transplant recipients have excellent short-term outcomes but long-term success is compromised by complications of chronic immunosuppressive medications and chronic allograft nephropathy. studies show that calcineurin inhibitors and steroids can be individually avoided in pediatric renal transplantation. building on that experience we designed this study to optimize short and long-term renal allograft function with minimal chronic immunosuppression using a steroid-free, calcineurininhibitor withdrawal protocol in low risk pediatric renal transplant recipients. methods: unsensitized pediatric recipients of a first living donor kidney transplant received doses of campath- h ® ( . mg/kg), day pre-and post-transplant. subjects received tacrolimus and mmf immediately post-transplant until week - when they underwent protocol renal biopsy and were changed to sirolimus and mmf if rejection free. the planned subjects have been enrolled; this report describes the clinical outcomes of the with year of follow up. results: the mean subject age is . yrs; . % are female and . % caucasian. at transplant, / were cmv seronegative and / were ebv seronegative. protocol therapy was discontinued in eight subjects due to: rejection ( ), mouth ulcers ( ), leukopenia ( ) , unrelated ( ). clinical acute rejection (ar) occurred in subjects ( %) and had subclinical ar; ar was cellular rejection in subjects, and humoral in at days post-transplant who had an undetected positive crossmatch to class ii hla. there were two graft losses, one due to recurrent fsgs and one due to medication non-adherence. there were no cases of ptld and no deaths. leukopenia occurred in subjects ( . %). there were infections of which were urinary tract infections ( . %) and were pneumonia ( . %). conclusions: minimization of immunosuppression using a steroid-free, calcineurinwithdrawal protocol in low risk pediatric renal transplant recipients appears to be well tolerated with acceptable rates of clinical ar and no serious infections months after transplantation. male; % white). substantial increases in bmiz were observed within the first mo.; no changes were seen from to mo. baseline bmiz category (<- . , - . to + . , >+ . ) influenced the pattern of change. subjects with low bmiz (<- . ) at baseline experienced the greatest increases in bmiz, but overweight was rare; increases tended to result in a bmiz in the healthy range. those with high bmiz (>+ . ) at baseline demonstrated no significant change in bmiz post-tx. younger age at tx (highest risk for those to y. at tx) more remote date of tx, and baseline bmi between the th and th percentiles were significant independent risk factors for unhealthy weight gain both at mo. and persisting at mo. post-tx. weight gains occur early after tx and tend to persist. counseling focused on prevention of weight gain should be a routine part of post-tx care, with the most intense efforts concentrated on the highest risk patients: young, healthy weight children. avoidance of early weight gains may have an important impact on bmi over the long term. referral. lindsey a. pote, jennifer trofe, erin h. wade, jorge baluarte, alden doyle, simin goral, karen warburton, robert grossman, jo ann palmer, roy d. bloom. pharmacy, hosp of the univ of penn; nephrology, univ of penn; transplant surgery, children's hospital of philadelphia. intro: few data exist on outcomes in pediatric kidney recipients who transition to an adult transplant program. purpose: to examine outcomes in pediatric kidney recipients who transition to an adult transplant program and to identify characteristics associated with non-adherence related graft loss. methods: retrospective, single center analysis of pediatric kidney recipients who transitioned to an adult program. results: for the cohort overall, transition to the adult program occurred a mean of ± . months following transplantation. mean serum creatinine at transition was . ± . mg/dl and mean patient age . ± . years. % of patients received living donor kidneys. within ± . months of transition, ( . %) of patients experienced graft loss. causes of graft loss included admitted non-adherence (n= ), recurrent disease (n= ), chronic progressive graft dysfunction (n= ) and bk nephropathy (n= ). graft loss occurred a mean of ± months post transition for non-adherent patients and . ± months for adherent patients (p=ns). the characteristics of the cohort is shown in the table according to whether or not patients had documented non-adherence related graft loss. conclusions: ) graft loss commonly occurs within years following transition and is attributable to both patient non-adherence and late referral by the pediatric transplant program, ) non-adherence related graft loss was more common in males, ) factors unassociated with non-adherence include ethnicity, prior transplantation, age at transplant, & duration of dialysis, ) the development of collaborative pediatric-toadult transition clinics may enhance adherence and lead to improved graft outcomes in this population. this -year, prospective randomized pilot study compares the effect of conversion to sirolimus (srl) vs. continued mycophenolate meofetil (mmf) in patients with chronic allograft nephropathy (can), on histological progression. we present -year data on safety and renal function. participants > year post-transplant with can (banff ≥ci , ct ) on tacrolimus, mmf and prednisone were randomized to continue mmf or convert to srl (target - ng/ ml). tac dose was minimized (target - g/l), and renal biopsy performed at baseline, , years. -month interval monitoring included adverse event (ae) reporting, egfr (schwartz) and immunosuppressant levels. / (mmf= , srl= ) have completed -year follow-up. baseline gender, ethnicity, previous acute rejection episodes, can grade, proteinuria (upcr) were similar. srl had lower baseline egfr ( ± vs. ± ml/min/ . m , p= . ). aes were reported (mmf= , srl= ), serious ae (sae: mmf= , srl= ). common saes were similar: dehydration & elevated creatinine ( ), gastroenteritis ( ) and rejection (ar). episodes ( patient) of ar occurred in mmf group and ( patients) in srl group, all attributable to non-adherence. there were no sustained change in electrolytes, hg and total wbc counts in the st year, except neutrophil counts were lower in the srl group ( mo: mean . vs. . , p< . ). platelets were significantly lower at months (srl) but not thereafter ( month: mean ± vs. ± x e ). cholesterol and tg increased early (p< . ), but only cholesterol persisted after years (mean . vs. . mmol/l, p< . ). in srl group only, upcr increased over years (∆upcr ± mg/mmol, p< . ). this was not associated with hypoalbuminemia at years. egfr was lower in srl vs. mmf after months (p< . ), but the rate of change in egfr from baseline was similar between groups (- ± vs. - ± ml/min/ . m , p=ns). serious adverse events did not differ significantly between the srl and mmf groups. sustained srl treatment was associated with mild neutropenia, hypercholesterolemia and proteinuria after years. egfr was reduced at baseline compared with mmf, and both groups had similar rates of decline in gfr over time. allograft recipients. maarten naesens, oscar salvatierra, li li, minnie sarwal. department of pediatrics, stanford university school of medicine, stanford, ca. background: in contrast to adult kidney recipients, little is known about the long-term evolution of tacrolimus pharmacokinetics in pediatric kidney transplant recipients. methods: one-hundred five pediatric recipients of a kidney allograft, all treated with a corticosteroid-free immunosuppressive protocol, were included. the evolution of tacrolimus doses and exposure was recorded at , , , , and months after transplantation, as well as all pre-dose trough levels (c ; n= ) obtained in the first years after transplantation. results: dose-corrected tacrolimus exposure (c /dose/kg) increased in the first years after kidney transplantation in pediatric recipients (table , figure ). this decrease in dose requirement by time was only significant in children older than years at the time of transplantation ( figure ). in addition, the younger patients had significantly higher dose requirements compared to older recipients, which translated in marked underexposure in - % of patients < years of age in the first days after transplantation. conclusion: pediatric kidney transplant recipients exhibit maturation of tacrolimus pharmacokinetics with time after transplantation. this can not be explained by differences in corticosteroid use, as all patients were treated with a corticosteroid-free protocol. the higher dose requirements for younger recipients and the absence of tacrolimus maturation in the youngest recipients suggest that age-dependent changes in tacrolimus intestinal first-pass effect, metabolism or distribution play a role. whether age-specific tacrolimus dosing algorithms will improve outcome needs further study. determinants of dose-corrected pre-dose trough levels (c /dose/kg) aih may present as acute hepatitis in % of patients (pts) and result in alf. early administration of corticosteroids may obviate the need for liver transplantation (lt), but features which identify aih in pts with alf have not been determined. aim: to identify clinical and histological features which distinguish alf due to aih. methods: / pts in the alf study group registry had no evidence of viral, metabolic, vascular, and drug/toxic liver injury. all had alf defined by acute disease, encephalopathy, and coagulopathy. based upon admission clinical features, / had probable aih, and were considered "indeterminate." liver biopsies (lbx) available from pts were reviewed by a blinded expert hepatopathologist. clinicopathologic correlations were analyzed retrospectively. results: all lbx available from pts with suspected aih had classical histologic features of aih. moreover, / ( %) lbx from pts with indeterminate alf also had aih features: extensive necrosis ( %), fibrosis ( %), cirrhosis ( %), interface hepatitis ( %), and plasma cell-rich inflammation ( %). of all lbx with aih features, centrilobular lesions associated with acute, severe aih (am j surg path ; : ) were frequent: plasma cellrich central venulitis ( %), exclusive centrilobular necroinflammation ( %), and pericentral dilatation/congestion ( %). clinical characteristics and outcomes of the pts with aih based upon laboratory and histologic findings differed significantly from pts whose alf remained indeterminate: aih pts were older ( v y), predominantly female ( v %), and had longer jaundice-encephalopathy interval ( v d) , lower alt ( v u/l), higher globulins ( . v . g/dl), lower creatinine ( . v . mg/ dl), and a higher prevalence of ana ( v %) and asma ( v %) (all p<. ). although spontaneous survival did not differ, more aih pts underwent lt ( v %), and more survived month after enrollment ( v %; p<. ). conclusions: using histologic criteria to classify pts with indeterminate alf, aih accounted for at least % of the alf study group registry, and represented % of indeterminate alf. lbx should be performed in all patients with alf of obscure etiology, in particular to identify centrilobular necroinflammatory lesions, which appear to be specific indicators of acute and severe aih. (u- from niddk). ( ) ( ) ( ) ( ) ( ) . mikel gastaca, miguel montejo, lluis castells, antonio rafecas, antonio rimola, ramon barcena, federico pulido, magdalena salcedo, martin prieto, manuel de la mata, jose r. fernandez, jose m. miro, the spanish lt in hiv-infected patients working group. hospital de cruces, bilbao, spain; hospital vall d´hebron, barcelona, spain; barcelona, spain; univ. of barcelona, barcelona, spain; hospital ramon y cajal, madrid, spain; hospital de octubre, madrid, spain; hospital gregorio marañon, madrid, spain; hospital la fe, valencia, spain; hospital univ. reina sofia, cordoba, spain. background and aim: we report on the preliminary results of the prospective multicenter spanish study in hiv- infected patients who underwent olt. methods: the prospective multicenter spanish study fipse-olt-hiv -gesida - was initiated in january . inclusion criteria follows the rules previously described in the spanish consensus document. hiv-infected patients transplanted between january and december were included in this study. results: olt were consecutively performed in hiv- infected patients in the period of the study. median (iqr) follow-up is ( - ) months. median (iqr) age was years ( - ), % of the recipients were male and former drug abuse was the most common hiv- risk factor ( %). % of the patients were transplanted due to hcv-related cirrhosis and % due to hbv cirrhosis. median (iqr) meld score was ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) . pre-olt median (iqr) cd cell count was ( - ) cells/mm and %patients had undetectable plasma hiv viral load. immunosuppression was based on tacrolimus in % of the patients. haart was re-started in a median (iqr) time of ( - ) days after olt and was based on efavirenz in % of the cases. acute rejection occurred in patients ( %). twenty patients died ( . %) mainly due to hcv recurrence ( %). antiviral therapy with peg-interferon plus ribavirin was initiated in patients obtaining a sustained viral response in of them ( %). patient survival ( % confidence intervals) at , , and years was % ( - %), % ( - %), % ( - %) and % ( - %), respectively. conclusion: for selected hiv- infected patients under haart therapy, olt is a safe and effective procedure at mid-term. as in the hiv-negative population, hcv recurrence is the mayor cause of concern and response to antiviral therapy is still disappointing. hepatitis c virus and objectives: describe the -year experience of a university hospital treating chronic hepatitis c relapse, histologically diagnosed after liver transplant. material & methods: from september through july , all the patients subject to liver transplant with histological diagnostics of chronic hepatitis relapse were submitted to antiviral treatment. treatment was suspended if there was a severe adverse reaction to the drugs used, non-adherence, severe rejection or no response after at least months. hcv positive serology patients, alt increase ( , x nl) and hepatic biopsy with metavir rating showing structural and/or periseptal or parenchymatous portal inflammatory activity /= were included. from to , all patients were treated with conventional interferon and ribavirine, regardless of the genotype. from october on, patients with genotype were treated with ribavirine and pegylated interferon and genotype were treated with conventional interferon and ribavirine. results: patients were treated during this time period, thirty-seven male ( . %). their average age was . . % were genotype , . % were genotype . average time between transplant and beginning of treatment was months. average treatment was ( - ) months. . % received conventional interferon, . %, pegylated alpha a and . %, pegylated alpha b. . % had one or more cellular rejection episodes before vhc relapse diagnostics and were treated with corticosteroids. sustained virological response (svr) occurred in . %. three patients had a virological response at the end of treatment (evr) and have not completed six months post treatment. out of patients with svr, . % were genotype , . % genotype and . % were not genotyped. considering genotypes, svr was detected in . % with genotype , in % with genotype . srv was detected when we examined hepatic biopsy, in . % of the f /f patients ( out of ), . % of the f patients ( ), . % of the f patients ( ) . survival period of years for svr patients was % ( out of ) and . % for patient without svr (p = . -kaplan-meier). five-year survival was . % for patients with genotype and % for patients with genotype . conclusions: our results show it is possible to get good five-year survival rates for treated patients. however, handling adverse reactions and long term treatment still pose difficulties in pursuing better svr rates. the impact of alcoholic liver disease (ald) and background: although liver transplant survival benefit has been shown to be associated with meld score, disease-specific analyses have not been reported. we evaluated, using srtr data, the effect of ald and/or hcv infection on transplant waitlist and post-transplant mortality, and survival benefit of deceased donor liver transplants. methods: , patients age ≥ , who were listed between sept and dec , and followed until dec , , were classified into cells according to hcv or ald status: hcv+, ald-: , ; hcv+, ald+: , ; hcv-, ald+: , ; hcv-, ald-: , ). cox regression was used to estimate waiting list mortality and post-transplant mortality separately. survival benefit, which encompasses both pre-and post-transplant events, was assessed using sequential stratification; an extension of cox regression which matches transplant recipients by meld and organ procurement organization to patients on active on the waitlist at the time of transplant. results: hcv significantly (p= . ) increased waitlist mortality, with a covariate-adjusted hazard ratio (hr) for hcv+ vs. hcv-of hr= . (p= . ). the impact of hcv+ was greater among ald+ candidates (hr= . ; p< . ), but was also significant among ald-candidates (hr= . ; p= . ). the contrast between ald+ and ald-waitlist mortality was only significant among hcv+ candidates (hr= . ; p= . ). post-transplant mortality was significantly higher among hcv+ vs. hcv-recipients (hr= . ; p=. ); there was no difference between ald+ vs. ald-recipients. survival benefit of liver transplantation was significantly lower among hcv+ compared to hcv-recipients with meld - , but significantly higher for hcv+ recipients with meld scores of ≥ . a diagnosis of ald did not influence the survival benefit of transplantation at any meld score. conclusion: despite higher waitlist mortality, hcv+ recipients had significantly lower liver transplant survival benefit than hcv-recipients, within categories defined by meld score. in contrast, transplant survival benefit was not influenced by ald. transplanted for hbv-related cirrhosis. giuseppe tisone, daniele di paolo, ilaria lenci, laura tariciotti, andrea monaco, manuele berlanda, linda de luca, giuseppe iaria, alessandro anselmo, irene bellini, mario angelico. hepatic surgery and transplantation, university of rome tor vergata, rome, italy. background and aim: post-transplant active immunization with hbsag vaccine is a potential prophylaxis strategy against hbv-recurrence after liver tranplantation due to hbv-related disease. previous studies showed conflicting results using standard vaccines, whereas the use of the new adjuvant -deacylated monophosphoryl-lipid-a (mpl) significantly increased patient's immunization rate. we investigated the efficacy of a long-term ( months) accelerated (monthly doses) vaccination schedule using the mpl-adjuvanted vaccine administered with and without concomitant hbig. methods: patients (m/f: / ) transplanted for hbv-related cirrhosis ± months earlier were recruited. all were hbsag and hbv dna negative in serum and cccdna negative in liver tissue; ( . %) were co-infected with hcv and ( . %) with hdv. study protocol consisted of consecutive monthly intramuscular vaccine doses (hbsag mg plus mpl mg) given together with lamivudine ( mg/daily). each of the initial doses (first cycle) was administered within days after iu hbig i.v. infusion, while the last doses were given after complete hbig withdrawal (second cycle). hbsab titre was determined before each vaccine dose and during the follow-up. all patients were maintaiened on low-level immunosuppression. preliminary results: all patients completed first vaccination cycle; ( . %) patients received adjuvanted vaccine doses (first and second cycles) and were monitored during months follow-up after vaccination end. no side effects occurred, nor evidence of hbv recurrence. at the end of first cycle all patients achieved an anti-hbs titre > iu/l (mean ± iu/l) and ( . %) a titre greater than iu/l. at the end of follow-up / ( . %) and / ( . %) had an anti-hbs titre greater than (mean ± ui/l) and iu/l, respectively. conclusions: nine months after hbig withdrawal more than half of the patients reached and maintained a protective anti-hbs titre (> iu/l). this intensive schedule using the mpl-adjuvanted vaccine, given in combination with hbig and lamivudine, seems to be more effective than previous hbv vaccination protocols, although a longer follow-up is needed to assess its final effectiveness. heterologous immunity via alloimmune responses to hepatitis c virus replication after liver transplantation. hideki ohdan, masahiro ohira, yuka tanaka, kohei ishiyama, nobuhiko hiraga, michio imamura, kazuaki chayama, toshimasa asahara. surgery, hiroshima university, hiroshima, japan; internal medicine, hiroshima university, hiroshima, japan. the immunosuppressive environment in liver transplantation (lt) recipients infected with hcv is believed to be associated with the progression of hcv reinfection. in the present study, we simultaneously monitored hcv levels and alloimmune status in hcv-infected lt recipients. for evaluating the immune status of hcv-infected lt recipients, we employed a mixed lymphocyte reaction (mlr) assay using a cfselabeling technique. the kinetics of the stimulation index of anti-donor reactive cd + t cells clearly mirrored that of the hcv rna titer, and a significant reverse correlation was observed between the parameters (r = . ). we did not observe a similar relationship between the stimulation index of an anti-third party cd + t cells and the hcv rna titer (r = . ). one possible explanation for this phenomenon might be that cytokines outputted by t cells responding to allostimulation display the anti-hcv activity either directly or indirectly through the activation of bystander immunocytes. to investigate such possibilities, we performed a transwell culture assay comprising a mlr culture in the upper chamber and genomic hcv replicon cell culture in the lower chamber to mimic the anatomical features of the interaction between hcvinfected hepatocytes and alloreactive t cells that infiltrate the portal area in the liver. when one-way mlr was performed using one-halpoidentical combination in the upper chamber, hcv replication was significantly suppressed in the lower chamber containing hcv replicon cells. hcv replication was further suppressed when mlr was carried out with a complete allogeneic combination. this inhibiting effect was dependent on their ifn-γ secreting activity. in addition, a similar result was obtained when ifn-γ-secreting nk/nkt cells stimulated with il- were cultured in the upper chamber. in conclusion, there was a close relationship between the anti-donor and the anti-hcv immune status in the lt recipients infected with hcv. cytokines such as il- and ifn-γ may be produced in response to allostimulation, and even these cytokines do not cause graft rejection, display anti-hcv activity. the elucidation of such a possible heterologous immunity via alloimmune responses to hcv replication might lead to the establishment of a novel method to prevent the progression of hcv reinfection in hcv-infected lt recipients. hepatitis c e region diversity after liver transplantation: a report from the hepatitis c iii trial. juan f. gallegos-orozco, hugo e. vargas, georges netto, gary l. davis, , and . % are morbidly obese . the goal of this study was to quantify the effect of bmi on access to transplantation (att), likelihood of receiving a transplantation (lrt), turndown rates (tr), and survival benefit (sb). methods: att was defined as either registering for the deceased donor waiting list or receiving a live donor transplant, and was analyzed based on the usrds registry using logistic regression. lrt was based on time spent in active status on the waiting list before receiving a transplant, and was analyzed based on the unos waiting list dataset using cox proportional hazards time-to-event analysis. tr was defined as the relative likelihood of being turned down for an organ offer by a provider other than the patient, and was analyzed based on the unos organ turndown dataset using negative binomial regression. sb was defined as survival after kidney transplantation versus survival on the deceased donor waiting list, and was analyzed based on the unos waiting list dataset using a time-dependent cox survival benefit model. all models were adjusted for known factors influencing those outcomes. lgbp lsg lsg mean follow-up (months) . (range - ) (range - ) . ( , ) patients > months since operation (n) / / / mean % ewl at > months (range - ) (range - ) ( , ) transplant candidate at > months / / / underwent transplant / / / complications developed in two patients (both with cirrhosis) and there was no mortality. mean follow-up was . months, and mean ewl at months or later was % (esrd), % (cirrhosis) and . % (esld). obesity associated comorbidities were improved or resolved in all patients. serum albumin and other nutritional parameters months or later after surgery were similar to preoperative levels in all groups. at most recent follow-up, out of patients ( %) have reached our institution's body mass index (bmi) limit for transplantation and are awaiting transplant; one patient with esld has undergone a successful lung transplant and one patient with cirrhosis has undergone a successful liver transplant. factors that contribute to inequitable access to the transplantation network (unos/ optn) include socioeconomic status, geographic location and delayed referral. the goal of the meld allocation system was to assign priority to the sickest candidate and assure equitable access to liver transplantation (lt). the meld has been validated as a reliable marker for liver disease severity. at the time of referral to the transplant center or listing a high meld (hm) is an indirect measure of delayed referral. therefore, the aim of this study is to identify factors associated with a hm at the time of listing for lt. method: using the unos database, we identified all adult candidates listed for lt from to . patients who received meld upgrade (i.e. hcc, fhf) and those listed for multi-organ transplant were excluded. the data collected included demographics, insurance payor, diagnosis, and meld score. meld score at time of listing was categorized as low (< ) and high (> ) and insurance type as private, medicaid and government (excluding medicaid) . results: during the study period , candidates were added to the lt waiting list. of these, there were , ( . %) males with a median age of (range - ) . caucasians were , ( . %), hispanic ( . %), african americans ( . %) and the rest ( . %). the underlying liver disease was hepatitis c ( . %), alcohol ( . %), alcohol/hepatitis c ( . %), pbc/ psc ( . %), cryptogenic ( . %) and others ( . %). age, gender and liver disease etiology were not associated with a hm at listing. a hm was associated with ethnicity: aa ( . %), hispanic ( . %), caucasian ( . %)[p< . ] and insurance type: medicaid ( . %), government ( . %) and private ( . %)[p< . ].there was no strong interaction between ethnicity/race and insurance in combination as predictors of hm i.e. both were independent. conclusion: ( ) aa and lt candidates with medicaid insurance are more likely to have a hm score at initial listing ( ) hispanics had the lowest rate of private insurance compared to caucasians with the highest rate. the above results suggest that type of insurance and ethnicity are independently associated with a hm (i.e. sicker patients) at listing. since a hm score is associated with increased mortality, implementation of strategies that result in timely and equitable access to the transplantation network regardless of the insurance type or ethnicity of the candidate(s) should be a priority. marked increase in the use of inactive status on the kidney transplant waiting list. kim nguyen, valarie ashby, , further wait time accrued only after active status was restored. on / / , the optn implemented a change in policy that provides for the accrual of waiting time during the entire interval that wl candidates are designated as s . methods: we investigated the impact of this policy change on the patterns of s designation, using the srtr/optn database. initial and subsequent status on the wl were determined for , candidates placed on the ki tx wl from / / - / / . the probability of becoming active (including receiving a living donor tx) on the wl was calculated for those who were s at listing before and after the policy change. results: table shows trends in the use of s before and after the policy change. of the , candidates listed as s before the policy change, % became active within months and % within year of wl. for the , candidates listed as s after the policy change, the corresponding figures are % and % respectively. figure demonstrates that, since the implementation of the new policy, the % of patients initially wl in s at most us tx centers has increased. conclusion: since the implementation of this policy, the number and % of pts wl as s at most ki tx centers, and the duration of s for those initially wl as s has increased dramatically. the implications of this practice on access to ki tx and survival warrants further investigation. can abstracts ( %) patients were retransplanted, and ( %) patients had panel reactive antibody > . the mean cumulative thymoglobulin dose administered was . mg/kg, and the primary maintenance immunosuppression started prior to discharge was a sirolimuscontaining regimen ( %). at the end of the follow-up period, ( %) patients had functioning grafts and ( %) patients experienced graft loss. of the patients with graft loss, ( %) patients experienced graft loss secondary to death. no pertinent differences were identified between groups. the graft survivals for african american recipients with african american or caucasian donors were % vs. % at year and % vs. % at year . conclusions: our study results suggest that donor race does not adversely affect graft outcomes in african american cadaveric kidney recipients with modern immunosuppression. donor racial differences may not play a primary role in the inferior graft outcomes of african american cadaveric kidney recipients. cardiac evaluation before kidney transplantation: are we screening too often or not enough? krista l. lentine, m. a. schnitzler, j. j. snyder, d. c. brennan, p. hauptman, p. r. salvalaggio, b. kasiske. evaluation for ischemic heart disease (ihd) is a common but non-standardized practice before kidney transplant. we retrospectively studied pre-transplant cardiac evaluation (ce) practices among a national sample of renal allograft recipients. methods: we examined usrds data for medicare beneficiaries transplanted in - with part a and b benefits from dialysis initiation through transplant. clinical traits defining "high" expected ihd risk were defined as diabetes, prior ihd, or > other coronary disease risk factors. pre-transplant ce were identified by billing claims for noninvasive stress tests and angiography. we quantified individuals with claims for coronary revascularization procedures between ce and transplant, and abstracted post-transplant acute myocardial infarction (ami) events from claims and death records. results: among , eligible patients, . % ( . % of high-risk and . % of lower-risk) underwent ce before transplant. overall, . % of patients who received ce also received pre-transplant revascularization, including only . % of lower-risk patients studied by ce ( table a) . the adjusted odds of transplant without ce (higher or for no ce, table b ) increased sharply with younger age and shorter dialysis duration. increased likelihood of transplant without ce also correlated with black race, female sex, and certain geographic regions. post-transplant ami rates in patients transplanted without ce allow assessment of whether ce was appropriately deferred in those who indeed face low ihd-risk after transplant. among patients transplanted without ce, the -yr incidence of post-transplant ami was % and % in lower and high-clinical risk groups, respectively, but varied by clinical traits within these groups. in lower-risk patients transplanted without ce, blacks patients faced increased ami-risk compared to whites (adjusted hr . , % ci . - . ). conclusions: observed ce practices demonstrate a low yield of pre-transplant revascularization but also raise concern for socio-demographic barriers to evaluation access. women who become pregnant in the first two years after kidney transplantation have a higher risk of graft loss. nadia zalunardo, olwyn johnston, caren l. rose, john s. gill. division of nephrology, university of british columbia, vancouver, bc, canada. existing information regarding the risks of pregnancy is derived from voluntary data sources. using medicare claims files from the usrds ( usrds ( - , we examined pregnancies (defined by presence of an inpatient icd- billing code for pregnancy or pregnancy-related complication) in the first years after kidney transplantation (ktx) among women aged - years whose primary insurance payor was medicare (n= , ) . patients were followed until graft failure, death or december , . there were pregnancies identified in women during the first post-transplant years. women who became pregnant during the st or nd post-transplant year had a shorter time to graft loss than women who became pregnant during the rd year (figure). to minimize the survivor bias among women who became pregnant during the rd posttransplant year, we determined the association between the timing of pregnancy and graft survival among the subset of women (n= ) who had graft survival of at least two years using a cox multivariate regression adjusted for age, race, cause of esrd, donor source, calendar year of transplant, dialysis vintage, maintenance immunosuppression, and gfr at months after ktx. pregnancy in the st year (hr . , % ci . - . ), and second year (hr . , % ci . to . ) was associated with an increased risk of graft loss compared to pregnancy during the third post-transplant year. we concluded that women who are able to wait should be counseled to become pregnant after the second post transplant year. the aging donor and recipient populations have led to new challenges in simultaneous kidney-pancreas transplantation (skpt). the purpose of this study was to retrospectively review our single center experience in skpt with respect to extended (ex) donor (d) and recipient (r) criteria. methods: over a month period, we performed skpts with enteric drainage ( portal venous drainage). ex ds were defined as age < (n= ), > (n= , mean age . yrs), or donation after cardiac death (dcd, n= ). all dcd donors were managed with extracorporeal support. ex rs were defined as age > (n= , mean age . yrs) or those with a pretransplant serum c-peptide level > . ng/ml (n= , mean . ng/ml). all rs received depleting antibody induction ( ratg, alemtuzumab) with tacrolimus, mmf, and tapered steroids ( steroid-free). results: a total of ds ( %) and rs ( %) met the above ex criteria. median waiting time was months, mean pancreas preservation was hours, and median length of stay was days. with a mean follow-up (f/u) of months, patient (pt) ( % ex d vs % non-ex d), kidney ( % ex d vs % non-ex d) and pancreas graft survival ( % ex d vs % non-ex d) rates were similar between d groups (all p=ns). the incidences of delayed kidney graft function ( % in each group) and early pancreas graft loss due to thrombosis ( % ex d vs % non-ex d) were also comparable between d groups. with regard to r groups, pt ( % vs %, p= . ) and kidney ( % vs %, p=ns) graft survival (gs) rates were slightly lower in the ex r group compared to the non-ex r group, respectively. however, death-censored kidney gs rates ( % ex r vs % non-ex r) were comparable between groups. uncensored pancreas gs rates ( % ex r vs % non-ex r) were similar. the incidences of acute rejection, surgical complications, infection, and other morbidity were comparable regardless of d or r group. at year (or latest) follow-up, renal and pancreas functional parameters were similar between d groups. however, the ex r group demonstrated slightly compromised renal and pancreas allograft function and a greater need for oral hypoglycemic agents. conclusion: intermediate-term outcomes in skpt from selected ex ds or rs have comparable outcomes, although ex r criteria may represent a risk factor for pt survival and functional outcomes. conclusion: spk recipients with functioning pancreas grafts have significantly improved kidney and patient survival compared to ld ka and dd ka. however, early pancreas graft failure results in kidney and patient survival rates similar to ka recipients. spk provides optimal outcomes for patients with dm , but long-term risk associated with early pancreas loss may be a consideration when selecting spk vs ka transplantation. the impact of long-term metabolic control on renal allograft and patient survival in type diabetes. christian morath, martin zeier, bernd dohler, jan schmidt, peter p. nawroth, gerhard opelz. nephrology, university of heidelberg; transplantation immunology, university of heidelberg; transplantation surgery, university of heidelberg; endocrinology, university of heidelberg. it is a matter of debate whether pancreas allografts independently contribute to renal transplant and patient survival in type diabetics who received a simultaneous pancreas kidney transplant (spk). using the data of the collaborative transplant study (cts), we studied type diabetic recipients of deceased donor kidneys (ddk), living donor kidneys (ldk), or spk performed during two time periods: to and to . we analyzed graft and patient survival rates for a maximum of years. ddk recipients showed inferior graft and patient survival compared to ldk and spk recipients in both time periods. ldk recipients had a superior graft survival rate initially, but the survival rate of kidneys in spk recipients reached the level of ldk toward the end of the follow up period. the results of patient survival paralleled those of kidney graft survival: an early advantage of ldk as compared to spk faded away during follow up. multivariate analysis, in which pretransplant cardiovascular risk assessment was appropriately considered, showed that patient survival of spk recipients was superior to that of ldk recipients beyond the tenth year after transplantation (hazard ratio hr = . , p = . ). this was reflected by a lower cumulative cardiovascular death rate in recipients of spk ( . %) compared to recipients of ddk ( . %) or ldk ( . %) . the early survival benefit of ldk compared to spk is lost during long-term follow up, probably related to improved glycemic control in spk recipients. proposal for a grading rejection schema in pancreas allograft biopsies from a multidisciplinary panel of pathologists, surgeons and nephrologists. .normal .indeterminate septal inflammation that appears active but the overall features do not fulfill the criteria for mild cell mediated acute rejection. active septal inflammation involving septal structures and/or focal acinar inflammation. -grade ii / moderate acute cell mediated rejection multifocal (but not confluent or diffuse) acinar inflammation with spotty acinar cell injury and drop-out and/or minimal intimal arteritis -grade iii / severe acute cell mediated rejection diffuse, (widespread, extensive) acinar inflammation with focal or diffuse multicellular /confluent acinar cell necrosis.and/or moderate or severe intimal arteritis and/or transmural inflammation -necrotizing arteritis chronic active cell-mediated rejection. chronic allograft arteriopathy (arterial intimal fibrosis with mononuclear cell infiltration in fibrosis, formation of neo-intima) .antibody mediated rejection =c d positivity + confirmed donor specific antibodies + graft dysfunction -hyperacute rejection -accelerated antibody mediated rejection severe, fulminant form of antibody mediated rejection with morphological similarities to hyperacute rejection but occurring later (within hours or days of transplantation). -acute antibody mediated rejection specify percentage of biopsy surface with interacinar capillaries positive for c d. .chronic allograft rejection/graft sclerosis stage i (mild graft sclerosis) < % of the core surface stage ii (moderate graft sclerosis) - % of the core surface. stage iii (severe graft sclerosis) > % of the core surface . other histological diagnosis e.g. cmv pancreatitis, ptld, etc. a simple, reproducible, clinically relevant and internationally accepted schema for grading rejection should improve the level of diagnostic accuracy and positively affect patient care. rank test). similar results were obtained when death was included as a cause of graft loss (data not shown). conclusion: par is associated with worse long term kidney graft survival than kar. it is likely that patients that experience par within the first year have also experienced undiagnosed sub-clinical kar. this adds associative data to the argument that the likelihood of concordance between par and kar is high and therefore, the need for increased monitoring of kidney function after par is warranted. the most efficacious immunosuppressive (immuno) regimen for spkt is debated, and information on the best regimen to prevent amr is particularly scarce. we performed a retrospective comparative cohort study that included spkt patients (pts) transplanted in - , who received maintenance immuno with tac, mmf and steroids. two groups were compared: pts who received induction with alemtuzumab (alem) (n= ) and pts who were induced with basiliximab (basilmab) (n= ). donor and recipient characteristics were similar. kt acute cellular rejection (acr) was more frequent with basilmab ( -yr . % vs . %, p= . ), but the incidence of biopsy-proven kt amr was similar ( -yr % with basilmab vs . % with alem, p=ns). no differences in prevalence were detected considering early amr (< th day) or late amr (> th day) separately. multivariate analyses showed that the only significant risk factor for amr was female gender (adjusted hr . , p= . ). the biopsy-proven kt rejection was associated with clinical pt rejection in out of the amr cases ( %), without differences between the groups. post-rejection kt graft survival was similar in both groups ( -yr basilmab/alem . / . %), but deathcensored kt survival was lower with alem ( / . %, p= . ). the predominant cause of kt loss in pts induced with basilmab was death-with-function (n= ), while in pts induced with alem acute or chronic amr was the most common cause of kt attrition (n= ). pancreas survival was similar between both groups. more pt losses due to ar occurred in alem-treated group than in the basilmab group ( vs ) . nearly all early episodes resolved with treatment and were not associated with worse graft survival. conversely, late amr episodes carried a worse prognosis, with decreased -yr kt ( . % vs . %, p< . ) and pt graft survival ( . vs . %, p< . ). late amr was associated with graft loss in multivariate cox models (kidney loss hr . , p= . and pancreas hr= . , p= . ) . amr is common in spkt recipients. acr is better prevented with alem than with basilmab, but no relevant difference is found between alem and basilmab in amr. late (as opposed to early) amr episodes are associated with significant reduction in spkt survival rates despite treatment. preventive and/or different treatment strategies are required to address late amr in spkt. intraoperative fluorescence imaging (ifi) in pancreas transplantation (pt) to determine vascular patency and allograft perfusion. edmund q. sanchez, srinath chinnakotla, marlon f. levy, robert m. goldstein, goran b. klintmalm. baylor regional transplant institute, dallas/ft. worth, tx. thrombotic complications of pt are well known. we report ifi using the spy device to assess immediate vascular patency and allograft perfusion. our controlled experience is presented here. methods: pts were imaged intraoperatively using the spy device under our irb approved study. indocyanine green solution ( . mg/ml) was injected into central venous catheters after completion of the vascular anastomoses of whole pancreaticoduodenal allografts. the pancreas transplants were performed in the retroperitoneal portal and enteric drained technique described by boggi, et al. imaging of the allograft vasculature, perfusion of the pancreas, and perfusion of the duodenal segments was performed and recorded intraoperatively. all video sequences were archived for later review. results: ifi on pancreas transplants ( simultaneous pancreas-kidney and pancreas after kidney) was performed and video sequences were recorded. all pancreas allografts demonstrated intraoperative vascular patency and complete pancreatic and duodenal perfusion. there were no side effects seen. all pancreas transplants had immediate graft function. one patient was re-explored on postop day due to persistent acidosis and hypotension. repeat ifi demonstrated vascular patency and perfusion, despite an ischemic external physical appearance. there were no vascular complications that required reoperation, nor were there any graft thromboses. characteristic slow venous outflow was seen in each case. conclusion: ifi with the spy device is a simple and effective method to determine immediate patency and perfusion of the whole pancreaticoduodenal allograft. its use is beneficial in re-exploration to rule out infarcted pancreas allografts. further development in quantification of vascular flow rates and allograft perfusion indices by this device is abstracts in progress. once these are established, this information will be studied in a controlled fashion, and will be compared with clinical outcomes. we have demonstrated safety and developed a protocol for using the spy device in ifi of pancreas transplants. tolerance/immune deviation i tolerance without immunosuppression: exploiting epigenetic regulation as a new approach to achieving donor-specific allograft tolerance. liqing wang, ran tao, joel a. friedlander, wayne w. hancock. pathology & immunology, children's hospital of philadelphia & upenn, philadelphia, pa. given toxicities of all current immunosuppressive agents, plus complications of malignancy, infection and chronic rejection, maintaining the search for new approaches to tolerance induction is essential. while weaning of immunosuppression is fraught with risks and costimulation blockade has not yielded the expected boon, use of a broad histone deacetylase inhibitor (hdaci), such as trichostatin a (tsa) or suberoylanilide hydroxamic acid (saha), promotes foxp acetylation of foxp and binding to target genes, leading to enhanced treg suppressive function, and wks of therapy with hdaci and low-dose rapamycin can induce allograft tolerance. we now show that in addition to acetylation, modulation of a second major epigenetic mechanism, dna methylation, has salutary effects, such that combined use of an hdaci and a dna methyltransferase inhibitor (dnmti, e.g. -aza- deoxycytidine) has potent effects. microarray analysis of the effects of hdaci on tregs showed down-regulation of multiple genes associated with dna methylation, including dnmt, methyl-cpgbinding domain and associated proteins, leading us to test the effects of dnmti use on treg function. dnmti administration decreased foxp gene methylation, enhanced treg gene expression, and increased treg suppression in vitro, and led to a dose-dependent prolongation of cardiac allograft survival (balb/c->c bl/ ) in vivo (p< . ). the combination of hdaci and lower doses of dnmti, administered for just wks, led to permanent engraftment (> d, p< . ), and the permanent acceptance of second donor allografts but acute rejection of third-party (cba) cardiac allografts indicated induction of donor-specific tolerance post-epigenetic therapy. analysis of long-surviving cardiac allografts showed a minor infiltrate consisting primarily of foxp + tregs and an absence of chronic rejection (transplant arteriosclerosis, myocardial fibrosis), whereas combined epigenetic therapy led to only minor prolongation of allograft survival in treg-depleted recipients. in summary, brief use of clinically approved agents (an hdaci plus an dnmti), can synergistically enhance treg function and induce tregdependent donor-specific allograft tolerance without use of any immunosuppression. we conclude that insights gained through epigenetic targeting may provide completely new approaches to the taming and regulation of otherwise powerful immune responses post-transplantation. a novel epigenetic approach to generate mouse and human cd + cd + foxp + regulatory t cells. girdhari lal, nan zhang, william van der touw, yaozhong ding, jonathan s. bromberg. gene and cell medicine, mount sinai school of medicine, new york city, ny. background: constitutive foxp expression is required for stable and suppressive cd + cd + regulatory t cells (treg) . we previously showed that demethylation of an upstream cpg island of the foxp promoter is characteristic of stable and suppressive treg. using dna methyltransferase inhibitors, we present a novel method to generate antigen-specific treg from mouse and human cd + cd -t cells. methods: naïve cd + cd -t cells and natural treg (ntreg) were purified from wild type or foxp -gfp transgenic mice. human naïve cd + cd -t cells were purified from pbmc. t cells were cultured with irradiated antigen presenting cells in the presence of il- , anti-cd ε mab, tgfβ or the dna methyltransferase inhibitor -aza- '-deoxycytidine (zdcyd), which demethylates the upstream promoter. foxp expression was determined by flow cytometry and qrt-pcr. results: naïve t cells cultured under stimulatory conditions with zdcyd express increased foxp mrna ( fold) and intracellular foxp protein ( % of cells vs % without zdcyd). foxp expression is synergistically enhanced with tgfβ ( ± . % vs . ± . % zdcyd alone or . ± . % tgfβ alone), similar to ntreg ( . ± . %). tgfβ in combination with other dna methyltransferase inhibitors (procainamide, hydralazine, rg ) also induces foxp . zdcyd plus tgfβ induced treg stably express foxp and similar surface markers and cytokine mrna as ntreg. zdcyd plus tgfβ induced treg suppress proliferation of cd + cd -t cells, prevent cd + cd -cd rb hi induced colitis in scid mice, and enhance islet allograft survival. zdcyd plus tgfβ induce foxp expression in antigen-specific wild type or t cell receptor transgenic cd + t cells stimulated with alloantigen or peptides, and in naïve cd + cd -t cells. in vivo administration of zdcyd preferentially preserves thymic cd + cd + foxp + treg generation. zdcyd alone or zdcyd plus tgfb induce strong foxp expression in human cd + cd -t cells compared to tgfβ alone. zdcyd plus tgfβ induced human treg suppress the proliferation of naïve cd + cd -t cells, whereas tgfβ-induced treg do not. conclusion: dna methyltransferase inhibitors induce stable and suppressive foxp + treg from peripheral cd + cd -t cells. these finding have important implications for understanding t cell development and differentiation, and provide a clinically applicable technique for manipulating epigenetic regulation for the generation of treg for tolerance. macrophages driven to a novel state of activation can promote tolerance. katharina kronenberg, seiichiro inoue, james a. hutchinson, beate g. brem-exner, gudrun e. koehl, hans j. schlitt, fred fandrich, edward k. geissler. surgery, university of regensburg, regensburg, germany; surgery, university hospital schleswig holstein, kiel, germany. background: the use of immunomodulatory cells in organ transplantation (tx) is a promising tolerance-induction strategy. here we describe a novel macrophage population capable of enriching t regulatory cells (treg) and promoting tolerance. methods: balb/c bone marrow, spleen and blood mononuclear cells were cultured with m-csf for days, with a hr pulse of ifn-γ on day ; the resultant adherent cells are referred to as ifnγ-induced monocyte-derived cells (ifnγ-mdc) . residual lymphocytes from these cultures were recultured with the adherent ifnγ-mdc for an additional days. ifnγ-mdc were phenotyped by facs and immunomodulation of lymphocytes was determined by cell counting and facs analysis for tregs. mouse heterotopic heart tx was used for in vivo testing. results: ifnγ-mdc highly express cd b/c, f / and pd-l . compared to classically-activated (m ) macrophages, ifnγ-mdc express less cd /cd , but higher cd c. ifnγ-mdc profoundly delete activated, but not resting, lymphocytes (> %), with cell contact (via transwell system) and caspases (via inhibitors) being essential. most interestingly, ifnγ-mdc highly enrich lymphocytes for cd + cd high foxp + treg ( ± %; n= ). the same, or higher, proportions of treg developed when ifnγ-mdc were produced from macs-purified cd b + and cd + cells ( : ) . ifnγ-mdc culture supernatant showed increased il- (elisa) vs. monocyte control cultures ( ± pg/ml vs. < pg/ml, respectively; n= ), suggesting il- as one potential mediator. ifnγ receptor signaling and cd interactions are required for treg enrichment, as confirmed using ifnγ receptor and cd knock-out mice (c bl/ mice). ifnγ-mdc derived from cd b + cells and lymphocytes of ova-transgenic otii mice showed enrichment of treg by -fold with high-affinity ova peptide ( - ) vs. treg levels using a low-affinity ova peptide (ova - ), suggesting ag-specific treg cells can be enriched with ifnγ-mdc. finally, a single post-heart tx (d+ ) i.v. injection of x donor (c h)-derived ifnγ-mdc prolonged allograft survival in balb/c recipients from . ± . d in controls (n= ) to . ± . d (n= ; p= . ). conclusions: ifnγ-mdc are a novel macrophage population capable of deleting activated t cells and highly enriching remaining lymphocytes for tregs. therefore, ifnγ-mdc could be useful in a clinical setting for promoting transplant tolerance. plasmacytoid dc therapeutic potential is independent of ido induction due to elevated dap expression. tina l. sumpter, bridget l. colvin, zhiliang wang, andrew l. mellor, angus w. thomson. , starzl transplantation institute, dept. of surgery, university of pittsburgh, pittsburgh, pa; immunology, university of pittsburgh, pittsburgh, pa; immunotherapy center, medical college of georgia, augusta, ga. optimizing the mechanisms of pdc tolerogenicity may facilitate their use in the development of cell-based tolerance induction strategies. pdc may undermine t cell function via inducible expression of ido, a tryptophan catabolizer that enhances t cell apoptosis. ido is negatively regulated by dap . in some systems, loss of dap correlates with immunostimulatory activation of pdc, while in others, its absence correlates with enhancement of pdc tolerogenicity through induction of ido. in these studies, we characterized the ability of wt and ido-deficient pdc to attenuate murine heart allograft rejection in order to define the contribution of ido and its regulation by dap relative to the immunoregulatory potential of pdc. methods: pdc and control myeloid (m) dc were generated from bm cultures of wt or ido ko c bl/ (b ; h b ) mice. wt mdc, pdc or ido ko pdc were pulsed with alloag (balb/c; h d ), stimulated with cpg, and then injected i.v. into syngeneic (b ) recipients d before heart transplant. donor ag-specific activation of t cells was analyzed by mlr and elisa. dap expression was evaluated by rt-pcr and abrogated with sirna. results: administration of ido ko pdc d before transplant prolonged graft survival beyond that seen with control mdc, but not to the extent seen with wt pdc, suggesting involvement of ido. pdc cultures from ido ko mice exhibited a more mature phenotype than their wt counterparts with reduced expression of co-regulatory molecules (e.g., icosl). although ido ko pdc induced enhanced t cell alloactivation compared to wt pdc, use of the ido inhibitor -mt indicated that wt pdc do not produce functional ido. further analyses showed wt pdc exhibited high levels of dap mrna expression. silencing dap had no effect on the ability of wt pdc to activate allogeneic t cell proliferation, but significantly enhanced ifnγ secretion. addition of -mt to mlr with dap -silenced pdc increased t cell proliferative responses to alloag, verifying ido induction in dap -silenced pdc. conclusions: these data underscore the prophylactic potential of pdc for cell-based therapy that may be independent of ido, due, in part, to elevated dap expression. additionally, our data support a role for dap in both immunostimulation and immunoregulation by pdc. rapamycin preferentially blocks the expansion of potentially tolerogenic plasmacytoid dendritic cells in vivo. heth r. turnquist, angus w. thomson. , starzl transpl inst and dept of surgery; dept of immunol, univ of pittsburgh sch of med, pittsburgh, pa. rapamycin (rapa), a 'tolerance-sparing' immunosuppressant with anti-proliferative properties, inhibits myeloid (m) dendritic cell (dc) differentiation/maturation in vitro. rapa decreases cd c + dcs in the mouse spleen and suppresses the expansion of total cd c + dc following administration of the dc growth factor, fms-like tyrosine kinase ligand (flt l). however, the influence of rapa on plasmacytoid (p) dc,the principal type- ifn producers in the body, known to regulate innate and adaptive immune responses, and reported to promote experimental transplant tolerance, has not been evaluated. methods: dc were propagated from bone marrow for days (d) in flt l, in the absence or presence of a clinically-relevant dose of rapa ( ng/ml). in addition, dcs were mobilized in c bl/ mice by i.p. flt l ( mg/d for d; d - ). untreated and flt l-treated groups also received rapa ( . mg/kg/d i.p.; d - ). on d , dc were isolated by density gradient centrifugation and/or cd c + positive selection. mdc (cd c + cd b + ) and pdc (cd c lo cd b -b + ) were identified by flow cytometry. results: rapa suppressed pdc and mdc generation in vitro; rapa-dc-treated cultures had only % of the pdc and % of the mdc found in control conditions. in normal mice, both mdc ( % of control) and pdc ( . %) numbers were reduced significantly by rapa administration. rapa, when given concurrently with flt l, blunted the typical profound expansion of mdc. specifically, flt l and rapa-treated mice displayed a ± x increase over control (steady state) numbers compared to a ± x increase in mice treated with flt l alone. flt l-induced expansion of pdc was to a much greater extent impacted by rapa, as absolute numbers of pdc increased only . ± . x over control numbers compared to ± x increase in absolute splenic pdc in flt l-treated mice. conclusion: these data identify rapa as a selective suppressor of pdc generation, as described for corticosteroids. this has significant implications, given the use of rapa following organ transplantation, and the suggested importance of secondary lymphoid tissue pdc for promotion of transplant tolerance mediated by treg. due attention to these disparate effects of rapa on regulating cell populations will be necessary to optimize therapeutic regimens for safe promotion of tolerance. the liver is tolerated better than other transplanted organs. this may reflect the inherent tolerogenicity of liver dendritic cells (dc) and interactions with foxp + regulatory t cells (treg). recent studies have shown that cd expression on dc correlates with induction of foxp + treg, and can be enhanced in the presence of transforming growth factor-β (tgf-β) and retinoic acid (ra), both of which are produced in the liver. this study evaluates cd expression on liver plasmacytoid (p) and myeloid (m) dc and the potential of liver dc subsets to induce tregs. methods: pdc and mdc were magnetically isolated from livers and spleens of c bl/ mice and co-cultured with cfse-labeled allogeneic (balb/c) cd + cd -t cells. after or d of co-culture, t cells were assessed for cfse dilution and intracellular foxp expression. rhtgf-β and either all trans or cis-ra were added to co-cultures. cd expression was evaluated by flow cytometry. results: cd expression was elevated on liver pdc and mdc compared to spleen pdc or mdc, with higher expression on liver mdc. pdc from the liver and the spleen were poor inducers of foxp in cd + cd -t cells compared to mdc. foxp induction was enhanced with tgf-β when splenic pdc were used as stimulators. however, when liver pdc were used as stimulators, tgf-β had no effect on foxp induction. ra (either all trans or cis) enhanced induction of foxp + cells in cd + cd -t cells in the presence of tgf-β when splenic pdc but not liver pdc were used as stimulators. tgf-β and tgf-β with ra also enhanced foxp induction in cd + cd -t cells when either spleen or liver mdc were used as stimulators, though splenic mdc were superior inducers of foxp . conclusions: many studies have focused on naturally-occurring foxp + tregs in systemic tolerance. our data show that liver mdc and pdc are poor inducers of foxp in t cells, even in the presence of tgf-β and ra. these data suggest that the inherent tolerogenicity of liver dc subsets may be independent of treg induction or that liver dc interact with treg through alternative mechanisms. background: t cell-mediated immune rejection occurs in organ transplantation. in addition to mhc-tcr signaling, t cell activation requires costimulation from antigen presenting cells. b molecules (cd /cd ) and cd are critical costimulatory molecules in t cell activation. insufficient or lack of costimulation results in inactivation or tolerance. we hypothesized that blocking the costimulation pathway using small interfering rna (sirna) expression vector can prolong allogeneic heart graft survival. method: vectors that express hairpin sirnas specifically targeting cd and cd were prepared. recipients (balb/c mice) were treated with cd and/or cd sirna vectors, and days prior to heart transplantation. control groups were injected with a blank vector and sham treatment (pbs). after sirna treatment, a fully mhcmismatched (balb/c to c /bl ) heart transplantation was performed. result: allogeneic heart graft survival (> days) was approximately % in the mice treated simultaneously with cd and cd sirna vectors. in contrast, allogenic hearts transplanted into recipients treated with blank vector and pbs stopped beating within days. hearts transplanted into cd or cd sirna vector-treated recipients had an increased graft survival time compared to negative control groups, but did not survive longer than days. real time pcr and flow cytometric analysis showed an upregulation of foxp expression in spleen lymphocytes and a concurrent downregulation of cd and cd expression in splenic dendritic cells of sirnatreated mice. an mlr, using splenic dendritic cells (dcs) isolated from tolerant recipients, showed a significantly lower t cell proliferation capacity in cd -and cd -sirna vector-treated mice, compared to control groups. tolerant dcs from cd -and cd -treated recipients promoted cd +cd +foxp + regulatory t cell differentiation. finally, tissue histopathology demonstrated an overall reduction in lymphocyte interstitium infiltration, vascular obstruction, and edema in mice treated with cd and cd sirna vectors. conclusion: this study demonstrates that the simultaneous silencing of cd and cd genes has synergistic effects in preventing allograft rejection, and may therefore have therapeutic potential in clinical transplantation. costimulation blockade (cob) of the cd /b pathway has long been suggested as a means of attaining indefinite, well-tolerated, antigen-specific prophylaxis from allograft rejection. although effective in rodent models, cd /b blockade is not alone sufficient to prevent rejection in more robust primate models. the relative presence of allocrossreactive memory t cells is one mechanism of cob resistant rejection. lfa -ig is an approved agent for the treatment of psoriasis, shown clinically to deplete tem cells in psoriatic lesions. we hypothesize that lfa -ig specifically targets cob resistance cells, including heterologous alloreactive t cell memory, while avoiding interference of peripheral mechanisms that foster cob-mediated allograft acceptance. rhesus monkeys underwent mhc mismatched renal allotransplantation. ctla -ig was given mg/ kg iv on days - , , , , , , and mg/kg iv on days , , , . lfa -ig, ( . mg/kg iv) was given on day - , , , then weekly ( wks). sirolimus was given orally ( mg/kg) days - and donor specific transfusion ( ml/kg iv) on day - . animals were followed by polychromatic flow-cytometry to quantify t cell subsets. lfa -ig synergized with ctla -ig successfully preventing renal allograft rejection in this model and leading to indefinite survival in / animals (table) . this enhanced survival was associated with a significant reduction in tem cells in animals treated with lfa -ig compared to animals without lfa -ig treatment (figure). lfa -ig withdrawal led to tem re-population and rejection. this therapy regimen, all clinically available agents, promotes cob-mediated graft acceptance without the use of calcineurin inhibitors, steroids, or gross t cell depletion. the aim of this study was to investigate the immunological pathways that regulate t cell dependant allograft responses in the hope of finding novel immunomodulatory compounds. islet (and skin) allografts were transplanted into full mhc-mismatched wild type (wt) and baff-transgenic (baff-tg) recipient mice. allograft function/ rejection was monitored by blood glucose levels and/or confirmed by nephrectomy. histology, splenocyte populations & t cell functions were analyzed. the b cell activation factor from the tnf family (baff) is critical for b cell survival. t cells express baff receptors suggesting that baff may also play a role in t cell responses. contrary to expectations, we found that baff-tg mice accepted a full mhc-mismatched islet allograft for > days. in addition, baff-tg mice also showed delayed skin graft rejection. this was due to a t cell intrinsic change in allo-responsiveness as shown by a failure of purified baff-tg t cells to reject an allograft in adoptive transfer experiments. however, baff-tg t cells were not anergic per se as they proliferated normally to mitogens in vitro and to antigenic challenge in vivo. intriguingly, baff-tg mice harbored an increased frequency of t regulatory cells in the periphery as compared to wt mice. elimination of cd + t cells restored normal allograft rejection to baff-tg mice, demonstrating that the increased number of treg cells were responsible for their altered allo-immunity. in a second approach, baff-tg t cells depleted of cd + cd + t cells were adoptively transferred to transplanted rag recipients, and in this case the baff-tg cd + cd -t cells rejected their allograft. proliferation assays showed that excessive baff was not promoting treg expansion by driving proliferation in the periphery. adoptive transfer of gfp expressing syngeneic splenocytes did not exhibit enhanced survival in the presence of excessive baff. however, analysis of treg cells in the thymus of baff-tg mice showed an increased intrathymic frequency. together these results demonstrate that baff-tg mice harbor an expanded number of treg cells that prevent th -type immune responses including allograft rejection. furthermore, we demonstrate that manipulating baff levels may provide a means to generate immunosuppression free dominant allograft tolerance. we have previously reported the outcome of pig-to-baboon thymokidney transplants from galt-ko miniature swine using an immunosuppressive regimen designed to facilitate the induction of tolerance. although the results were superior to results using hdaf thymokidneys, there was a high rate of early post-operative complications. we investigated whether the elimination of steroids and whole body irritation (wbi) from the treatment protocol would decrease the complication rate in the perioperative period. methods: baboons received thymokidney transplants from galt-ko miniature swine. the immunosuppressive regimen was based on the previously published protocol, but eliminated steroids and substituted rituximab for wbi. it consisted of thymectomy day - , splenectomy day , atg, rituximab, mmf and anti-cd mab. renal function was assessed by serum creatinine levels. evidence for baboon thymopoiesis in the pig thymus was assessed by facs and immunohistochemistry. results: one animal died due to a drug reaction at pod with normal renal function and no evidence of infection. the remaining baboons showed no signs of rejection although igm deposition was observed, likely due to preformed non-gal nab. there were no deaths due to rejection. although early infectious complications were not seen, late complications leading to mortality were still observed, including systemic cmv infection (pod ), pleural effusions (pod , ) , ards with pulmonary hemorrhage (pod , ) and acute myocardial infarction (pod ). two animals showed evidence for early thymopoiesis in the pig thymus by facs and immunohistochemistry. cd +/cd + thymocytes were seen in the thymic grafts, indicating t cell development was supported by the pig thymus. the average survival of the steroid-free group was days including the animal with the drug reaction and days excluding it, compared to . days in the regimen that included steroids/wbi. conclusions: a steroid-free and wbi free regimen designed for the induction of tolerance across the pig-to-baboon xenogeneic barrier had fewer early post-operative complications than previous regimens. greater than -day average survival of recipients of life-supporting xenogeneic thymokidneys was observed without rejection. this regimen also appears to permit baboon thymopoiesis in the pig thymus. purpose: the effects of human decay accelerating factor (hdaf) addition to the galactosyl transferase knock-out (galt-ko) background in transgenic pig kidney xenotransplantation in baboons has not been previously studied. methods: baboon recipients of galt-ko (n= ) or galt-ko/hdaf (n= ) pig kidneys received flolan, heparin and/or aspirin. steroids (s), cobra venom factor (v), atg (a), mmf (m), and/or a low-dose cd blockade (c) regimen were given. results: pre-transplant (tx) anti-non-gal antibody (ab) titers were inconsistently associated with early galt-ko kidney xenograft failure. high d-dimer levels hours post-tx were closely associated with early galt-ko graft loss but not when assessing levels of c a, btg, or increased cd p expression on circulating platelets. regimens lacking mmf and/or cd blockade elicited by day - large anti-donor non-gal ab responses . post-operative creatinine levels for the galt-ko/hdaf recipients were lower along with superior graft survival compared to galt-ko; d-dimer, anti-donor non-gal ab, c a, btg, and f + measurements are in progress for the galt-ko/hdaf recipients. galt-ko/hdaf v,a,m,s,c > * tbp . ; . * graft survival at time of abstract submission as graft still functioning at time of abstract submission; tbp --to be processed ! --anuric; non-life supporting, but appeared viable, pink, and well-perfused until day conclusion: early failure of vascularized galt-ko organs is closely associated with activation of coagulation pathways; whether this is triggered primarily by anti-non-gal ab or by other mechanisms is not addressed by our study. preliminary results using galt-ko/hdaf pig kidneys show promise in attenuating or possibly preventing these and/or other such mechanisms. the established efficacy of cd -based costimulation blockade with mmf to prevent induced anti-non-gal antibody elaboration is confirmed by our preliminary findings. recovery of cardiac function after pig-to-primate orthotopic heart transplant. christopher g. a. mcgregor, william r. davies, keiji oi, henry d. tazelaar, randall c. walker, krishnaswamy chandrasekaran, guerard w. byrne. mayo clinic, rochester, mn. xenotransplantation has been proposed as a method to alleviate the shortage of donor organs. we report survival of up to weeks of three orthotopic transplants after pig-tobaboon cardiac xenotransplantation. pig-to-baboon orthotopic transplantation was performed using an adult baboon recipient and cd transgenic pig. the recipients were treated with an α-gal polymer to block the effects of anti-gal antibody and with atg induction and a tacrolimus and sirolimus based maintenance immunosuppression. heart function was continuously monitored by intramyocardial electrocardiography and every - days by serial echocardiography. standard hematological, serum chemistry and cardiac troponin levels were monitored every - days. the animals survived , , and days in a healthy condition. mortality was a result of pneumonitis, respiratory failure and bowel infarction respectively. one recipient (survival days) showed impaired perioperative left ventricular function with an ejection fraction of % on echo. over the next two weeks, the ejection fraction in this animal returned to normal ( %) and troponin levels normalized. this study shows the longest survival of orthotopic cardiac xenografts to date with recovery of impaired heart function after early ischemia reperfusion injury. this significant improvement in ventricular function suggests that the normal reparative processes appear to function across the xenotransplantation barrier, supporting the potential clinical potential of cardiac xenotransplantation. purpose: to determine whether the galactosyl transferase gene knock-out (galt-ko) protects pig lungs from hyperacute lung rejection (halr) by human blood. the effects of adding human decay accelerating factor (hdaf) to the galt-ko background will also be examined for the first time. methods: heparinized fresh human blood was used to perfuse galt-ko pig lungs (n= ). lung function was assessed by flow, pulmonary vascular resistance (pvr), oxygen transfer, and tracheal edema using pre-defined survival endpoints. historical wild-type (wt) pig lungs (n= ) provide context for analysis. additionally, two pig lungs expressing galt-ko/hdaf were recently examined, one of which received treatment with xigris. results: median lung survival in galt-ko lungs was minutes (range min to > min) vs. minutes in wt lungs (range min to min, p= . ); / galt-ko/hdaf pig lungs survived > min. pvr at minutes for galt-ko lungs was ± mmhg-min/l vs.wt lungs ( ± mmhg-min/l at minutes) vs. mmhgmin/l for each galt-ko/hdaf lung. complement activation (∆c a) at minutes was significantly lower with galt-ko (∆c a ± ng/ml) than wt lungs (vs. ± ng/ml, p= . ). galt-ko was also associated with reduced platelet activation: only ± % of circulating platelets express cd p at min. vs. ± % in the wt group (p= . ); ∆βtg at min [ ± iu/ml (galt-ko) vs. ± iu/ ml (wt)], and less thrombin formation (∆ f + ) at minutes (galt-ko: ± . nm vs. wt: ± nm). ∆c a, % of circulating platelets expressing cd p, ∆βtg, and ∆ f + measurements are in progress for galt-ko/hdaf. platelet sequestration was delayed as mean % of the initial platelets remaining in the galt-ko lung perfusate was ± % at min vs. ± % in the wt lung group (p= . ). neutrophil sequestration was also diminished in galt-ko ( ± % residual) vs. wt lung ( ± % residual, p= . ). conclusion: galt-ko pig lungs are significantly protected from several facets of halr: complement and coagulation cascade activation, neutrophil sequestration, and platelet activation are all partially attenuated by this modification alone. preliminary results suggest that galt-ko/hdaf lungs offer even further protection. disseminated intravascular coagulation is associated with tissue factor expression on recipient platelets and monocytes. chih che lin, , , mohamed ezzelarab, corin torres, david ayares, anthony dorling, david k. c. cooper. thomas e. starzl transplantation institute, university of pittsburgh, pittsburgh, pa; revivicor inc., blacksburg, va; department of immunology, imperial college london, hammersmith hospital, london, united kingdom; department of surgery, chang gung memorial hospital, kaohsiung, taiwan. purpose acute humoral xenograft rejection (ahxr), frequently associated with disseminated intravascular coagulation (dic), remains a challenge in pig-to-primate xenotransplantation (tx). a previous in vitro study showed that recipient platelets and monocytes were induced to express tissue factor (tf) after incubation with porcine endothelial cells, and we speculated this may contribute to thrombosis. the present study investigated whether circulating (extragraft) monocytes and platelets express tf and whether this relates to the development of dic after pig tx. methods baboons (n= ) received an aortic patch or kidney from either a wild-type (wt) or α , galactosyltransferase gene-knockout (gtko) pig, with or without immunosuppressive therapy. baboon monocytes and platelets were isolated from blood before and after tx. surface tf phenotype was determined by flow cytometry. functional tf activity was determined by clotting time assays after mixing monocytes and platelets with recalcified factor vii (fvii)-deficient plasma with or without added fvii. before tx, monocytes and platelets did not express tf or display tf-dependent procoagulant activity. after gtko aortic patch tx, the baboons were euthanized by day without developing dic. however, by day , circulating platelets (but not monocytes) expressed tf and promoted tf-dependent clotting in recalcified plasma. in the absence of immunosuppression, wt kidneys survived < day before the onset of dic, at which time tf activity was detected on both platelets and monocytes. after gtko kidney tx in immunosuppressed baboons, platelets expressed tf as early as day . in contrast, monocytes began to express tf only at the onset of dic. this study links expression of tf on recipient monocytes and platelets with the development of dic. expression on platelets appeared to predict the subsequent development of dic, whereas that on monocytes was associated with the onset of dic. whilst our observations do not establish a causal relationship, they provide the basis for further study. international human xenotransplantation inventory. antonino sgroi, leo buhler, megan sykes, luc noel. surgical research unit, department of surgery, geneva university hospital, geneva, switzerland; transplantation biology research center, massachusetts general hospital, boston, ma; world health organization, geneva, switzerland. background: xenotransplantation carries inherent risks of infectious disease transmission to the recipient and even to society at large, and should only be carried out with tight regulation and oversight. a collaboration between the international xenotransplantation association, the university hospital geneva and the world health organization has established an international inventory (www.humanxenotransplant. org) aiming to collect basic data on all types of xenotransplantation practices on humans that are currently ongoing or have been recently performed. methods: we collected information using publications in scientific journals, presentations at international congresses, internet-based information, and declarations of ixa members. an electronic questionnaire is available on the website www. humanxenotransplant.org, which can be filled out and sent to the office in geneva. results: we identified a total of recent or current human applications of xenotransplantation, eight were currently ongoing and one will start soon. the source animal was: pig (n= ), sheep (n= ), calf (n= ), rabbit (n= ), blue shark (n= ), hamster (n= ), and unknown (n= ). all trials transplanted xenogeneic cells, i.e. islets of langerhans (n= ), hepatocytes (n= ), kidney cells (n= ), chromaffin cells (n= ), embryonic stem cells (n= ), fetal (n= ) and adult cells (n= ) of various organs. the treatments were performed in different countries, in europe, in russia, in asia, mexico, in usa and in africa. six countries had no national regulation on xenotransplantation. conclusion: several clinical applications of cell xenotransplantation are ongoing around the world, often without any clear governmental regulation. this information should be used to inform national health authorities, health care staff and the public, with the objective of encouraging good practices, with internationally harmonized guidelines and regulation of xenotransplantation. cells from α , -galactosyltransferase gene-knockout pigs additionally transgenic for human membrane cofactor protein demonstrate resistance to human complement-mediated cytotoxicity. hidetaka hara, cassandra long, mohamed ezzelarab, peter yeh, carol phelps, david ayares, david k. c. cooper. surgery, thomas e. starzl transplantation institute, university of pittsburgh, pittsburgh, pa; revivicor, inc., blacksburg, va. purpose: ( ) to compare the antibody binding and complement-mediated cytotoxicity (cdc) of human sera to pig peripheral blood mononuclear cells (pbmc) and porcine aortic endothelial cells (paec) from (i) wild-type (wt), (ii) α , -galactosyltransferase gene-knockout (gtko), (iii) human membrane cofactor protein transgenic (mcp) and (iv) gtko/mcp pigs. ( ) to investigate the effect on binding and cdc of human sera following activation of paec. methods: pooled human serum was tested by flow cytometry for binding of igm and igg ( % serum concentration) and cdc ( % serum concentration) to pbmc and paec from wt, gtko, mcp, and gtko/mcp pigs. paec from all pig types were activated by ifn-γ, and again tested for antibody binding and cdc. results: there was higher binding of igm and igg to wt and mcp than to gtko and gtko/mcp pbmc and paec, but there were no differences in binding (i) between wt and mcp cells or (ii) between gtko and gtko/mcp cells. cdc of wt pbmc and paec was significantly greater than of gtko, mcp, and gtko/mcp pbmc (wt %; gtko %; mcp %; gtko/mcp %) and paec (wt %; gtko %; mcp %; gtko/mcp %) (both p< . ). importantly, there was no lysis of gtko/ mcp paec. after activation of paec, although cdc was increased against wt and gtko paec, mcp and gtko/mcp paec demonstrated significant resistance to lysis (wt %; gtko %; mcp %; gtko/mcp %). conclusions: ( ) cdc of all types of genetically-engineered pbmc and paec was significantly lower than of wt pbmc and paec, with lysis of gtko/mcp paec being significantly lower than to gtko or mcp cells. ( ) paec from both mcp and gtko/mcp showed resistance to lysis even after activation of the cells. ( ) organs from gtko/mcp pigs should provide considerable protection against cdc. role for cd -sirpα signaling in human t cell proliferation in response to stimulation with porcine antigen presenting cells. hiroyuki tahara, hideki ohdan, kentaro ide, toshimasa asahara. department of surgery, hiroshima university, hiroshima, japan. we have previously proven that genetic induction of human cd on porcine cells provides inhibitory signaling to signal regulatory protein(sirp) α on human macrophages; this provides a novel approach to preventing macrophage-mediated xenograft rejection. a recent report indicated that the similar cd -sirp system negatively regulates the functions of both t cells and antigen presenting cells(apcs) in humans. we hypothesize that the interspecies incompatibility of cd may also act as an additional barrier of t cell-mediated xenograft rejection. we have analyzed the frequency and proliferative activity of human t cells responding to either porcine or allogenic apcs by using in vitro mixed lymphocyte reaction (mlr) assay with a cfse-labeling technique. irradiated stimulator porcine or human peripheral blood mononuclear cells(pbmcs) were cultured with cfse-labeled responder human pbmcs. by fcm analysis, the number of division precursors was extrapolated from the number of daughter cells of each division and from the mitotic events, and precursor frequencies in cd + and cd + t cell subsets. using these values, stimulation index(si) was calculated. the frequencies of alloreactive and xenoreactive cd + t cell precursors were almost identical, . ± . % and . ± . %, respectively (n= - , for each type of precursor). however, the si of xenoreactive cd + t cells was significantly higher than that of alloreactive cd + t cells, indicating a stronger reaction by a single xenoreactive cd + t cell. in the presence of human cd -fc(containing the extracellular domain of human cd fused to the fc portion of human ig, µg/ml), the si of xenoreactive cd + t cells was significantly reduced to a level similar to that of alloreactive cd + t cells (n= , p< . ), although the frequencies of their precursors were absolutely uninfluenced. the frequencies of alloreactive and xenoreactive cd + t cell precursors were also identical, i.e. . ± . %, . ± . %, respectively (n= - ). the si of both alloreactive and xenoreactive cd + t cells did not differ: however, that of xenoreactive cd + t cells was significantly suppressed in the presence of human cd -fc (n= , p< . ). these results suggest that the t cell responses to porcine cells are stronger than those to allogeneic cells because of the interspecies incompatibility of cd . moreover, genetic manipulation of porcine apcs to induce human cd expression might attenuate human t cell-mediated xenograft rejection. reactivation of human cytomegalovirus (hcmv) is a potential risk following the clinical application of pig-to-human xenotransplantation. since little is known about undesirable side-effects arising from hcmv infection of porcine organs, we investigated the capabilities of various hcmv strains to infect porcine endothelial cells (pec) and putative immunological consequences thereof. pec from different anatomical origins were incubated with the hcmv laboratory strains ad and tb /e or a clinical isolate at a multiplicity of infection (moi) ranging from . to . viral replication kinetics, evolution of cytopathology, and lytic end points were analyzed. consequences of pec infection on human nk cell activation were evaluated using xenostimulation assay assessing nk cell ifng secretion and cytotoxicity. infection was evident and a maximum percentage of infected cells was reached at a moi of . hcmv replicated in all tested pec types, with a fraction of infected cells ranging from % to %. ad infection of a (microvascular ec) resulted in cytopathic effect (cpe) development by dpi and in lysis of % of the cells at dpi. contrary, no cpe was observed in ped (aortic ec) up to dpi. infection of a with tb /e was non-lytic and resulted in accumulation of both extra-and intracellular virus. virus titers reached a maximum at dpi, peaking at levels -fold higher than residual input virus. we then determined whether infected pec supported a complete replication cycle and produced viral progeny. after dpi pec supernatants and lysates revealed the production of significant amounts of virus. preliminary results showed that coculture of human nk cells and infected pec resulted in increased nk killing and ifng production. altogether, these findings provide evidence that pec are permissive and support the complete productive replication cycle of hcmv. however, cpe are cell-type and strain dependent. moreover, pec infection leads to modification of the xenogeneic cytotoxicity mediated by human nk cells. our findings allow a better estimation of the potential role of hcmv cross-species infection following xenotransplantation and may be crucial to guide future clinical trials. chronic rejection/injury: innate and adaptive immunity i testing for donor-specificity of antibodies post-transplantation increases the predictability of chronic rejection. mikki ozawa, arundhati panigrahi, paul i. terasaki, narindar mehra. one lambda, inc., canoga park, ca; new delhi, india; terasaki foundation laboratory, los angeles, ca. purpose: post-transplant hla antibodies have been shown to be linked to chronic graft failure, yet some recipients do well in the presence of antibodies. we investigated whether testing for donor-specificity of antibodies improves the predictive value of antibodies in regards to chronic rejection. methods: in a prospective study of renal transplant recipients, post-transplant sera were tested for hla and mica antibodies, and positive sera were tested by single antigen beads to determine whether the antibodies were donor-specific. one year after testing, biopsy and clinical data on the graft status were collected. patients who did not have follow-up or died with function were excluded from analysis. results: of the patients with follow-up and abdr typing info, ( %) were found to have antibodies. sixteen of those had donor-specific a, b, or dr antibodies (dsa), while had non-donor specific ab's (ndsa). sixty patients had only dp, dq, cw, or mica antibodies, and were grouped into "donor-specificity unknown", as these typing were not available at the time of this report. table summarizes the antibody groups and transplant outcome one year later. interestingly, the mean serum creatinine values at the time of antibody testing were very similar among the groups, regardless of the presence of antibody or the type (ranged . to . mg/dl). one year later, however, a striking % of dsa patients had either returned to hemodialysis, were regrafted, had died, or had biopsy-proven chronic rejection, compared to only % in ndsa patients and % in those who were negative (dsa vs. neg, p= . ). mica antibodies also showed significant association with graft failure or can (p= . ), and donor mica typing is underway. conclusion: in this prospective study of renal patients, dsa detected posttransplantation were highly correlated with chronic rejection. inclusion of specificity analysis in post-transplant antibody monitoring would significantly improve the predictability of chronic rejection. donor cd t cells within solid organ transplants contribute to graft rejection. thet su win, sylvia rehakova, margaret negus, kourosh saeb-parsy, martin goddard, eleanor bolton, andrew bradley, gavin pettigrew. university of cambridge; papworth hospital, united kingdom. this study examines the contribution of donor cd t cells within heart allografts to the development of graft vasculopathy, by providing help to recipient b cells for generating effector autoantibody responses. b mice were transplanted with mhc class ii-disparate bm hearts. the allo-and auto-antibody responses were quantified, and their contribution to allograft vasculopathy assessed by incorporating b cell deficient mice as recipients. the role of donor cd t cells in providing help for autoantibody was examined by removing cd t cells from donor hearts before transplantation by three approaches: treating with anti-cd mab, administering gy lethal irradiation, and using bm donors that are genetically deficient in t cells. the contribution of donor cd t cells to autoanitbody development was further assessed by challenge with bm cd t cells two weeks prior to transplantation. bm heart grafts developed progressive vasculopathy and were rejected slowly (mst= days, n= ). the contribution of antibody-mediated effector mechanisms was confirmed by histopathological evidence (fibrinoid necrosis and vascular proliferation), by complement c d staining, and by a reduction in the severity of vasculopathy and long-term graft survival in b cell deficient recipients. surprisingly, no alloantibody was detected, but recipients instead developed autoantibody. the autoantibody response was completely dependent upon the provision of help from donor cd t cells; it was abrogated by transplanting cd t cell deficient hearts. to further confirm an effector role for autoantibody in vasculopathy development, b mice were primed for humoral autoimmunity, but not for alloimmunity, by injecting with highly purified bm cd t cells prior to transplantation. heart grafts were rejected more rapidly (mst= days, n= , p< . ), and demonstrated severe vasculopathy. finally, bm-chimeric recipients that lacked mhc class ii expression only on b cells did not develop autoantibody, suggesting that cognate interaction between the donor cd t cells and mhc class ii of recipient b cells is crucial for post-transplant humoral autoimmunity. our results demonstrate the novel finding that help for autoantibody production is provided by graft-versus-host cognate recognition of recipient b cell mhc class ii by donor cd t cells. this autoantibody contributes to the development of vasculopathy independently of alloantibody. allograft rejection. gang chen, huiling wu, jainlin yin, josette m. eris, steven j. chadban. collaborative transplantation research group/renal medicine, university of sydney/rpah, sydney, nsw, australia. toll like receptors (tlrs) are innate immune receptors that play an important role in innate immunity but also provide a link between innate and adaptive immunity. myd is a key tlr signal adaptor. a recent clinical study reported that activation of innate immunity in heart-transplantation recipients through tlr contributes to the development of chronic rejection after cardiac transplantation. in this study, we aimed to determine whether tlr -myd signaling is required for the development of chronic allograft damage using tlr -/and myd -/mice in a murine model of chronic cardiac allograft rejection. methods: cardiac transplants were performed: b .c-h- bm (b .h- bm ) hearts to wt, myd -/and tlr -/mice (all c bl/ -h- b -single mhc class ii mismatch) as allografts (n = - /group) and c bl/ to c bl/ isografts (n = ). blood and tissue were harvested at day after transplantation. cell infiltration, fibrosis and vasculopathy were assessed histologically (grade - ). cd + foxp + cells in the blood and spleen were measured by flow cytometry analysis. results: all hearts remained pulsatile until sacrifice. histology of tlr -/and myd -/recipients showed protection from leukocyte infiltration ( . ± . & . ± . vs . ± . , p < . ), fibrosis ( . ± . & . ± . vs . ± . , p < . ) and vasculopathy ( . ± . & . ± . vs . ± . ) at day post transplantation compared to wt recipients. by facs, the ratio of cd + foxp + regulatory t (treg) cells to total cd + cells at day post transplantation in spleen was significantly increased in tlr -/and myd -/recipients versus wt allograft and isograft mice ( . ± . & ± . % vs . ± . & . ± . %, p < . ). conclusion: absence of tlr and myd signaling reduces chronic allograft damage in a murine model of chronic cardiac rejection. one mechanism of protection may be enhancement of regulatory t cell function. promotion of treg generation via the tlr / myd pathway may be one important consequence of cross-talk between innate and adaptive immunity. pathway: implication in transplant arteriosclerosis. thibaut quillard, stéphanie coupel, flora coulon, juliette fitau, maria-cristina cuturi, elise chiffoleau, béatrice charreau. inserm u , itert, nantes, france. endothelial cell (ec) activation, injury and apoptosis are the key events associated with transplant arteriosclerosis (ta) and chronic allograft rejection (cr). notch is a major signalling pathway controlling vascular function and injury. nonetheless, the involvement of notch, at endothelial level, in both ta initiation and progression remains unknown. using a fully mhc mismatched rat cardiac allograft model of cr, we found that ta at day correlates with a strong decrease in both expression and activity of the notch pathway in transplant as compared to tolerant and syngeneic controls. the present study investigates the contribution of notch activity to ec survival. in this purpose, a recombinant adenovirus, encoding the notch intracellular domain (nicd) and the reporter gfp cdnas was constructed and used to maintain notch activity in cultured primary human arterial ec. our results demonstrate that nicd protects ec from cell death induced by tnfα in the presence of chx or by anoïkis as measured by annexinv and dna content staining. nicd mediates cytoprotection through the regulation of key-apoptosis molecules. using an apoptosis-dedicated qpcr array, we found that out of a panel of apoptosis-related genes were significantly regulated. nicd upregulated bcl , bcl a and bcl-xl ( . -, . -and . -fold compared to noninfected cells, respectively). in addition, pro-apoptotic genes bim, drp , bok and cd were markedly downregulated in transduced cells ( . -, . -, . -and . -fold decrease, respectively). western blotting analysis confirmed the induction of protective molecules (bcl and bcl-xl) and the inhibition of pro-apoptotic proteins (bad, bak and bax). consistent with these data, nicd conducted phosphorylation of akt as well as a reduction of pten expression, suggesting that the cytoprotective activity of nicd may be mediated by recruitment of the pi k survival pathway. to conclude, our findings indicate that ta correlates with a decreased notch signaling in transplant and that activation of the notch pathway in vascular ec prevents apoptosis by promoting protective gene expression and survival pathway. these data suggest that controlling notch activity may prevent ec dysfunction associated with ta and cr. donor age intensifies the early immune response. christian denecke, xupeng ge, irene kim, daman bedi, anne weiland, anke jurisch, steven mcguire, johann pratschke, stefan g. tullius. div. of transplant surg, bwh, transplant surg res lab, boston; dept. of surgery, charite berlin, germany. increasing numbers of organs from elderly donors are currently utilized for transplantation. advanced donor age may not only be associated with physiological impairments but also with a modified immune response of the recipient. we hypothesized a more potent early immune response following the transplantation of elderly donor organs and we analyzed the immune response in a mouse heart transplant model. young b mice received heart allografts from , and mths old bm donors. the recipients immune response and intragraft changes were analyzed. elderly, non-manipulated hearts contained overall significantly elevated frequencies of cd + and cd + t-cells and dcs (cd c + ) ( mths vs. mths: cd + : . % vs. . %, cd + : . % vs . %, cd c + : . % vs. . %, p< . ). following engraftment of mths old heart grafts numbers of activated dc's (cd c + i-a b+ and cd c + cd + ) had significantly increased in recipient spleens (day :p< . ). in parallel, frequencies of effector/memory phenotype t-cells (cd + cd high cd l low and cd + cd high cd l low ) were significantly elevated with increasing age ( vs. vs, mths: cd + cd high cd l low : . % vs. . % vs. . %, respectively, p< . ). in addition, tregs (cd + cd + foxp + ) were also elevated ( vs. vs, mths: . %vs. . %vs. %,p< . ) t-cell alloreactivity, as measured by ifnγ-production, increased with donor age ( mths vs. mths vs. mths: . ± . vs . ± . vs. . ± . ifnγ-producing spots/ x cells, p< . ). mixed lymphocyte reaction (mlr) at day revealed a gradual increase in splenocyte proliferation with advancing donor age (p= . ) indicating an enhanced immunogenicity of older organs. immunohistochemical staining confirmed augmented cd + and cd + t-cell infiltrates and an intense ki positivity of gics in mths old heart grafts, emphasizing an intensified immune response towards organs from old donors. in summary, old native heart transplants contain an overall higher number of passenger leukocytes contributing to an increased immunogenicity of these organs. after transplantation, a more potent dc and t-cell activation and intragraft t-cell infiltration was observed in elderly organs. nox and chronic kidney allograft interstitial fibrosis. shannon reese, madhu adulla, surmeet bedi, jose torrealba, deb hullett, arjang djamali. nephrology, uw madison, madison, wi. to determine the role of oxidative stress (os) in kidney allograft fibrosis, we are developing strategies that would decrease the generation of reactive oxygen species (ros) instead of using ros scavengers, the standard approach to antioxidant therapy so far. we therefore started to examine the expression of nadph oxidase (nox) subunits (nox , nox , p and p phox) and their distribution in human and rat kidney allografts with chronic interstitial fibrosis (iftanos). using double-staining immunofluorescent studies in human allografts (n= ) we showed that nox was present in injured tubules costained with αsma ( figure . ) similarly, interstitial macrophages (cd + -nox + ) and myofibroblasts (αsma + -nox + ) but not cd + t cells or s a + fibroblasts expressed high nox levels, suggesting that nox is involved in the pathogenesis of allograft fibrosis via epithelial-tomesenchymal transition (emt), macrophage and myofibroblasts activation. we then examined the coexpression of nox , nox and p phox in normal and transplant kidneys with iftanos and showed that these molecules were upregulated in the latter group and that nox and nox were associated with p phox expression. these results were confirmed in the fisher to lewis rat kidney transplant model (figure . ). immunoblot analyses at weeks and months showed that nox and nox levels were increased in the allogeneic (n= ) compared to syngeneic transplants (n= ). greater nox levels were composite tissue allotransplantation (cta) is a recently introduced option for limb replacement and reconstruction of tissue defects. as other allografts, a cta grafts can undergo immune-mediated rejection, and standardized criteria are required for characterizing and reporting severity and types of rejection. this manuscript documents the conclusions of a symposium on cta rejection held at the ninth banff conference on allograft pathology in la coruna, spain on june , , and proposes a working classification scheme, the banff cta- , for the categorization of cta rejection. this classification was derived from public international consensus discussions regarding all published scoring systems for cta rejection. given the current limited clinical experience in cta, a formal histological classification was established for acute skin rejection with the understanding that other types of rejection involving other tissues will be developed with periodic review of this emerging field. it was agreed that the defining features to diagnose acute skin rejection would include inflammatory cell infiltration with epidermal and/or adnexal structure involvement, epithelial apoptosis, dyskeratosis, and necrosis, and that severity of rejection will be graded under five categories. this classification refines proposed schemas, represents international consensus on this topic, and establishes a working collective classification system for cta reporting. the role of skin biopsies in diagnosing clinical rejection in hand composite tissue allotransplantation (cta). christina l. kaufman, , ruben n. gonzales, kadiyala v. ravindra, , brenda w. blair, joesph f. buell, , warren c. breidenbach. , , christine m. kleinert institute, louisville, ky; kleinert kutz and associates, louisville, ky; jewish hospital transplant center, louisville, ky; surgery, university of louisville, louisville, ky. aim: traditionally allograft biopsy has dictated treatment of allograft rejection. we hypothesize that histological grade of the biopsy may over diagnose rejection in cta hand cta is unique in that the organ is external and early signs of rejection can be viewed directly. skin is the first tissue in the cta graft to show rejection. methods: for this study, rejection by defined by requirement of treatment. these episodes were compared with histological grade, and hand appearance. we reviewed skin biopsies taken during , and years of follow up. results: three observations surfaced. first, a rejection grading scale for cta is still evolving. a review of the literature showed at least four different criteria are in use. the criteria used to grade biopsies at our center changed over the year follow up. bias towards calling higher grades of rejection, and more aggressive treatment occurred in the first two patients. a re-read of random biopsies taken from the first two patients showed a down-grade of rejection to grade ii or i in five cases. secondly, concomitant cmv infection in the last patient prevented the aggressive treatment of rejection. despite high grade histology, the swelling, rash and redness responded to topical immunosuppression. systemic treatment was not necessary. analysis indicated that swelling and/or rash, and the percentage of skin involvement seemed to be more informative markers of existing (or resolving) alloreactivity than was histology. the biopsies taken from the graft shown below showed a grade iii histology. thirdly, changes in hand function were not associated with changes in biopsy grade. conclusion: the histologic grade of skin biopsies from hand allografts appears to over estimate rejection. alterations in immunosuppression shoud be based on appearance of the allograft, and clinical course as much as on the histologic grade of the biopsy. expression of molecular mechanisms of lymphozyte trafficking correlates closely with skin rejection in human hand transplantation. theresa hautz, bettina zelger, gerald brandacher, hans g. mueller, andrew w. p. lee, raimund margreiter, stefan schneeberger. dept. of general and transplant surgery, innsbruck medical university, austria; dept. of pathology, innsbruck medical university, austria; dept. of dermatology, innsbruck medical university, austria; div. of plastic surgery, university of pittsburgh. introduction: to understand in greater depth the molecular mechanisms involved in skin rejection in hand transplantation, we investigated key molecular markers of lymphocyte trafficking, cellular rejection and antibody mediated rejection in human hand transplantation. methods: a total of skin biopsies taken from three bilateral hand transplants were assessed by h&e histology (grades as per previously a published classification - b) as well as immunohistochemistry using antibodies for following markers: lymphocyte function-associated antigen (lfa)- = cd a, intercellular adhesion molecule (icam)- = cd , selectin e = cd e, selectin p = cd p, ve-cadherin = cd , human leukocyte antigen (hla) ii (dp, dq, dr), psoriasin = s a and c d. levels of expression were assessed ( , +, ++, +++) and read in the light correlated with the rejection as well as time after transplantation. results: rrejection ranged between grade and a with an average score of . . in healthy skin, none of the markers investigated was consistently up-regulated. upon rejection, cd , cd e and cd p staining in endothelial cells was significantly increased. expression of cd e and cd p correlated well with severity of rejection. the majority of infiltrating lymphocytes stained positive for cd a. interestingly, also kerationcytes were highly positive for cd a at the onset of rejection. cd was detected on endothelial cells, but its occurrence did not correlate with rejection. psoriasin expression was observed in keratinocytes in a basal and focal pattern and correlated well with rejection. for c d, no consistent staining pattern was observed indicating that antibody mediated rejection did not play a role in these patients. conclusion: molecular markers involved in lymphocyte trafficking are up-regulated upon skin rejection after hand transplantation and represent promising target for prophylaxis and treatment of rejection in composite tissue allotransplantation. investigation of the immunomodulatory phenotype of infiltrating lymphozytes in skin rejection of human hand allografts. theresa hautz, gerald brandacher, bettina zelger, hans g. mueller, andrew w. p. lee, raimund margreiter, stefan schneeberger. dept. of general and transplant surgery, innsbruck medical university, innsbruck, austria; dept. of pathology, innsbruck medical university, austria; dept. of dermatology, innsbruck medical university, austria; div. of plastic surgery, university of pittsburgh. introduction: skin rejection has complicated the postoperative course in human hand transplantation. to better define the characteristics of the lymphozytic infiltrate human hand transplant biopsies have been investigated for expression of foxp and indoleamine , -dioxygenase (ido), a key regulatory enzyme to induce t-lymphocyte unresponsiveness. methods: a total of skin biopsies taken from three bilateral hand transplant recipients during the first years after transplantation were assessed by h&e histology (graded as per previously published classification - b) as well as immunohistochemistry for ido and foxp . levels of expression were assessed ( , +, ++, +++) and interpreted in the light of clinical courses, time after transplantation, severity of rejection as well as markers for lymphozyte migration. results: overall, rejection ranged between grade and a with an average score of . . ido was found constitutively expressed in the endothelium independent of rejection. ido expression in the cellular infiltrate was significantly increased upon and correlated well with severity of rejection (rejection grade , . +/- . : rejection grade , . +/- . p< . ). foxp positive t-cells were mainly found in severe rejection (rejection grade , . +/- . : rejection grade , . +/- . p= . ). ido expression correlated well with foxp expression, although the overall staining intensity for foxp was lower. a strong tendency towards higher expression of ido as well as foxp towards later time-points after transplantation was observed (year -foxp . +/- . , ido . + /- . [n= ] , year -foxp . +/- . , , year -fox . +/- . , ). expression of ido correlated closely with expression of e-selectin, p-selectin, icam and lfa- . conclusion: characteristics of the cellular infiltrate indicate a strong tendency towards self limitation of the alloimmune response towards the skin with both time after transplantation as well as severity of rejection in human hand transplantation. further studies are warranted to clarify the clinical relevance of these findings. prospective analysis of the immunologic profile of a hand transplant recipient in the first year. kadiyala v. ravindra, warren c. breidenbach, joseph buell, suzanne t. ildstad. department of surgery, university of louisville, louisville, ky; kleinert, kutz, and associates, louisville, ky; institute for cellular therapeutics, university of louisville, louisville, ky. introduction: a major obstacle to the wider application of hand transplantation is the long term complications associated with immunosuppression. minimization of immunosuppression is an important goal in all transplant recipients. currently there are no accurate tools to evaluate the immunological responsiveness which might help tailor the level of immunosuppression for an individual patient. the response of recipient lymphocytes to pha, candida, and alloantigen may represent laboratory tool towards this end. it has been reported that these responses are hierarchical with response to alloantigen being the first to be lost, followed by candida and finally pha. a year old male received a proximal forearm transplant in november . immunosuppression included induction with a single mg dose of alemtuzumab and maintenance with tacrolimus and mycophenolate mofetil. the patient developed an episode of cytomegalovirus infection followed by acute rejection after reduction of his immunosuppression during the rd post-operative month. these were successfully treated with ganciclovir and topical tacrolimus & steroids respectively. blood samples were drawn at selected time points, and subjected to phenotyping of lymphocyte subsets and immune monitoring for circulating peripheral blood regulatory t cells (t reg ) and proliferative responses to phytohemagglutinin (pha), candida, and alloantigen. results: alemtuzumab induction resulted in profound lymphopenia. at week and month, the response to pha was intact (stimulation index and respectively), but response to alloantigen and candida suppressed (si < ). a similar immunologic profile persisted up through months. at year, the pha and candida responses are robust (si and respectively), but alloresponses have not returned. current immunosuppression consists of tacrolimus ( - ng/ml) and mycophenolate mofetil ( mg b.i.d.). there is no gross evidence of acute or chronic rejection. conclusions: induction with alemtuzumab alters the recovery of immune response: recovery to candida was delayed beyond months and to alloantigens beyond a year. in light of this, further reduction of immunosuppression may be contemplated in future hand transplants without the risk of rejection. composite tissue allotransplantation has achieved significant clinical advances despite an adequate preclinical model to study technical and immunosuppressive strategies. we have developed a non-human primate model of facial composite tissue allografts (cta). unilateral lower hemi-facial cta (bone, muscle, skin) transplants were performed between mismatched cynomolgus monkeys. immunosuppression consisted of days of continuous iv tacrolimus monotherapy followed by tapered daily im doses. six animals received prophylactic gancyclovir. all animals had serial transplant biopsies. ten transplants have been performed, with one loss secondary to line infection on day without evidence of rejection. two ctas had evidence of chronic rejection (day , ); with development of alloantibody after days. five ctas had prolonged survival (day , , , , ) , but developed ptlds resulting in experimental endpoints. all animals had clinically normal grafts, but animals showed histological evidence of mild rejection not treated with any additional immunosuppressive therapy. ptld tumors were analyzed using short tandem repeats (str) to define donor or recipient origin. str analysis demonstrated donor origin of ptld tumors and recipient origin in animal. none of the ptld animals had clinical evidence of rejection of skin, bone, or muscle. ebv was not detected in the serum of tested animals, and ganciclovir therapy had no effect on the development of tumors. tacrolimus levels of ptld animals were higher than animals with rejected grafts ( vs. ng/ml; p= . ). two additional animals have healthy grafts (day +, +) without evidence of rejection or ptld, and have been converted to rapamycin after day . we have developed a preclinical model for facial cta transplantation that achieves prolonged graft survivals with tacrolimus monotherapy. the high incidence of ptld tumors of donor origin represents an outcome similar to bone marrow transplantation in contrast to its rarity in solid organ transplantation. our findings are a cautionary note regarding ctas that include vascularized bone elements. immunosuppressive protocol modifications have been made in an effort to decrease the incidence of these donor-derived ptlds. heterotopic heart transplantation: the united states experience. jama jahanyar, tarek a. sibai, matthias loebe, michael m. koerner, guillermo torre-amione, george p. noon. dept. of surgery, baylor college of medicine, houston, tx. heterotopic heart transplantation (hht) is utilized in patients (pts) who do not qualify for standard orthotopic heart transplantation (oht). specific indications include refractory pulmonary hypertension and a donor-recipient size mismatch. the objective of this study was to analyze the unos database and compare outcomes of hht to oht. the unos database with more than pts undergoing thoracic organ transplantation in the u.s. between and was reviewed (based on optn data as of may , ) . primary endpoint of this study was overall survival and subgroup survival [pts with transpulmonary gradient (tpg)> , ischemic (icm) and dilated cardiomyopathy (dcm)]. secondary endpoint was assessment of pretransplant criteria. exclusion criteria were retransplantation and missing transplant dates. of who underwent oht and who underwent hht, and respectively, were enrolled in this study. [ ] [ ] [ ] [ ] [ ] [ ] survival after oht is superior to hht. this survival benefit however, disappears in pts with a tpg> . overall the survival after hht is superior to the reported survival in pts who undergo lvad implantation as destination treatment (rematch-trial/ year survival of %). thus in selected pts, especially those with elevated tpgs, hht should be considered a viable option with overall good results. ventricular assist device as a bridge to heart transplantation in children: can we afford it? william t. mahle, glen ianucci, robert n. vincent, kirk r. kanter. sibley heart center cardiology, emory university school of medicine, atlanta, ga. ventricular assist devices (vads) allow children with severe heart failure to be bridged to successful heart transplantation (ht). vads are being used with increasing frequency in the pediatric population and newer devices allow even young infants to be supported. vad implantation and maintenance, however, is quite expensive and the cost-effectiveness of vad use in adults has been questioned. to date, an economic analysis of vad support in children has not been undertaken. methods: we used pediatric health information system (phis), an administrative database of the child health corporation of america (a consortium of children's hospitals in north america), to determine the costs related to vad use in children. data on subjects < yrs of age from - were reviewed. hospital charges were converted to costs based on hospital-specific cost-to-charge ratios. projected survival for subjects who were successfully bridged to ht was derived from published data. costutility was expressed as cost per quality-adjusted life years (qalys) saved, expressed in us dollars. all future costs and benefits were discounted at %. results: the median age at implantation from the phis database was . years, range days to yrs. the mean hospital cost per patient was $ , . estimated survival to heart transplantation was % and estimated successful explantation without transplantation was %. the calculated cost-utility for vad as a bridge to transplantation was $ , /qaly saved. if one assumes that the children who survived to vad explantation would otherwise have a high risk of hospital death ( %) without vad support, then the calculated cost-utility would be $ , / qaly saved. even if abstracts survival to transplantation exceeds %, vad implantation does not achieve a favorable cost-utility ratio. vad support in pediatric heart only becomes cost-effective if recovery and explantation can be achieved in over % of subjects. conclusions: vad supports serves as an effective bridge to heart transplantation in children. however, the cost-utility of this strategy is above the generally accepted threshold for cost-effectiveness ($ , /qaly). in a setting of limited healthcare resources vad as a bridge to transplantation may not be justified. purpose: heart transplantation (tx) in patients with hla sensitization presents challenges in organ allocation. virtual crossmatch (vxm), in which recipient hla antibodies, identified by labscreen pra beads, are compared to the prospective donor hla-type, could increase the use of allografts from distant donors (dd). accuracy of vxm and outcomes of this approach in heart tx are not known. methods and materials: to increase time-efficiency at the time of organ allocation, crossmatch testing is frequently initiated on pre-set trays which contain sera of multiple prospective sensitized recipients. we used results from these studies to determine expected accuracy of vxm. we assessed outcomes of allocation algorithm implemented in in sensitized patients. conventional prospective crossmatch was done when allografts were procured from local donors (ld), while vxm was utilized when allografts were offered from dd. there were direct t-cell ahg crossmatch tests done with sera of potential allograft recipients who had preformed hla-antibodies of known class i hla specificities. as shown in table , the positive and negative predictive values (ppv, npv) of vxm were % and %. table shows outcomes in sensitized patients who were eligible for vxm approach. received allografts from ld with negative prospective crossmatch while ( %) received allografts from dd with negative vxm. three dd patients had a positive retrospective crossmatch -npv of vxm was % in this cohort. vxm has high negative and positive predictive values, accurately predicting results of standard direct crossmatch in most patients. vxm allows use of allografts from dd and is likely to improve organ allocation in the disadvantaged group of sensitized patients. single center experience with new heart allocation system implemented by united network of organ sharing. biljana pavlovic-surjancev, nilamkumar patel, linda dusek, james sinacore, jennifer johnson, cassie bessert, alain heroux. heart failure/heart transplant program, loyola university medical center, maywood, il. purpose: in july , united network of organ sharing (unos) implemented a new allocation system for adult heart transplant (tx) candidates with the following sequence: local status a→local status b→zone a status a→zone a status b→local status . the purpose of this study is to evaluate the impact of new system on heart tx patients at a single center in region . methods: patients transplanted during year (y) prior to new system ( - - to - - , group , n= ) and y following new system ( - - to - - ,group , n= ) were compared for unos status at the time of transplant, waiting time, ischemia time, length of the hospital stay (los) before and after transplant, donor age, procurement-team travel distance and cost. results: new system significantly decreased median waiting time, but increased median ischemia time without affecting short-term survival: patient died in each group. number of transplants increased % in group mostly due to increased number of status a patients supported by iabp without change in number of status b and patients. median pre-transplant los increased -fold in group (p= . ), whereas mean pre-transplant los increased by days. in group , thirteen patients had donor heart procured in zone a and thirteen patients had donor heart procured locally, whereas in group , all donor hearts were procured locally. median procurement-team travel distance increased -fold and median travel cost -fold in group (p< . ). clinical outcomes associated with simultaneous heart-kidney transplantation. tariq shah, , , , suphamai bunnapradist, jagbir gill, steven k. takemoto. the figure below indicates recipients of shk transplants (open symbols) had lower rates of rejection when compared to heart (square) or kidney (diamond) recipients. survival rates were initially higher for kidney recipients with rates for shk similar to heart recipients. the hazard ratio (hr) for graft loss was higher for shk recipients compared to kidney, but lower when compared to heart recipients. the lower hazard for shk in heart allografts might be attributed to risk associated with pretransplant dialysis (hr= . , . - . , p< . ) . approximately % ( , ) of cardiac transplant recipients initiated dialysis prior to transplantation. the differing rates of shk rejection observed in the heart and kidney analytic files might be attributed to susceptibility or treatment of heart and kidney allografts, rejection monitoring or reporting. conclusion: retrospective examination of data provided to the optn indicates simultaneous heart-kidney transplantation seems to be effective for cardiac transplant candidates who require dialysis. abstract# objective: krp , a novel structural analog of fty , has been documented to display -fold greater selectivity of agonism for sphingosine- -phosphate (s p) type (s p ) receptor compared with s p receptor. clinical trial has shown that fty produced dose-limiting toxicity-bradycardia, due to its effect on s p receptor. we have tested the effect of krp on the survival of islet allografts either alone or combined with local delivery of cd + cd + foxp + t regulatory (treg) cells. previous studies using knockout mice have documented a key role for the integrin cd in promoting allograft rejection. these data are consistent with a critical role for cd expressing cells in this process. however, a direct test of this hypothesis has proven problematic due to the lack of mabs that efficiently deplete cd + cells in wild type hosts. to circumvent this problem, we conjugated the non-depleting anti-cd mab, m , to the toxin, saporin (sap), to produce an immunotoxin (m -sap) that selectively depletes cd -expressing cells in vivo. treatment of naive mice with m -sap selectively depleted cd + cd + cells and dramatically reduced the overall frequency of cd + lymphocytes in diverse compartment including intestinal intraepithelial lymphcyte,spleen and mesenteric lymph node. m -sap also depleted cd + dendritic cells (cd c + ) and t regulatory cells (tregs, cd + cd + ) in the above compartments. in the thymus, m -sap depleted cd -expressing cells in both cd -cd and cd -cd + subpopulations, both of which express cd , leading to a dramatic reduction in the number of thymocytes( . ± . × vs. . ± . × , p< . , in control vs. treated respectively). we next assessed the effect of m -sap in a fully allogeneic islet transplantation model (balb/c→c bl/ ). m -sap produced long-term (lt) graft survival (> d, n= ,vs. untreated median survival time d, n= ). unconjugated m or isotype control (igg-sap) did not significantly prolong islet allograft survival. graft histology showed little, if any, lymphocyte infiltration surrouding islet transplants in lt mice. in contrast, intense lymphocyte infiltration with disruption of islet morphology was observed in untreated mice. pretreatment of donor islets with m -sap did not significantly prolong allograft survival indicating that the immunosuppressive effect of m -sap resides at the level of host. interestingly, we found a - fold increase in both percentage and number of foxp + cd + cd + cells in the spleen and draining lymph node from lt mice, though it remains to be determined whether such cells account for graft acceptance in m -sap treated recipients. in summary, these data document that depletion of cd expressing cells promotes long-term islet allograft survival. these findings point to a novel strategy for therapeutic intervention in islet allograft rejection. pancreatic objective: to engineer pancreatic islets in a rapid and efficient manner with a novel form of fasl protein chimeric with core streptavidin and test the efficacy of engineered islets for long-term survival in allogeneic hosts. methods: balb/c pancreatic islets were engineered first by cell surface modification with biotin followed by the display of a chimeric form of fasl protein that consists of extracellular domain of fasl fused c-terminus with core streptavidin (sa-fasl). sa-fasl-engineered islets were transplanted into streptozotocin diabetic c bl/ mice under transient cover of rapamycin. unmodified islets or those engineered with streptavidin protein (sa) served as controls. results: all the islets showed effective engineering with sa-fasl, which persisted on the surface of islets for weeks in vitro as assessed by confocal microscopy. all the islets (n= ) engineered with sa-fasl survived over the observation period of - days without detectable signs of rejection. in marked contrast, all the unmodified (n= ) and sa-engineered (n= ) islets underwent acute rejection within days. the observed tolerance was localized to the engineered islets as unmodified second set of islets transplanted under contralateral kidney of long-term (> days) graft recipients were rejected in a normal tempo (mst= ± days) without any effect on the survival of primary islets. conclusions: engineering pancreatic islets with exogenous immunomodulatory molecules, such as sa-fasl, in a rapid (∼ hrs) and efficient ( % of targeted islets) manner represents a novel means of immunomodulation with considerable therapeutic potential for the treatment of type diabetes. supported in parts by nih (r dk , r ai , r ai , r hl ), jdrf ( - - ) the ability of embryonic stem (es) cells to form cells and tissues from all three germ layers can be exploited for the generation of cells that can be used to treat diseases. in particular, successful generation of hematopoietic cells from es cells could provide safer and less immunogenic cells than bone marrow cells, that require severe host preconditioning, when transplanted across mhc barriers. in the past, it has been difficult to derive hematopoietic cells from es cells. it has now become clear that this was due to the lack of self-renewal properties by these newly developed progenitor cells. here, we exploited the self-renewal properties of ectopically expressed hoxb , a homeobox transcription factor, to generate hematopoietic progenitor cells (hpcs) that successfully induce high level mixed chimerism and long-term engraftment in recipient mice. hoxb -transduced svj es cells (h b ) were allowed to form embryoid bodies. these were dismantled after days and the cells treated with a cocktail of hematopoietic cytokines. by day , es cells had formed hpcs. these newly generated hpcs were cd +, cd +, cd + but poorly expressed mhc class i molecules and no class ii. the hpcs fully restored splenic architecture in rag -/-γ c -/immunodeficient mice, abstracts comparable to bone marrow. additionally, hpc-derived newly generated t cells were able to mount a peptide-specific response to lymphocytic choriomeningitis virus (lcmv) and specifically secreted il- and ifn-γ upon cd stimulation. further, hpc-derived antigen presenting cells (apcs) in chimeric mice efficiently presented viral antigen to wild type (wt) t cells. in syngeneic recipient mice, hpcs engrafted and formed more robust t and b cell populations. the majority of the hpc-derived cells were however, gr- + , suggesting a bias towards myeloid cells by the hoxb . interestingly, these cells successfully engrafted in allogenic mrl (h k ) and balb/c (h d ) recipients without the need for immunosuyprresion. this ability to form mixed chimerism across mhc barriers is a consequence of their lack of mhc, cd and cd expression. our results demonstrate for the first time that leukocytes derived from es cells ectopically expressing hoxb are immunologically functional and escape immunological rejection when transplanted across mhc barriers allowing the induction of mixed chimerism. normalization the de is derived from the anterior segment of the primitive streak which corresponds to the early and mid-gastrula organizer during early embryo development, from which many of the major visceral organs, including the liver, pancreas, lung, thyroid and intestines are derived. es cells were cultured in a serum-free medium containing activin a and bfgf for - days, the differentiated cells developed into an epithelial monolayer yielding more than % of cxcr expressing cells. cxcr has been reported as a cell surface marker of the definitive endoderm. molecularly, the differentiated es cells express typical definitive endodermal genes in particular, foxa , sox- , gsc, and hnf α. the cxcr + definitive endodermal cells were further purified using immunomagnetic bead separation to more than % purity in order to eliminate teratoma-forming cells. to study their engraftment and regenerative capacity, these newly differentiated cells were intravenously infused into a mouse with carbon tetrachloride-induced liver injury. harvested livers from these animals showed large de-derived cells positive for albumin suggesting that they were de novo generated hepatocytes. a second cell type was ck- expressing, suggesting that the engrafted cells also differentiated into cholangiocytes. therefore, we further transplanted these de cells into a factor viii null mouse and asked whether these cells could correct factor viii activity. plasma factor viii activity (coamatic assay) fully normalized to that of wild type mice and has remained stable over days. these data suggest that es cell-derived de progenitor cells can restore factor viii activity in hemophilia a mouse model, presumably through protein production in de novo generated hepatocytes. more importantly, none of these animals developed teratomas. thus, es-cell derived cells can potentially be coaxed to form cellular transplants with curative capabilities. objective: we have established a novel approach, protex, to rapidly and efficiently engineer primary cells, tissues, or organs to display on their surface exogenous proteins of interest for immunomodulation. this approach involves generation of chimeric proteins with core streptavidin, biotinylation of cells, and the transient display of chimeric proteins on the cell surface. in this study, we displayed a chimeric form of fasl (sa-fasl) on the surface of bone marrow cells and tested the efficacy of these cells to establish mixed chimerism in allogeneic hosts under nonmyeloablative conditions. methods: balb/c bone marrow cells were engineered with sa-fasl and million of these cells were transplanted into c bl/ mice subjected to various doses of total body irradiation days earlier. a short course of rapamycin was used to enhance the tolerogenic effect of sa-fasl. bone marrow cell recipients were typed for multilineage chimerism at various times post-transplantation and tested for donor-specific tolerance using skin grafts. results: all the animals (n= ) treated with cgy total body irradiation and transplanted with sa-fasl-engineered donor cells showed significant levels of chimerism ( - %) on day post-transplantation that showed a steady increase overtime and reached to - % on day post-transplantation. in marked contrast, none of the control animals (n= ) receiving bone marrow cells and a short course of rapamycin showed detectable chimerism. chimerism was multilineage and associated with donor specific tolerance since chimeric animals accepted donor, but rejected third party skin grafts. conclusions: engineering bone marrow cells in a rapid (∼ hrs) and efficient ( % targeted cells) manner with exogenous proteins having immunoregulatory functions provides a new and effective means of immunomodulation to establish mixed allogeneic chimerism under nonmyeloablative conditions with significant potential in clinical bone marrow transplantation. funded in parts by nih (r dk , r ai , r ai , r hl ), jdrf ( - - ) , ada , and aha fellowship b. chronic allograft dysfunction is still a major clinical problem in organ transplantation. morphologically it is characterized by changes suggestive of an alloantibody mediated mechanism such as glomerulopathy and vasculopathy or by non-specific changes such as interstitial fibrosis and tubular atrophy. alloantigen dependent as well as -independent factors contribute to the pathogenesis of these changes. in this study we analysed allospecific t cells from the peripheral blood of kidney transplant patients under different immunosuppressive protocols with or without chronic allograft dysfunction. renal allograft recipients of our renal transplant clinic were screened at least six months after transplantation. all patients were on a calcineurin-based immunosuppressive protocol consisting of cyclosporine (csa)/mycophenolate mofetil (mmf)/steroid, tacrolimus (tac)/mmf/steroid, or csa/steroid. patients had to be mismatched for one or more of the five candidate hla-dr antigens for which synthetic peptides were available (dr , dr , dr , dr , and dr ). patients with biopsy proven chronic allograft nephropathy (can)with an elevated serum creatinine level of ≥ . mg/dl were compared with patients with stable allograft function (serum creatinine < . mg/dl). t cell lines were generated from peripheral blood lymphocytes of renal transplant recipients against donor-derived hla-dr peptides presented by self apc. t cell lines generated from patients with can produced significantly more ifn-γ, while those generated from stable patients produced il- associated with a low proliferation index in response to the donor-derived mismatched hla-dr allopeptide in vitro. moreover, significantly more cd +cd +foxp + t cells were found in stable patients on tac and mmf as compared to patients on csa and mmf. interestingly, a higher gene expression of cd , cd , ctla- , foxp , and il- was observed in those patients. taken together a th (ifn-γ) alloimmune response is deleterious and promotes chronic graft damage, while a th (il- ) response seems to be associated with a lower incidence of chronic allograft nephropathy. an immunosuppression based on tac and mmf seems to favour cd +cd +fosp + t cells and to allow long-term engraftment with stable renal function. cyclosporin induces epithelial to mesenchymal transition in renal grafts. the expression of epithelial to mesenchymal transition (emt) markers is a reliable predictor of the progression towards interstitial fibrosis and tubular atrophy of the renal grafts. in vitro experiments suggest that calcineurin inhibitors (cni) can induce emt of tubular epithelial cells. although no evidence was ever provided in vivo, this suggests that emt could be involved in the pathogenesis of renal fibrosis induced by cni. we have previously reported the results of a prospective randomized trial comparing the elimination at month of either cyclosporine (csa, n= ) or mycophenolate (mmf, n= ) from a triple drug regimen in de novo renal transplant patients. all of them had systematic graft biopsies at months and post engraftment. in the leftover material, we retrospectively detected in tubular cells and by immuno-histochemistry the expression of two validated markers of emt: the de novo expression of vimentin (vim) and the cytoplasmic translocation of b-catenin (cat). we were able to measure the emt score at both months and in a total of patients ( in each group). in the csa group, the vim and cat scores had progressed between and months from . calcineurin inhibitors (cni) are efficacious but nephrotoxic immunosuppressives. arteriolar hyalinosis is one of the characteristic histological correlates of this toxicity. yet, time course of this lesion, its reversibility, dose-dependency and the discrimination between drug-related effects, diabetic and hypertensive vasculopathy remain unclear. aim of this study was to evaluate the prevalence and time course of cni-related vascular changes after renal transplantation (tx) in protocol (pbx) as well as in indication biopsies (ibx) and to correlate this to the cni blood levels, blood pressure and diabetes. from patients, a total number of pbx, taken at weeks (n= ), (n= ) and months (n= ) after tx and ibx were classified according to banff-criteria. assessement of cni toxicity included: isometric vacuolisation of tubules (isovac), intimal arteriolar hyalinosis (iah) and nodular arteriolar hyalinosis (nah) and vacuolisation of small vessel smooth muscle cells (vsm). % of patients received either cyclosporine or tacrolimus. in pbx, isovac was present in %, % and % of patients at weeks, and months post-tx, respectively; iah in %, % and %; nah in %, % and %; vsm in %, % and %. in late ibx (> years post-tx) the prevalence of the analyzed parameters apart from isovac ( %) was markedly higher: % for iah, % for nah and % for vsm. in pbx, vsm, iah and nah were associated with each other but not significantly dependenct on blood pressure, rejection episodes or diabetes. through levels of cnis were not different between patients with and without vascular hyalinosis. in patients with vsm and iah in late ibx one third had these lesions already present in earlier biopsies. conclusion: the prevalence of presumed morphological signs for vascular cni-toxicity in pbx is low and constant and apparently, not associated with cni blood levels, hypertension or diabetes. in contrast, in ibx later than two years after transplantation, prevalence of vascular cni-toxicity signs is much higher. this emphasises that cni reduction protocols should be regarded within the first six months after transplantation, when vascular changes are still marginal. further elucidation of precursor lesions in pbx would help to find out patients at risk for cni-induced vascular changes. donor introduction: achieving donor specific tolerance has been the goal of the transplant community. success has been reported with donor stem cell transfusion in animal studies and prevention of chronic rejection in cardiac allograft recipients in the clinic. this report details the results of the initial phase of a study in humans. methods: a prospective phase i/ii fda approved pilot protocol was initiated to evaluate the effects of donor graft facilitating cell (fc)/stem cell infusion in kidney transplant recipients. conditioning was performed with cgy of total body irradiation. bone marrow processed to remove gvhd-producing cells but retain cd + /tcr -fc and stem cells was infused hours post-operatively. the dosage of stem cells was limited by the t cell dosage. the starting dose was x t cells. this was increased in steps of x per patient. as the study spans a -year period, the immunosuppression changed: patients received cyclosporine (cya), mmf and prednisone; were induced with basiliximab and maintained on cya( )/fk( ), cellcept and prednisone; and received alemtuzumab induction and maintenance with fk and mmf. all patients underwent tolerance testing and immunoprofiling studies. results: of the patients, received live donor kidney transplants. one graft was lost from arterial thrombosis on day . delayed graft function was seen in patients. good long-term graft function was seen in the other patients. acute rejection was noted only in and infectious complications (cmv- , histoplasma- ) in patients. two patients died with functioning grafts -one at years from lung cancer and another from complications from diabetes at years. six patients are alive with functioning grafts at mean follow-up of . years with a mean creatinine of . mg/dl. no gvhd was detected in any of the patients. macrochimerism was not detected in any of the patients at any point. notably, in spite of the fact that durable engraftment was not yet achieved, none of the patients were sensitized as a result nor did they experience immunologic sequelae. conclusions: in our patients there were no untoward sequelae related to either the conditioning regimen or marrow infusion. the incidence of acute rejection even on longterm follow up has been low. we currently propose to reduce the immunosuppression further in these patients. a pilot study was performed to evaluate whether immune cell depletion with alemtuzumab would permit post-transplant weaning of maintenance immunosuppression in well-matched renal transplant recipients. patients received alemtuzumab mg intravenously on the day of the transplant and the subsequent days while sirolimus and tacrolimus were started on day . tacrolimus was discontinued at day in all patients. extensive immune monitoring was performed at year. at current follow-up ( to months), all patients are alive with a functioning graft (median mdrd gfr= ml/ min). one patient experienced clinical and biopsy-proven rejection at months. all other patients remain on sirolimus monotherapy. four patients have been weaned to mg of sirolimus daily as their sole immunosuppressive agent, with resulting blood levels of - ng/ml. these patients have no evidence of donor-specific alloantibody, are unresponsive or hyporesponsive to donor cells by the cytokine kinetics test, and have a regulator phenotype to soluble donor antigens by trans-vivo dth. flow cytometry of peripheral blood demonstrated increased foxp expression in the cd +cd + population (p= . ). naive b cells (cd /cd neg) cells increased in of patients (p= . ) and memory b cells increased in all recipients (p= . ) when comparing pretransplant to year timepoints. other than the patient with rejection, -month protocol biopsies did not show any evidence of rejection, although of showed focal c d positivity and diffuse positivity. these patients also had evidence of alloantibody by luminex xmap testing. in conclusion, the cytokine kinetics test, alloantibody testing, and trans-vivo dth assay abstracts results correlated with clinical evolution of patients who successfully weaned both tacrolimus and sirolimus without rejection or alloantibody. the flow cytometry findings described occurred regardless of clinical evolution and may represent alterations of the immune system inherent in the treatment protocol independent of individual patient responses to the graft. however, the functional assays of cytokine kinetics assay and trans-vivo dth may be of potential use to correlate with the clinical immune status of the kidney transplant recipient. we have previously reported the short-term results of alemtuzumab (campath- h) pre-conditioning with tacrolimus monotherapy and subsequent spaced weaning in living donor kidney transplantation (ldkt). we report here our year experience. methods: we performed consecutive unselected ldkt (donor kidneys were removed laparoscopically) from / / to / / using mg ( . mg/kg) alemtuzumab and tacrolimus monotherapy. at months post-transplant and every to months interval, we used clinical data (including elisa antibody titers, cylex t-cell activation assay, and identification of donor specific antibodies) to wean tacrolimus when possible (bid-->qd-->qod-->tiw-->biw-->qwk). the recipients included hiv+, pediatric recipients, and re-transplants. the mean follow up was . + . days. results: actuarial recipient survivals at -, -, -years were . %, . %, and . %, respectively. graft survivals at -, -, -years were . %, . %, and . %, respectively. the mean creatinine (mg/dl) at -, -, -years were . + . , . + . , and . + . , respectively. the mean gfr (ml/min/ . m ) at -, -, -years were . + . , . + . , and . + . , respectively. the cumulative incidence of acute cellular rejection (acr) at -, -, -, -, -, -, -, and > conclusions: in the current era low risk patients infrequently have ar, and have excellent short and long term graft survival without the use of depleting antibodies. given the increased costs of these drugs, the indications for using depleting antibodies in low risk ktrs of scd kidneys should be further clarified. kidney transplantation prolongs survival in hepatitis c virus-positive (hcv+) patients with end-stage renal disease (esrd). however, the effects of induction therapy and chronic immunosuppression are unknown on the course of hcv infection and potential for cirrhosis in renal transplantation (rtx) recipients. we have retrospectively assessed parameters of liver function, child-pugh (cp) and meld scores in hcv+ esrd patients who received induction therapy with t-cell depletion (group : thymoglobulin, n= ) or an il- inhibitor (group : basiliximab, n= ). pre-rtx liver biopsies were similar in group and . patients were followed for a mean of days (range to days) following rtx and received tacrolimus, mycophenolate mofetil, and sometimes steroids post-transplant. overall graft survival was % in group and % in group (p > . ). data were analyzed pre-rtx, at and days and at time of last follow-up. serum ast, alt, platelets, inr, albumin and bilirubin did not change following rtx in either group. cp scores in group were not significantly changed after rtx ( . ± . to . ± . at last follow-up, p= . ). group patients on steroid-free protocols (n= ) demonstrated declining cp scores from . ± . to . ± . at last follow-up that were not statistically significant (p= . ). group patients on steroids showed opposite trends of cp: . ± . pre-rtx vs . ± . at last follow-up that similarly did not reach statistical significance (p= . ). group cp scores declined from . ± . to . ± . at last follow-up (p= . ). there was no difference between cp or meld scores at any point between the groups. as expected, meld scores improved significantly following rtx and remained low up until the final visit (p= . ); this was attributed to the drop in serum creatinine post-rtx. neither the use of thymoglobulin or basiliximab resulted in acute hepatitis resurgence or the development of cirrhosis post-transplant. we have not identified any association between choice of induction agent or maintenance immunosuppression regimens, including steroid withdrawal, with impaired hepatic function or progression to liver cirrhosis in hcv+ rtx patients. t cell depletion was well-tolerated by hcv+ rtx patients and resulted in good graft outcomes. interestingly, cp scores declined after renal transplantation in the basiliximab induction group. kidney: complications i prevalence background: a few years ago we observed an expansion of blood gd t cells following cytomegalovirus (cmv) infection in kidney transplant recipient (ktr). we recently demonstrated that these cells share a strong reactivity against cmv infected cells and tumor epithelial cells in vitro. an implication of gd t cells in the immune surveillance against cancer has been demonstrated in mouse and strongly suggested in human. we tested here the hypothesis of a protective role of cmv-induced gd t cells against neoplasia in ktr through: / a longitudinal case / control (ktr with cancer / ktr without cancer) study where gd t cell percentages were determined before and after cancer diagnostic (n= ), / a retrospective follow-up of ktr for . years looking for risk factors for malignancy. results:the median of gd t cell percentage in patients with malignancies was significantly lower when compared to control patients , and months before the diagnostic of the cancer (p< . ). using a conditional logistic model, we determined that patients with a gd t cell percentage above % were protected from cancer (p< . ). a significant association between increase of the vd neg gd t cell subset and lower cancer occurrence was only retrieved in the ktr who experienced pre-or post-graft cmv infection. finally, using univariate and multivariable analysis, absence of pre-or post-graft cmv infection in ktr was associated with a risk of cancer . times more elevated (p= . ). this study reveals an unexpected protective role of cmv against cancer in ktr most probably via the expansion of gd t cells cross-reactive against cmvinfected and tumor cells. background: viral infection (vi) is a morbidity factor in transplant recipients (tx pts). induction therapy (ind-rx) is a known risk factor for vi. although cam is thought to be a more potent ind-rx than zen, we have previously shown similar cmv infection rates in each. we have also shown that cmv-tc analyzed by cytokine flow cytometery (cfc) are consistently detectable in cmv sero(+), but not sero(-) individuals. cmv-tc(-) was associated with persistence of cmv infection in tx pts. here, we report on the effect of ind-rx on cmv-tc in kidney tx pts. methods: pre-tx samples from cmv-sero(+) pts and post-tx samples from pts were submitted for cmv tc-cfc. whole blood was incubated with a pooled overlapping peptide mixture consisting of peptides from cmv pp and brefeldin a at degrees for hours and room temperature overnight. ifnγ+cd + cells were enumerated by cfc and results were expressed as ifnγ+cd + cell%. results > . % were considered as (+ infection associated graft loss during the entire study period is shown in figure . infections contributing to renal allograft loss increased significantly from to . this may be due to increase use of both induction agents and potent maintenance regimens. this is an important cause for poor long-term graft outcome despite decreasing rejection rates and a balance has to be maintained between prevention of rejection and avoidence of infection. serum creatinine (scr) at procurement was ± µmol/l. the incidence of donor hypertension, diabetes, and death from cerebrovascular origin was %, %, and % respectively. multivariate analysis showed that the only clinical parameters associated with a low egfr were donor scr and donor hypertension. nyberg or pessione scores were not significantly associated with a low egfr. regarding d biopsies, univariate analysis showed that % of sclerotic glomeruli (sg, p= . ), arteriolar hyalinosis (p= . ), mean remuzzi score (p= . ) and mean cadi score (p= . ) were all significantly associated with a low egfr. a logistic regression showed that an integrated score including: i) donor scr (± µmol/l), ii) hypertension, and iii) sg (± %) had the highest performance in predicting a low egfr at yr compared to clinical or histological parameters alone. using this composite score, the adjusted or for the prediction of a low -yr egfr ranged from if none of the factors were present, to . (if sg > % was associated with one of the clinical factors), and to . (if the factors were present, p= . ). conclusion: this study highlights that d biopsies are useful to predict graft outcome particularly in md population, and may perform better than clinical scores alone. in this population, a simple and routinely applicable integrated scoring strongly predicts a poor graft outcome, which may allow an optimized allocation of marginal donors. prospective kidney transplantation from small pediatric donors is increasingly being utilized as a means to optimize the organ supply, however the single most common specified reason for the discard of pediatric kidneys is vascular damage, such as shortening of the suprarenal aorta or injury to the renal artery orifices, which often precludes en bloc transplantation (ebk). at our center, damaged kidneys were salvaged by transplantation as singles (sk background: in an effort to maximize the number of recipients transplanted per donor, transplant centers in our donor service area (dsa) voted to preferentially allocate local and imported en-bloc pediatric donor renal allografts to centers willing to transplant two individuals with single allografts. after year of implementing this policy into action, we report on our initial experience. methods: from july to june we reviewed our experience with adult single allograft recipients of pediatric donors less than months of age. there were no exclusions based on age or size with exclusion criteria consisting of donor age < months and single allograft size < cm. all but recipient received rabbit anti-thymocyte globulin induction, tacrolimus, mycophenolate mofetil, and rapid steroid withdrawal. results from this cohort were compared to consecutive recipients of adult single allografts from standard criteria donors with the same immunosuppression protocol used as historical controls. results: pediatric single allografts with median donor age of months (range - ) were transplanted into adults with median age years (range - ). showed that non-white race was associated with increased risk of death (p< . ). this effect of race was attenuated when ltx center was taken into account [b] . finally, with the addition of meld in the model, the effect of race on waitlist mortality all but disappeared [c] . conclusions: on the surface, minority patients may appear to have higher mortality on ltx waitlist compared to caucasian counterparts. however, this association is predominantly a result of minority patients having a higher meld score, although ltx center-specific mortality may contribute. these data suggest that waitlist outcome may be improved by optimizing referral of minority patients. background: in cirrhotic patients awaiting liver transplantation (lt), low serum sodium (na) predicts short term pre-lt mortality, independently of meld. incorporation of na into meld has been recommended to improve prognostic accuracy (meld-na; gastroenterology : gastroenterology : , ). however, short term interventions such as water restriction that improve na may have little effect on prognosis. hypothesis: the lowest level of serum sodium in the preceding (na ), (na ) or days (na ) may be better than the current serum sodium (nac) for predicting pre-lt cirrhotic mortality. methods: we reviewed electronic records of cirrhotic veterans referred for consideration of lt, / / - / / . date of most recent na at referral was chosen as time zero for determining nac, na , na , and na and for assessing subsequent survival. findings: within days, patients died pre-lt ( %) and underwent lt ( %). na at all time points was associated strongly (p<. ) with prelt death (censored at lt). areas under receiver operating characteristic curves (aurocs) for na , na and na as predictors of d prelt mortality (mean±se) were . ±. , . ±. and . ±. , respectively, compared to . ±. for nac (all p<. vs. nac). on multivariable logistic regression analysis, meld and na were independent predictors of d prelt mortality, with best discrimination given by the following model: meld-na = meld + ( -na )* . with value of na capped at . aurocs for meld-na , meld-na, and meld were . ±. , . ±. and . ±. , respectively. findings were similar when patients with hcc at referral (n= ) were excluded (aurocs . ±. , . ±. and . ±. , respectively), and when day survival endpoint was changed from "death censored at lt" to "death or lt" (auroc's . ±. , . ±. , and . ±. , respectively). conclusion: short term improvement in na may mask true prelt mortality risk. the lowest na in the preceding days is a better prognostic indicator than current sodium. substitution of meld-na for meld would permit more accurate "sickest first" organ allocation, while at the same time allowing prelt correction of na without loss of priority. prospective validation of meld-na , in comparison to meld-na and meld, is warranted. hyponatremia does not affect survival following liver transplantation. byung cheol yun, w. ray kim, y. s. lim, joanne t. benson, walter k. kremers, terry m. therneau. gastroenterology and hepatology, mayo clinic college of medicine, rochester, mn. background: hyponatremia is a common yet important complication of cirrhosis. serum sodium (na) has been found to be an important predictor of survival in patients with cirrhosis. models incorporating na have been proposed for liver allocation. concerns have been raised, however, that liver transplantation (ltx) in hyponatremic patients will adversely affect the outcome. in this work, we assessed the effect of pre-ltx na on the short term survival following ltx. methods: patient-level data on all waitlist registrants in the us for and were obtained from the organ procurement and transplantation network. demographic, clinical and laboratory data at the time of ltx and outcomes following ltx were extracted. the relationship between na pre-ltx and survival post-ltx was analyzed using multivariable regression analyses. results: there were primary transplants that met the inclusion criteria between and . the median na in meq/l at the time of ltx was (interquartile range, iqr: - ). there were patients who had a na ≤ meq/l. the mean meld score was . (sd . ). median follow up was (iqr: - ) days. the overall -and -day survival post-olt was % and %, respectively. in a multivariable logistic regression model, meld was associated with . -fold increase in -day mortality ( confidence interval: . - . ), while na did not have impact on survival (hr= . , % ci: . - . ). the figure represents the risk of -day mortality according to na after adjustment for meld, which clearly shows absence of mortality increase over a wide range of na. conclusions: hyponatremia at the time of ltx has no detrimental impact on short term patient survival following ltx. although these data do not address morbidity (e.g., central pontine myelinolysis), there is no evidence that incorporation of na in organ allocation will lead to diminished survival. objective. this study examined the relationship between meld at liver transplantation (lt) and post-lt quality of life (qol). methods. adult lt recipients (n = ) at two centers completed the sf- and transplant symptom frequency questionnaire (tsfq) -year post-lt. high sf- scores indicate better qol; high tsfq scores indicate more symptomatology. clinical (lab) meld at lt, demographic characteristics, presence of ascites, encephalopathy, and variceal bleeding pre-lt, current employment status, presence of co-morbid medical conditions, and bmi were collected from medical records. results. primary lt indication was viral hepatitis ( %), cholestatic liver disease ( %), or hepatocellular disease ( %), and % had ascites, % encephalopathy, and % gastroesophageal bleeding. mean meld at lt was ± . there was almost no correlation between meld and sf- physical (r = . ) and mental (r = . ) functioning. statistically significant yet weak correlations were found between meld and physical functioning (r = - . ) and role functioning -physical (r = - . ). meld was not significantly correlated with any other sf- scales (r's - . to - . ). meld was not significantly correlated with any tsfq domains: affective distress (r = . ), neurocognitive symptoms (r = . ), gastrointestinal distress (r = - . ), physical appearance changes (r = . ), appetite and weight changes (r = . ), and miscellaneous symptoms (r = . ). older age (ß = - . ), female sex (ß = - . ), viral hepatitis (ß = . ) or cholestatic disease (ß = . ), higher bmi (ß = - . ), and > medical co-morbidity (ß = . ) were significant predictors of lower qol as measured by the sf- (adj r = . , f = . , p < . ). older age (ß = . ), female sex (ß = . ), higher bmi (ß = . ), history of variceal bleeding (ß = . ), and > medical co-morbidity (ß = . ) were predictive of more symptoms on tsfq (adj r = . , f = . , p < . ). meld was not predictive of qol. conclusions. higher disease severity, as measured by meld, at lt does not portend a worse qol outcome for patients -yr after transplantation. other pre-lt indicators of decompensation also do not predict post-lt qol. post-lt qol is affected more by other variables, including age, sex, bmi, and medical co-morbidities. introduction: racial disparities in access to cadaveric renal allografts have been well described for renal transplantation. however, little is known about differences in orthotopic liver transplantation (olt) rates for patients of minority racial groups following listing. the purpose of the current study was to determine if there is difference in rate of transplantation among racial groups and to examine the potential reasons for the disparity. methods: the united network for organ sharing (unos) database was obtained. data was extracted for adult olts greater than years of age performed from / - / . transplants for which recipient race or model for end-stage liver disease (meld) score at listing were unknown and patients active on the list were excluded. rates of transplantation as well as differences in reasons for de-listing (transplantation, death/deterioration, and improvement) were examined. in an effort to examine only patients with chronic liver disease, further analysis was performed excluding patients with acute fulminant liver failure and retransplants. results: the database contained complete meld and race information on , olts. seventy-four percent of patients were caucasian, % hispanic, % african-american, and % were asian. as seen in table , laboratory meld score at removal differed between racial groups. examining pair-wise comparisons of the three minority groups to caucasians, only hispanics differed in reason for delisting (table ) . subgroup analysis excluding acute hepatic failure patients and retransplants showed similar results with hispanic patients being more likely to die/deteriorate as compared to other racial groups ( % deaths vs. % deaths for caucasians), and being less likely to receive a transplant ( % of hispanics vs. % of caucasians, p< . ). conclusion: hispanic patients, although listed with higher meld scores, are transplanted less often than caucasian patients and are more likely to die/deteriorate while awaiting olt. reasons for this discrepancy are unclear and merit further attention. background: since the implementation of the model for end-stage liver disease (meld) for liver allocation, an increasing number of candidates with renal insufficiency have undergone orthotopic liver transplantation (olt). since candidates with renal insufficiency have higher post-transplant morbidity and mortality, meld-based allocation may be shifting some waiting list mortality to the post-transplant period in these candidates. the objective of this study was to evaluate the survival benefit among candidates with renal insufficiency who underwent olt. methods: scientific registry of transplant recipients data for adult candidates age ≥ initially listed for olt between / / and / / (n= , ) were analyzed. the effect of serum creatinine on the survival benefit (contrast between waiting list and post-transplant mortality) was assessed by sequential stratification, an extension of cox regression. each recipient was matched with candidates active on the waiting list in the same organ procurement organization with the same meld score. results: for meld scores - , the survival benefit of olt significantly decreased as serum creatinine increased. among candidates transplanted at meld - , the % with serum creatinine > . mg/dl ( %) experienced no significant survival benefit ( figure) . candidates transplanted at meld ≥ experienced significant olt benefit irrespective of serum creatinine level. conclusions: comparing two patients with meld ≥ , the patient with higher creatinine experiences significantly less survival benefit from liver transplantation. almost onequarter of patients transplanted at meld - experienced no survival benefit from olt based on years of follow-up. therefore, more careful assessment of candidates is required in order to maximize the survival benefit gained by the wait-listed end-stage liver disease population as a whole. liver: living donors and parial grafts i assessment introduction: consideration of the risks and benefits of a procedure are critical in medical decision making. however, relatively little is known about risk tolerance amongst donors and transplant professionals in live-donor liver transplantation (ldlt). we conducted confidential semi-structured interviews in a convenience sample of donors, non-donors (individuals who had been assessed for donation but did not donate) and transplant team members. in addition to examining issues surrounding decision making for ldlt donation, we sought to assess the tolerance of participants, above which they would no longer contemplate donation, for a number of potential outcomes following ldlt. the outcomes that participants were asked to consider included their tolerance for risk of donor death, risk of serious donor complication, as well as risk of recipient death following transplantation. the interviews were conducted sequentially, data was coded quantitatively, and the study terminated once saturation was reached. (pre, weeks , , , and post-donation) . ambivalence detected by staff or described by donor was recorded. donor and recipient characteristics were examined and compared between ambivalent and non-ambivalent groups. results: staff identified and self identifed ambivalent donors were not equivalent. staff assessments indicated ambivalent donors ( male, female). donors self-identified as ambivalent ( male, female); donors were on both lists ( male, female). the combinations of brother to brothers and sons to fathers were the most common pairs among ambivalent donors and more common than in total donor cohort. recipient diagnosis of alcohol or hepatitis c related liver disease was more common in ambivalent donors. ambivalent donors were more likely to be college educated and to express significant religious affiliations than the total rhl donor group. all but ambivalent donor indicated that they would donate again on the year qol survey. conclusions: ambivalence about rhl donation is present in approximately % of candidates who complete donation. staff-identified and self-identified groups showed only % overlap; however, both groups showed similar characteristics. brother-tobrother and son-to-father pairings and recipients with perceived self-induced liver failure were more common in both groups compared to total donor cohort. ambivalent donors had more education and stronger religious or spiritual identification than the entire cohort. only donor indicated persistent doubt about donation. these results suggest that expressed or perceived donor candidate ambivalence may represent a process of careful consideration and should not be used sole basis for donor disqualification. the impact of donor age on recipient outcome for adult right-lobe living donor liver transplantation (rldlt) is unclear. aim: to analyze the effect of donor age on recipient outcome following rdldt. methods: since we have performed rldlt (mean donor age years, range - years), including donors age years or older. we analyzed the effects of donor age, as a continuous or categorical (< vs > years) variable, on recipient outcome. recipient outcome measures included biochemical markers of hepatocytes injury (ast, alt) and graft function (inr, bilirubin), postoperative infections, bleeding, biliary complications, acute cellular rejection, as well as patient and graft survival. analyses were carried out stratified for higher recipient meld scores (< vs. > ), recipient age (< vs. > years), and hepatitis c virus (hcv) infection (presence vs. absence). results: -year patient and graft survival after rldlt was % and %, respectively. donor age as a continuous variable was associated with increased ast (p= . ) and alt (p= . ) release after transplantation, while no effect was observed on inr or bilirubin. rldlt using donors above years of age resulted in an increased incidence of biliary strictures ( % vs. %, p= . ), postoperative cholangitis ( % vs. %, p= . ). no effect of donor age was found for the following recipient outcome measures: the number of bile ducts supplying the graft, type of biliary reconstruction required; rejection, hemorrhage, pulmonary or urinary tract infections, renal failure, or length of hospital stay. -year patient survival was identical for patients receiving grafts from donors below or above years of age ( % vs %, p= . ). similarly, -year graft survival was comparable for young and old grafts ( % vs %, p= . ). recipient age (< vs > years), recipient meld score (< vs > ), or hepatits c status of the recipient did not impact on the effect of age on patient or graft survival. conclusion: in this single center series of rldlt, the use of selected older donors did not impair graft and patient survival, but was associated with an increased rate of biliary strictures. background: biliary stricture rate after living donor liver transplant (ldlt) in adults remains relatively high in comparison to the stricture rate after adult cadaveric liver transplant or ldlt in pediatric patients. the etiology or risk factors for biliary stricture development at present time are uncertain. purpose: to determine the risk factors for biliary stricture after right lobe (rl) ldlt. methods: from / to / , ldlt procedures were performed in adult recipients. eleven patients were excluded from analysis due to < days follow up or need for retransplant. the following data was prospectively collected: . demographics, . acuity of illness, . number of bile ducts, . type of biliary reconstruction, . graft to recipient weight ratio, . hemodynamic parameters, . outcomes. these parameters were compared in patients with and without strictures. results: mean follow-up for patients is days (range: - ). of patients died during the follow-up range and required whole liver re-transplants. patients ( %) developed a biliary strictures during the follow-up period. comparison of risk factors in patients with and without strictures revealed the following results: mean meld > bile duct grwr* < . neither meld score, number of bile ducts or type of biliary reconstruction appear to be contributing factors to the development of bile duct stricture following rl ldlt. the biliary stricture rate was related to the volume of transplanted liver and post transplant graft recovery. therefore, the development of biliary strictures in some patients may represent yet another feature of small-for-size syndrome. background: ox and cd can be expressed by both foxp + tregs and activated t effector cells. however, the question as to how ox and cd function, individually or collectively, in regulating such functionally different t cell subsets in transplant models remains poorly understood. in some models, blocking cd costimulation is remarkably effective in prolonging graft survival, but targeting cd alone rarely creates tolerance. but the role of ox in regulating the cd blockade induced tolerance is completely unknown. in the present study we critically examined the role of ox in the activation of cd deficient t effector cells as well as in the regulatory function of foxp + tregs. we also examined the effect of ox on the induction of new foxp + tregs/th cells from activated cd deficient t effector cells. the impact of ox in the induction of allograft tolerance was examined using an islet transplant model. we found that cd deficient foxp + tregs constitutively expressed ox on the cell surface, but the cd deficient t effector cells did not. however, when the t effector cells were sorted and stimulated in vitro, ox expression could be abstracts readily induced on the t effector cells. to further examine how ox regulates such functionally different t cell subsets, we found that ox delivers potent costimulatory signals to t effector cells, which prevent the induction of new foxp + tregs from activated t effector cells but promote their differentiation to th cells but not th cells. surprisingly, ox costimulation to cd deficient foxp + tregs completely inhibited their regulatory functions. in an islet transplant model, we showed that cd deficient mice can reject the dba/ islet allografts, but blocking ox costimulation readily induced donor specific tolerance (mst> days), and this tolerant status was critically dependent on the induction of foxp + tregs. in contrast, treatment of cd deficient recipients with a agonist anti-ox mab precipitate rapid islet allograft rejection, suggesting that ox costimulation is critically important in the induction of transplant tolerance. conclusions: our data suggest that ox is a costimulatory molecule to t effector cells but a powerful negative regulator for foxp + tregs. thus, a key role for ox in the induction of transplant tolerance is the control of t cell mediated regulation. background: foxp is a winged-helix family transcription factor that is the master regulator for the development and function of regulatory t cells (treg). we investigated the molecular mechanisms important for regulation of foxp expression, and defined the structure of the active foxp promoter in cd + t cell lineages. methods: purified cd + cd -foxp -gfp -t cells (naïve) and cd + cd + foxp + gfp + treg were cultured with antigen presenting cells in the presence of il- , anti-cd ε mab, tgfβ or the dna methyltransferase inhibitor -aza- '-deoxycytidine (zdcyd). foxp promoter structure and activity were monitored with methylation-specific pcr, disulfite-sequencing, chromatin immunoprecipitation (chip) assays, electrophoretic mobility shift assay (emsa) and luciferase promoter assay. the foxp promoter has an upstream cpg island ∼ kb from the transcriptional start site. disulfite-sequencing and methylation-specific pcr analysis showed that this region is heavily methylated in naïve cd + t cells and tgfβ induced peripheral treg, but demethylated in thymic derived natural treg (ntreg). chip analysis showed that the methylated cpg island is bound specifically by the dna methyltransferases and b. zdcyd causes demethylation of the cpg island, and in combination with tgfβ, synergistically induces foxp expression. chip assays for acetylated histone and sp , both markers of gene activation, showed that the cpg island is acetylated and bound by sp in ntreg and zdcyd plus tgfβ induced treg, but not in activated cd + t cells or tgfβ induced treg. emsa likewise shows the cpg island binds sp . in contrast to the upstream promoter, the structure of the first intronic promoter differs markedly between ntreg and tgfβ induced treg, but is not affected by zdcyd. the upstream cpg island also possesses enhancer activity that is repressed by dna methyltransferases. zdcyd plus tgfβ induced treg have stable foxp expression and enhanced suppressive functions in vitro and in vivo. conclusion: these results demonstrate that ntreg and tgfβ induced treg are genetically distinguished from each other by the epigenetic structure of a unique upstream cpg island of the foxp promoter. the function of this region is regulated by dna methylation and histone acetylation. zdcyd demethylates the promoter, leading to enhanced and stable expression of foxp and suppressor activity, similar to ntreg. this has important implications for biology, and generating treg for tolerance. chemokine background: trafficking of lymphocytes through lymphatics to secondary lymphoid organs is crucial for immune responses. we previously showed that regulatory t cell (treg) function required trafficking from the inflammatory graft site to the local draining lymph node (dln). since the mechanisms that regulate migration through afferent lymphatics are poorly understood, we explored the role of chemokine receptors on treg for afferent lymphatic migration in an islet transplantation model. methods: islets were transplanted from balb/c mice into foxp gfp c bl/ mice. treg from wild type, ccr -/-, ccr -/-, ccr -/-, or ccr -/-c bl/ mice were isolated, labeled with red dye pkh , and transferred intravenously, or locally into the islet allograft. treg migration to islet grafts and dln was determined by flow cytometry and immunohistochemistry. endogenous foxp gfp+ treg and transferred pkh labeled treg were sorted from the islet grafts and the dln, and chemokine receptor and sphingosine -phosphate receptor (s p ) expression were determined by rt-pcr. islet allograft survival was determined by measurement of blood glucose. results: freshly isolated treg expressed s p and the chemokine receptors ccr , ccr , ccr , and ccr . endogenous treg, and both intravenously and locally transferred treg, that were recovered from islet allografts and dln expressed similar levels of ccr and ccr . ccr was expressed preferentially on islet migrating treg, while s p and ccr were expressed preferentially in dln migrating treg. locally transferred treg migrated to the dln, but ccr -/-treg were not able to migrate to the dln. ccr -/and ccr -/-treg were impaired in their ability to migrate to the dln. this suggested that these three chemokine receptors all regulated treg entry into afferent lymphatics and migration from the graft to the dln. in contrast, ccr -/-treg migrated normally from the islet to the dln. importantly, ccr -/-, ccr -/and ccr -/-, but not ccr -/-treg, were impaired in their ability to prolong islet allograft survival when transferred locally in the islet allograft. conclusion: treg migrate from the inflammatory site of the allograft to draining secondary lymphoid tissue through afferent lymphatics. this process depends on ccr , ccr , and ccr ; and is crucial for full treg function in vivo. these results demonstrate a novel role for sequential migration from the graft to the dln in treg function and suppression. epigenetic regulation of gene expression provides a major, and especially beyond oncology, largely unexplored means to regulate host immune cell functions. our ongoing analysis of histone deacetylase (hdac) expression by foxp + naturally occurring murine regulatory t (treg) cells showed tcr-activated tregs had - fold more hdac mrna than corresponding resting treg or non-treg cells. in various cell types, hdac deacetylates alpha-tubulin, cortactin, and hsp , abrogates formation of the aggresome, and blocks the unfolded protein response, though nothing is known regarding these pathways in tregs. we found that an hdac -specific inhibitor, tubacin (but not the control compound, niltubacin), increased treg suppressive function in vitro (p< . ), in association with increased expression of ctla, il- , gitr, pd- and other treg-associated genes (p< . ), and increased treg foxp protein (though not mrna) expression. tubacin enhanced the conversion of cd +cd -cells into cd + foxp + treg in vitro, and globally decreased cytokine production, with the exception of il- and il- mrna. comparable and dose-dependent effects were seen using the hsp inhibitor, geldanamycin, suggesting that the effects of hdac inhibition were mediated, at least in part, by blocking the chaperone effect of hsp . use of tubacin in vivo significantly decreased the severity of colitis in two murine inflammatory bowel disease models (p< . ), dextran sodium sulfate-induced colitis and the cd +cd lhigh adoptive transfer model of colitis, as assessed by standard clinical and histologic criteria. in addition, days combined use of tubacin and a subtherapeutic dosage of rapamycin led to significantly prolonged cardiac allograft survival (balb/c->c bl/ ) compared to use of either agent alone (p< . ). our data show that use of the first known small molecule inhibitor of one specific hdac has important therapeutic effects, including enhancing the production and suppressive function of tregs. while ongoing studies are directed towards unraveling the interactions of hdac -dependent pathways and treg functions, the current data indicate the importance of understanding the functions of hdacs to the development of entirely new ways to regulate host immune responses. dendritic cells supply paracrine il- for treg cell functional activity. regulatory cd +cd + t cells (tregs) are important for the maintenance of immune tolerance, and immunotherapy with tregs is being explored for organ and cell transplantation. treg development, expansion and function depend on il- . because tregs do not make il- , they must obtain il- from another cell. although cd + teffectors are a logical candidate, the identity of the paracrine source of il- for tregs is not substantiated. we explored whether dendritic cells (dcs) could serve as the paracrine source of il- for treg and rd , a cd +cd + regulatory hybridoma. using four dimensional live cell imaging we demonstrate that treg and rd cells establish tight contact with dcs, and cd is localized at these contacts. using the il- elispot and real-time rt-pcr we found that splenic dcs and the jawsii dc cell line constitutively make il- . lps and cpg increases dc production of il- . co-culture with jawsii dc cell line significantly upregulates cd expression on alloreactive do . tregs and rd cells, but not on do . cd + teffector cells. tregs and rd cells are functionally suppressive after activation by wild type but not il- knock-out allogeneic dcs, and anti-cd inhibits the function of treg and rd cells in a dose response fashion. in contrast, wild type and il- knock-out dcs are equally able to activate alloreactive cd +cd -cells. supplemental il- at high ( u/ml) but not low doses ( u/ml) restores the function of alloreactive tregs and rd that were activated by il- ko dcs. these data indicate that treg cells acquire il- from dendritic cells for their gain of function and validate dendritic cells as a paracrine source of il- for treg. introduction: previously, it has been demonstrated that foxp , a gene required for the development and function of regulatory t cells, was highly expressed in the graft during cardiac rejection, suggesting infiltration of regulatory t cells in the transplanted organ during an allogeneic response. in this study, we investigated whether graftinfiltrating t cells expanded from rejecting human cardiac allografts exhibit immune regulatory activities. methods: graft-infiltrating lymphocytes (gils) cultured from endomyocardial biopsies (emb; n= ) with histological signs of acute cellular rejection were expanded in the presence of donor-antigens in il- /il- -enriched medium for - weeks. flow cytometry was used to analyze the expression of cd , cd , cd and foxp . to analyze the immune regulatory function, we performed mlrs with peripheral blood mononuclear cells (pbmc) of the patients and irradiated donor or third party spleen cells in the absence and presence of gils (ratio : ). results: of the cd + gils, % (median; range: - %) stained positive for foxp . this foxp expression was detected in both cd + and cd + t-cell population (median: % and %, respectively). functional analysis demonstrated that gils suppressed the antidonor proliferation of responder t cells (range % inhibition: - %). interestingly, this suppression was predominantly achieved by cd + gils: depletion of cd + cells from the gils population diminished the inhibitory effect, whereas addition of solely cd + gils to the mlr abundantly suppressed the anti-donor response (range % inhibition: - %). in contrast, gils did not inhibit the proliferation of t cells stimulated with third-party antigens. the figure below depicts a representative example. graft-infiltrating lymphocytes expanded from rejecting cardiac allograft exhibit donor-specific immune suppressive activities. these results suggest that during acute cellular rejection, graft-infiltrating lymphocytes not only consist of graft-destructing effector t cells, but may also comprise immune regulatory cells of the cd + phenotype. the context: it has been previously suggested that a liver allograft is immunoprotective and able to decrease the rate of rejection of a donor-specific allograft of another organ. it has been recently proposed that allografts other than the liver may also be immunoprotective. objective: the aim of this analysis was to examine one year rejection rate and the incidence of rejection free survival of all combined transplants in the collective us experience to gain insight to any possible protective effect of one organ for another. methods: the united network of organ sharing (unos) provided de-identified patientlevel data. analysis included all recipients transplanted between january , and october , who were years or older (except intestinal transplants). rejection at one year was defined as treatment for one or more episodes of rejection. results: analysis included a total of , patients who received either one, or combined, simultaneous or sequential, organ transplants in all possible combinations. results are summarized in figure (one-year organ allograft rejection rate). the collected data demonstrate that the rejection rate of donor-specific organ allografts which accompanied primary liver, kidney, and heart transplants was significantly lower in combined transplants as compared to that of the primary allograft transplanted alone. this was not true, however, for intestinal and pancreatic allografts where protection for the accompanying organ was not observed. we further demonstrate that transplantation of two organs of the same type (double kidneys or double lungs), i.e. increase in antigen load, also leads to decreased rates of rejection of the allografted organs. conclusions: in combined simultaneous transplants, the heart, liver, and kidney allografts appear themselves to be protected, and to protect the other organ from rejection. increased antigenic load of identical antigens in case of double lung and double kidney transplants appears to also offer immunologic protection against rejection, perhaps by different mechanisms. background: a all ( -center adult-to-adult living donor liver transplantation cohort study) has identified risk factors for mortality after aaldlt, including center experience. the aim of this study was to determine if a all findings are reflected in the national experience. methods: aaldlt at a all (n= ) and non-a all centers (n= ) from / / to / / in the scientific registry of transplant recipients database were analyzed. cox regression models adjusted for recipient and donor characteristics were fitted to test associations with mortality risk after aaldlt, including center type (a all vs. non-a all) and case number (for each aaldlt at each center). results: aaldlt were performed at a all and non-a all centers. there was no significant difference in overall mortality risk between a all and non-a all centers. significant predictors of death (both groups combined) included donor age (hazard ratio (hr)= . per years, p= . ), recipient age (hr= . per years, p< . ), diagnosis of hcv (hr= . , p= . ) or hcc (hr= . , p< . ), and earlier center experience (aaldlt case number ≤ , hr= . , p< . ). there was no significant effect of transplant year after adjusting for experience. cold ischemia time > . hours was associated with higher mortality (hr= . , p= . ); this effect was similar in a all and non-a all centers. there were no significant interactions between center type and any predictor except center experience ( figure) . compared to later experience, earlier center experience was associated with significantly higher mortality risk in both a all (hr= . , p< . ) and non-a all centers (hr= . , p< . ). survival during early experience was significantly worse at a all vs. non-a all centers (hr= . , p= . ), but survival in later experience was similar. conclusions: after the first cases, aaldlt survival was similar at a all and non-a all centers, and similar significant mortality risk factors were identified, including center experience. these analyses support the generalization of findings from a all centers to others performing aaldlt. abstract# rejection with hemodynamic compromise (hc) and chronic allograft vasculopathy (cav) impact survival in pediatric heart transplantation (phtx). we showed that high pro-inflammatory / lower regulatory cytokine gene polymorphism (gp) profile increased the risk for acute rejection. in this analysis, we assessed the effect of genetic factors on hc and cav. methods: phtx with clinical and gp data for cytokines (tnf-α a- g; inf-γ t+ a; il- g- a, c- t, c- a ; il- c- t; il- t- c; il- g- c), growth factors (tgfβ- t+ c, c+ g; vegf a- c, c- t, g+ c), effector molecules (fas a- g; fasl c- t) and pharmacogenomics (abcb c t, g t/a) were analyzed regarding hc and cav. results: adjusting for recipient black race and age with cox regression models, we identified the following risk factors: il- high was associated with lower rates of hc. low th (inf-γ, tnf-α) with high th (il- , il- ) cytokine gp profiles were protective for hc in combination with il- high. carriers of fas high experienced higher rates for hc and cav and high fas-fasl combination doubled the relative risk for cav. abcb cc/ gg genotypes were also associated with lower rates of hc (table ) . conclusion: in this large multi-center study gps with higher regulatory profiles and increased drug transport were associated with a lower incidence of hc. a genetic proapoptotic profile might contribute to the pathogenesis of cav. sponsorship: this work was supported by p hl - from the national heart lung and blood institute, national institutes of health. it has recently been reported that cd d-restricted nkt cells that express invariant tcr (inkt cells) play an important role in the production of autoantibodies through the interaction with b- cells. this observation prompted us to investigate the possible role of inkt cells in the production of antibodies (abs) against transplant-related antigens, such as abo blood group carbohydrates and histocompatibility complex allopeptides, in a mouse model. we have previously demonstrated that b cells with receptors for blood group a carbohydrates were found exclusively in a cd b + cd + b- subpopulation of mice, resembling humans with blood group o or b. immunization with human blood group a red blood cells (a-rbcs) elicited the extensive production of anti-a igm and igg. furthermore, the number of b- cells with receptors for a carbohydrates increased in the peritoneal cavity. in cd d -/and vα -/-balb/c mice, which lack inkt cells, such elicited production of anti-a igm was not observed, even after immunization with human a-rbcs. however, class ii -/-balb/c mice, which lack cd + t cells but maintain normal levels of inkt cells, exhibited levels of anti-a igm production comparable to those in wild-type (wt) balb/c mice. moreover, anti-a igg production was absent in cd d -/-balb/c mice even after the immunization, indicating that although inkt cells crucially contribute to anti-a igm production and igg class switching, helper t cells do not. notably, the proportion of b- cells in the livers of cd d -/-balb/c mice was significantly reduced ( . ± . %, n = ) when compared to that in wt mice ( . ± . %, n = ). we next immunized cd d -/and wt balb/c mice twice with × allogeneic b mouse thymocytes, and thereafter detected the anti-b (allopeptides) abs by flow cytometry. in the cd d -/mice, anti-b igm production was comparable to that of wt mice, and igg class switching also occurred normally. these findings indicated that inkt cells play a pivotal role in the production of abs specific for blood group carbohydrate determinants that are believed to be t cell independent, but are not required in the production of abs for allopeptides that are believed to be t cell dependent. the depletion of inkt cells or the suppression of their function might constitute a novel approach for preventing antibody-mediated rejection in abo-incompatible transplantation, or in xenotransplantation, which involves similar carbohydrate antigens. background static cold storage (cs) is the most widely used organ preservation method for deceased donor kidney grafts. retrospective analyses have indicated that preservation by hypothermic machine perfusion (mp) may lead to improved outcome after renal transplantation. however, there is a lack of sufficiently powered prospective studies to test the presumed superiority of mp. in an international prospective randomized controlled trial we enrolled kidney pairs of consecutive deceased donors and randomly assigned one organ to mp and the contralateral kidney to cs preservation. follow-up was directed at all recipients of these grafts. the primary endpoint was delayed graft function (dgf). mp significantly reduced the risk of dgf (or . ; p= . ) and more than halved the incidence of primary non-function after transplantation, when compared to cs ( . vs. . %; p= . ). furthermore, mp significantly reduced the risk of graft failure in the first months post-transplant (hr . ; p= . ). in recipients who developed dgf, -month graft survival was better if their transplanted kidney was machine perfused ( vs. %; p= . ). hypothermic machine perfusion reduces the risk of delayed graft function, primary non-function, and graft failure in deceased donor kidney transplantation when compared to static cold storage. furthermore, mp alleviates the deleterious effect of dgf on graft survival. we investigated the trafficking of cells after skin and heart transplantation in a dynamic fashion through the use of in vivo microscopy. antigen presenting cells were followed using mhc-cl-ii-gfp and cd c-gfp transgenic mice. vascularized and non vascularized skin grafts as well as heart transplants were used in syngeneic as well as allogeneic settings. after syngeneic non-vascularized skin transplantation, we observed an early and massive cellular infiltration of host cells into the graft as early as hours post-transplant with a gradual accumulation in the dermis. the accumulation of host-derived cells was accelerated after graft vascularization at day / post transplantation. this graft infiltration by recipient cells was more pronounced with vascularized skin grafts, and to a higher degree in heart transplants. recipient cells similarly infiltrated allogeneic grafts early on and in larger numbers than for syngeneic grafts by day / post-transplantation. when visualizing mhc-cl-ii-gfp recipient cells in a syngeneic skin transplant, recipient dcs invaded the graft early on and, by weeks post transplant gradually replaced graft dcs in the dermis (dermal dcs) and the epidermis (langerhans cells). donor dcs could still be seen in the graft up to days post transplant. however, virtually all donor langerhans cells were eventually replaced by recipient ones in a concentric fashion suggesting that the new langerhans cells originate from the recipient skin adjacent to the graft and not from centrally-derived precursor cells. the vascular endothelium of a syngeneic transplant was partially replaced by recipient vascular endothelial cells in a centripetal fashion with more recipient-derived vascular endothelium present at the periphery of the graft and more donor-derived endothelium remaining in the center of the graft. therefore, the graft can be seen as a "chimera" of cells from donor and recipient origin. the presence of recipient endothelial vascular cells and dcs within the graft may be important for maintaining the indirect response thought to be responsible for chronic rejection. objective: maturation resistance and tolerogenicity can be conferred on dendritic cells (dc), -crucial regulators of t cells, by exposure to rapamycin (rapa), a tolerance-sparing immunosuppressant. the mechanisms underlying this acquired unresponsiveness, typified by diminished responses to toll-like receptor (tlr) or cd ligation, have not been identified. thus, our objective was to elucidate a molecular basis for rapa-induced dc maturation resistance. methods: rapa administration was used to condition splenic dc in vivo and bone-marrow derived dc in vitro. dc maturation was monitored by assessment of co-stimulatory molecule expression, cytokine production, and t cell allostimulatory capacity. to identify negative regulators of maturation, microarray analysis and quantitative rt-pcr was completed, and findings confirmed via western and flow cytometric analyses. results: in vitro or in vivo exposure of myeloid dc to rapa elicited de novo production of il- β by otherwise immature dc (cd lo ). interestingly, dc il- β production, acting in an autocrine/paracrine fashion, promoted dc overexpression of the il- receptor(r) family member, st l, and enhanced its surface expression. st l is the receptor for il- , an il- family member, and has also been implicated as a negative regulator of tlr signaling. consistent with this regulatory function, il- β-induced st l expression suppressed the responsiveness of rapa-conditioned dc to tlr or cd ligation. conclusion: rapa causes de novo production of il- β by immature dc, upregulating st l, and establishing a barrier to dc maturation following exposure to tlr or cd ligation. as such this work identifies a novel mechanism by which a clinically-important immunosuppressant impedes the capacity of dc to mature and consequently stimulate effector/adaptive t cell responses. these findings are particularly relevant to the potential use of rapa-conditioned dc as "negative" cellular vaccines to block alloag-specific responses, as exposure to endogenous and exogenous inflammatory stimuli can induce dc maturation and negate the tolerogenic properties of immature dc. exosomes are nanovesicles ( - nm) released to the extracellular milieu by different cell types. exosomes secreted by dendritic cells (dcs) and other apcs express mhc ag, adhesion molecules and costimulatory molecules oriented on the membrane surface with their binding domains facing outwards. thus, exosomes released by graft-infiltrating leukocytes (gils) could function as "ag-presenting vesicles" or as vehicles to transfer alloag between recipient's apcs during elicitation of t-cell allo-immunity. aims: to test if (i) gils activate anti-donor t-cells in secondary lymphoid organs by releasing exosomes with alloag into systemic circulation; or (ii) gils that traffic to the spleen as passenger leukocytes use exosomes as a local mechanism to transfer alloag to recipient's dcs. methods: exosomes were isolated from supernatants of bm-derived [c bl/ (b ), ia b ] dcs pulsed with the balb/c iea - allopeptide and purified by ultra-filtration and ultra-centrifugation on a %sucrose/d o gradient. we used pkh + exosomes and cd . congenic b mice for traffic studies, heart (heterotopic) and skin transplantation models (balb/c→b , thy . + ), and cfse-labeled h . tcrtg cd t-cells (thy . + ) specific for ia b (b ) loaded with iea - (balb/c). dcs were genetically engineered to release exosomes expressing green fluorescent protein (gfp). we have previously shown that blood-borne exosomes carrying balb/c alloag are reprocessed by different subsets of splenic dcs for presentation to indirect pathway h . cd t-cells. here, we demonstrated that although gils of cardiac and skin allografts release exosomes ex vivo, they did not secrete enough concentrations of exosomes with alloag into circulation to stimulate donor-reactive t-cells in lymphoid organs. instead, our findings indicate that migrating dcs (generated in vitro or isolated from gils), once homed in the spleen, they transfer exosomes expressing gfp and carrying allopeptides to spleen-resident dcs of the recipient, identified by the congenic marker cd . . conclusion: exchange of exosomes between dcs in lymphoid organs might be a mechanism by which passenger leukocytes transfer alloag to recipient's apcs in secondary lymphoid organs. t cell activation is critical in initiating adaptive immunity, and pkcθ, a novel member of the pkc family, mediates non-redundant functions in the t cell receptor; however, its role in the mediation of allograft rejection remains unclear. this study is aimed at investigating whether alloimmune response can be alleviated by a deficiency of the pkcθ molecule, and whether transgenic expression of anti-apoptotic bcl- methods. wild-type (wt) cardiac allografts were transplanted into pkcθ -/mice, with or without sub-therapeutic anti-cd mab. purified pkcθ -/or pkcθ -/-/ bcl-x l t cells were adoptively transferred into rag -/mice engrafted with cardiac allografts. lymphocyte proliferation assays were performed (cfse). nf-kb activation was assessed by bioluminescence imaging (bli) using luciferase transgenic mice under the control of a nf-kb promoter. results. the cardiac allografts were rejected in a delayed fashion in pkcθ -/mice with increased nf-kb activation; however, sub-therapeutic anti-cd mab (that normally delays rejection of cardiac allograft) induced long-term survival of cardiac allografts. the cardiac allografts were permanently accepted in rag -/mice with adoptive transfer of pkcθ -/-t cells, and the rejection can be elicited by transfer of pkcθ -/-/ bcl-x l t cells. in a lymphocyte proliferation assay, pkcθ -/-t cells displayed greatly reduced proliferation. in response to cd and cd stimulation, pkcθ -/-t cells underwent accelerated apoptosis and reduced th , th , and treg subsets compared to the wt t cells. bcl-x l restored the survival of the pkcθ -/-t cells. conclusions. the results suggest that pkcθ mediates the alloimmune response. bcl-x l transgene prevents pkcθ -/-t cell apoptosis and re-elicits allograft rejection. tolerogenic dendritic cells (dc) are immature, maturation-resistant(mr) or alternatively-activated dc that express mhc molecules and low levels or absent costimulatory signals. although mrdc administration has successfully prolonged allograft survival in murine models, the mechanism of action in vivo remains unknown. aim: to test in vivo if the down-regulation of the anti-donor response induced by donor-derived tolerogenic dc is due to: (i) direct interaction of the tolerogenic dc with donor-reactive t cells or (ii) by reprocessing of the tolerogenic dc into alloantigen (alloag) by recipient apc for interaction with indirect pathway t cells. methods: dc were generated in vitro by culturing balb/c bone marrow cells for - days in medium with gm-csf + il- supplemented with nm α, -( h) vitamin d (vd ). we used a model of heterotopic vascularized allogeneic heart transplantation [balb/c into c bl/ (b )] and cd t cells from h . tcrtg mice that recognize b ia b loaded with the balb/c allopeptide ieα - (indirect pathway) . results: we demonstrated that vd renders dc maturation resistant (vd -mrdc) as vd -mrdc fail to up-regulate co-stimulatory molecule expression, release il- p , or stimulate allo-responsive t cells after challenge with potent dc-maturation stimuli. adoptive transfer (i.v.) of balb/c vd -mrdc (day - ) significantly prolonged survival of balb/c heart grafts in b mice in the absence of immunosuppressive therapy. interestingly, we found that in vivo, balb/c vd -mrdc induced proliferation of indirect pathway h . cd t cells in the spleens of b recipient mice, indicating that reprocessing of the balb/c dc by host (b ) apc does occur. proliferation of h . cd t cells in response to balb/c vd -mrdc resulted in defective activation (cd l high , cd low ) of h . t cells, leading to their peripheral deletion and outgrowth of cd + foxp + treg cells. reprocessing of balb/c vd -mrdc was performed by recipient splenic cd c high cd α neg dc, and donor alloag continued to be presented through the indirect pathway for days after donor dc administration. conclusion: these results suggest that dc-based therapies downregulate t cell allo-immunity and prolong allograft survival, at least in part, through reprocessing of the tolerogenic dc into alloag by recipient apc. early introduction: alloreactive memory t cells are present in all transplant recipients due to prior direct sensitization or heterologous immunity. these cells are known to circumvent tolerance induction and/or prevent indefinite graft survival in several models, but mechanistic details of their function are unknown. the goal of this study was to test the hypothesis that cd memory t cells initiate alloreocognition and express effector functions within hours of reperfusion. methods: syngeneic or a/j (h- a ) hearts were transplanted into wt c bl/ (h- b ), cd -/-, cd -/-, or rag -/-recipients. rna and protein were prepared from total graft homogenates and analyzed by qrt-pcr and elisa. rag -/-mice received x wt or ifng-/- c cells and were used as recipients weeks after reconstitution. donor-specific cd memory cells were purified from wt spleens weeks after a/j skin grafting, and donor-specific effector cd cells were purified from spleens of cd . mice days after a/j heart transplantation. flow cytometry was used to quantify graft infiltrates. results: allografts contained elevated levels of ifng and cxcl mrna at , and hrs post-transplant vs. isografts. detectable cxcl protein was produced in allografts from wt and cd -/-recipients but not in isografts or allografts from cd -/-or rag -/recipients. treatment with ctla -ig and mr failed to reduce cxcl production. reconstitution of rag -/-mice with ifng sufficient or deficient c tcr transgenic cd cells indicated that early allospecific cxcl production absolutely requires ifng made by recipient cd cells. although donor-specific ifng production was undetectable in splenocytes until day - post transplant, graft-infiltrating cd hi cd l lo cd t cells were present as early as hrs post-transplant. in adoptive transfer studies, effector-memory cd t cells reconstituted early allospecific cxcl production in cd -/-mice. lastly, primed cd t cells adoptively transferred at day post-transplant readily infiltrated allografts in control but not cd depleted recipients. conclusions: cd memory t cells infiltrate allografts rapidly post-transplant, produce ifng, and propogate an inflammatory environment which optimizes recruitment of primed effector t cells. successful neutralization of this early allorecognition pathway should provide valuable adjunctive therapy to improve graft function and survival. background: allogeneic t cell stimulation requires not only antigen-specific signals but also costimulatory signals, most importantly between cd / on the antigen presenting cell (apc) and cd and ctla on the t cell. engagement of the t cell receptor without costimulation can lead to anergy and the induction of regulatory t cells (tregs). t cell activation is also controlled by expression of the tryptophan-catabolising enzyme indoleamine , -dioxygenase (ido). depletion of this essential amino acid, and/or the production of tryptophan metabolites inhibits t cell proliferation. methods: a genetic approach to confer tolerogenic properties on murine dendritic cells (dcs) has been explored using lentiviral vectors, based on the equine infectious anaemia virus. firstly, an intracellular method that prevents costimulation has been developed: a fusion protein consisting of ctla and kdel [an endoplasmic reticulum (er) retention signal] is expressed in dcs. the ctla -kdel binds to cd / in the er and prevents expression of these proteins on the dc surface. a second approach uses an elevated expression of the ido enzyme by transduced dcs. results: ctla -kdel-or ido-transduced dcs were unable to induce allogeneic t cell proliferation. however, using two-stage dc:t cell co-culture assays, it was shown that ctla -kdel-, but not ido-transduced dcs, can induce donor-specific t cell anergy in vitro and in vivo. tolerance to both the direct and indirect pathways was shown using ctla -kdel-transduced dcs. linked suppression was mediated by the generation of donor-specific tregs. ido-transduced dcs did not generate tregs. furthermore, it was shown separately that dcs expressing ido whilst lacking cd / expression for potential ligation by ctla (although ctla -cd / ligation upregulates ido, it downregulates t cell activation) failed to generate or even sustain foxp + treg populations. the ability of the transduced dcs to induce tolerance to allografts was assessed in a complete mismatch and cbk→cba (indirect pathway) corneal graft model. these results support a clinical strategy to induce treg-mediated, donorspecific transplantation tolerance using ctla -kdel-, rather than ido-expressing dcs. indirect cd t cells that recognise processed alloantigen on recipient apc can provide help to alloreactive cytotoxic cd t cells that recognise intact mhc i alloantigen on donor apc, but exactly how such 'un-linked' help is provided is not clear. the respective abilities of direct and indirect pathway cd t cells to provide help for cytotoxic cd alloimmunity were examined in a mouse model of heart graft rejection in which the recipients contain only monoclonal helper cd t cells, specific for self-restricted h-y antigen (female b mar/rag -/mice). mice were additionally reconstituted with b cd t cells, and then challenged with female balb/c (no cd t cell help), or male balb/c (indirect pathway help), or male b xbalb/c f hearts (direct pathway help) . un-reconstituted mar/rag -/mice lack effector b and cd t lymphocytes, and consequently all heart grafts survived indefinitely. in contrast, reconstituted mar/ rag -/mice rejected male f grafts rapidly (mst d), whereas female balb/c grafts survived indefinitely, confirming a cd -dependent effector role for the transferred cd t cells. cd t cell help through the indirect pathway, although sufficient to elicit graft rejection, was less efficient than direct pathway help, because male balb/c grafts were rejected more slowly than the f grafts (mst d, p< . ). we next considered whether indirect pathway cd t cells provide help through recognition of mhc ii complexes on the surface of alloreactive cd t cells, in analogous fashion to the cognate interaction between b and t lymphocytes. in support, reconstitution of mar/rag -/recipients with instead, mhc ii-deficient cd t cells, resulted in slower rejection of male balb/c hearts (mst d, p< . ), whereas male f grafts, that still permit provision of linked help, were rejected at the same tempo. most tellingly, mar/rag -/mice that received simultaneously a female balb/c heart and male b apc (to activate mar cd t cells) rejected their grafts rapidly when reconstituted with male cd t cells (mst d). in contrast, grafts survived indefinitely when female cd t cells were transferred. flow cytometric analysis of mitogenstimulated cd t cells revealed surface mhc ii expression. indirect allorecognition can provide help for generating cytotoxic alloimmunity, but not as effectively as through the direct pathway. indirect pathway help is potentiated by linkage through recognition of cd mhc ii. purpose: tolerogenic properties of dendritic cells (dc) are supported and preserved by conditioning with the immunosuppressant rapamycin (rapa). the ability of rapaconditioned, recipient-derived dc pulsed with alloantigen (alloag) to suppress both direct and indirect alloag-specific t cells in the absence of immunosuppression has been demonstrated in a murine allograft model. dc can acquire intact mhc from cells or cell lysates. however, the ability of alloag-pulsed rapa-dc to immunomodulate directly-reactive alloag-specific t cells has not been formally demonstrated. methods: dc were generated from c bl/ (b ; h b ) bone marrow cells in gm-csf and il- . rapa was added to indicated cultures (rapa-dc) beginning on day (d) . on d , cd c + bead-purified rapa-dc or non-treated control dc (ctr-dc) were incubated with balb/c (h d ) splenocyte lysates ("alloag pulsing"). following incubation, the dc were harvested and the level of donor and recipient mhc molecules on cd c + cells determined by flow cytometry and immunofluorescent imaging. surface levels of cd , b -h (programmed death ligand- ; pd-l ), and fas-l were compared. pulsed-dc were also incubated with cd + t cells from rag -/- c mice for d. c cd + cells express t cell receptors specific for h -l d , a mhc class i molecule of balb/c. following incubation, c cell proliferation and apoptosis were both assessed. results: ctr-and rapa-dc presented detectable levels of directly-transferred mhc class i and ii on their surface after incubation with allogeneic balb/c cell lysate. donor mhc presented by "pulsed" recipient dc stimulated directly-reactive, alloag-specific c t cell proliferation. however, only rapa-dc induced apoptosis in the overwhelming majority of these cells responding via the direct pathway. induction of apoptosis correlated with an increased level of surface fas-l on rapa-dc and their comparatively low level of cd relative to pd-l . conclusions: rapa-conditioned dc can present intact mhc molecules acquired from lysates of allogeneic splenocytes and concurrently induce apoptosis of directlyreactive alloag-specific cd + t cells. as such, we provide mechanistic insight into a mechanisms by which alloag-pulsed, recipient-derived rapa-dc may facilitate allograft tolerance. background: t regs actively regulate alloimmune responses and promote transplant tolerance. atg, a widely used induction therapy in organ transplantation, depletes peripheral t cells but may preferentially spare t regs . sirolimus is thought to expand natural t regs . b t cell costimulatory blockade inhibits effector t cell (t eff ) expansion and may promote regulation. we investigated the effect of combining mouse atg (matg), ctla ig and sirolimus on stringent skin allograft survival, and studied the mechanisms by determining t reg /t eff balance in vivo using a unique model (abm-tcrtg-foxp /gfp reporter mouse conclusion:this is the first report to establish that t cell depletion with matg combined with ctla ig and sirolimus synergize to prolong stringent fully allogeneic skin allograft survival by promoting regulation and tipping the t reg /t eff balance by both preserving t regs and facilitating generation of new t regs by a conversion mechanism. these results provide the rationale for translating such a novel therapeutic combination to promote regulation and tolerance in primates and human organ transplantation. expansion of cynomolgus cd +cd +foxp + regulatory t cells using low dose anti-thymocyte globulin. to test low dose atg in vivo, mg/kg ( % of depleting dose) was administered thrice (day , and ) to a naïve monkey and to a monkey that was treated concurrently with sirolimus (trough - ng/ml) after heart transplantation. in the naive monkey, lowdose atg led to expansion of cd +cd +foxp + tregs in peripheral blood (baseline . %, day = . %, day = . %) and in lymph nodes (baseline . %, day = . %, day = . %) without causing t cell depletion. similarly, in the transplanted monkey peripheral blood tregs expanded from . % at baseline to . % on day . low dose atg is not only able to expand tregs ex vivo by proliferation of natural cd +cd + cells, but can equally induce tregs in vivo without lymphodepletion. these findings provide the rationale for development of tolerance inducing strategies based on enhancing regulatory mechanisms in human transplant recipients. immunological background⁄aim: previously, we have shown that combination of human anti-cd mab, d and tactolimus exerts additive immunosuppressive effect and markedly prolongs renal allograft survival in cynomolgus monkeys. in this study, we further evaluated the immunological aspects among these transplant recipients. method: kidney transplantations were performed across mhc mismatched cynomolgus monkeys. transplant recipient was given either no-treatment, tacrolimus ( mg⁄kg⁄day, po), d ( mg⁄kg, iv) or tacrolimus+ d (n= ⁄group). peripheral lymphocyte population, mlr and serum anti-donor antibody levels and graft histology were assessed. results: mean graft survival for no-treatment, tacrolimus, d and tacrolimus+ d treatment groups was . ± . , . ± . , . ± . and . ± . days, respectively. peripheral cd + cells partially declined in both d alone and d + tacrolimus given animals at the early post-operation period, although the numbers recovered thereafter. cd + and cd + cells were unaffected. cd + effector memory population was reduced by addition of tacrolimus to d (fig. a ). mlr against donor and rd party antigens were suppressed in both d and tacrolimus+ d groups (fig. b) . addition of tacrolimus further reduced graft cd + , cd + and cd + cellular infiltration (fig. c ). anti-donor antibodies were detected in sera during the treatment course of d ; however, they did not develop under the tacrolimus+ d treatment. graft c d deposition correlated with serum anti-donor antibody levels. the d inhibits both cellular and humoral responses against donor antigens. addition of tacrolimus strengthens these immunosuppressive effects of d , leading to further prolongation of graft survival. objectives: allogeneic islet transplantation offers the potential for cure from diabetes. application of this therapy, however, is limited by immunologic mechanisms requiring medical therapy to prevent rejection of the islets. costimulatory blockade of the cd / cd /cd and the cd /cd pathways has shown promise in ameliorating the immune response to allow engraftment and function of islets. we have evaluated a new drug regimen consisting of induction therapy with a , a murine anti-cd antibody, and basiliximab and maintenance treatment with ctla ig and sirolimus in diabetic rhesus macaques which received allogeneic islets. methods: allogeneic rhesus macaque islets ( , ie/kg ± , ) were transplanted intraportally into diabetic rhesus macaques (n= ) under the following immunosuppressive regimen: short term administration of anti-il- receptor (basiliximab) and anti-cd ( a ), with maintenance immunosuppression using sirolimus for days and abatacept (ctla ig) for long term therapy. weekly peripheral blood flow cytometric and cmv viral load monitoring was performed. results: recipients treated with this immunosuppressive regimen had immediate return to normoglycemia following islet transplant. the graft survival in the first three animals was , and days. the fourth animal continues to exhibit good glycemic control at his current post-operative day . each of these animals had monthly intravenous glucose tolerance tests with monitoring of blood glucoses and c-peptides with further evidence of glycemic response and c-peptide generation. flow cytometry confirms cd blockade during the administration of a and return of cd after cessation of therapy. the treatment was well tolerated with minimal evidence of cmv reactivation and no evidence of thrombocytopenia or thromboembolism. conclusions: these preliminary results indicate that cd /cd costimulatory-based immunosuppressive regimens can protect allogeneic islets from rejection. furthermore, a appears to adequately block cd to facilitate this engraftment and function as demonstrated by flow cytometry. iwami, , qi zhang, osamu aramaki, nozomu shirasugi, katsuya nonomura, masanori niimi. surgery, teikyo university, tokyo, japan; renal and genitourinary surgery, hokkaido university, sapporo, japan. many studies have shown immunosuppressive effects of dietary intake of fish oil containing eicosapentaenoic acid (epa) in various models such as autoimmune diseases and transplantation. however, its mechanisms remain uncertain. furthermore, there have been no studies examining the effect of purified epa. here we determined the ability of purified epa to inhibit alloimmune response in mouse cardiac transplantation model. methods: cba recipients (h- k ) were given single injection of purified epa intraperitoneally on the same day as transplantation of a heart from c bl/ donors (h- b ). mixed leukocyte reaction (mlr) assay and enzyme linked immunosorbent assay (elisa) were also performed to evaluate the effect of purified epa on cell proliferation and cytokine production. to determine the presence of regulatory cells, adoptive transfer study was conducted. results: untreated cba recipients rejected c bl/ cardiac allografts with median survival time (mst), days. in contrast, cba recipients treated with purified epa ( . g/kg) had significant prolongation of allograft survival (mst, > days). cba recipients treated with . g/kg purified epa eventually rejected allografts (mst, days). in mlr assay, treatment with . g/kg purified epa suppressed alloproliferation of splenocytes in the recipients. the treatment also inhibited production of il- , il- and ifng by the splenocytes in the recipients. when splenocytes were harvested from the recipients treated with . g/kg purified epa days after cardiac allografting and were adoptively transferred into naïve secondary recipients, the adoptive transfer induced significant prolongation of cardiac allograft in nave secondary recipients (mst > days, compared to that in the recipients with adoptive transfer of naïve splenocytes, mst, days). conclusions: purified epa induced significantly prolonged survival of fully mismatched cardiac allografts, and generated regulatory cells. background: chronic allograft nephropathy (can), the most common cause of late kidney allograft failure, is not effectively prevented by the current regimens. activation of extracellular signal-regulated kinases / (erk / ) mediating intracellular signal transduction from various growth factor stimuli is required for tgf-β production, which plays a key role in the development of can. hence, the therapeutic potential of disruption of erk / signaling to prevent can was examined in an experimental model. methods: kidney donors from c bl/ j mice (h- b ) were transplanted to bilaterally nephrectomized balb/c recipient mice (h- d ). the recipients were treated with ci (mek-erk / inhibitor) or vehicle after days post-transplantation for days. can was evaluated with the banff working classification. results: all six allografts receiving ci treatment were survived, while two out of seven grafts were lost in vehicle-treated group. at the end of experiment, the function of grafts in ci treated recipients had been maintained, indicated by lower levels of serum creatinine and bun ( ± µm and ± mm, n= ) as compared to those ( ± µm and ± mm, n= ) in vehicle group (creatinine, p= . ; bun, p= . ). pathological evaluation indicated that ci reduced can, reflected by a lower can score in ci -treated group ( . , n= ) as compared to that ( . , n= ) in vehicle controls (p= . ). further examinations showed that ci treatment resulted in inhibition of phosphorylation of erk / and reduction of tgf-β levels in grafts. in vitro ci potently suppressed not only growth factors-stimulated erk / activation and tgf-β biosynthesis in renal tubular epithelial cells, but also attenuated alloantigenstimulated t cell proliferation. conclusion: our data suggest that interference of erk / signaling with pharmacological agent (i.e. ci ) has therapeutic potential to prevent can in kidney transplantation. objective: this is the first study to investigate the role of a novel jak and sykinhibitor, r , in the prevention of obliterative airway disease (oad), the major obstacle after lung transplantation. methods: trachea from brown-norway (bn) donors were heterotopically transplanted in the greater omentum of lewis (lew) rats. recipients were treated for days with r ( , , , or mg/kg), rapamycin ( . or mg/kg), or left untreated. allografts were recovered and processed for histological evaluation determining degree of luminal obliteration, percentage of respiratory epithelial coverage, and mononuclear cell infiltration. donor reactive (igg) antibodies from the recipient's serum were determined using flow cytometry. results: r at , , and mg/kg significantly inhibited luminal obliteration in a dose dependent manner ( ± %, ± %, ± %; p= . vs. no medication). rapamycin in both concentrations significantly inhibited luminal obliteration ( ± %, ± %; p< . vs. no medication) similarly to r at and mg/kg. r at and mg/kg significantly preserved respiratory epithelium compared to r at and mg/kg ( ± %, ± % vs. ± , ± %; p= . ) and was superior to rapamycin in epithelial preservation ( ± %, ± % vs. ± %, ± %; p= . ). all r and rapamycin-treated recipients expressed decreased numbers of peritracheal mononuclear cells in a dose dependent manner (p< . ). r , , and mg/ kg treated recipients had significantly reduced igg levels versus untreated recipients ( ± , ± , ± vs. ± ; p< . ). all r treated recipient thymus and spleen weights were significantly lower compared to the untreated group (p= . ). bun, cr, and cholesterol levels were unaffected in r treated recipients. conclusion: r potentially exhibits its inhibitory effect by preserving the respiratory epithelium, rather than by rapamycin's mechanism of reduced smooth muscle cell (smc) proliferation. r occupies a beneficial pharmacokinetic profile, lacks nephrotoxic and atherogenic properties, and provides a favorable alternative to rapamycin in the treatment of chronic rejection in lung transplant recipients. genz- is a novel, oral immune-modulatory agent identified in a high-throughput screen designed to find inhibitors of tnfα-induced apoptosis. the molecular target of the compound remains under investigation but is likely downstream of the tnfα cell surface receptor. in vitro studies have shown genz- to be an effective inhibitor of the tnfα-triggered caspase cascade but not anti-cd or fas-mediated apoptosis, and thus may act by inducing allograft resistance to immune attack rather than suppressing the alloimmune response per se. it has been shown to synergize with sirolimus in murine heterotopic cardiac allotransplant models. in order to test this promising new agent in a more clinically relevant model of solid organ transplantation, we studied genz- in a mismatched nhp (rhesus macaque) renal transplant model. genz- (n= ) was administered ( mg/kg, iv, days - ) with sirolimus ( mg/kg, po, days - ). five control animals received only sirolimus and vehicle ( mg/kg, po, days - ). all animals were followed serially by polychromatic flow cytometry to determine the relative and absolute number of cd + and cd + t cell subsets. time to allograft rejection, the primary end point, was determined by a significant rise in serum creatinine and bun, as identified with biweekly monitoring. after diagnosis of rejection, allografts were removed for histological and transcriptional studies, along with splenocytes for immune function assays. in this pilot study, prolongation of rejection-free survival was significantly improved with genz- and sirolimus combined vs. sirolimus alone ( . days vs. . days, respectively, p = . ). given these initial results, we have initiated a larger study (n= ) to optimize the dose and duration of genz- . five animals, transplanted within the past month remain alive and well in this study. further investigation of this agent will allow us to better understand the benefit of inhibiting tnfα-mediated apoptotic effects in both cellular alloimmune response and allograft injury in solid organ transplantation. targeting purpose: allospecific t memory cell responses are present in transplant recipients from exposure to cross-reacting antigens. we have previously reported that lfa- inhibition suppresses primary cd -dependent rejection responses which are not controlled by any conventional immunosuppressive strategy. these studies were conducted to analyze the efficacy of this anti-lfa- ab for control of cd -dependent responses in sensitized hosts. methods: fvb/n (h- q ) donor hepatocytes were transplanted into c bl/ (h- b ) or cd ko (h- b ) recipients. memory responses were analyzed by retransplantation with a second fvb/n allogeneic hepatocyte transplant. cohorts of mice were treated with anti-lfa- mab and observed for hepatocyte survival or magnitude of cd + t cell mediated allospecific cytolytic activity. results: the untreated secondary cd ko and c bl/ recipients rejected hepatocyte allografts with enhanced kinetics in comparison to the primary graft (mst= day vs day , and mst= day vs. day , respectively; p < . ). anti-lfa- mab treated cd ko recipients demonstrated delayed rejection (mst= day vs day ; p= . ) compared to secondary rejection in untreated cd ko hosts. anti-lfa- mab treatment did not delay rejection in sensitized c bl/ recipients (mst= day ) but did significantly reduce the in vivo allospecific cytotoxic effector function in c bl/ secondary recipients ( . ± . %; p= . ) as compared to untreated controls ( ± . %). the residual cytotoxicity observed in anti-lfa- mab treated c bl/ recipients is comparable to the in vivo cytotoxicity of cd -depleted c bl/ secondary recipients ( . ± . %) and is likely mediated by alloantibody. in fact, the level of allospecific cytotoxicity in anti-lfa- mab treated sensitized c bl/ recipients correlated with the amount of alloantibody present in recipient serum. conclusion: in conclusion, treatment with anti-lfa- mab delayed (cd -independent) cd -dependent rejection in sensitized recipients but did not delay rejection in sensitized cd -sufficient c bl/ recipients. despite the efficacy of treatment with anti-lfa- mab to significantly reduce the in vivo allospecific cytotoxic effector function in sensitized c bl/ mice this strategy did not delay rejection. this is likely due to alloantibody mediated rejection in sensitized c bl/ (but not cd ko recipients) which is not suppressed by treatment with anti-lfa- mab. abstract# cytomegalovirus (cmv) represents a major cause of infectious complications after transplantation. recently, chronic infections with lcmv, hiv or hcv were shown to be associated with functionally anergic t-cells characterized by high expression of the programmed death (pd)- molecule. this study was carried out to characterize functional exhaustion of cmv-specific cd t-cells as determinant of impaired cmv-control and to elucidate whether the pd- pathway may be operative in active cmv-infection after renal transplantation. cmv specific cd t cells from controls, hemodialysis patients, and renal transplant patients were quantified using flow cytometry and analysed for their expression of pd- and cytokines ifnγ and il . cmv specific proliferation was analysed by cfda-se dilution. in viremic transplant-recipients, a significantly higher proportion of cmv-specific cd t-cells were pd- positive (median . %) as compared to non-viremic transplant patients ( . %), dialysis-patients ( . %) or controls ( . %, p< . ). in line with functional impairment, pd- positive t-cells produced significantly less ifnγ per single cell as compared to pd- negative t-cells (mean fluorescence intensity . ± . versus . ± . , p< . ). moreover, unlike controls or non-viremic patients, the majority of cmv-specific t-cells from viremic patients showed a long-term loss of il- production. interestingly, functional anergy of pd- positive cmv-specific cd t-cells was reversible in that antibody-mediated blockade of pd- signaling with its ligands pd-l /-l led to a fold increase in cmvspecific proliferation. in conclusion, expression of pd- defines a reversible defect of cmv-specific cd t-cells, and blocking pd- signaling may provide a potential target for enhancing the function of exhausted t-cells in chronic cmv-infection. differential background: some patients with cmv disease may be simultaneously infected with multiple viral strains. it is unknown if different strains clear differently with the commencement of antiviral therapy. we assessed response to antiviral therapy in patients with simultaneous co-infection with multiple strains of cmv. methods: pcr-based strain typing of cmv was performed using the glycoprotein b gene of cmv (gb - ) in a cohort of organ transplant recipients with cmv disease. from this, patients were identified that had simultaneous infection with ≥ cmv strains. quantitative assessment of each of the strain types was performed at regular intervals after starting antiviral therapy. results: the different types of multi-strain infections were gb +gb ( / , %), gb +gb ( / , %), gb + gb ( / , %), gb +gb ( / , %), gb +gb ( / , %) and gb +gb ( / , %). / ( %) were simultaneously infected with or different genotypes. within individual patients, there was trend for gb cmv load ( . log genomes) to be lower than the other genotypes (p= . - . ) at the onset of disease. decay kinetics for all genotypes showed a bisphasic response with a st phase decline of ∼ . days and a nd phase of ∼ days. st phase delines were fastest for gb (p= . vs gb and ) while gb decline was slower than gb during the st phase. nd phase declines were similar between gb and ( days and . days) but were slower for gb and ( . days; p= . ). there was a significant correlation between st phase decline and log decline from baseline by day (r= . ; p= . ). relative fitness calculations revealed complex fitness dynamics between genotypes although gb was always less fit than gb , and , and gb and were always less fit than gb . conclusion: in patients with cmv disease who have simultaneous coinfection with multiple strains, the st and nd phase declines in gb are significantly slower than either gb or and have a lower log decline from baseline by day . these data indicate that either a significant fitness difference exists between cmv strains or that antiviral control of replication may be linked to cmv gb genotype and should aid our understanding of treatment success and failure. one introduction: parvovirus b (pvb ) is a single-stranded dna virus that was first reported to affect transplant (tx) recipients around years ago. in the kidney tx setting pvb has been reported to cause anemia and proteinuria. reported incidence in a general kidney transplant cohort has been reported to be between %- % and as high as % in an anemic kidney tx population. here we report our incidence of pvb infection over a year span. patients and methods: all records of kidney tx recipients from until were reviewed for the presence of pvb infection. there were kidney tx performed during this period. diagnosis of pvb infection was made either by detection of pvb via pcr in a blood/tissue sample or by detection of virus on renal tissue by immunostain. in patients found to have pvb infection; presence of anemia, proteinuria, concurrent infection and acute rejection rates were examined. response to treatment with ivig was also evaluated. results: incidence of infection was . % as patients were found to have evidence of infection. average time from tx to diagnosis of infection was . months (range days- months). average creatinine at diagnosis was . mg/dl. anemia was present in % of patients with an average hematocrit of . %. proteinuria was present in % of patients with evidence of pvb infection. co-infection was noted in patients ( cmv, ebv) and acute rejection was noted in % of individuals within months of diagnosis. collapsing glomerulopathy (cg) was present in patients and they all had subsequent graft loss at an average of months after diagnosis. of the patients with cg had proteinuria along with anemia and were caucasian. % ( / ) of all patients with evidence of pvb infection received ivig and cleared their infection. one of the remaining pts without ivig spontaneously cleared their virus. conclusion: although the incidence of pvb infection in our kidney tx cohort was very low, its presence portends an unfavorable outcome. the presence of cg associated with pvb is an especially devastating lesion with very poor outcomes. response to treatment with ivig and reduction of immunosuppression is variable. based on our data it seems reasonable to screen all tx patients with unexplained anemia and concurrent proteinuria as early detection of pvb may be crucial. background: prior to transplant, screening for latent tuberculosis (ltbi) by tuberculin skin test (tst) is recommended. the accuracy of tst in end stage renal disease however may be limited. the quantiferon®-tb gold assay (qft) detects interferon-δ produced by peripheral blood t-cells in response to tb specific antigens and may be more accurate for diagnosis of ltbi. methods: this prospective single center study compared the tst to qft for the diagnosis of ltbi in a cohort of adult patients listed or undergoing workup for renal transplantation. all patients had both tst and qft performed. additional data collected included demographics, tb risk history and chest x-ray results. based on demographic and radiographic findings, patients were classified as high or low risk for ltbi. a positive tst was defined as ≥ mm and positive qft as ≥ . iu/ml. results: a total of patients were enrolled. complete data was available for subjects ( did not return to have tst read). the mean age was . +/- . years with ( . %) males and ( . %) females. the most common etiologies of renal diseases were diabetes ( . %) and glomerulonephritis ( . %). most subjects ( of ) were on renal replacement therapy (hemodialysis in . % and peritoneal dialysis in . %). twenty ( . %) subjects had received bcg and ( . %) were born in or lived in a country in with tb prevalence rate > / population. fifteen ( . %) subjects were considered to be at high-risk for ltbi. overall ( . %) had a positive tst and ( . %) had a positive qft. the qft was indeterminate in subject due to a low mitogen response. agreement between the tests was % (k= . , p< . ). in low-risk subjects (n= ) the tst was negative in all and the qft was negative in and indeterminate in . in clinically high-risk subjects, ( %) had a positive tst and ( %) had a positive qft. the subjects with discordant results, both from tb endemic countries, had both completed treatment for ltbi years prior and remained tst positive, but were qft negative. in renal transplant candidates, the tst and qft are comparable for the diagnosis of ltbi. the qft has the advantage of being completed in a single visit and in our cohort indeterminate results were uncommon. optimal utilization of htlv i/ii positive organs -a nationwide survey. objective: we recently presented data from the unos database that demonstrated no significant difference in graft or patient survival between htlv i /ii (+) and (-) liver recipients. several organ procurement organizations (opo) including our own, do not offer htlv i /ii positive organs while many others find it difficult to place them. despite this, the number of htlv (+) organs is increasing with utilized in alone. this prompted us to evaluate the practical difficulties in placing these organs so as to improve utilization of these "high risk" life saving organs. medthod: a telephone/email survey of all the opos in usa was done over a month period from october to november . results: of the opos, responded. all screen patients for htlv i/ii with elisa. centers confirm with repeat elisa, confirm with western blot and centers do not pursue further. of the centers offer the htlv i/ii positive organs. there were a total of positive donors in the past years of which organs from donors ( . %) were placed. centers offer all the organs while offer one or more organs selectively based on accepting centers. none have been able to place the pancreas. only liver and kidney were commonly accepted. several centers noted a high false positive rate. based on the unos regional analysis data, % of the organs are utilized in ny state alone. many opos did not know which particular centers accept these organs and consequently spend a lot of time and effort in order to place them. a majority wanted to have a list of transplant centers that accept these organs. conclusions: htlv i/ii organs are being underutilized. moreover, our prior analysis of unos data shows that these life saving organs are shared more nationally than loco-regionally which is associated with a poorer outcome. increased knowledge of successful htlv (+) donation and the centers that are willing to utilize these organs in the appropriate setting will help expand the donor pool and decrease mortality on the waiting list. increasing traditional two-drug chronic immunosuppression (is) used in organ transplantation (tx) is associated with development of ebv-driven complications because of impairment of anti-viral cd + t cell surveillance. since the long-term impact of alemtuzumab preconditioning combined with tacrolimus monotherapy on ebv immunity after tx has not been studied, here we aim to analyze the frequency and function of peripheral blood ebv-specific cd + t cells. thirteen ebv + stable kidney transplant (ktx) recipients and ebv + healthy controls were recruited to this cross-sectional study. all patients received alemtuzumab preconditioning, followed by tacrolimus monotherapy. blood samples were collected at least year post-tx to allow immune reconstitution. the ebv-specific cd + t cell phenotype and function were screened by flow cytometry and ifng elispot assay. hla-a restricted ebv-lytic (bmlf ) and latent (lmp a) peptides were used to generate tetramer (tmr) probes, and for functional screening in elispot. circulating cd + t cells from ktx patients had recovered by year, and were comparable to those of healthy controls ( . %± . vs % ± . , p= . ). moreover, the memory distribution and the frequency of ebv-specific cd + t cells detected in patients and controls (bmlf -specific: . %± . vs . %± . , p= . , and lmp specific: . %± . vs . %± . p= . ) were similar. in contrast, the frequency of functional type- (ifn-g producing) ebv-specific cd + t cells was significantly lower in ktx patients than in healthy controls (bmlf : ± spots/ cd t cells vs ± p= . , and lmp : ± vs ± p= . ). accordingly, on average, only - % of circulating ebv-specific cd + t cells from ktx patients produced ifn-g, while - % of effector cells were functional in healthy controls. addition of il- ( iu/ml) during the elispot assay reversed the hypo-responsiveness of type- (ifn-g) ebv-specific cd + t in patients (range - fold increase), suggesting that these effector cells were anergic. these results support the notion that alemtuzumab-induced lymphocyte depletion followed by tacrolimus monotherapy renders ebv-specific cd + t cells anergic in vivo, a state that can be readily reversed by cytokines such as il- , which are commonly released during immune activation. background: the epidemiology of the transmission of cytomegalovirus (cmv) from organ donors to recipients is not completely understood. we studied donor to recipient transmission patterns by analyzing viral genomic variants through the use of cmv glycoprotein b (gb) genotyping by real-time pcr. polymorphisms in gb ul allow discrimination of distinct genomic variants (gb - ). methods: organ transplant recipient pairs or triplets were included in the study if: a) they had cmv infection, b) they received an organ from a cmv seropositive donor, and c) there was at least one other recipient from the same donor that also developed cmv infection. genotyping (gb - ) was performed by quantitative real-time pcr on stored blood samples. clinical charts were reviewed to evaluate the clinical characteristics and outcome of cmv infection. results: of the cmv seropositive donors screened, were multiple organ donors for which or more of their recipients developed cmv infection. the total number of recipients from these donors was (median of recipients per donor). of these recipients, ( %) had cmv infection ( recipient pairs and recipient triplets). the prevalence of genotypes was gb (n= ; %), gb (n= ; %), gb (n= ; %), gb (n= ). mixed infection with two concurrent genotypes was present in patients ( %). overall concordance between cmv gb genotype in recipient pairs was . % ( / ). if both recipients were cmv seronegative (d+/r-) the gb concordance in recipients was % ( / pairs). gb concordance was % ( / pairs) if one of the recipients was seronegative and the other seropositive. concordance was % ( / pair) if both recipients were seropositive. concordance between genotypes was seen in / ( %) recipients triplets. in seropositive recipients with cmv viremia, the origin of the cmv strain was thought to be donor derived in / ( %) and of reactivation of the recipients own virus in / ( %) of the cases. no difference in clinical outcome or organ tropism was seen between genotypes. based on an analysis of strain concordance within recipients from common donors, transmission patterns of cmv can be assessed. in d+/r+ transplant patients, donor strain superinfection accounts for the approximately two-thirds of cmv infection. backgroud: although map kinases have been implicated in the pathophysiology of liver iri, their functional significance in the mechanism of tlr mediated pro-inflammatory immune regulation, remains to be elucidated. methods: map kinase activation in a murine model of liver warm iri ( min. ischemia, h reperfusion) was determined by western blots. chemical inhibitors of erk (u , µm in vitro or mg/kg in vivo), jnk (sp , µm or mg/kg), and p (sb , µm, mg/ kg) map kinases were utilized in vitro in primary bm-derived macrophage cultures stimulated with lps ( ng/ml); or in vivo in liver pro-inflammatory immune responses induced by lps ( µg/mouse, i.p.) or iri. results: erk and jnk, but not p , map kinase activation were readily detected in liver iri. in primary macrophage cultures, lps induced pro-and anti-inflammatory genes, including tnf-α, il- β, il- , il- , inos and cxcl . erk inhibitor mainly suppressed il- β and il- ( % and % resp), whereas jnk inhibitor suppressed the majority of genes. in lps-induced liver inflammation, erk inhibitor suppressed il- , il- β, inos and il- by > %, but failed to affect tnf-α/cxcl . jnk inhibitor, on the other hand, preferentially inhibited pro-inflammatory genes, but marginaly affected il- (< %). and produced comparable suppression of pro-/anti-inflammatory genes (figure ). interestingly, tnf-α was the least responsive gene subjected to map kinase regulation. conclusion: erk and jnk map kinase activation: / are required for tlr activationinduced pro-inflammatory gene induction; / play critical role in the development of ir-mediated liver immune response/tissue injury. background: the jak/stat signaling is one of the major pathways for cytokine signal transduction. the signal transducer and activator of transcription (stat ) is mainly activated by ifn-α/ß/ifn-γ. the activation of stat by ifn-γ has been implicated in hepatic inflammation. we have shown that activation of toll-like receptor (tlr) complex initiates pro-inflammatory response leading to liver ischemia/reperfusion abstracts injury (iri). indeed, tlr signaling in vitro activates stat , which in turn triggers production of type- ifn-dependent cxcl (ip- ). this study was designed to analyze the cross-talk between stat and the map kinase (erk) downstream of jak/ stat signaling pathways. methods & results: we used a mouse liver model of partial warm ischemia ( min), followed by reperfusion ( h) . first, we employed stat ko (n= ) and control wt (n= ) mice. the hepatocellular damage, as measured by salt levels (iu/l), was significantly decreased in / stat ko mice (p< . ); the remaining / of stat ko showed salt levels comparable with wt. hence, we distinguished two groups of stat "protected" vs. "nonprotected" ko recipients. histology revealed minimal sinusoidal congestion without edema/vacuolization or necrosis in stat ko "protected" group. the induction of mrna coding for tnfα/ il- was higher in stat ko "nonprotected" livers. the expression of cxcl , the product of stat activation downstream of tlr in type i ifn pathway, was profoundly and selectively depressed in livers from stat ko "protected" mice, as compared to iri susceptible livers. similarly, western blot-assisted phospho-erk expression was up regulated selectively in the stat ko "protected" group. in the second series, c bl/ mice were treated h prior to liver ischemic insult with jak- inhibitor (tyrphostin ag ; mg/kg, i.p.; n= ), or vehicle (n= ). the hepatocellular damage, as measured by salt levels (iu/l), and histology was significantly decreased in ag group, as compared with controls (mean = vs. ; p< . ). the disruption of jak/stat signaling by inhibiting jak uniformly ameliorates the inflammatory immune response in liver iri. however, the blockage of stat alone is insufficient to reproducibly exert cytoprotection. as jak is upstream of stat as well as upstream of map kinase (erk), this study highlights the role of both signaling pathways in hepatic iri. purpose: tlr is required for maximal ischemic injury of the heart, liver, lung, and kidney. to better understand the mechanisms of tlr action, we investigated a murine model of ischemic kidney disease and examined endothelial tlr expression. methods: . animal ischemia reperfusion injury(iri): the right kidney of wildtype(wt) c bl/ , or tlr -deficient(ko) c bl/ scn mice was removed. the left pedicle was clamped for min, followed by hr reperfusion. sham animals were controls. . bone-marrow chimera: four groups of bm chimeras were created: wt→wt; ko→wt; wt→ko; ko→ko ( recipients/grp). wks later, chimerism was confirmed by tail and blood genotyping, and mice subjected to renal iri. . tlr mrna detected by dig-labeled antisense; tlr on endothelium by anti-tlr and anti-cd . . total genome mrna expression on ischemic vs. sham wt mice, ischemic vs. sham ko mice( mice/grp) determined using affymetrix mouse genome . genechips followed by genesifter analysis. quantitative real-time rt-pcr confirmed candidate genes. . ms endothelial cells were treated with h o ( um) for min, cultured for hr and then expression of tlr and endothelial genes determined. results: tlr -deficient mice had less renal injury as assessed by pathology and function. radiation chimeras showed that radioresistant parenchymal cells and radiosensitive leukocytes were both required for maximal injury. immunohistology and in situ hybridization identified endothelia in the outer medulla as a major tlr expressing cell type at hr post-reperfusion. genechip analysis revealed a panel of cytokine, chemokines, proinflammatory and cell-cycle related genes that were differentially expressed on iri versus sham kidneys. real-time rt-pcr confirmed that the following pro-inflammatory endothelial genes increased after ischemia in wildtype but not tlr ko mice: tlr , pentraxin related gene(ptx ), and endothelial cell-specific molecule (esm ). real-time rt-pcr showed that in vitro h o treatment of endothelial cells induced expression of tlr ( . -fold), ptx ( . -fold), and esm ( -fold). we found that endothelia in the outer medulla increase their expression of tlr after ischemia, and that the endothelial genes ptx and esm increase only in wt ischemic kidneys. we also found that h o , which mimics reactive oxygen species generated during iri, directly increases these same endothelial genes in vitro. nkg d is an activating receptor expressed on nk cells and cd + t cells. ligands for nkg d including rae- , mult- and h in mouse, may be upregulated by tissues in response to stress. a study in mouse macrophages described upregulation of rae- in response to stimulation through tlr by lps. we have shown that tlr mediates kidney ischemia reperfusion injury (iri) and also observed rae- upregulation in iri kidneys. we now determined whether: ) kidney iri could induce the expression of nkg d ligands: ) expression of nkg d ligands is tlr dependent; ). bone marrow (bm) derived cells or parenchymal kidney cells express nkg d ligands. methods. kidney-ischemia was induced in tlr -/-, myd -/and wt mice for min. blood and tissue were harvested at days , and . primary cultures of mouse tubular cells (tecs) were also subjected to ischaemia (mineral oil overlay for hr) or tlr activation (lps stimulation). bm chimeric mice were generated by transplanting bm into irradiated recipient mice before iri was induced. results. rae- mrna level in iri kidney was increased from day to day , peaking at day compared to sham operated kidney ( - fold increase, p < . ) measured by real time pcr. rae- protein was detected in renal tubular cells from ischemic kidney but not from shamoperated control by flow cytometry. mult- mrna expression was also increased from day to day ( - fold increase, p < . ). both rae- and mult- mrna levels were reduced in tlr -/and myd -/-iri kidneys ( - fold reduction) versus wt controls (p < . ). tlr -/and myd -/primary cultured tecs submitted to iri in vitro also showed less rae and mult- mrna expression than wt controls (p < . ). lps stimulated rae- and mult- expression in wt but not in tlr -/-tecs. tlr -/mice bearing wt hemopoietic cells had significantly lower kidney rae- and mult- mrna expression after iri versus wt mice with tlr -/-bm (p < . ). conclusion. kidney iri causes rae- and mult- expression and kidney parenchymal cells are the dominant source. tecs can be stimulated to express rae- and mult- by ischemia or lps via the tlr pathway in vitro and deficiencies in this pathway provide protection against iri and rae- and mult- expression in vitro and in vivo. thus, kidney iri causes upregulation of nkg d ligands by parenchymal kidney cells via tlr . background: intestinal ischemia/reperfusion injury (iri) is a major clinical problem. although toll-like receptor (tlr ) has been implicated as a potential link between the innate and adaptive immunity, little is known on its role in intestinal iri. our preliminary research in intestinal iri has shown that, compared to sham controls, wt mice had decreased survival, worse tissue injury/apoptosis, increased pmn infiltration, increased cd + cell infiltration, and increased production of tlr , chemokines, and adhesion molecules. here we used tlr ko mice to further investigate the role of tlr in intestinal iri and its effects on cytokine/chemokine programs and apoptotic signaling. methods: c bl wt and tlr ko mice underwent min of total jejunoileal warm iri by clamping of the sma. separate survival and analysis groups were performed. intestinal tissue was harvested at h and h. tissue analysis included histopathology, cd immunostaining, myeloperoxidase (mpo) activity, rt-pcr for chemokines/ cytokines, and western blots for apoptotic and ho- protein expression. results: tlr ko had superior survival compared to wt ( % vs. %, p< . ). on histopathology tlr ko had near normal-appearing villous architecture, while in contrast, wt showed mucosal erosions and villous congestion/hemorrhage. tlr ko had reduced cd + cell infiltration as compared to wt ( . ± . vs. . ± . per hpf at h, p< . ; and . ± . vs. . ± . per hpf at h, p< . ). early mpo activity was also reduced in tlr ko ( . ± . vs. . ± . u/g at h, p< . ). rt-pcr analysis demonstrated decreased production of mrna for ip- , mcp- , rantes, and ifn-γ and increased production of il- in tlr ko. there was decreased protein expression of caspase- and increased expression of bcl- and ho- in tlr ko mice. conclusion: the genetic absence of tlr exerts protection against intestinal iri, demonstrating for the first time that tlr is required for intestinal iri. the absence of tlr signaling reduces iri through reduced neutrophil and t cell chemotaxis, and up-regulation of protective molecules. these results support data that tlr is a mechanistic link between the innate and adaptive immunity, implicating tlr as a potential therapeutic target for the prevention of intestinal iri. it is well known that liver steatosis increases hepatic vulnerability to ischemia/ reperfusion (i/r) injury as part of the transplantation process. endotoxin (lps) is thought to be a major contributing factor to the pathogensis of i/r. during portal occlusion, lps is translocated across the mesenteric tissue barrier into the portal circulation, and is delivered as a large bolus to the liver at the point of reperfusion. at this time, lps is mainly recognized by toll-like receptor (tlr ). this leads to downstream signaling and the production of proinflammatory products that ultimately lead to cellular inflammation, necrosis, and apoptosis. it is well known that steatotic livers are highly sensitive to endotoxin as compared to their lean counterparts post-i/r, and we have previously seen that monoclonal antibody blockade of endotoxin dramatically improves animal survival after i/r. therefore, we propose the novel hypothesis that tlr signaling is a major contributor to cellular damage after steatotic hepatic i/r. to test this hypothesis, we subjected male -week-old c bl/ j (control) or c bl/ scn (tlr deficient, tlr ko) mice to a high-fat diet (hfd) for four weeks. then, we subjected the animals to minutes of total hepatic ischemia and or hours of reperfusion. there was a dramatic improvement in animal survival in the hfd tlr ko animals versus control hfd animals at hours ( % vs. % in control hfd animals, p< . ). there was significantly more liver necrosis (as measured by a grading scale from - ) in the control hfd animals as compared to the tlr ko hfd animals ( . ± . in control vs. . ± . in tlr ko, p< . ). in addition, we see significant increases in the message level of the proinflammatory cytokines il- , il- , and ifn-γ at one hour in the control hfd animals that is abrogated dramatically in the tlr ko hfd animals. we do not see these dramatic changes in the control animals fed a normal diet. despite the significant increases in inflammation in the control hfd animals versus the tlr ko hfd and normal diet control animals, we do not see changes in the tlr message level or endotoxin boluses, implying an increased sensitivity in the absence of an increased number of receptors. tlr is a critical molecule in the pathogenesis of steatotic liver ischemia/reperfusion, and represents a potential therapeutic target for expansion of the donor pool. the background: neutrophils are considered crucial effector cells in the pathophysiology of organ ischemia and reperfusion injury (iri). particularly, neutrophil elastase (ne) accounts for a substantial portion of the neutrophil function. this study was designed to explore the role of, and mechanism by which ne exerts its function in a mouse model of liver warm iri. methods: partial warm ischemia was produced in the left and middle hepatic lobes of c bl/ mice for min, followed by - h of reperfusion. mice were treated with ne inhibitor (nei; mg/kg p.o.; gw a; n= ) or control (n= ) at min prior to the ischemia insult. after h or h of reperfusion, sast/salt levels and intrahepatic neutrophil accumulation (myeloperoxidase [mpo] activity) were assessed. the pro-inflammatory cytokine (tnf-α, il- ), chemokine (cxcl- , cxcl- , ip- ) and toll-like receptor (tlr) gene expression profiles were screened by rt-pcr. liver samples were collected for histological grading, and detection of neutrophil infiltration by the naphtol as-d chloroacetate esterase stains. results: nei treatment significantly reduced sast/salt levels, as compared with controls ( ± vs ± ; p< . / ± vs ± ; p< . at h, and ± / ± vs ± / ± ; p< . at h). the expression of pro-inflammatory cytokines, and chemokines was significantly reduced in the nei treatment group (tnf-α in h; p< . , il- in h and h; p< . and p< . , cxcl- in h; p< . , cxcl- in h and h; p< . and p< . , ip- in h; p< . ). the mpo activity (u/g) was also significantly reduced following nei treatment ( . ± . vs . ± . ; p< . ). tlr expression was selectively diminished in nei pretreated livers ( h; p< . ). histological examination of liver sections has revealed that unlike in controls, nei treatment markedly reduced edema, diminished centrilobular ballooning/sinusoidal congestion, ameliorated hepatocellular necrosis, and decreased local neutrophil infiltration. conclusion: the inhibition of ne ameliorated hepatocellular damage, reduced local inflammatory responses, and neutrophil activity/infiltration in a stringent mouse liver model of warm iri. interestingly, it also downregulated the innate tlr signaling. this study documents the previously unrecognized ne -tlr cross talk, and implies neutrophil elastase in the signal transduction pathway instrumental for liver iri. lymphocyte lymphocytes are involved in the early pathogenesis of ischemia-reperfusion injury (iri) in kidney; however, their role during healing is unknown. this has direct clinical consequence since lymphocyte-targeting agents are currently administered to prevent rejection during recovery from iri in renal transplants. c bl/ mice underwent unilateral clamping of renal pedicle for min, followed by reperfusion, and were sacrificed at day . mice were treated with saline (c), methylprednisolone (pred) or mycophenolate mofetil (mmf) i.p. daily from day until sacrifice (n= /group). lymphocytes were isolated from the kidneys, counted and stained with monoclonal antibodies. kidney damage (% damaged tubules) and proliferation (ki assay) were assessed. flow cytometry analysis demonstrated increased numbers of tcrβ + cd + and tcrβ + cd + t and tcrβ -nk . + nk, but not cd + b cells at day in the ischemic (ir) kidneys compared to contralateral. regulatory t cells, tcrβ + cd + cd + foxp + , and t cell subsets tcrβ + cd + cd + and tcrβ + nk . + also increased. moderate tubular damage in cortex, severe injury in outer medulla and increased proliferation in both compartments characterized the repair phase. pred improved histological damage in ir kidneys, while mmf worsened it. proliferative index correlated with histology in outer medulla. pred reduced the total counts and activation of tcrβ + cd + and tcrβ + cd + t cells in ir kidneys, and increased the percentage of tcrβ + cd + cd + among total tcrβ + cd + t cells. mmf reduced all lymphocyte subsets, decreased the percentage of tcrβ + cd + cd + foxp + among total tcrβ + cd + t cells, and lowered il- tissue levels. il- and platelet derived growth factor-bb protein levels were also decreased in ir kidneys from mmf-treated mice. in conclusion, specific trafficking and phenotypic changes of kidney-infiltrating lymphocytes occur during recovery from renal iri, and lymphocyte-targeting agents, pred and mmf, alter tubular cell structure, proliferation, and inflammatory response in the repair phase. background: ho- plays an important cytoprotective role in a variety of organ injury models. we have shown that ho- exhibits potent cytoprotective effects against liver i/r injury. this study explores the function and mechanism of ho- in liver i/r injury by using sirna that suppress ho- expression both in vitro and in vivo. methods: using a partial liver warm ischemia model, c bl/ wide-type (wt) mice (n= /gr) were injected with ho- sirna/nonspecific control sirna ( mg/kg, i.v. at day - ) or ad-ho- /ad-β-gal ( . x pfu, i.v. at day - ). sham control wt underwent the same procedures, but without vascular occlusion. mice were sacrificed at h of reperfusion; liver tissue and blood samples were collected for future analysis. in in vitro studies, ypen- endothelium cells were transfected with ho- sirna ( nm) or ad-ho- / ad-β-gal. results: ho- sirna treated mice showed significantly increased sgot levels (iu/l), as compared with nonspecific control sirna or ad-ho- ( ± vs. ± and . ± , respectively; p< . ). these correlated with histologic suzuki's grading of liver i/r injury, with ho- sirna showing significant edema, sinusoidal congestion/cytoplasmic vacuolization, and severe hepatocellular necrosis; nonspecific control sirna showed moderate edema, sinusoidal congestion/cytoplasmic vacuolization. in contrast, ad-ho- revealed only minimal sinusoidal congestion without edema or necrosis. ho- sirna significantly increased local neutrophil accumulation and caspase- activity, and increased the frequency of apoptotic cells ( . ± . vs. . ± . and . ± . , respectively; p< . ), as compared with nonspecific control sirna or ad-ho- . both ypen- endothelium cells and wt mice treated with ho- sirna revealed markedly increased caspase- activity and reduced ho- expression. in contrast, ad-ho- significantly decreased caspase- activity and increased ho- and anti-apoptotic bcl- /bcl-xl expression. conclusion: this study provides evidence that ho- exerts cytoprotection against i/r injury by regulating liver apoptosis and inhibiting caspase- activation pathway. organ specific sirna is not only a powerful tool to study local gene function, but it may also provide novel therapeutic application in transplant recipients. islet cell transplantation has recently emerged as one the most promising therapeutic approaches for diabetic patients to improve glycometabolic control. one major problem of the procedure is the requirement of an immunosuppression protocol capable of counteract both auto and allo-immune response. recent data suggest that anti-thymoglobulin (atg) can halt efficiently the mounting of an alloresponse and the recurrence of autoimmunity in nod mice by expanding antigen specific t-regulatory cells. we retrospectively reviewed our casuistry type diabetic kidney-transplanted patients who underwent islet transplantation using an immunosuppressive protocol based on atg or daclizumab (as induction treatment) plus cyclosporine and mmf as maintenance therapy. patients underwent islet after kidney transplantation in our center. thirty-four patients received a time course of atg as induction ( mg per day for - days), (number of islet infused= , ± , ), and patients received daclizumab at induction ( mg/kg every weeks for weeks); (number of islets= , ± , ). no major adverse events were recorded in our center; no malignancies or infections outbreaks were evident. patients in the atg induction group showed a better islet survival rate compared to daclizumab (p= . ), according to c-peptide> ng/ml. a sustained and prolonged c-peptide secretion was evident in the atg group; while in the daclizumab group only patient was functioning at year. interestingly, in the atg, which reached a longer follow-up, we cannot observe a loss of beta cell mass according to our metabolic test, suggesting the preservation of islet mass. in conclusion, atg can provide a good protection towards both allo and auto immune response. the next step will be to use atg in a calcineurin free protocol, allowing a better expansion of t-regs. introduction: despite consistent achievement of insulin-independence, recent data indicate that the long term success of islets transplants using the edmonton protocol is < % at years. the cause of the nearly universal late islet allograft failure remains unknown but hypotheses include: allo or autoimmune injury, marginal mass exhaustion, hepatic site related dysfunction, and immunosuppression toxicity. we examined the series of islet transplants performed at our institution and noted marked differences in the outcome and complications in ia and iak groups. our results may provide insight as to the cause of chronic islet loss. methods: thirty-one islet infusions were administered to ia (n= ) and iak (n= ) type- diabetics between / - / . ia and iak had similar demographics and transplanted islet mass ( , vs , ieq/kg). ia received edmonton like immunosuppression with zenapax induction and cni/srl, whereas iak patients received zenapax and: cni/mmf/pred ( ), cni/mmf ( ), cni/srl ( )). results: insulin-independence was achieved in all but patients who completed therapy ( others withdrew). compared with ia, iak exhibited better glycemic control ( -month mean hba c . vs . , stimulated c-peptide . vs . ), and improved islet survival; all ia eventually failed and only / were insulin free for > -years, whereas only / iak grafts have failed with exhibiting continued robust function at > ,> ,> , and > months with / fully insulin independent. in addition, all ia patients demonstrated immune sensitization post graft failure versus / iak. all ia developed mouth ulcers versus only / iak. conclusions: ia and iak exhibit striking differences in outcome and complications. the absence of mouth ulcers and lack of sensitization in iak may relate to steroid use and continued immunosuppression for the kidney graft, respectively. the superior outcome of the iak cohort may be a result of differences between the two groups including the use of maintenance steroids, prior exposure to thymo or the chronically immunosuppressed state of the iak recipient. perhaps the most interesting correlation with outcome is the absence of the anti-proliferative agent sirolimus in the iak group. our results provide clues to the cause of chronic islet transplant failure and may lead to novel approaches to avoid it. islet background: although islet transplantation has become an option for treatment of type diabetes, all currently used immunosuppressive protocols have significant renal and islet toxicity. we describe a novel immunosuppressive protocol using sirolimus and the anti-lfa antibody efalizumab that permits prolonged islet allograft survival without the need for steroids or calcineurin inhibitors (ci). methods: between february and august , consecutive type diabetic patients with hypoglycemic unawareness and normal renal function received allogeneic pancreatic islet transplants. induction immunosuppression consisted of doses of thymoglobulin given on pre-transplant days - and - , efalizumab ( mg/kg sq/week starting on d - ), and sirolimus. maintenance immunosuppression consisted of sirolimus and efalizumab. results: all patients achieved insulin independence after single islet infusions (mean ieq/kg= , ). three of remain insulin independent or more months after transplant (table ) . patient resumed low dose insulin (approximately % of original dose) weeks after transplant and is awaiting a second islet infusion. her blood glucose control is markedly improved and she has not experienced any hypoglycemic episodes. all patients show persistent c-peptide secretion and have stable renal function. side effects due to efalizumab were limited to transient irritation at the injection site. conclusions: thymoglobulin induction followed by sirolimus and efalizumab maintenance is well tolerated and allows prolonged islet allograft survival. this protocol is the first ci/steroid free islet regimen resulting in insulin independence with a single donor islet infusion. by eliminating ci, this protocol minimizes renal and islet toxicity and may thus improve long-term islet survival and function. long . clinical and metabolic profiles were assessed every months for months. results: si-exn group was on this drug for median time of days pre-si. both groups were similar except for duration from post-completion to graft dysfunction ( ± vs ± in si-exn and si-c group respectively, p< . ) and duration of graft dysfunction before si ( ± vs ± , p= . ). si-c and si-exn groups received mean of ± and ± ieq/kg, respectively (ns). only / of si-c patients achieved insulin independence for , and > days after si. all subjects in si-exn group achieved insulin independence for more than , , , days. at months insulin independence was % in si-c and % in si-exn group. comparing pre and post-si, si-exn group had significantly lower a c at - months and lower auc glucagon at and months (p< . ). auc c-peptide in si-exn group was significantly higher than si-c at and months. intravenous glucose tolerance test showed significantly increased acute insulin responses to glucose at - months in si-exn and at and months in si-c group. si-exn group had more acute c-peptide response to glucose than si-c at and month (p< . ). acute exenatide administration during intravenous glucose tolerance test at month in si-exn revealed significantly increased acute insulin and c-peptide response to glucose which indicates improved first phase insulin release. conclusion: supplemental islet infusions under exenatide lead to insulin independence, restore first phase insulin secretion and result in long term insulin independence. exenatide this prospective phase / trial aimed to demonstrate safety and reproducibility of allogeneic islet transplantation (tx) in type diabetic (t dm) patients and implement a strategy to achieve and maintain insulin-independence with minimal islets. ten c-peptide negative t dm subjects with hypoglycemic unawareness received - intraportal allogeneic islet tx. four subjects (group ) received the edmonton immunosuppression regimen (daclizumab, sirolimus, tacrolimus). the next subjects (group ) received etanercept, exenatide and the edmonton regimen. we followed all subjects for months after the first tx. the primary efficacy end point was insulin independence. secondary endpoints were hba c, fructosamine, ogtt, mixed meal test, glucagon stimulation test, ivgtt and hypoglycemia. to study the effect of exenatide, we compared frequently sampled ivgtt, c-peptide, proinsulin, amylin and glucagon with and without exenatide. two self-limiting bleeds occurred in infusions. all subjects became insulin independent. group received a mean total number of islets (ein) of , , ± , in (n= ) or (n= ) tx, whereas group became insulin independent after tx ( , ± , ein, p= . ). all group subjects remained insulin free through the -month follow-up. two group subjects resumed insulin: one after immunosuppression reduction during an infectious complication, the other with severe gastroparesis and exenatide intolerance. hba c reached normal range in both groups ( . ± . at baseline to . ± . after - tx in group vs. . ± . to . ± . after tx in group ). baseline hba c was significantly higher in group than group (p= . ). pre-and post-tx hypo scores were . ± . and in group vs . ± . and . ± . in group . glucagon levels decreased significantly in all subjects. in group , the decrease in glucagon levels after challenge tests with and without exenatide was . fold more significant after exenatide in all subjects ( background istx has been investigated as treatment for type dm. however, the hope this approach would result in long-term freedom from exogenous insulin has failed in practice. techniques for isolating islets have advanced and with availability of new is agents, strategies can now be developed specifically for istx that will provide greater immunologic protection w/o diabetogenic side effects. rejection/ vascularization still remain major limitations for success of istx. we hypothesize that bm is an accessible, immunologic privileged space with natural well-developed vasculature and may be a suitable site for istx. method wistar rats were used as donors/ recipients. dm was induced by iv-streptozocin. rats who had morning glc levels > mg/dl on two separate occasions were used as recipients. ptx was performed as normal rodent standard. islets were isolated from pancreas by distending pancreatic duct with liberase. islets were separated on a discontinuous histopaque density gradient, further purified, counted, divided into aliquots transplanted into different sites (liver, bm). bw, glc and c-pep were measured in all groups before/after tx. background: in february , the islet community was notified of a possible bovine product contamination in the collagenase enzyme (liberase hi) used for human islet isolations. to eliminate the potential hazard of bovine spongiform encephalopathy, we successfully adapted our human islet processing procedure to utilize a different gmp collagenase and neutral protease (nordmark/serva). here we describe what we consider the most important factors for achieving reproducible and clinically useable islet isolations. methods/results: a standard isolation protocol involving controlled enzymatic digestion followed by density gradient purification was used. seventeen donor pancreata were processed and ten were ultimately used for clinical transplantation. eight of these successful isolations were performed using the nordmark/serva enzymes (table ) . the following factors were identified as being important for ensuring successful islet yields: ) donor age and size: male donors - years old who were tall (> cm) and heavy (> kg) conclusions: incorporation of several important modifications into our existing islet isolation protocol has allowed us to routinely obtain high quality islet isolations using an alternative, bovine product-free gmp enzyme. (n= )], and in pts immuno was cni-free. tac-treated pts were younger, more sensitized and more frequently re-kt. dgf was more frequent in pts receiving cni-free immuno. as a group, these pts were older, more frequently received a kt from a donor after cardiac death and less frequently from a living donor. acute cellular rejection (acr) was diagnosed in pts ( . %), occurred before day th (e-acr), and after this date (l-acr). c d-positive amr was diagnosed in pts ( . %), of which were early (e-amr) and were late episodes (l-amr rates of acute rejection (ar) and background: an increasing number of hs patients are being transplanted using desensitization protocols. these patients are considered high risk for ar, particularly antibody mediated rejection (amr). here we examined our ar rates and treatment outcomes for our hs kidney transplant (kt) recipients using our most current desensitization strategies. methods: between june (when rituximab was introduced to our desensitization and treatment protocols) to july , hs kt patients were transplanted using combinations of high-dose ivig, rituximab, and plasmapheresis (pp) for desensitization. we examined the overall ar, c d-cell-mediated (cmr), and c d+amr rates. amr episodes were treated with steroids, high-dose ivig, and rituximab. refractory and rapidly progressive amr was treated with pp. treatment outcomes and differences in ar rates between deceased (dd) and living donors (ld) was examined. recent reports suggest that treatment with the monoclonal anti-cd antibody, rituximab (rtx) may improve renal graft survival in antibody mediated rejection (amr); however optimal dosing for this indication is unknown. we examined the efficacy of a single low dose ( mg) of rtx for treatment of refractory amr in order to limit significant infective complications associated with conventional dosing regimens ( mg/m ). rtx was used in seven consecutive patients who had refractory amr as judged by ongoing biopsy evidence of amr after weeks of standard therapy consisting of pulse methylprednisolone and plasma exchange with low dose ivig ( mg/kg) (pe/ivig). all patients received tacrolimus and mycophenolate mofetil. amr was defined as ) characteristic histology on biopsy (banff criteria), ) graft dysfunction and ) presence of a donor specific anti-hla antibody (dsab). b-cell counts (cd ) and serum creatinine (scr) were monitored. pe/ivig was ceased in all cases after rtx dosing. the average follow-up since rtx dosing is . months (range . - . mths). all patients still have functioning grafts ( % month graft and patient survival), with current mean scr levels ( ± µmol/l) significantly lower than mean peak rejection levels ( ± µmol/l) p= . . cd counts fell to zero and remained < x /l for > months in all patients. dsabs remain detectable by luminex flow beads in all patients despite stabilization of scr. two of patients developed an infective complication post rtx dosing. one patient developed bacterial pneumonia requiring hospital admission whilst a second patient developed cmv viraemia and later bk nephropathy which has not led to significant graft dysfunction. hence, infectious complications are far less than those reported for multiple standard dose regimens in similar patient groups. large clinical trials are required to confirm the efficacy of rtx in treating amr as well as optimal dosing. standard dosing is based on oncology treatment regimens which are likely to be excessive for amr in patients already markedly immunosuppressed. our data suggests single low-dose rtx for refractory amr results in excellent patient and graft survival and leads to low rates of serious infective complications in the short-term. renal the demographic and clinical data were similar between +cxm and negative cxm groups. the rejection rate was significantly higher and the length of stay was significantly longer in +cxm group as shown in the table. the ra survival rates were %, % and % lower at , and years post transplant respectively among + cxm recipients. however, this did not reach significance mostly due to small sample size (p= . ). the inferior ra outcome is seen during the initial few years after transplantation that disappears after years. in clkt, pre-transplant +cxm can result in higher ra rejection rate and longer length of stay in hospital. la may not always confer immunological protection to ra in + cxm clkt and its true burden may be under recognized since cxm is not routinely performed in all clkt. study of antibody specificities or la volume to determine the effect of +cxm on ra outcome is essential. skpt in patients with positive cdc b-cell and/or flow cytometry crossmatch is associated with high amr rate despite low dose ivig and r-atg/alemtuzumab induction. pancreas graft survival is inferior in patients with positive crossmatch while kidney graft and patient survival are similar to that of negative crossmatch recipients. majority of amr can be reversed with treatment but further long-term follow-up is needed to determine the impact on graft survival. ten the first european phase iii trial with tacrolimus in the early ´s clearly showed advantages for tacrolimus in terms of acute rejection (ar) vs the original cyclosporin formulation. however, no advantages were seen in survival rates. the present investigator-initiated, observational follow-up study collected data on patients included in the original cohort of patients who participated in this large study and who were randomised to a triple immunosuppressive regimen consisting of tacrolimus, azathioprine and steroids (tac/aza/ster, n= ) or cyclosporine, aza and steroids (csa/aza/ster, n= ). efficacy and safety parameters assessed at follow-up included: acute rejection; patient and graft survival; renal function, vital signs, basic lab results and immunosuppressive regimen for the patients years after completion of the original study. results: currently, data are available from % of the patients. all assessments were conducted following the intent-to-treat principles. more patients in the tac than in the csa group remained on the randomised treatment. while kaplan-meier (k-m) estimates for patient survival at year show comparable ( % tac vs %csa) results, k-m estimates for graft survival demonstrate an advantage for the tacrolimus cohort ( % tac vs % csa). graft half-life estimates (gjertson and terasaki method) yielded overall results of . years (tac) and . years (cs). in both treatment groups, ar was associated with inferior long-term results. for patients with ar, half-life estimates were . and . years for tac and csa, respectively, while in patients without ar half-lives were . (tac) and . (cs) years. mean serum creatinine after years was significantly lower in the tac cohort vs the csa group ( . mg/dl tac vs . mg/ dl csa, p< . ). mean glomerular filtration rates (mdrd estimate) were . ml/ min in tac and . ml/min in cs. after ten years of follow-up, the mean (c- ) levels were . mg/ml (tac) and mg/ml (cs). conclusion: analysis of this long term follow-up of a large study confirms the benefits of a tacrolimus-based therapy. it also confirms the concept that freedom from early acute rejection is associated with superior long term results in renal fuction. therefore, long term renal allograft function is remarkably good in tac patients. cyclophosphamide a novel therapy for ivig/plex-resistant antibody-mediated rejection. (c)). moreover subgroup analyses within the different groups were made to identify potential differences. groups were analyzed for survival, graft rejection and graftvasculopathy (cad). kaplan-meier analysis was used and log-rank test was performed to detect differences. results: a total of transplants ( . %) were blood group compatible. the majority (n= ) were a transplants ( %) followed by b (n= ; %), aab (n= ; %), bab (n= ; %) and ab (n= ; %). overall survival comparison showed no significant difference in long-term survival ( -year) recent data has shown the utility of urinary biomarkers to predict delayed graft function early following renal transplantation, but their ability to predict longer-term allograft function is less clear. we evaluated whether urinary biomarkers measured in the early post-transplant period would predict allograft function at and months after renal transplantation. urinary biomarkers including n-acetyl-β-d-glucosaminidase (nag), kidney injury molecule- (kim- ) and matrix metalloproteinase- (mmp- ) were measured in patients at hours , , , and days and following renal transplantation. glomerular filtration rate (gfr) at and months were calculated using the mdrd equation. all patients had functional allografts at months and patients lost allografts after months. levels of individual biomarkers at different post-transplant time points were correlated with and month gfr using spearman's correlation (rho) and are shown in the urinary nag levels at post-transplant hours , , and days and showed significant negative correlation with -month gfr. nag levels at hour and days and maintained the significant negative correlation with -month gfr. urinary kim- levels at the hour point showed significant negative correlation with -month gfr and kim- levels at hour and day displayed significant negative correlation with -month gfr. urinary mmp- levels at no time points had significant correlation with either -month or -month gfr. urinary biomarkers particularly nag shows promise as a tool in the early prediction of longer-term allograft function following renal transplantation. in kidney disease chemokines are involved in the recruitment of leukocytes causing kidney damage and progression to endstage renal failure. similar mechanisms are proposed for chronic allograft failure in renal transplant recipients. the chemokine attracted leukocytes produce proinflammatory and profibrotic cytokines contributing to fibroblast proliferation, matrix production and tubular atrophy. the role of chemokines in the acute post-ischemic tubular necrosis early after renal transplantation and their impact on long term allograft outcome has not been investigated yet. methods patients ( m, f, mean age . yrs) with dgf (with a median number of . dialysis sessions) developed biopsy proven acute tubular necrosis during the first three weeks after transplantation. the biopsies were studied by immunostaining for ki , ccr , ccr , rantes, mcp- , cd and cd , respectively. the patients were followed for a mean time of . yrs. none of them developed a rejection episode in the follow up time. after follow up the mean serum creatinine was . mg/dl ( . - . the goal of a tx is for the recip to achieve long-term survival (surv.), with continued graft function, equivalent to the gen. population. we studied subsequent outcome in , -yr kidney tx survivors (tx - ) ; % living donor (ld); % st tx; % female; % type diabetes; mean age at tx (±se), ± yrs. actuarial -yr surv. is shown in table ; ld patient, graft, and death-censored graft surv. is signif. ↑vs. deceased donor (p<. ). mean creatinine (± se) at yrs was . ±. ; yrs, . ±. ; yrs, . ± ; yrs, . ± . the major causes of late graft loss (gl) were death with function (dwf) and chronic rejection (can). by multivariate analysis, risk factors for gl after yrs were age ≥ yrs at tx; type diabetes, retx, hla mm ≥ ; ≥ acute rejection episode, and pretx cardiac, peripheral vascular, or liver disease (p<. ). the most common cause of dwf was cardiovascular disease (cvd); however, > % deaths were due to malignancy. the use of kidneys from donors with a positive serology for hcv into hcv(+) recipients still remains controversial. in , our units adopted this policy, subsequently modified in , so these kidneys were limited to recipients with a positive rna hcv before transplantation. the aim of the present analysis was to review the long-term safety of our policy. since january to february , hcv(+) patients with a negative hbsag and not treated with interferon received a kidney transplant in our units. patients were transplanted from an hcv(-) donor(group ) and from an hcv(+) donor(group ). median follow-up time was (ir - ) months. remarkably, group showed a significantly higher donor age ( . ± . versus . ± . years;p< . ) and recipient age ( . ± . versus . ± . years;p< . ), as well as a worse hla compatibiliy than group . immunesuppressive therapy did not significantly differ between the two groups. group notably, only patients died because of a liver disease ( in group versus in group ) and only patients developed a hepatocarcinoma (both in group ). in summary, no significant differences were observed in hcv(+) recipients according to hcv serology of the donor, in terms of death censored graf survival, patient survival and evolution of liver disease. therefore, our experience clearly demonstrates that the use of kidneys from hcv(+) donors into hcv(+) recipients is a safe strategy in the long-term and a wise way of using these kidneys, that otherwise would be lost at a moment of shortage. introduction: deceased donor kidneys are allocated to adult candidates in the u.s. primarily by waiting time and hla similarity. we investigated two alternative allocation systems, lyft-scd and lyft-dy, that prioritize offers of kidneys to adults based in part on lyft. methods: based on characteristics of each candidate and donor, lyft was calculated as the difference between expected median years of life for the candidate with that kidney donor transplant (tx) versus without a kidney. years on dialysis were weighted at . of years with a functioning graft. the relative risk of graft failure for each donor was calculated from donor factors using a cox model and the donor profile index (dpi), the percentile score of that risk among kidneys used in ( %=greatest risk of graft failure). using actual candidates and deceased donors and acceptance patterns from , we modeled allocation with the kidney-pancreas simulated allocation model (kpsam). kpsam sequentially offers kidneys to candidates according to user-specified allocation rules, and models the probability for kidney placement and post-tx survival. the lyft-scd model allocated scd kidneys by lyft and ecd kidneys by dialysis years (dy). the lyft-dy model allocated organs according to the formula [. lyft( -dpi)]+[dy(. dpi+. )]+[ pra/ ] to adult kidney tx candidates. results: the table compares a year's worth of kidney-alone recipient characteristics, lifespan after tx and additional lifespan (v. dialysis) due to tx among all kidney (including spk) recipients, and the donor/recipient age correlation (r) under current national kidney allocation, allocation using lyft for scd, and allocation using lyft-dy within each donation service area. conclusions: allocation systems incorporating lyft have the potential to increase the number of years of life attainable from tx and are being considered for the allocation of kidneys within the u.s. introduction : organ shortage for transplantation has led to the use of marginal kidneys, which has been associated with poorer but still acceptable results. in our center, the transplantation of two very marginal kidneys into a single recipient was used as a strategy to increase the number of organs available. the present study reports renal function and survival of dual-kidney transplants (dkt) performed at our institution. methods : from october to june , transplants were performed at our center, from which were dkt. kidney selection for a dkt was based on refusal of the kidneys for single kidney transplantation (skt), donor's age ≥ years and ≥ % of glomerulosclerosis on pre-implantation biopsy. the calculated creatinine clearance (crcl, ml/min), graft and recipient survival from dkt were compared to those of skt from ecd (based on unos criteria, n= ) and ideal donors (id, aged to y, n= ) over a seven-year period. results : dkt donors were significantly older than the two other groups (dkt: year old, ecd: , id: ). dual transplants were offered to older recipients by design ( , , ). delayed graft function was more prevalent for dkt and ecd than with id ( %, %, %). twelve, and -month crcl were similar for dkt and ecd but lower than id (twelve months: , , ; months: , , ; months: , , , respectively). patient survival, actuarial graft survival and actuarial graft survival censored for death were similar between the groups. conclusion : to our knowledge, this study reports short and long-term outcome of the largest cohort of dkt performed at a single institution. it shows that dual-kidney transplantation is a more complex intervention with more risks of surgical and post-op complications (data not shown). however, with adequate surgical expertise, dkt patients can expect long-term results comparable or even better than ecd. in our center, the use of dkt has increased the number of transplantations by % for the period of study. it allowed older patients to receive a graft within shorter delays. moreover, it permitted a more judicious allocation of a resource that is still limited. an results: very few differences resulting from donornet were seen. for regionally exported kidneys, cold ischemia time (cit) was . h before donornet (bd) and . h after donornet (ad); . % of regionally exported kidneys had cit> h bd, as compared with . % ad. for nationally exported kidneys, cit was . h bd and . h ad; . % of nationally exported kidneys had cit> h bd, as compared with . % ad. the same hucs of exported kidneys bd were also hucs ad. of the top centers utilizing exported kidneys bd, only one was not within the top centers utilizing exported kidneys ad. when we looked at cit for kidneys exported to hucs, again there was little difference before or after donornet, with a trend to possibly longer cit for hucs. for kidneys regionally exported to hucs, cold ischemia time (cit) was . h before donornet (bd) and . h after donornet (ad); % of regionally exported kidneys had cit> h bd, as compared with . % ad. for kidneys nationally exported to hucs, cit was . h bd and . h ad; . % of nationally exported kidneys had cit> h bd, as compared with . % ad. conclusion: the first months of national implementation of donornet were not successful in decreasing cit for regionally or nationally exported kidneys. furthermore, the centers to which a high proportion of these kidneys were exported did not change as a result of donornet, and for these centers allocation efficiency is no better and might even be worse than before. machine cold machine preservation has been shown to improve outcome following transplantation of deceased heart-beating donor kidneys. to determine whether cold machine preservation also improves outcome of kidneys donated after cardiac death (dcd) we undertook a multicentre randomized controlled trial in the uk comparing machine perfusion with simple cold storage. one kidney from each dcd donor was randomized to machine perfusion (organ recovery systems lifeport device with kps- solution), the other to perfusion with uw (viaspan) solution followed by simple cold storage. the primary outcome measure was the need for dialysis in the first days (delayed graft function, dgf); secondary outcome measures included gfr at week (mdrd technique); and month graft and patient survival. at the time of writing, one week outcome data are available for all patients. a sequential design was used with the data inspected after transplants and then every . all recipients received basiliximab, mycophenolate sodium, tacrolimus and prednisolone. the significance of the initial results has been tested using paired t tests and mcnemar's exact tests. recruitment was stopped after donors when the unadjusted sequential analysis concluded that there was no difference in the incidence of dgf. of the kidneys were transplanted; one was not used for anatomical reasons. one kidney suffered primary non function; it had been machine preserved. the results shown below represent intention to treat. introduction: split liver transplantation has been adopted as an alternative to expand the organ donor pool. while the adult/child split liver (a/csl) in which an extended right graft (erg) is transplanted into an adult and a left lateral segment (lls), transplanted into a child, has gained a wide acceptance within the transplantation community, adult/ adult split liver (a/asl) in which the liver is split into a full right (fr) and full left (fl) is still performed very rarely. we analyzed the experience at our center with split liver grafts over a period of years. material and methods: between october and october we performed a total of liver transplants in recipients ( / children and / adults). among the ( children and adults) recipients of a primary isolated liver transplant, ( children and adult) were transplanted with a a/c or a/a sl. the recipients of a whole size graft ( adults and children) were used as a control. results: adults: patients received a split liver graft ( erg graft and fl/fr grafts) and a whole liver graft. overall the incidence of biliary complications using a split liver graft was % and with a whole liver graft % (p= , ).for the recipients of a split liver graft and years patient and graft survival was %/ % and %/ %. recipients of a whole liver graft had a and years patient / graft survival of %/ % and %/ % respectively. children: children received a split graft ( lls, erg and fl) and a whole size graft. biliary complications occurred in % of the recipients of a split liver graft and % of the recipients of a whole size graft (p < , ). among the recipients of a split graft and years patient / graft survival was %/ % and %/ % respectively. the recipient of a whole size graft had a and years patient / graft survival of %/ % and %/ %. conclusion: incidence of biliary complications is significantly higher using a split graft. at a high volume center, use of split liver grafts reached comparable and even better results of a whole liver graft in terms of patient and graft survival. thus, the use of split grafts should be strongly encouraged to expand the donor pool. a groups of primary ldlt and ddlt recipients were identified by matching diagnosis, meld score, and recipient age. cost data, acquired from the hospital financial database and inflation adjusted and clinical outcomes were compared between the groups. background: liver transplant recipients with (gw/rw) < . % are thought to have a higher incidence of post-operative complications, including small for size syndrome (sfss), and overall inferior outcomes. we analyzed a cohort of partial liver graft recipients and compared those with gw/rw < . % to those with gw/rw > . %. results: between and , adult patients underwent partial graft liver transplant. seventy-six grafts were from live donors (ldlt) and the remaining were from deceased donor split-liver transplants (slt). of these, patients had gw/ rw < . % ( ldlt, slt), and had gw/rw > . % ( ldlt, slt). median follow-up was . months and did not differ between the two groups. baseline demographics including donor and recipient age, and meld at the time of transplant also did not differ between groups (table) . three-month and -year graft survival for the two groups was comparable. three recipients with gw/rw < . % developed sfss ( . %); all three had inflow modification with splenic artery ligation (sal), and of the grafts was lost at one-year. eight recipients with gw/rw > . % developed sfss ( . %) (p = ns); six underwent sal and none had graft loss at one-year. there was a trend toward increased hepatic artery thrombosis with the smaller grafts that did not reach statistical significance (p = . ). the incidence of other surgical complications was similar between the two groups (table) . the diagnosis of rejection in post-transplant kidney disease depends largely on the assessment of biopsy pathology, which suffers from arbitrary scoring, subjectivity, and sampling variance. with microarray gene expression data from biopsies for cause, we used predictive analysis of microarrays (pam) to build a rejection classifier. because of the imperfect gold standard, the classifier cannot and should not have extremely high predictive accuracy. the goal was to see if microarrays could detect a robust rejection signature despite this uncertainty, and to identify the top genes distinguishing rejecting from non-rejecting biopsies. by examining discrepancies between pathology diagnoses and pam predictions, and using clinical follow-up information, we combine the strengths of each method to produce a more accurate diagnostic system. biopsies with rejection plus clinical episodes (fig. ) have higher positive predictive value than those lacking episodes. the majority of episodes occur in the top part of fig. , indicating that a valid rejection signature is being detected. of the exceptions, most had been treated with steroids before the biopsy, suppressing their gene expression patterns. samples called borderline rejection separated into two distinct high/low probability groups using pam, with all the cases classified as clinical rejection episodes occurring in the high probability group. of the genes chosen by the classifier, had previously been annotated in our studies of mouse kidney graft rejection. gzma, gzmb, and prf ranked th, nd, and th respectively. the top five genes were: cxcl , gbp , cxcl , indo, and cxcl , all previously annotated in rejecting mouse kidneys as interferon-γ induced genes. in summary, gene expression-based classifiers, combined with histopathology, promise more accurate diagnostic assessment of the state of kidney transplants at the time of biopsy. moreover our data driven classifier independently identifies the genes previously annotated in experimental studies. influence of donor background: renal-cell associated tlr activation was found to be important in mediating ischemia reperfusion injury to murine kidneys. we hypothesized that genetic variations within the tlr gene of the kidney donor affects the rate of delayed graft function (dgf). methods: we genotyped the functional tlr polymorphisms (snps) d g (rs ) and t i (rs ) in kidney donors from centers and correlated them with the occurrence of dgf. dgf was defined as the need of dialysis within days post-transplant or less than % drop in creatinine within the first hours after transplant. in a sample of patients from one center, we analysed intragraft hmgb- (high mobility group box protein- ) gene expression in pre-and post-implantation biopsies. statistical analyses were performed using the spss statistical package. results: both tlr snps were in hardy-weinberg equilibrium, and were combined for further analysis. recipients of a tlr mutated kidney showed a significant lower rate of dgf (p= . ), which was persistent after correction for known donor-derived risk factors (donor age, ecd vs. dcd-kidney, cold ischemia time, p= . , hr . , cl . - . ). additionally, we confirmed the presence of a possible tlr ligand, hmgb- , in pre-and post-implantation biopsies. we also confirmed the presence of a functional tlr receptor in human proximal tubular cells which expressed mcp- , il -β, il- and tnf-α after specific tlr agonist treatment. conclusion: human renal tubular epithelial cells express tlr and a functional tlr snp in donor kidney is associated with lower rate of delayed graft function. background: methylation of promoter cpg islands has been associated with gene silencing and demonstrated to lead to chromosomal instability. we postulate that differences in methylation patterns observed in tissues ranging from normal to cirrhosis to hcc may lead to the discovery of direct precipitating events that contribute to tumorigenesis in hcv-hcc patients. methods: dna from hcv-hcc tumors and corresponding non-tumor hcv cirrhotic tissues as well as independent hcv cirrhotic tissues and normal liver tissues were bisulfite treated and hybridized to the illumina goldengate methylation beadarray cancer panel i for interrogating cpg sites in the promoter regions of distinct genes. for each cpg site, a summary statistic representing "percent methylated" was estimated. for each cpg site, a jonckheere-terpstra test was applied to identify whether there was a significant monotonic trend in percent methylated across the independent normal, cirrhotic, and hcc tissues. in addition, the paired hcc and non-tumor cirrhotic tissues were analyzed using a paired t-test. the q-value method for estimating gene-wise false discovery rates (fdr) was used to adjust for multiple comparisons; cpg sites with an fdr< . were considered significant. to identify if serological responses to allogenic non-hla renal compartmentspecific antigens can be detected after renal transplantation (txp). methods: paired pre-and post-transplant (mean time months) serum samples from pediatric kidney allograft recipients were used for identification of de novo non-hla antibody formation assessed using invitrogen protoarray (v ), measuring signal post-pre. probes from the protoarray and cdna microarray platforms were re-annotated to current ncbi entrez gene identifiers using ailun. cdna arrays (gse: ) from normal kidney regions (inner and outer cortex, inner and outer medulla, papillary tips, renal pelvis and glomeruli; see table) were analyzed for compartment-specific genes by sam, and the highest-ranked genes in each compartment (ks two-sample test p < . ) and were ranked against each individual patient's numerical antibody response across the proteins on the protoarray. results: in % of patients ( / ), kidney-specific serological responses against the renal pelvis were the first to be detected, followed by responses against renal cortex. control gene expression data sets from other solid organs (gse: ; lung, heart, pancreas) did not demonstrate immune-response enrichment. the presence of these antibodies was not associated with donor specific hla class i or ii antibody levels. conclusion: we demonstrated de novo formation of circulating non-hla non-abo antibodies after txp, irrespective of hla antibodies, are detected against kidneyspecific protein targets. as these antibodies are not seen against other solid organs, the response is likely allogeneic. the renal pelvis and renal cortex have the highest immunogenic potential. we conclude that urinary cell mrna profiles that include levels of mrna encoding tubular proteins and mrna encoding proteins implicated in emt offers a noninvasive means ascertaining renal allograft status with respect to presence or absence of can. functional functional annotation of these genes identified cell cycle pathway and carboxylic acid metabolism pathway as potentially relevant for both datasets. altogether, these data suggest that as compared with recipients requiring on-going immunosuppression, operationally tolerant liver recipients exhibit traits of immunological quiescence and activation of natural killer related pathways. liver and kidney tolerant states appear to be fundamentally different biological processes, although some common pathways could be relevant to both settings. genetic introduction: both normal and fibrotic renal allografts develop a large number of persistent changes in pro-inflammatory gene transcripts early after transplantation ( ) . the aim of the current study was to determine if this "transplant effect" is attenuated by immunosuppression, fibrosis or hla-match. methods: we studied histologically normal implantation biopsies (t ) and m protocol biopsies (t ) from living donor kidney transplant recipients who had no identifiable post-transplant complication (no acute rejection, polyoma virus, etc). patients were stratified into distinct clinicopathologic groups. the first three groups had normal histology at both t and t and included: ) hla identical-tac treated (n= ); ) non-hla identical-tac (n= ); ) non-hla identical srl (tac-free, n= ). a fourth group was histologically normal at t but developed mild fibrosis by t (n= ). mrna expression from these biopsies was measured using the u plus . microarray and custom taqman low density arrays (tlda). data for each group was tested using a generalized linear model and changes common to each dataset identified (p< . ). results: in addition to transcript changes specific for each clinicopathologic condition, a large number of transcripts (n= ) were altered in all groups. % (n= ) had higher expression in all comparisons at t versus t , including fibronectin and collagen iv. % (n= ) of transcripts were considered down-regulated, including interleukin and β. gene ontology and signaling pathway analyses showed many of the transcripts to be involved in pro-inflammatory and immune response signaling pathways (ex. tlr, il- /- , etc). custom tldas were used to study several genes considered not detected by microarray. this included tgf-β , cd- e/- /- and vegf, all of which were significantly up-regulated in multiple comparisons. conclusions: persistent changes in pro-inflammatory transcripts occur in all renal transplants. this "transplant effect" cannot be explained by calcineurin-inhibitors (occurs in tac-free immunosuppression), increased alloreactivity (similar in hla identical/ non-identical) or fibrosis. a likely cause is persistent changes from ir injury. the role of the "transplant effect" in long-term allograft survival is unclear, but it likely interacts with other forms of graft injury. background: a critical gene-set for acute renal rejection (ar) diagnosis in peripheral blood has been previously identified by our group using cross-platform hybridization of unique peripherla blood samples with matched biopsy diagnosis, with ar (n= ) and without ar (sta; n= ), with cross-hybridization across human microarray platforms: k cdna lymphochip, k agilent and k affymetrix hu plus. in this study we proposed to verify and validate this gene-set for ar diagnosis and prediction by peripheral blood pcr-based analysis. method: / peripheral blood samples from the microarray gene-set were selected for -well format multiplex abi-taqman qpcrv validation of ar diagnosis across the gene-set. an independant set of new, time and immunosuppression matched, peripheral blood samples ( sta, ar) were used as a test-set for blinded prediction of ar diagnosis using qpcr across the most informative genes. sequential samples from ar patients (at ar, - months prior to, and after ar) were also examined by qpcr for these genes for blinded ar prediction, prior to biopsy proven ar. prediction models were generated using logistic regression analysis and p< . considered significant. result: in the validation set of samples, / genes were highly significant for confirming peripheral blood-ar diagnosis (p< . ). in the test group of samples, ar was predicted with a very high level of sensitivity and specificity (ppv and npv> %). in the group of ar patients with sequential samples pre-and post-ar, expression for these genes were significantly elevated in the pre-ar samples (vs. sta, p< . ) but were not different between pre-ar and ar values. conclusion: a highly specific and sensitive biomarker panel of genes has been derived and tested by cross-platform microarray analysis. thsi gene-set has now been validated and further verified by multiplex pcr, for ar diagnosis and prediction, even months prior to the rejection event. utility of this gene-set should be further validated in real-time by serial longitudinal peripheral blood testing, for more senstive and specific minimally invasive monitoring for graft rejection. tolerance/immune deviation ii background: our previous studies showed that regulatory t cells (treg) execute suppressive function in both the inflammatory site of the allograft and the draining lymph node (dln) to suppress allograft rejection. we explored how treg influenced the migration and function of antigen specific effector t cells and dendritic cells (dc) at these two sites in an islet transplant model. methods: treg were sorted from wild type or ccr -/-c bl/ mice, labeled with pkh , and transferred directly to islet allografts (balb/c into c bl/ ). effector cd t cells (te) from alloantigen specific t cell receptor transgenic mice were labeled with csfe and transferred intravenously. treg and te migration to islet grafts and dln were analyzed with flow cytometry and immunohistochemistry. chemokine expression was determined by rt-pcr. cx cr gfp mice, in which dc are internally labeled with gfp, served as islet donors, and donor dc migration was tracked with fluorescence microscopy. results: during priming and rejection, islet allografts expressed a panel of inflammatory chemokines shortly after transplantation that recruited te to the islets. te accumulated and proliferated in both the islet allograft and the dln. concomitantly, donor derived dc migrated from the islet to the dln as early as day after transplantation. local transfer of treg into the islet allograft inhibited islet chemokine expression, inhibited donor dc migration in an il- and tgfb dependent fashion, and reduced te migration to and proliferation in the graft. the transferred treg also migrated from the islet allograft to the dln, and directly inhibited te accumulation and proliferation in the dln, and migration of te to the islet. ccr -/-treg, which could not migrate to the dln but were retained within the graft, and were far less effective than wild type treg in inhibiting te migration and proliferation into both the islets and dln. conclusions: treg migrate to the islet and suppress parenchymal cell chemokine expression, dc migration, and te responses in the islet allograft. treg also migrate to the dln to suppress te proliferation and migration. treg trafficking from the allograft to the dln is crucial for suppressive function in order to target the separate effector mechanisms. these results demonstrate novel and important functions for migration in treg induced suppression and graft survival. tregs cd +foxp + regulatory t cells (tregs) play an important role in transplant tolerance, yet basic aspects of treg biology including the mechanisms involved in their induction remain unclear. α-cd rb is a potent tolerogenic agent that induces a x increase in number of tregs in wt mice, even in the absence of allo-antigen. here we use foxp red fluorescent protein reporter knock-in mice to study treg homeostasis and identify the mechanisms by which α-cd rb induces tregs. cfse-stained highly sort-abstracts purified foxp + or foxp -cells were adoptively transferred into fully replete naive wt congenic mice. whereas only - % of transferred foxp -cells undergo homeostatic proliferation (hp) over d, - % of foxp + cells proliferate -a surprisingly high rate of hp. moreover, treatment with α-cd rb markedly enhanced hp by transferred foxp + cells, resulting in a x increase in cell number. α-cd rb induced de novo foxp expression in - % of transferred foxp -cells (many of which subsequently underwent hp). thus, α-cd rb induces both conversion of foxp -to foxp + cells and promotes hp in foxp + cells in the absence of exogenous antigen. we then addressed the signals controlling basal hp of tregs and both hp and conversion mediated by α-cd rb. cfse-stained congenic cd + cells adoptively transferred into naive wt mice were untreated, or received csa, α-cd rb, or both and foxp + and foxp -cd cells were assessed on d . calcineurin inhibition greatly reduced basal hp of transferred foxp + cells compared to untreated mice. moreover, csa completely abrogated increased treg hp induced by α-cd rb, although conversion still occurred. next we assessed the role of tgfβ. we found that blocking tgfβ signaling with α-tgfβ shortened allograft survival, but had no effect on basal treg hp, or on treg hp or conversion by α-cd rb. finally, although exogenous antigen is not required, basal and α-cd rb-induced hp may still require recognition of self-ag bytregs. indeed, preliminary results reveal that when cfse-labeled cd + cells are transferred into mhcii ko mice, basal hp by tregs is reduced and not restored by α-cd rb. thus, α-cd rb alters normal controls regulating hp by treg. moreover, both basal and α-cd rb-mediated hp requires intact calcineurin activity and tcr signaling, but not tgfβ. understanding the signals controlling hp in treg may reveal new therapeutic targets for tolerance induction. we report the first demonstration of a tolerance-inducing strategy based on posttransplant administration of allo-ag-specific treg (aastreg) and rapamycin (rapa), in a fully allogeneic, unmanipulated mouse heart transplant model. enrichment of naturally-occurring aastreg represented the first step to counterbalance the high frequency of alloreactive t cells. selection was achieved by co-incubation of freshly-isolated cd + cd + t cells with donor bone marrow-derived dendritic cells (dc). the source of stimulatory factors necessary to sustain treg proliferation was the supernatant of cd + cd -t cells co-cultured with allogeneic dc (mlrsup). use of mature (cd high ) dc favored extensive expansion of foxp cells capable of il- production (t h ). co-culture of treg with immature dc in the presence of mlrsup rendered a t cell population with regulatory phenotype: foxp + , gitr + , ctla- + , ccr + , cd l -. these cells inhibited in vitro effector t cell proliferation at : and : treg:t cell ratios. the suppressive activity was ag-specific; no significant inhibition was evident when effector t cells were stimulated by third party dc. aastreg were then tested for their ability to induce transplant tolerance in a mouse heterotopic heart allograft model (balb/c to c bl/ ; unmanipulated). rapa was used: i) to inhibit effector t cell proliferation, while sparing treg activity and thus enhancing the in vivo treg:effector t cell ratio; ii) to promote resolution of the inflammatory state and preserve the susceptibility of conventional t cells to treg suppression. graft recipients received rapa ( mg/kg/d; d - ) and x aastreg i.v. on d . in comparison to the untreated group (median survival time, mst= d; n= ), rapa alone extended mst to d (n= ), with no long-term graft survival. under cover of rapa, aastreg exerted a profound tolerogenic effect: > % recipients exhibited longterm graft survival (mst> d; n= ). this effect was stronger than polyclonal treg administration: % long-term survivors (mst> d; n= ). moreover, the tolerogenic effect was ag-specific, as aastreg selected against third party dc (c h/hej) did not prolong graft survival in comparison to the rapa-only control group. these results indicate the feasibility and therapeutic potential of ag-specific tolerogenic cell therapy based on post-transplant administration of selected aastreg. direct intravenous immunoglobulins (ivig) is an effective treatment for t-cell mediated graft rejection and autoimmune diseases. ivig treatment is associated with rapid clinical improvements without the side effects of global immunosuppression. this prompted us to ask whether ivig might enhance directly the suppressive function cd +cd +foxp + regulatory t cells in vitro and in vivo. in vitro, mouse cba/ca (h k ) total cd + or cd + cd responder cells were stimulated with c bl/ (h b ) splenocytes, and human cd + or cd + cd cells were stimulated with allogeneic antigen presenting cells. in vivo, * total cd + or cd + cd -t cells of cba/ca mice were adoptively transferred into cba/rag -/mice, and one day later transplanted with a tail skingraft of c bl/ mice. ivig was administered i.v. on day , , , and . human serum albumin (hsa) was used as a control. binding of ivig and activation status of foxp + cd + t cells were determined. in vivo, only when total cd + t cells, but not cd -t cells, were adoptively transferred, ivig protected against t-cell mediated rejection of the fully mismatched skingraft (mst: ivig > vs hsa days, p< . ). ivig binds to ± % of foxp + t cells, while binding to foxp -t cells was minimal. this binding was partially fcγ receptor mediated. furthermore, ivig treatment resulted in activation of cd + t cells as detected by increased expression of phosphorylated zap /syk, which could be abrogated by specific tyrosine kinase inhibition. in vitro, ivig inhibited the alloproliferative response of murine cd + t cells by ± %, but after depletion of cd + t cells, this inhibition decreased to ± % (n= , p< . ). similar results were found in human mlr, where depletion of cd + t cells resulted in twofold reduction in ivig mediated suppression. significantly, incubation of human cd + cd + with ivig enhanced their ability to suppress allogeneic t-cell proliferation (ivig ± % vs hsa ± % of inhibition, n= , p< . ) . our results identify a novel pathway through which ivig treatment induces direct functional activation of both mouse and human regulatory t cells. immediate binding and activation of regulatory t cells is one of the mechanisms in the immunomodulatory repertoire of ivig, which allows rapid inhibition of allogeneic responses, and therefore can be a valuable tool after organ transplantation. generation foxp is a dna-binding protein that is necessary for regulatory t cell (treg) function, though recent data indicate that detection of foxp mrna or protein alone does not necessarily indicate whether the associated foxp + cell is functional or not. to that end, our analysis of foxp sequence showed the presence of an rxxr motif, conserved between mice and humans, that is located amino acids (aa) from the carboxy terminus of foxp and is a potential recognition sequence for cleavage by proprotein convertase enzymes. we found several proprotein convertases are expressed by naturally occurring tregs, with further upregulation upon tcr activation. we generated an antibody against the aa carboxyl peptide of foxp , and by western blotting detected a peptide of about . -kda, consistent with a -aa long carboxy-terminal foxp cleavage product. both cleaved and uncleaved forms of foxp were resolved by sds-page, and the cleaved form was found only in the dna-bound fraction. the requirement of proteolytic cleavage, at the intact tetrabasic rxxr motif ( rkkr ), for full treg function was shown by abolishing the motif through mutagenesis, followed by retroviral expression of mutants, and analysis of proteolytic cleavage by western blotting. assays of treg function by cells expressing either long-or short-foxp mutants showed short-foxp (missing the carboxy-terminal -aa) suppressed teff cell proliferation significantly more effectively than wt foxp or an engineered and cleavage-resistant mutant, termed long-foxp (p< . ). moreover, adoptive transfer of cells expressing short-foxp prevented experimental colitis more effectively than wt-foxp (p< . ), and both were more effective than cells expressing long-foxp . animals that received cells expressing short-foxp gained weight and were protected from disease. thus, function of foxp is regulated at a post-translational level by proteolytic cleavage and the short form of foxp represents the active form. our data shows proteolytic cleavage of foxp is key to the generation of functional tregs, with enzymes(s) of the proprotein convertase family likely playing an important role in this process and providing an extra and hitherto unrecognized important level of regulation for foxp . blocking since tgf-β is secreted in a latent form and active tgf-β is rapidly cleared from circulation. in order to make long-lasting active form of tgf-β, the mutant tgf-β was fused to a human igg fc component. the secreted human mutant tgf-β/fc (hmtgf β /fc) fusion protein stained with both anti-human tgf-β and anti-human igg antibodies, confirming the cytokine and isotype specificity of tgf-β moiety and fc domain. moreover, in vitro bioassay, hmtgf-β/fc inhibited il- dependent ht- cell proliferation in a dose dependent manner and had a circulating half-life of hours in mice. in vitro, anti-cd and anti-cd triggered cd + or cd + t cell proliferation, measured by h-thymidine uptake, could be synergistically inhibited by tgf-β and rapamycin. at the same time, the two reagents when added together could synergistically promote the induction of cd + foxp + t cells from peripheral naïve cd + foxp -t cells. in a pancreas islet transplantation model in which the fully mhc mismatched dba/ islets were transplanted into c bl/ mice rendered diabetic by streptozotocin, mean survival time of islet was days in control recipients (n= ). administration of muttgf-β/fc resulted in a delayed islet allograft rejection (mst; days, n= ). treatment with rapamycin prolonged islet grafts survival in % of recipients. in contrast, combined treatment with tgf-β/fc and rapamycin by four doses produced indefinite islet allograft survival in % cases. we have produced the long-lasting active hmtgf-β/fc fusion protein. the tgf-β/fc is synergistic with rapamycin to convert naïve cd +foxp -t cells into cd +foxp + tregs and promote long-term islet allograft engraftment. the the recognition of microbial motifs by the innate immune system leading to the stimulation of adaptive immune responses may explain the relationship between infections and susceptibility to graft rejection. a number of infectious agents have been reported to prevent the induction of allograft tolerance, however, none of these can reverse established tolerance, a situation that is highly relevant to clinical transpalntation. we here report that established allograft tolerance (induced with anti-cd + donorspecific transfusion) can be reversed in a cardiac transplantation mouse model following infection with listeria monocytogenes. this reversal of tolerance was dependent on the presence of both cd + and cd + cells, as well as of the tlr/il- /il- adaptor molecule, myd . we hypothesized that the reversal of tolerance requires that alloreactive conventional t cells (tconv) escape dominant regulation by pre-existing tregs. these alloreactive tconv can then proliferate resulting in an increased ratio of tconv:tregs favoring the reversal of established tolerance. indeed, we observed that tolerant grafts harbored low numbers of infiltrating cd + and cd + t cells ( - . x /heart), with - % of the infiltrating cd + cells co-expressing foxp (n= ). following the reversal of tolerance and allograft rejection, a - fold increase in cd + foxp and cd + foxp -tconv was observed in the graft, but no increase in the foxp + subset. in contrast, we observed no significant changes in the splenic t cell subsets, raising the possibility that the escape from tregs occurs locally within the graft. infection of tolerant il- -or ifnar -deficient recipients with listeria monocytogenes failed to reverse tolerance, consistent with a hypothesis that the initial escape of tconv from regulation may depend on il- , while their proliferation may be type i ifn-dependent. in summary, the conditions for the reversal of tolerance is more stringent than the prevention of tolerance, and requires myd -signalling and the presence of both cd + cells and cd + cells, as well as il- and type i ifn signaling. these studies point to the potential impact of bacterial infections on established tolerance, and to novel strategies to monitor and facilitate the maintenance of tolerance in the clinic. th our laboratory has identified kα tubulin, as an epithelial autoantigen to which immune response occurs following human ltx. further, we have shown a strong correlation between the presence of antibodies (abs) to kα tubulin and development of chronic rejection ie bronchiolitis obliterans syndrome (bos). goal of our study is to test the hypothesis that epithelial damage due to allo immunity results in remodeling exposes otherwise cryptic self antigens including kα tubulin and collagen type v (coll v) leading to cellular immune reactivity and ab production against these auto-antigens which may play a role in the pathogenesis of bos. patients who developed anti-hla ab and patients who had no detectable anti-donor hla abs post-ltx by luminex assay were analyzed for development of abs to kα tubulin and coll v using elisa method developed in our laboratory. the elisa for kα tubulin used recombinant protein ( µg/ml) purified on affinity resin. the elisa for collagen v uses commercially available human collagen v ( µg/ml). elispot assay for ifnγ and il- were performed for bos-and bos+ patient pbls (at the time of bos diagnosis), after stimulations with kα tubulin protein. the levels of anti-tubulin abs and anti-coll v abs were increased significantly in bos+ ltx recipients with anti-hla compared to those with no anti-hla, table (p< . ). . surprisingly, both cd and cd subsets induced long-term survival of secondary test grafts (> days). however, only the cd + t-reg prevented tvs (ni= + , + % of vessels), as compared to cd + t-reg (ni= + in + % of vessels). the cytokine profile indicated a dominant il- response. allo-antibody analysis showed up-regulation il- /il- dependent igg and igg c. conclusion: allochimeric protein-therapy and csa treatment generate t-reg that prolong graft survival, but only cd + t-reg generated from allochimeric protein-therapy prevent tvs. this cd + t-reg may be responsible for long-term graft maintenance through its unique ability to control anti-inflammatory and alloantibody responses. immunodominant h background: minor histocompatibility ags (mihas) are self peptides, derived from proteolytic processing of normal cellular proteins. miha incompatibility can induce t cell response to dominant mihas and facilitates expansion of ctls and graft rejection. however, the contribution of ctls recognizing these mihas in solid organ transplantation has not been fully evaluated. methods: balb.b (h- b ) donor hearts were transplanted heterotopically to c bl/ (h- b ) recipients. in vivo alloreactive cd t cells were monitored with peptide/mhc multimers. α-lfa- mab was given to recipients to prevent rejection. various numbers of h -specific cd t cells or cd t cells lacking h specificity were adoptively transferred into balb.b graft bearing b .scid recipients. results: % of transplantation recipients developed acute rejection and showed markedly increased numbers of h -specific cd t cells at day - in spleen. abundant cd infiltration was found and selective infiltration of h -specific cd t cells in the graft was confirmed with flow cytometry and in situ tetramer staining. α-lfa- mab treatment prevented acute rejection (mst> ) and allogeneic t cell expansion. it also profoundly attenuated neointimal hyperplasia ( graft-bearing b .scid mice. we found that the degree of acute rejection positively correlated with the number of h -specific cd t cells transferred. however, transferred h -specific cd t cells did not cause chronic rejection. interestingly, greater numbers of cd t cells with h immunodominance were found in spleen, blood and graft at days after adoptive transfer of cd lacking h specificity compared to h -specific cd t cell transfer. conclusion: h responses dominate other miha immune responses during acute rejection after balb.b to b cardiac allograft. we confirmed a role of immunodominant h specific cd t cells as pathological effector t cells in acute rejection but not in chronic rejection. maintaining a h response may be beneficial in allotolerance to suppress miha responses that could induce chronic rejection in long-term grafts. chronic lung allograft rejection, bronchiolitis obliterans syndrome (bos), affects up to % of transplant survivors years post-ltx. human neutrophil peptides (hnp - / α defensins), have been identified in the lavage fluids from patients with chronic rejection by proteomics. goals of our study were to determine the role of anti-hla abs and defensins in bos and to define the interactions between α- antitrypsin (aat), and defensins in regulating inflammation leading to epithelial cell proliferation and bos pathogenesis. bal and serum samples (post-ltx and pre bos) from bos+, bos-patients and normals were analyzed by elisa for α defensins (hnp - ), human β defensin (hbd ) and aat. small airway epithelial cells (saec) were treated with hnp or , with or without equimolar aat or with anti-hla abs and analyzed for levels of hbd production (elisa), cytokine and chemokine (luminex), and cell surface adhesion molecules (icam, vcam) by facs. bal and serum from bos+ patients had high levels of hnp - and hbd compared to bos-recipients or normal sera (p< . ) ( table ). there was also a significant decrease in aat levels in bos+ compared to bos-or normal serum (p= . ) ( table ) . saec produced human β defensins following anti-hla ab stimulation or by hnp treatment (table ) . there was increase in adhesion molecules (icam, vcam, folds) cytokines {il- , il- r α ( folds), il- β, il- ( folds)}, il- ( folds decrease), chemokines (il- , mcp , - folds increase) and growth factors (egf and vegf, . folds increase) in response to treatment with hnp or hnp compared to untreated saec, that was inhibited by aat. increased defensins and decreased aat levels were seen in lavage and serum of ltx recipients with bos. anti-hla abs further stimulate defensin production by saec. we conclude that chronic stimulation of epithelial cells both by defensins and anti-hla abs can lead to increased growth factor production contributing to the pathogenesis of bos. under tubular atrophy/interstitial fibrosis (ta/if) remains a major cause of late kidney allograft loss and epithelial mesenchymal transformation (emt) is now appreciated as a key feature in this process. we hypothesize that macrophages play a direct role in the development of ta/if by creating a profibrotic microenvironment and stimulating emt within the allograft. in human kidney allografts with ta/if (n= ), macrophage infiltration detected by immunostaining was significantly upregulated (mean score . ± . ) compared to biopsies without corresponding pathological changes from recipients with stable function (n= ; . ± . ; p< . ) and the extent of this staining also correlated to the magnitude of graft dysfunction (p< . ). rt-pcr in ta/if grafts also showed marked upregulation for emt markers αsma ( . ± . -fold; p< . ), s a ( . ± . -fold; p< . ), and vimentin ( . ± . -fold; p< . ), compared to stable function allografts. to explore the macrophage-emt relationship, we co-cultured freshly isolated human monocytes over a primary culture of human proximal tubular epithelial cells (ptecs) using culture inserts allowing media exchange but forbidding contact between the two cell populations. by rt-pcr, co-cultured epithelial cells showed significant downregulation of bmp ( . ± . -fold; p< . ), a negative regulator of emt, and epithelial marker e-cadherin ( . ± . -fold; p< . ), with marked upregulation of emt genes s a ( . ± . -fold; p< . ) and αsma ( . ± . -fold; p< . ) compared to ptecs cultured in media alone. investigating the mechanism of monocyte driven emt, we evaluated the effects of am , a synthetic retinoid that also inhibits il- and vegf signaling. am markedly reversed the transcriptional profile with reduction of emt markers s a ( . ± . -fold; p< . ), αsma ( . ± . -fold; p< . ) and vimentin ( . ± . -fold; p< . ), and a simultaneous increase in expression of bmp ( . ± . -fold; p< . ), thus reversing the pro-emt milieu. these results indicate that human monocytes induce transcription of emt related genes in ptecs, even in the absence of physical contact. moreover, this phenomenon appears to be mediated in part by il- , vegf, and other pathways mediated by the retinoic acid receptor. thus, in addition to their typical immune functions, macrophages support a milieu within allografts that promotes ta/ if in humans. further investigation into this novel pro-ta/if pathway could ameliorate chronic injury and improve graft survival. old donor kidneys are more likely to develop long-term graft failure, especially if they are exposed to stresses (e.g. rejection). this limited ability of old tissue to withstand stress may be due to its reduced capacity of replication and regeneration. telomere shortening determines lifespan and regenerative capacity. we showed that telomere shortening occurs in old human kidneys. late-generation terc ko mice with critically short telomeres have a reduced lifespan, show an accelerated aging phenotype and are an ideal model to resemble the situation in old human donors. this study addressed the question whether iri causes greater damage in kidneys from late-generation terc ko mice. we studied terc wildtype (wt), early-(g ) and late-generation (g ) terc ko mice day (wt:n= ; g :n= ; g :n= ), (wt:n= ; g :n= ; g :n= ), (wt:n= ; g :n= ; g :n= ) and days (wt:n= ; g :n= ; g :n= ) after iri. acute tubular necrosis was found mainly on days and and was significantly higher in terc ko g kidneys. tubular atrophy (ta) and intersitial fibrosis (if), reflecting chronic damage, were first detected at day and increased in all mice with a significantly higher extent of ta/if in terc ko g kidneys at day ( fig.) . the cell cycle inhibitor p , a downstream mediator of senescence induced by telomere shortening, was significantly upregulated in all groups after iri. p levels were highest in terc ko g kidneys at day ( fig.) . proliferative capacity, as measured by ki- immunostainings at day , was significantly lower in tubular, glomerular and interstitial cells of kidneys from terc ko g mice. we show that late-generation terc ko mice with critically short telomeres have a greater susceptibility towards acute injury and develop more chronic renal damage. this is likely due to the reduced capacity of these kidney to proliferate and thereby to regenerate. our data strongly suggest a pathogenetic role of telomere shortening, an important senescence mechanism, for the development of long-term graft failure. results: belatacept treatment had no effect on the number of peripheral blood treg cells and t cell suppression assays. the percentage of foxp cells was significantly elevated in rejecting kidney allografts in belatacept-treated patients compared to cnitreated patients. conclusions: following chronic belatacept therapy, the number and function of peripheral blood treg cells are maintained in kidney transplant patients. belatacept enhances the treg population in the allograft in patients with acute rejection. therefore, our data suggest that co-stimulation blockade with belatacept does not affect treg homeostasis. the increased number of treg cells in rejecting allografts in belatacepttreated patients may provide a novel mechanism whereby belatacept can mitigate the severity of acute rejection and improve graft survival. the steady-state auc of n-desmethyl-aeb was minor in comparison to aeb and similar between the patient groups: ± ng.h/ml in transplantation vs ± ng.h/ml in psoriasis (p= . ). demographic covariates: in the first week posttransplant with patients receiving fixed-dose aeb , intersubject variability for c was % and for auc was %. aucs were similar in men vs women ( ± vs ± , p= . ). age, which ranged from - years, did not influence auc based on regression analysis (r = . , p= . ). there was a borderline-significant negative correlation between weight (range, - kg) and auc (p = . ); however, its clinical relevance was low in that it could explain < % of the variability in auc (r = . ). there was a significant positive correlation between aeb c and auc (r = . , p< . ). conclusions: ( ) in the first week posttransplant, patients achieved aeb blood levels anticipated for this regimen. ( ) there was notable intersubject pharmacokinetic variability at this time but it was not attributable to standard demographic factors such as sex, age, or weight. ( ) a good correlation was noted between c and auc suggesting that c might serve as a marker for total drug exposure. a we studied which is protocol would be best after rapid discontinuation of p. between / and / , st and nd kidney tx recips were randomized: csa-mmf (n= ) vs. high tac-low srl (n= ) vs. low tac-hi srl (n= ) (for tac and srl levels, high = - , low = - ). all received thymoglobulin (tmg) doses, and p for days; tmg was continued in dgf. min f/u = yr; mean = ± mos. there was no diff between groups in recip age, gender, ethnicity, prim dis, donor source, % retx, pra; or in donor age, gender, ethnicity. there was no signif diff. between groups (intention to treat) in actuarial patient, graft, death-censored (dc) graft, acute rejection (ar), or biopsy-proven chronic rejection rates (cr), serum cr level or calculated (mdrd) gfr or in studied side effects. cr(sd) . (. ); . (. ); . ( . ); . ( ); . (. ) . (. ); . ( ); ( . ); . ( . ); . (. ) . (. ); . (. ); . (. ); . (. ); . (. ) mdrd gfr(sd) ( ), ( ), ( ), ( ), ( ) ( ), ( ), ( ), ( ), ( ) ( ) the most common cause of graft loss was death with function (csa %, high tac %, low tac %). the majority of recips in each group remained p-free (csa %, high tac %, low tac %) but a large % were not on the medications which they were randomized to (csa %, high tac %, low tac %). we found a signif ↑ of new onset diabetes (nodm) (p=. ) in the tac-srl groups: csa %, high tac %, low tac %. there was a trend towards more use of lipd lowering medications in the tac/srl groups. in summary, all is protocols were effective; with min f/u yr for each group, patient and graft survival and ar rates are similar. we found an increased nodm and possibly hyperlipidemia in the tac/srl groups. purpose: we present preliminary -year results from a worldwide trial comparing srl regimens with tac+mmf. methods: renal transplant recipients (n= ) were randomly assigned to the following treatment regimens: group : srl ( - ng/ml, then - ng/ml after week ) + tac ( - ng/ml) with elimination at weeks (n= ); group : srl ( - ng/ ml through week , - ng/ml thereafter) + mmf (up to gm/day) (n= ); or group : tac ( - ng/ml through week , - ng/ml thereafter) + mmf (up to gm/day) (n= ). all patients received corticosteroids and daclizumab. in june , group was terminated (after all patients were accrued) because of increased acute rejection (ar) rates. results: demographic characteristics were similar between groups except for more females in group . patient and graft survival were also similar among groups (see table) . biopsy-confirmed ar (bcar) was significantly greater in group compared with groups and , p< . , and most occurred in the first months posttransplant with preponderance during the first months. subtherapeutic srl trough concentrations were reported in a large number of rejectors in group . most of the rejections in group occurred within the first months before tac was eliminated. all ars were mild to moderate; grade ii ars were proportionally greater in group . mean nankivell gfr was numerically higher in group . preliminary results at years show excellent patient and graft survival and similar renal function among treatment groups, despite higher ar rates in group . early adequate exposure to sirolimus is mandatory to achieve desired (low bcar rates) results. the goal of rapid discontinuation of prednisone (rdp) after kidney transplantation is to minimize prednisone-related side effects without increasing acute rejection (ar) rates or decreasing long-term graft survival. to date, studies have shown that rdp is associated with decreased prednisone (p)-related sided effects, and randomized trials of rdp (vs. long-term p) have shown little or no ↑ in ar rates. however, concern remains that long-term graft survival will be worse with rdp protocols. we studied t½ (the time it takes for ½ of the grafts surviving at year to subsequently fail) for st tx recipients treated with rpd ( - ) (n = ) (antibody, cni, antimetabolite [mmf or srl] , and rdp) vs. st tx historical controls ( ) ( ) ( ) ( ) ( ) ( ) (n = ) treated with a protocol of antibody, cni, antimetabolite [mmf] , and long-term p. table shows characteristiscs of the groups. pra; rdp were more likely to get a living unrelated donor transplant. t ½ is shown separately for living (ld) and deceased (dd) donor transplants in table . there was no significant difference in t½ between groups. we conclude that, compared to historical controls, rapid discontinuation of prednisone can be done without any detrimental impact on long-term outcome. a prospective, randomized study to confirm this observation is necessary. successful a) . we enrolled patients (table ) with an average follow-up of . + . months. subjects - years old included primary and re-transplants, with primary endpoints of renal function and can. follow-up averaged . + . months in patients withdrawn from ci ( from group , from group ). we found no increased rejection after ci discontinuation and that both ratg induction dosing regimen and ci discontinuation significantly impacted renal function and the development of can. we successfully discontinued both calcineurin inhibitors and steroids with either single or divided-dose ratg induction, and single-dose ratg induction independently associated with improved renal function and reduced can. study demographics single-dose ratg ( mg/kg x ) divided-dose ratg ( . mg/kg x ) group (n = ) group (n = ) group (n = ) group (n = ) age . ± . . in subset analysis, we also find an enhanced negative impact of srl when combined with steroids (str) in patients without dgf. conclusion: this data supports previous findings that srl may be associated with a sustained survival disadvantage apparent early post transplant, and that this effect appears exacerbated when combined with dgf or str. several potential explanations for this effect may include srl-associated prolonged early graft dysfunction, hyperlipidemia or proteinuria. however, patients initiating srl after discharge may not evidence this survival disadvantage, and this question was not addressed here.these findings may be of particular importance to centers employing combined srl and str therapy, as well as to define the best mode of support during recovery from dgf. prospective randomized studies would be necessary to evaluate these. table shows cai in both groups from to years. in slr group the doses of cin were significantly lower from one through years (p= . ). cai due to interstitial fibrosis/ tubular atrophy was significantly lower in slr group at years. our data shows that year patient and graft survival, graft function, bpar and scar were comparable between mmf and slr groups despite lower prevelance of interstitial fibrosis/tubular atrophy in slr group. registry analyses suggest that tac/mpa immunosuppression is associated with superior kidney graft survival vs. tac/srl. large single-center experience may assist in clarifying these findings, by examining outcomes related to specific utilization practice. we retrospectively examined the outcomes of consecutive first renal transplants ( % deceased donor, % living donor) at a single center, treated with tac/srl or tac/mpa. graft and patient survival, acute rejection rates, and yr egfr were analyzed by era of transplant ( - vs. - ) . changes in tac/srl utilization between eras included elimination of the srl loading dose and a reduction in tac target trough concentrations. summary. compared to tac, srl+cya was associated with a % decreased risk of skin ca that was statistically significant. although srl+cya was associated with a % decreased risk of de novo solid ca, it did not reach statistical significance. this reduced risk may not reflect the unique aspects of srl itself, but may be a reflection of practice pattern, patient selection or center effect. the backgrounds: a number of studies have observed increase of malignancies following renal transplantation. however, the incidence and the site of malignancies were quite different by the follow-up times, the era and the region. methods: we reviewed the records of renal transplant recipients in our institute between and and recorded the incidence and types of de novo malignancies. they were divided into two groups by immunosuppressive era; azathioprine (aza) era ( . - . : n= ) and calcineurin inhibitor (cni) era ( . -: n= ) . results: a total of ( in aza era and in cni era) kidney recipients out of developed malignancies. the tumors included gi-tract cancers, liver cancers, skin cancers, tongue cancers, breast cancers, renal cell carcinomas, thyroid cancers, leukemia, lymphoma, one lung cancer, one uterus cancer and one kaposi's sarcoma (ks). the average interval between transplantation and development of malignancy was ± ( - ) months. mortality was high in liver cancer ( %) and leukemia ( %). cumulative incidence of malignancies of all recipients in , , , years were . %, . %, . % and . %, respectively. graft-loss censored cumulative incidence, which was calculated to see the incidence among graft survivors under continuing immunosuppression, of all recipients in , , , years were . %, . %, . % and . %. that of , and years in cni era was . %, . % and . %, while that in aza era was . %, . % and . %, showing early higher incidence in cni era outstripped by aza era by years. site of malignancy in cni era occurring within years, which was never observed in aza era, was focused on liver, leukemia (including atl), ks and ptld. discussions:our results demonstrated that recent potent immunosuppressive regimen shortened the interval between transplantation and viral-related malignancies. however, long-term incidence of whole malignancies has been decreasing by minimizing chronic immunosuppression in our institute. the impact of transplant center practice on the association between pulsatile perfusion and delayed graft function. jagbir gill, david gjertson, suphamai bunnapradist, michael cecka. ucla, la, ca. the use of pulsatile perfusion (pp) is increasing in the us, but practice varies widely among transplant (tx) centers. we describe the variability of pp use and its impact on post transplant outcomes. methods: we identified all cadaveric kidney tx from - using optn/unos data. the cohort was stratified by tx center pp use as follows: low pp centers ( - % pp use), med pp centers ( - % pp use), and high pp centers (> % pp use). donor characteristics and the incidence of dgf were compared between and within each strata. results: pp was used by % of centers, however most centers used pp < % of the time ( . %). compared to low and med pp use centers, kidneys pumped in high pp centers were from donors that were younger, had a lower mean terminal serum creatinine, and had a lower incidence of cva and hypertension. the overall incidence of dgf was lowest in the high pp centers ( . %), compared to the med ( . %) and low pp ( . %) centers. the rates of dgf within each strata (high, med, and low pp centers) for tx performed using pp versus cold storage (cs) are outlined in the table below . within each strata, the rate of dgf did not differ between tx performed with and without pp. in ecd transplants, pp was associated with lower rates of dgf across all center groups. however, the impact of pp on scd and dcd transplants was less significant and varied across centers by pp use. hla sensitized patients ( - %) in our dsa are now transplanted at a rate of %, which is significantly higher (p = . ) than the % rate when ua weren't entered into unet. furthermore, of kidneys imported into our dsa and allocated to the dsawide renal candidate list (since ua entry started), % ( / ) were transplanted into sensitized candidates ( % to %), each of whom had a negative flow or ahg t cell igg crossmatch. conclusion. the higher transplantation rate for hla sensitized patients in our dsa shows that virtual a, b, & c crossmatching yields a dsa-wide ranked list of sensitized candidates likely to have a negative final class i (t cell) crossmatch and be transplanted. the data also lend support to the notion that sharing kidneys across dsa boundaries for hla sensitized candidates, based on a negative virtual hla class i crossmatch, has merit. predicting introduction: predicting graft outcome after renal transplantation based on donor histological features has remained elusive and is subject to institutional variability. we propose a pre-transplant donor path scoring system that reliably predicts graft outcome regardless of recipient ® characteristics. methods: we retrospective analyzed imported cadaveric renal transplants which were initially rejected by other centers due to donor parameters between / - / . all kidneys were re-biopsied at our center prior to implantation. morphometric analysis performed consisted of measuring glomerular-size arterioles, interlobular, and arcuate/ interlobar arteries and wall to lumen ratio (wlr) was calculated; calculating % glomerulosclerosis (gs); the presence of arteriolar hyalinosis (ah), scar, periglomerular fibrosis (pfg), and acute tubular necrosis in the biopsies. the patients were followed for a mean of months. multivariate cox analysis was done to evaluate the predictive value of these path variables to graft outcome.results: ah, gs> %, wlr> . , pgf, and scar were found to independently predict graft outcome. the unos board of directors has approved the change from using panel reactive antibodies (pra) to calculated pra (cpra). the cpra is a formulated pra based upon the frequency of the specificities of hla antigens found in the donor pool and is expected to standardize the degree of patient sensitization. donor organs expressing unacceptable antigens will not be offered to a recipient with donor (hla) antigen specific antibodies (dsa). highly sensitive, single antigen bead and solid phase assays (flow pra and luminex) are used to identify these hla antibodies (abs). each transplant center can determine the criteria used for identifying an unacceptable antigen, for example, based upon dsa titer or the fluorescence intensity (fi) coming from the donor-specific single antigen bead. it is unclear whether abs identified by these techniques are clinically relevant for organ allocation. we retrospectively evaluated flow-pra, flow cytometry crossmatching (fcxm), hla ab specificities and titers of pre-transplant (tx) sera from transplant recipients of deceased renal allograft donors transplanted following a negative cytotoxic-anti-human globulin crossmatch. the two year graft survival of % for the recipients ( / , %) with low-titer (≤ : ) donor specific hla ab and a negative (-) fcxm was significantly better when compared to the % two year graft survival of the % ( / ) of recipients presenting with (+) dsa but a (+) fcxm (p< . ). recipients ( / , %) with non-donor abstracts specific hla abs (high or low titer) and a (-) fcxm also experienced a better two year graft survival of % compared to the % for the other % of recipients with (+) fcxms (p < . ). these data suggest that in the presence of donor-specific or non-donor-specific hla abs you can not predict the crossmatch outcome without actually performing the crossmatch which will then influence donor organ allocation and graft survival outcome. in the face of low-titer dsa and a (-) fcxm recipients experienced excellent graft outcome when compared to recipients with (+) fcxms. therefore, donor organ allocation based on cpra (ab specificity and unacceptable ags) utilizing highly sensitive single antigen bead and solid phase luminex assays may disadvantage recipients (no donor crossmatch) who could otherwise be successfully transplanted. aim: to assess vegfr expression in hcc and the adjacent benign cirrhotic parenchyma and its correlation with tumor differentiation, vascular invasion, and tumor morphologic parameters. background: hcc is a highly vascular tumor in which angiogenesis is mediated in part by vegf. vegf is highly expressed in hcc and mediates its effects through multiple receptors including vegfr . the tyrosine kinase inhibitor sorafenib inactivates the vegfr receptor and exhibits anti-tumor effects in hcc. clinical significance of vegfr expression with respect to tumor parameters has not been evaluated. patients and methods: immunohistochemical staining for vegfr was performed in hcc and corresponding adjacent cirrhotic liver from patients undergoing liver transplant. patients had hcc within mc. stains were scored by estimating the % of positive surface area in veins, arteries, and sinusoidal lining cells. data are presented as median [p , p ]; wilcoxon signed rank and wilcoxon rank sum tests were used. results: vegfr levels in hcc were significantly correlated to levels in adjacent non-tumorous liver. higher levels of vegfr in non-tumorous liver were associated with higher levels in hcc from the same patient. vegfr levels were significantly higher in hcc compared to adjacent areas (p< . ). vegfr levels in hcc were not significantly different between patients who fell within mc and those beyond mc. however, vegfr levels were significantly higher in the adjacent arteries of nontumorous liver ( . [ , ] vs. [ , ]; p= . ) in those patients with hcc beyond mc. subjects with moderate or poor differentiation had significantly higher levels of vegfr in sinusoids and veins of hcc and in the sinusoids of adjacent non-tumorous liver. there was no correlation with vascular invasion. conclusions: elevated vegfr in hcc correlates with elevated vegfr in adjacent cirrhosis, suggesting that high expressing hcc arise in a high vegfr expression, pro-angiogenic environment. moreover, higher vegfr expression in background cirrhosis correlates with advanced hcc (beyond mc), suggesting that anti-angiogenic agents may prevent tumor formation or progression in cirrhotic patients. this novel concept warrants further study. . we further attempted to find a subgroup of patients combining tumor size, number of nodules and various levels of afp which together would correlate strongly with pdiff tumors. however, the distribution was erratic and even in extreme outliers (> nodules, > cm in maximum size, with or without high afp), where transplantation is currently not indicated according to any current expanded tumor inclusion criteria, the incidence of pdiff did not exceed %. conclusions: pdiff is most highly associated with tumors that exceed the milan criteria and the expanded criteria currently used for liver transplantation. thus, tumor biopsy would not appear to be of benefit except perhaps in those patients with a high afp level. however, if tumor criteria are further extended in the future, tumor biopsy may be justified in those with higher tumor burdens. < cm) ), , ( . %) at ls=t ( tumor < cm or tumors ≤ cm), and ( . %) with ls> . results: overall survival at months was . %, with significant differences seen for listing stage (ls = . %, ls = . % ls> = . %, p= . ) and by histologic stage as determined by pathology (p<. ). survival for patients with histologic stage b hcc was only % at months, versus % for stage . patients with tumors greater than cm both by listing stage and by pathology fared poorly compared to those with smaller tumors (p= . . ). patients listed who received ablation treatment (at) preoperatively and who had their tumors "down-staged" had better results (p= . ). we observed no difference in at types (tace vs rfa). those with micro or macrovascular invasion had lower survival rates, at . % and . %, respectively (p <. ). afp > continues to be a significant predictor of lower post-transplant survival, with a survival rate of . % at -months (p<. ). conclusions: we conclude that listing and histologic tumor size, presence of micro/ macrovascular invasion, and high afp are associated with poorer lt results. at is emerging as potentially effective treatment for improving lt outcomes for hcc recipients. introduction: so far, milan criteria are used to select patients with hepatocellular carcinoma (hcc) for liver transplantation (lt). herein we compare prognostic markers in patients with pretreatment by transarterial chemoembolization (tace) to a second cohort transplanted without tace pretreatment. patients and methods: between september and october , patients with hcc underwent lt at our institution. eighty-two patients were pretreated by repeatedly performed tace whereas in patients none or other forms of pretreatment had been used (non-tace group). tace was performed using lipiodol and mitomycin. every weeks tace was repeated until transplantation. tumor response was assessed by ct scans ( -week intervals). results: sixty-seven percent of the patients transplanted after tace pretreatment exceeded the milan criteria compared to % in non-tace patients. the proportion of recurrence-free patients was comparable in the tace and non-tace group ( . % and . %, respectively). in the univariate analysis grading, angioinvasion and progress-free tace were significant predictors for recurrence after lt in patients with tace pretreatment. progress-free tace was the only significant predictor of recurrence in the multivariate analysis. in the non-tace group t classification, number of nodules, grading, angioinvasion, milan criteria and underlying disease (hcv versus all other diseases) were significant. after tace pretreatment freedom from recurrence was . % in patients with stable disease or regress but only . % in patients with progress during tace. conclusions: tace pretreatment is capable of selecting a biological entity of tumors which differs significantly from untreated tumors. this statement is deduced from the remarkable differences of predictors for tumor recurrence in patients with and without tace pretreatment. moreover, tace patients can be separated into two groups: those who experienced tumor progress during repeatedly performed tace and those who did not. stable disease during the continued tace before transplantation resulted in remarkably low tumor recurrence. liver with a median follow-up of months, there was no statistical difference in the year overall survival in the m ( %) and m+ ( %) groups (p= . ). the -year disease free survival was significantly higher in the m ( %) vs. m+ ( %) groups (p= . ). when stratifying for ucsf criteria tumors, the -year disease free survival was milan ( %) vs. ucsf ( %) vs. beyond ucsf ( %) groups (p= . ). univariate analysis demonstrated the following factors to be associated with disease recurrence: intermediate waiting time - months (p= . ), preoperative tace (p= . ) or resection (p= . ), more than tumors (p= . ), and max tumor size over cm (p= . ). multivariate analysis controlling for age, gender, and waiting time demonstrated that preoperative resection hr . ( %ci . - . ), more than tumors hr . ( %ci . - . ) and max tumor size greater than cm hr . ( %ci . - . ) were independently associated with disease recurrence. conclusions: the overall survival after liver transplantation is excellent in the m and m+ groups, far exceeding survival rates that can be obtained via any other modality. the current unos hcc algorithm should be reconsidered, to allow extra listing points for selected patients with hcc that exceed both the milan and ucsf criteria. postoperative use of intense insulin therapy in liver transplant recipients. lama m. hsaiky, iman e. bajjoka, dhaval patel, marwan s. abouljoud. transplant institute, henry ford hospital, detroit, mi. hyperglycemia and insulin resistance are common post liver transplant, even in patients with no history of diabetes. in the general surgical intensive care unit (sicu) population, the use of intensive control of blood glucose has recently been shown to reduce both morbidity and mortality. thus far, limited data exist in the liver transplant population. purpose: assess the impact of intensive insulin therapy to maintain blood glucose at or below mg/dl immediately post liver transplantation in the surgical intensive care unit (sicu). methods: a retrospective evaluation of liver transplant recipients who received two different insulin protocols in the sicu was performed. prior to january , patients were assigned to receive sliding scale insulin therapy (ssit) to maintain blood glucose (bg) less than mg/dl. the intensive insulin therapy (iit) was implemented in august with a goal bg level between - mg/dl. the following data was analyzed: bg ranges, need for mechanical ventilation, blood transfusions, infection rate in the sicu, rejection episodes and patient survival. results: a total of liver transplant patients were evaluated; of which patients were in the ssit group and the other in the iit group. demographic characteristics were comparable between the two groups. in the iit, % of the bg readings were maintained at < mg/dl versus % in the ssit. the incidence of hypoglycemia (bg < mg/dl) was less than % in both groups. the need for mechanical ventilation was . days vs. . days and the overall number of blood transfusion was on an average of . vs. units (p< . ) in the ssit vs. iit group, respectively. iit also reduced overall sicu infection rate by % (p= . ). the rate of acute cellular rejection at months post transplant was less in the iit, % vs. % in the ssit group (p= . ). moreover, mortality during hospital stay was reduced from % in the ssit to % in the iit group. conclusions: the use of intense insulin therapy immediately post liver transplantation has resulted in reducing infection rate and rejection episodes and a trend for reduced morbidity and mortality among post surgical liver transplant recipients without the adverse effects of hypoglycemia. medical epidemiology of patients surviving ten years after liver transplantation. kerri a. simo, stephanie e. sereika, david a. gerber. abdominal transplantation division, department of general surgery, university of north carolina, chapel hill, nc. background: as the population of long term survivors of liver transplantation (olt) grows, their medical epidemiology has become increasingly important. the goals of this study were to define a collective profile of liver transplant recipients ≥ years post olt and to compare their co-morbidities with those of the general population. in , the national health survey reported that % of the us population had hypertension, . % had diabetes/impaired fasting glucose, and . % had chronic kidney disease. methods: a retrospective review of a prospectively collected database of adult patients who underwent olt at a single transplant center from september , to october , was performed. inclusion criteria consisted of survival ≥ years post olt with > year follow up. results: seventy-one patients met inclusion criteria. ninety percent of patients had ≥ years follow up. the mean age at transplant was (range - ). the mean calculated meld score was (range - , median= ). indications for olt were hcv( %), alcohol( %), cryptogenic( %), autoimmune( %), hbv( %), psc( %), pbc( %), and other( %). seven patients required retransplant during the first years. an additional patients underwent other operations: arterial or biliary revisions, hernia repairs and non-transplant related ( abdominal). during analysis, the following medical co-morbidities were found: patients( %) had hypertension ( new onset), ( %) diabetes ( new onset), ( %) renal insufficiency and renal failure ( new onset), ( %) cardiovascular disease (all new onset). nine patients ( %) were diagnosed with de novo cancer. medications for chronic health problems included patients on diabetic medications, on antihypertensives and on lipid lowering agents. initial immunosuppression consisted of % on steroids, % on cyclosporine, % on tacrolimus, % on mycophenolate mofetil (mmf) and % on azathioprine. immunosuppression at years consisted of % on cyclosporine, % on tacrolimus, % on steroids, % on mmf, % on sirolimus, % on mycophenolate sodium, % on azathioprine. no patients were on triple therapy, were on dual therapy, and were on monotherapy. summary: patients alive years post olt have a significantly higher incidence of hypertension, diabetes, and renal disease than the general population. this study supports conscientious medical follow up to ensure continued meaningful survival. liver transplantation in the morbidly obese (bmi> ). c. quintini, l. kauzman, k. hashimoto, p. ding, t. doago uso', n. sopko, j. rosenblum, f. aucejo, c. winans, d. kelly, b. eghtesad, d. vogt, j. j. fung, c. miller. general surgery -liver transplant service, cleveland clinic oh. morbid obesity (mo) is a problem seen with increasing frequency among candidates for olt. mo is considered a contraindication for olt in some centers without clear evidence to support such a practice. our aim is to describe outcomes for olt in patients with a bmi> kg/m in a single center. methods. between / and / , olts were performed in patients. / patients with a bmi> were compared to all other olt patients with a bmi< . we analyzed patient and graft survival, operative time, blood transfusion requirements, and post operative events (icu and overall length of stay los, surgical complications and infections). we also analyzed the post transplant weight records of our study group at - and months. results. results are summarized in table . outcomes of olt in the morbidly obese are no worse than those of other patients undergoing olt. bmi is often artificially elevated in end-stage cirrhotic due to severe fluid retention. these patients exhibit rapid weight loss following transplantation that is likely due to extra-cellular fluid loss from the improved homeostatic milieu provided by the new liver and possible improvement in renal function. the presence of obstructive cad was not associated with increased peri-operative morbidity or mortality. these patients experienced similar patient and graft survival irrespective of the degree of cad. significant unmodified cad should not represent an absolute contraindication to liver transplantation. olt was also examined. we found that using a trj of . m/s as a cut-off created two age-matched groups with significantly different survival curves at one year (p = . ) with a relative risk of mortality of . for trj ≥ . m/s. this study underscores the importance of screening tte for the presence of pph in the evaluation of patients for olt, suggesting that clinically significant pph may be present at lower trj than typically prompt right heart catheterization. some of the limitations of the study are the variability of the time from pre-olt echo to transplant, and the assumption of rap = without assessment of ivc size. we are using these data as a framework for further prospective trials to better assess the clinical applications of the findings. hyperlipidemia has been shown to predict faster chronic kidney disease (ckd) progression over the long term in lung allograft recipients. it is unknown whether disordered lipid metabolism may also aggravate the early loss of renal function often seen in this patient population in the immediate post-operative period. we studied lung allograft recipients transplanted between january and december . pertinent demographic and clinical variables were recorded at baseline and one month post-transplant, including creatinine levels and fasting lipid panels. logistic regression models were created to investigate an independent association between lipid levels and change in renal function by one month post-transplant. mean +/-sd baseline creatinine was . +/- . mg/dl and low density lipoprotein (ldl) was +/- mg/dl, the latter remaining unchanged at one month. in contrast, by one month post-transplant the mean creatinine level of survivors increased significantly to . +/- . mg/dl (p < . ), with a overall mean increase of % above baseline. the highest quartile of patients that fared the worst experienced a rise in creatinine > % above baseline. on univariate analysis, there was a strong trend toward those with one month ldl values in the highest quartile (i.e., > mg/dl) having an increased odds of experiencing a rise in creatinine by one month > % above baseline (or . , p= . ). after controlling for age, gender, pre-transplant creatinine, bmi, and the presence of diabetes prior to transplant an ldl value in the highest quartile by one month post-transplant was the only variable independently predictive of a rise in creatinine > % above baseline at one month (or . , p= . ). in summary, hyperlipidemia occurring early post-lung transplant predicts faster loss of renal function soon after surgery. though speculative, given the known beneficial effects of lipid lowering agents such as statins on the rate of ckd progression, perhaps timely initiation of these medications after surgery may also attenuate the early decline in renal function often observed in this patient population. the risk for acute cellular rejection (acr) following lung transplantation (ltx) is still a problem despite heavy multidrug immunosuppression. induction therapy with potent t-cell depleting agents have facilitated the implementation of minimal post-transplant immunosuppression.the impact of this protocol on the activation of proinflammatory cytokines and effector molecules that affect the cellular rejection process is not well determined. in this study, we evaluated the relationship between upregulation of t-cell and macrophage-dependent inflammatory cytokines detected by molecular methods and the clinical status of ltx patients. we studied ltx patients who received anti-lymphocytic induction therapy(thymoglobulin n= or campath- h n= ) followed by maintenance immunosuppression with tacrolimus and low-dose steroids. we analyzed bronchoalveolar lavage (bal) mrna samples ( - per ltx patient) by real-time pcr for gzmb, ifn-γ, il- , mcp- , rantes, tnf-α, and gapdh (control). the abi prism sds and fast real-time pcr systems were used and data were analyzed by the dct and -ddct methods. we determined the relationship of categorical outcomes (rejection-no rejection) with continuous variables (number of cycles) by anova. early acr that occurred within the first days post-ltx was associated with an increase (> - fold) of macrophage-specific mrna (mcp- , rantes, tnf-α). / thymo-treated ltx patients experienced early acr while only / campath- h-treated patients had early acr (p< . ). in contrast, late acr that occurred greater than days post-ltx was associated with a significant upregulation ( - fold) of t-cell dependent mrna (gzmb and ifn-γ) in addition to increases of rantes and mcp- mrna. overall, ltx patients who experienced early or late acr (n= ) exhibited higher mrna levels for the above mediators compared to stable patients (n= ). our data indicated that in early post-t-cell depletion, the acr phenotype was characterized mainly by macrophage activation. in contrast, greater than months post-ltx with the recovery of cd + t-cells, the acr phenotype was associated with cytotoxic t-cell activation. furthermore, sensitive molecular methods may detect the activation of pro-inflammatory mediators within the allograft prior to the diagnosis of rejection by transbronchial biopsies and may impact optimal patient management. antibody-mediated rejection (amr) is defined by the presence of donor-specific alloantibodies, markers of complement activation and the clinical phenotype of organ dysfunction. compared to renal and cardiac allografts, very few amr cases are documented in lung transplantation (ltx). methods. we report here on seventeen ltx patients exhibiting: ) donor-specific hla antibodies (dsa); ) linear, continuous subendothelial c d deposition in lung allograft; ) lung allograft dysfunction. the presence and specificity of dsa were determined by elisa and/or luminex. c d deposition was assessed by immunohistochemistry in transbronchial biopsy paraffin blocks. allograft dysfunction was considered when either biopsy-proven acute cellular rejection (acr, ≥ ishlt a ) or bronchiolitis obliterans (bos) were diagnosed. the average detection of dsa occurred in the first year post-ltx, on ± postoperative day (pod), range to days. thirteen ( %) of amr patients were females. an anamnestic humoral response was encountered in cases (one pregnancyrelated and three re-transplant patients), while de novo dsa were detected in cases ( %). the percent-reactive antibody (pra) was lower in de novo cases ( %) when compared to memory response ( %, p< . ). anti-class i dsa were found in cases, anti-class ii in cases, while patients exhibited both class i and class ii dsa. specific vascular c d deposition was detected in ( %) patients. lung allograft dysfunction was considered in ( %) cases, while three patients with dsa and specific c d deposition fulfilled the criteria of sub-clinical amr. in patients where plasma-exchange/ ivig were applied, antibody titers dropped from : to : or : in two cases; in a third case, antibody titer remained high post-pheresis ( : ) and the graft failed. conclusions: both anti-class i and anti-class ii, low pra or high pra, pre-formed or de novo dsa can be detrimental for lung allograft. the presence of donor-specific alloantibodies, vascular c d deposition and allograft dysfunction shows that amr criteria can also be met in ltx. rationale: recently, inhaled cyclosporine has been shown to reduce mortality and bos. previously, we demonstrated that apically-dosed cyclosporine poorly transmigrated differentiated human airway epithelial cells in vitro (using a transwell system). only ∼ % of the apically deposited cyclosporine passed through the epithelial layer whereas most of the drug accumulated within the epithelium. thus inhaled cyclosporine may not be capable of inactivating airway allogeneic t cells since it may not reach the airway wall in high concentration. in this study, we hypothesized that inhaled cyclosporine alters airway epithelial signaling cascades that may ultimately result in reduced inflammatory cell recruitment to the lung. methods: human tracheobronchial epithelial (htbe) cells from healthy donors were grown in ali media to confluence at an air-liquid interface in millicells. differentiated htbes were treated on their apical surface with cyclosporine concentrations of and , ng/ml or vehicle for hr to mimic the effects of inhaled cycolsporine. the basilar and apical compartments (n= - for each) were then assayed for cytokine secretion (il , il , il , il- p , tnf, eotaxin, mcp- , gm-csf, rantes and egf) by luminex assays in pg/ml. results: , ng /ml of cyclosporine markedly blunted the basilar secretion of il- ( ± vs ± , p= . ), rantes ( ± vs ± , p= . ), gm-csf ( ± vs ± , p= . ) and mcp- ( ± vs ± , p= . ). il and eotaxin were unaffected; tnfα, il β and il p were undetectable. at ng/ml cyclosporine, cytokine secretion was decreased to a lesser extent. a similar pattern of diminished cytokine secretion was seen in the apical compartment of this system. last, apical, but not basal, egf secretion was augmented by cyclosporine ( ± vs ± pg/ml, p= . ). conclusion: cyclosporine decreased the secretion of critical cytokines and chemokines from human airway epithelial cells. these mediators are known to enhance mononuclear and t cell recruitment in a large variety of animal models of disease as well as in clinical studies. inhaled cyclosporine may work by reducing cell recruitment. this hypothesis will require in vivo testing. funded by: cf foundation. background: cytomegalovirus (cmv), human herpes virus - and - (hhv- and - ) are β-herpesviruses that commonly reactivate and have been proposed to trigger acute rejection and chronic allograft injury. the role of these viruses in the development of bos after lung transplantation remains unclear. we assessed the contribution of β-herpesvirus infection in the allograft by a prospective molecular assessment of serial broncho-alveolar lavage (bal) samples in lung transplant recipients. methods: quantitative real-time pcr of bal samples were performed for cmv, hhv- and hhv- in a prospective cohort of lung transplant recipients. a time-dependent cox regression analysis was used to correlate the risk of bos and acute rejection in patients with and without β-herpesviruses infection. results: patients were included in the study over a period of years. a total of samples from bal were obtained (median per patient). / patients ( %) had at least one positive result for one of the β-herpesviruses: patients ( %) for cmv, patients ( %) for hhv- , and patients ( %) for hhv- . median time to detection was days (range - ) for cmv, days (range - ) for hhv- , and days (range - ) for hhv- . median peak viral load was , copies/ml (range - , , ) for cmv, copies/ml (range - , ) for hhv- , and copies/ml (range - , ) for hhv- . acute rejection (≥ grade ) occurred in % and bos (≥ stage ) in %. in the time dependent cox regression model, the relative risk of bos or acute rejection was not increased in patients with cmv, hhv- , or hhv- reactivation. for example, the hazard ratio of cmv and bos was . ( % ci . - . , p= . ) and for cmv and acute rejection was . ( % ci . - . , p= . ). in many of the patients, β-herpesvirus reactivation occurred after the acute rejection episode likely reflecting augmented immunosuppression. abstract# mannose binding lectin in this large cohort of lung transplant recipients, local reactivation of cmv, hhv- and hhv- in the allograft was very common. however, despite high viral loads in many patients, infection was not significantly associated with the development of acute rejection or bos. introduction: the role of chemokine receptors in regulating donor-specific responses to allografts is poorly understood. cd + cd + t cells regulate alloreactive cd t cell responses and acute rejection of single class ii mhc-disparate cardiac allografts in c bl/ mice. ccr is expressed by a small proportion of cd + cd + t cells but the requirement for these cells in regulating alloreactive t cell responses remains poorly understood. the goal of this study was to investigate the role of ccr + t regulatory cells in acute rejection of single class ii mhc-disparate cardiac allografts. methods: wild-type c bl/ (h- b ) and b .ccr -/received heterotopically transplanted b .h- bm mice heart grafts. the presence of cd + foxp + t cells in the recipient spleen and in heart allografts was determined by flow cytometry. foxp mrna expression in the heart grafts was analyzed by qrt-pcr. donor-specific cd + t cells producing ifn-g or il- in allograft recipient spleens were enumerated by elispot assay. cell sorted naïve wild-type cd + cd + t cells and cd + cd -t cells were adoptively transferred to wild-type and ccr -/mice before the cardiac transplant. results: in wild-type recipients > % b .h- bm cardiac grafts survived more than days whereas ccr -/recipients rejected the allografts within days ( days mean survival) with intense cd + t cell infiltration in the graft. donor-reactive ifn-g and il- producing cd + t cell numbers were increased -fold in the spleens of ccr -/vs. wild-type recipients at day post-transplant and in contrast to wild-type recipients these numbers were sustained for at least more days. allograft infiltrating cd + cd + foxp + cells and intra-graft foxp mrna expression were clearly present in allografts from wild-type recipients and were virtually absent in allografts from ccr -/recipients. transfer of purified wild-type cd + cd + t cells to ccr -deficient mice resulted in the long-term survival of % of b .h- bm cardiac allografts. conclusion: ccr + regulatory t cells control the magnitude and function of the alloreactive t cell immune response to single class ii mhc-disparate cardiac allografts. profile that were distinct from those of cd +cd + tregs, naïve cd +cd -t-cells, and activated cd + t-cells. furthermore, the cd + converted dn t-cells were highly potent in suppressing antigen specific alloimmune responses in vitro. in this study, we further characterized and test the functional potential of the converted dn t-cells in vivo. we showed that the converted dn t-cells retained a stable phenotype after re-stimulation in vitro and in vivo. il- was capable of breaking the anergic status and reserving the suppressive function of dn t-cells. in an immunocompetent mhc completely mismatched islet transplant model, the transfer of x dn t-cells (converted from cd +cd -t-cells of naïve c bl/ mice by co-culture with mature dba/ dc plus ril- in mlr for days) resulted in a statistically significant prolongation of alloantigen specific dba/ strain, but not third party c h strain, islet allograft survival in c bl/ recipients in comparison with that of untreated control group. as il- was capable of breaking the anergic status and reserving the suppressive function of dn t-cells, we added il- /fc, a long-lasting form of il- , and low dose rapamycin with dn t-cells in a mhc mismatched skin allograft model. the single transfer of x dn t-cells plus days il- /fc and low dose rapamycin treatment significantly prolong dba/ skin allograft survival in c bl/ recipients in comparison with untreated group (mst days vs. days, p= . ) and il- /fc plus rapamycin treated group mst ( days vs. days, p= . ). the results of using ex vivo cd + t-cells converted dn t-cells in skin and islet transplantation models support the concept and the feasibility of potentially utilizing this novel cell-based therapeutic approach clinically for the prevention of allograft rejection. jessamyn bagley, jonathan g. godwin, joren madsen, john iacomini. introduction: it has been suggested that natural killer (nk) cells are critical mediators that connect the innate and adaptive immune response. cytokine production by nk cells contributes to the polarization of immune responses to t helper , and nk cells express co-stimulatory molecules that may affect t cell proliferation. recent work has shown that nk cells are involved in the chronic rejection of parental cardiac grafts by f recipients. we hypothesized that given the role of nk cells in t cell activation and proliferation, nk cells may play a role in the development and function of t regulatory cells (treg) which control alloreactive responses. methods: nk, cd + t cells and cd +cd + treg were purified from the spleens of c bl/ j mice using macs bead separation followed by fluorescence activated cell sorting. naïve cd +cd -t cells from c bl/ j mice (h- b) were placed in culture with tgf-beta in the presence or absence of nk cells, and the development of treg was monitored by assessing foxp expression by intracellular cytokine staining and flow cytometry. in addition, the effect of nk cells on the function of treg was measured by elispot assay. results: following days of culture with tgf-β and anti-cd antibody, cd +cd -t cells acquire foxp expression. the addition of activated nk cells to cultures with cd +cd -t cells and tgf-β prevented the acquisition of foxp expression by cd cells. tregs induced by stimulation of cd +cd -t cells with anti-cd in the presence of tgf-β are capable of suppressing the production of il- , ifn-γ and il- by cd t cells in response to fully allogeneic balb/c stimulators. however, in the presence of activated nk cells, induced tregs fail to function, and production of il- , ifn-γ and il- by cd t cells is restored. these experiments were repeated with natural cd +cd + tregs isolated directly from mice without induction, and found that the ability of natural treg to suppress a cd t cell response to alloantigen was impaired in the presence of activated nk cells. conclusions: the presence of activated nk cells in culture can prevent the development of induced treg. furthermore, the presence of activated nk cells interferes with the function of mature treg in culture and allows a productive cytokine response by cd effector cells in response to alloantigen. results: both the syngeneic b cells and allogeneic dba/ cells survived nicely in the rag-/-il- rg-/-mice. however, the allogeneic dba/ cells, but not the syngeneic b cells, were readily killed by the nk cells in the rag-/-mice. however, both rag-/-and rag-/-il- rg-/-mice accepted the dba/ skin allograft long term without any sign of rejection (> days). thus, nk cells by themselves, though cytolytic to dba/ cells, fail to reject the dba/ skin allografts. to test the hypothesis that the activation status of nk cells may dictate their alloreactive potential, we treated the rag-/-mice with il- /il- ra complex to maximally stimulate the nk cells in vivo. we found that il- is remarkably potent in stimulating nk cells in vivo; and nk cells stimulated by il- express an activated phenotype and are surprisingly potent in mediating acute skin allograft rejection in the absence of any adaptive immune cells, as il- treated rag-/-, but not the rag-/-il- rg-/-mice, readily rejected the dba/ skin allografts (mst= days). nk cell-mediated graft rejection doesn't show features of memory responses. and suggests that the fate of the allografts may depend on the activation status of nk cells and the availability of nk stimulating cytokines. background: t-bet is a transcription factor that promotes th development. both t-bet and the cytokines ifng and il- have been implicated as negative regulators of th . in contrast, il- promotes th development. il- production is associated with granulocytic pathologies in several disease states. hence, this study assessed the relationship between t-bet, ifng, il- and il- in th induction and granulocytic infiltration of cardiac allografts. (ifng-/-) mice were transplanted with balb/c cardiac allografts. recipients were left untreated, depleted of cd + or cd + cells, treated with anti-cd l mab, and/or treated with neutralizing anti-il- or anti-il- mab. graft histology was assessed and primed donor-reactive th responses were quantified by elispot. intragraft expression of il- and the th transcription factor rorgt were quantified by real-time rt-pcr. results: wt and t-bet-/-mice rejected their allografts at a similar tempo but with distinct pathologies: allografts in t-bet-/-recipients were heavily infiltrated with granulocytes while graft infiltrating cells in wt recipients were primarily mononuclear. while th and th responses were readily detectable in t-bet-/-recipients, th dominated the response in wt mice and th were not detectable. depletion of cd + cells prolonged graft survival in wt, but not in t-bet-/-recipients suggesting that cd + cells mediated rejection independent of cd + help in t-bet-/-mice. cd + cells were the source of il- in t-bet-/-recipients. anti-cd l therapy promoted long-term allograft survival in wt recipients, but not t-bet-/-mice unless cd + cells were depleted. additionally, anti-cd l therapy inhibited th responses in both wt and t-bet-/-recipients, but not the cd + th response in t-bet-/-mice. eliminating ifng and il- failed to induce il- production, while neutralizing il- reduced the th response in t-bet-/-mice. conclusions: while cd + th have been described in detail, cd + th have received less attention. in t-bet-/-allograft recipients, cd + th emerge independent of cd + help. cd + th are resistant to anti-cd l therapy and are associated with granulocyte infiltration of the graft. these data implicate t-bet, as opposed to ifng and il- , as a negative regulator of the th response while il- is required for cd + th induction. nan zhang, bernd schroppel, girdari lal, jordi c. ochando, jonathan s. bromberg. background: cd + cd + foxp + regulatory t cells (treg) are important in suppressing immunity to prolong allograft survival. treg migration and its effects on suppressive activity are poorly understood. we determined treg migration patterns and effector function in an islet allograft model. ccr -/-, ccr -/-, ccr -/-, ccr -/-, l-selectin (cd l) -/-, and fucosyltransferase (fuct) iv-vii -/-c bl/ mice were used to generate treg. treg were transferred intravenously, or locally to islet allografts, following islet transplantation (balb/c into c bl/ ). transferred treg were labeled with red dye pkh and migration to islet grafts, draining lns (dln), peripheral lns and spleen were tracked with fluorescence microscopy and flow cytometry. islet allograft survival was determined by measurement of blood glucose. results: treg expressed p-selectin ligand, cd l, and a panel of chemokine receptors similar to other t cell subsets. intravenously transferred wild type treg migrated to both islet grafts and dln, and prolonged allograft survival. cd l -/or ccr -/-treg, which migrated to islets but not dlns, prolonged allograft survival as potently as wild type treg. fuct iv-vii -/-treg, which lack e-selectin and p-selectin ligands, migrated to dln, but not islets, and did not prolong graft survival. similarly, ccr -/-, ccr -/-, and ccr -/-treg migrated to dln, but not islets, and did not prolong graft survival. when locally transferred to the islet graft, treg also migrated from the allograft to the dln, and prolonged graft survival even longer than after intravenous transfer. locally transferred ccr -/-, ccr -/-, or ccr -/-treg were not able to migrate from the islet to the dln, and were impaired in their ability to prolong islet survival. conclusion: treg migration to allografts is essential for their suppressive function; migration to lymphoid tissues alone is not sufficient to prolong graft survival. treg migration from the islet allografts to the dlns, via afferent lymphatics, is also required for optimal suppressive function and graft survival. the sequential migration from the site of inflammation and then to dlns is necessary for treg to execute fully their suppressive program. these results demonstrate a novel and important aspect of migration in treg suppression and tolerance. abstracts failure is unknown. methods: patients with iothalamate gfr < ml/min (n= ) or on dialysis (n= ) at the time of liver transplant evaluation had undergone a percutanous ct guided renal biopsy. prior to the biopsy an inr ≤ . and platelet count ≥ , / ml were achieved in the majority of cases. all patients were monitored overnight for complications. candidates were listed for slk if pathology showed ≥ % glomerulosclerosis (gs) or ≥ % interstitial fibrosis (if). results: patients were eligible for slk and for lta.creatinine was higher in slk candidates but not clinically different. background -it is well known that delayed graft function (dgf) is costly for those who received a renal transplant. however, the true cost of dgf is unknown. methods -we estimated the cost of dgf for adult cadaveric renal recipients in the usrds - who had medicare as their primary payer. those included were restricted to single organ recipients as well as those who had no previous transplants. cost was defined as the accumulated average cost per day for everyone with a functioning graft on that day. we examined the total cost of dgf, cost associated with dialysis, and non-dialysis cost for all patients combined and separately by donor type; standard criteria donors (scd), expanded criteria donors (ecd), and non-heart beating donors (dcd) as well as time to graft failure or no graft failure. background: based on adverse outcomes during the first year post-transplant, the optn membership and professional standards committee (mpsc) peer-review process flags transplant programs for further review using one of two methods. programs performing at least transplants during at . year cohort are flagged based on the comparison of observed to expected event counts (death or graft failure) and the corresponding p-value (< . ). programs performing or fewer transplants are flagged if they have any adverse events. this leads to different flagging rates for programs of different sizes. the p-value has low sensitivity to identify poorly performing programs with a "moderate" number of transplants ( to ) whereas flagging every event results in a high false positive rate for "small" centers with < transplants. during the july review, % of "small" centers and % of centers with + transplants were flagged for review (all organs). the scientific registry of transplant recipients (srtr) has developed alternative approaches for flagging centers with more consistent flagging rates. methods: the new method would allow for different choices of sensitivity (rr) and specificity (p-value) for different purposes and would flag program if either {p-value < . (a different value could be chosen)} or {observed / expected > rr}. the resulting false negative rate is less than % for centers with the given rr. this approach avoids an arbitrary definition of small versus large programs, and has sensitivity (or power) > % to flag programs with the selected rr, regardless of size. results: the following discussion: this approach gives a more balanced distribution of flagging across programs of different sizes and has sensitivity > % for transplant programs of all sizes. with the choice of rr= . , the overall number of centers flagged would be nearly unchanged from current methods. center performance ratings are of increasing importance to the transplant community with the introduction of the cms final rule. ongoing debate exists regarding how much center ratings are directly a reflection of quality of care or whether ratings can be substantially influenced by exogenous factors. the study examined data from the national srtr database from - . centers' semi-annual graft and patient survival were calculated along with a comparison with expected outcomes adjusted for covariates used by the srtr. centers meeting three criteria for poor performance were categorized within each cohort. patient characteristics at each center were compared with performance evaluations. overall, half of transplant centers met criteria for low performance in at least one of the semi-annual intervals. several center factors investigated were not significantly associated with the likelihood to meet low performance criteria including the proportion of older, obese and privately insured patients. in contrast, centers with higher levels of ecd transplants (most ecds= % vs least ecds= %), african american recipients (most aas= % vs least aas= %) and patients with low albumin level (lowest albumin = % vs highest albumin = %) were more likely to meet low performance criteria. approximately half the centers that initially met criteria for low performance no longer met criteria when excluding ecd transplants and african american recipients from the performance assessment. conclusions given the extreme implications of performance ratings for transplant centers including possible loss of funding to centers with low performance, it is critical that we recognize potential weaknesses and biases of performance ratings. our results are important towards understanding factors related to performance ratings and raising questions as to whether risk adjustment techniques are adequate for fair transplant center performance evaluations. rather than only risk adjustment, stratified evaluations may be a partial solution to remove disincentives to performing higher risk transplants. it is also important to recognize factors not associated with low performance such that centers do not unnecessarily limit access to groups based on perceived deleterious impact on ratings. model. olaf boenisch, takaya muramatsu, francesca d'addio, robert padera, hideo yagita, nader najafian. transplantation research center, brigham and woman's hospital and children's hospital, boston, ma; immunology, juntendo university of medicine, tokyo, japan. t cell immunoglobulin and mucin domain (tim)- , a molecule expressed on terminally differentiated th cells, is an important regulator of th autoimmunity and in induction of transplantation tolerance. its functions in alloimmune responses during acute and chronic rejection are unknown. tim - , a novel blocking antibody of tim- , was administered to recipients in various donor-recipient strain combinations on days ( ug), , , , and ( ug) after transplantation. the frequency of ifng-, il- -, and granzyme b-producing splenocytes was measured by elispot. these data establish the regulatory functions of tim- in alloimmune responses in solid organ transplantation models. the inhibitory actions may be secondary to modulation of effector or regulatory t cells and appear to dominate in conditions of low levels of t cell activation, due to a restricted degree of allogeneic mismatch or absence of cd costimulation. the i-r injury in transplanted kidney is a major cause of dgf, an event associated with an increased risk of acute rejection. adaptative immunity was suggested to play a role in the pathogenesis of renal i-r injury, although the influence of the th /th bias in this scenario is still debated. thus, the aim of the present study was to evaluate the features of t cell response during i-r injury at the peripheral and tissue level in renal graft recipients with dgf. the mrna levels of specific th (t-bet) and th (gata- ) transcription factors were evaluated in circulating lymphomonocyte of kidney transplant recipients with early graft function (egf) (n= ) and dgf (n= ), before (t ) and hours after transplantation (t ) by real time pcr. infiltrating lymphocytes were characterized in graft biopsies of patients with dgf (n= ) and in a control group of patients with tubular damage by acute cni toxicity (n= ) by immunohistochemistry. in addition, we evaluated the th /th bias at the renal level in a pig model of i-r injury. t-bet/gata- mrna ratio was similar in the groups of patients at t . at t the dgf group presented a significantly higher increase of t-bet/gata- ratio compared with the egf group ( ± vs ± % of t , p< . ). moving to the tissue level, dgf patients presented a number of interstitial cd + ( . ± . vs . ± . , p= . ) and cd + ( . ± . vs ± , p= . ) t cells significantly higher compared to the control group, while no significant differences were observed in cd + cells number between the two groups. also at the tissue level the ratio between t-bet + and gata- + cells was significantly higher in the dgf compared with the control group ( . ± . vs . ± . , respectively, p= . ). to confirm that these changes were due to i-r, we investigated the presence of t-bet + and gata- + cells in a pig model of i-r injury. interestingly, the ratio was significantly increased after hours of reperfusion (basal . ± . vs hours . ± . ; p= . ). in conclusion, our results suggest that kidney transplant recipients with dgf present a bias toward a th -driven immune response both at the peripheral and at the tissue level. this event, due to the i-r process, as suggested by the animal model, may represent a link between dgf and acute graft rejection. as expected a strong negative association between duration of dialysis and patient survival was seen in caucasian recipients. in contrast the relationship between patient survival and duration of dialysis was u shaped in minorities with the worst patient survivals seen among preemptive transplants and those patients with over months of dialysis. the difference in outcomes between caucasians and minorities could be related to the biologic differences in the effect of dialysis on subsequent transplantation or to a differential selection bias introduced by duration of dialysis in the two populations. anti-carbohydrate natural antibodies. lorenzo benatuil, jonathan g. godwin, shamik gosh, john iacomini. transplantation research center, boston, ma. background. we constructed immunoglobulin gene knock-in mice lacking expression of the αgal epitope that carry rearranged vh and vl genes encoding antibodies specific for the carbohydrate antigen galα - galβ - glcnac-r (αgal). here, we describe two novel populations of b cells in the spleen of these m vhvlgt mice and their role in the production of αgal specific antibodies. methods. m vhvlgt mice were sacrificed and single cell suspensions from spleen, bone marrow, peripheral lymph nodes and peritoneal cavity were prepared and stained with different antibodies and with fluorescently labeled αgal-bsa for flow cytometric analysis. using multiparameter cell sorting, we purified marginal (mz) and follicular (fo) zone b cells, in addition to two novel splenic b cell populations. cells were adoptively transferred into b cell deficient µmt/gt double knock-out mice. serum samples were collected, and production of αgal specific igm antibodies was assessed by elisa. to investigate these b cell tolerance mechanisms, we employed mice with naturally occurring antibodies (abs) against human blood group a carbohydrates in their sera and possessing b cells with receptors for blood group a determinants. b cells with receptors for a carbohydrates in mice belong to the cd + b- subset, with phenotypic properties similar to those of human b cells. when these cells were temporarily eliminated by injecting synthetic a carbohydrates, subsequent treatment with cyclosporin a or tacrolimus, which blocks b- cell differentiation, completely inhibited the reappearance of b cells with receptors for a carbohydrates in mice. it is probable that calcineurin inhibitors used for preventing t cell-mediated rejection simultaneously suppress b- cell differentiation. however, despite a very limited dose of calcineurin inhibitors, the b cell tolerance toward blood group a antigens was persistently maintained in the blood group a-to-o liver transplant recipient. b cell tolerance after abo-incompatible transplantation might be a consequence of presentation of blood group carbohydrate antigens by cells in the engrafted liver. immune fluorescence staining of the human liver reveals that blood group antigens are predominantly expressed on the liver sinusoidal endothelial cells (lsecs). we have previously proven that lsecs, which constitutively express fas-l and pdl- have the capacity to tolerize alloreactive t cells. taken together, we hypothesize that blood group antigen-reactive b cells are also tolerized through the interaction with the lsecs. in order to address this possibility, we used α- , galactosyltransferase-deficient mice. when the α-gal-expressing lsecs isolated from wild-type mice were adoptively transferred via the portal vein into the splenectomized congeneic α-gal-deficient mice, these mice lost the ability to produce anti-α-gal abs (n = ). this finding suggests that the lsecs expressing blood group carbohydrates play a pivotal role in the tolerization of newly developed b cells specific for the corresponding carbohydrate antigens after abo-incompatible liver transplantation. success in kidney transplantation has resulted from control of t-cell-mediated acute rejection. however, little has been done to improve the fate of patients who possess pretransplant donor-specific antibody (dsa), and no proven therapies exist to specifically prevent dsa formation post-transplant. while methods to detect and characterize dsas are clearly useful, the b-cell subsets that produce dsas or more importantly sustain their production are poorly characterized. this study set out to investigate the fundamental mechanisms determining the formation of donor-specific memory b cells and plasma cells in novel mouse models of dsa formation. we have developed two complementary and novel systems to track the phenotypic and functional properties of polyclonal donor-specific b-cells. the first system involves allosensitization between normal c bl/ and balb/c mice. we used advanced flow cytometric methods to track donor-specific b cells with h- k b or h- k d tetramers. tetramers are comprised of four identical h- molecules bound to a fluorescently labeled steptavidin molecule that is able to bind the donor-specific b-cell antigen receptor (surface immunoglobulin). in our second system, we use donor mice that constitutively express full-length membranebound chicken ovalbumin (mova) protein in all tissues. analogously, ova specific-b cells can be analyzed flow cytometrically with the use fluorescently-labeled ovalbumin. both systems allow us to identify donor-specific memory b cells ( aad -cd -cd -ova/ tetramer + igd -b + cd -) and plasma cells ( aad -cd -cd -ova/tetramer + igd -fb lo cd + ) during skin graft rejection (day post transplant). we were also able to track the development of a stable memory cell population in the bone marrow and spleen for > days cells.for the ova system quantitative elisa and elisot measurements for serum dsa and dsa-secreting cells, respectively, correlated with the development of donor-specific memory b cells. using these assays we will be evaluate polyclonal donor-specific b memory subsets under multiple conditions of immunity and tolerance and for the first time, characterize their functional properties and migration patterns. these experiments will provide new information on the basic biology of the memory b-cell response to allografts in mice and facilitate the development of these methods in sensitized patients that may lead to critical therapeutic opportunities for dsa production. in the tnf-related b-lymphocyte survival factor, blys/baff, is critical for primary follicular (fo) and marginal zone (mz) b-cell survival. in vivo neutralization of blys/ baff, using a newly developed monoclonal antibody, depletes the fo and mz b-cell compartments. here, we hypothesized that targeting b-lymphocytes, via the blys/baff pathway, could promote humoral tolerance to islet allografts. cohorts of stz-diabetic c bl/ mice were transplanted with isolated islets from balb/c donors. treatment with anti-blys/baff alone ( mcg/mse x doses+ mcg/wk/mse) did not protect islet allografts from acute rejection (mst= d; n= ) . on the other hand, a -day course of rapamycin ( mg/kg) prevented acute allograft rejection (mst= d; n= ). when rapamycin was combined with the anti-blys regimen, islet allograft survival was markedly prolonged (mst> d; n= ). importantly, islet allograft survival was coincident with the absence of detectable serum alloantibodies and blunted donor specific t-cell responses. following treatment with anti-blys, b-lymphocyte compartment reconstitution was detectable at days following treatment and was characterized by stringent selection at the transitional→fo b-cell tolerance checkpoint. overall, these data indicate that the blys/baff pathway may be a logical target of immunomotherapy for the achievement of humoral transplantation tolerance. reza tavana background: highly sensitized patients' ability to be transplanted is severely compromised because of high level of antibodies against various hla antigens. interleukin- is a type i cytokine that signals through a receptor composed of the il- r and the common cytokine receptor -chain ( c ). it is produced by t-cells and has been shown to be the contributing factor in terminal differentiation of memory b-cell to anti-body producing b-cell and plasma cells. objective: we evaluated the effect of il- co-stimulation on antibody production capacity of b-cells and also measured the expression of il- r on b lymphocytes of highly sensitized patients compared to non-sensitized patients on the kidney transplant list. methods: patients with a pra level > % (sensitized) were compared to non-sensitized patients from the transplant waiting list. after consent was obtained, peripheral blood was taken from patients before initiating hemodialysis. leukocytes were labelled with anti-cd , anti-il- antibodies and paraffin fixed after rbc lysis. il- r expression was measured by flow-cytometry over facscan machine on the same day. the expression of the il- r is significantly higher on b-lymphocytes of highly sensitized patients (hsp) compared with non sensitized patients (nsp) (fig .p< . ). in-vitro co-stimulation of isolated peripheral b-cell with il- and anti-cd results in higher igg production compared with anti-cd- or il- stimulation alone (fig ) . conclusions: il- is an important cytokine in b-lymphocyte stimulation and increases igg production. il- receptor on b-lymphocytes is up-regulated in sensitized kidney transplant recipients. il- to il- r interaction between t and b-lymphocytes may be an important pathway in antibody production in highly sensitized renal transplant recipients. we created mixed bone marrow chimeras in irradiated µmt (b cell-deficient) recipients by transplantation of syngeneic bone marrow from µmt, wildtype (wt) and mhcko (lack mhc i & ii expression on all cells) mice. µmt+mhcko chimeras lack expression of mhc i & ii on b cells but not other professional apcs such as dcs hence antigen presentation specifically by b cells is disrupted. allograft rejection and development of alloreactive memory t cells in µmt+mhcko chimeras was compared to µmt+wt chimeras that had intact antigen presentation by both b cells and apcs. skin allograft rejection was comparable between µmt+mhcko and µmt+ wt chimeras (mst = and days, respectively, p = . , n = /grp). however, heart allograft rejection was significantly delayed in the µmt+mhcko chimeras compared to µmt+ wt chimeras (mst = and days, respectively, p= . , n = / grp). development of alloreactive memory t cells was assessed in chimeras at weeks after skin allograft rejection by quantitation of antigen-specific ifnγ producing t cells. alloreactive cd and cd memory t cells were significantly fewer in µmt+ mhcko chimeras ( -fold fewer cd , p = . , and -fold fewer cd , p = . , n = /grp) than in µmt+ wt chimeras. these results show that the disruption of antigen presentation by b cells significantly delays heart but not skin allograft rejection. development of alloreactive cd and cd memory t cells is significantly impaired in the absence of antigen presentation by b cells. conclusions: antigen presentation by b cells accelerates heart allograft rejection and leads to development of alloreactive memory t cells. these findings emphasize antibodyindependent functions of b cells in promoting alloimmune responses and highlight the need for b-cell targeted therapies to improve long-term allograft survival. purpose: to test whether depletion of cd + b cells at the time of engraftment alters the prevalence of anti-donor alloantibody (ab)or severity of cav in the context of therapeutic immunosuppression with (csa) or high dose cd inhibition. methods: forty-five mlr-mismatched heterotopic cardiac cynomolgus allograft recipients were treated with high dose anti-cd monotherapy (αcd ; n= , with atg induction) or αcd with additional anti-cd therapy (rituximab mg/ kg q wk for weeks: αcd + αcd ; n= , with atg). thirteen other animals received therapeutic csa (target trough > ng/ml), five of which received additional αcd . graft survival was censored at days. ab was deemed (+) if present (by flow cytometry) around time of explant. results: animals died with beating grafts, mainly with atg-associated lung pathology or infectious etiologies and are excluded from this analysis. animals that had sustained αcd levels < µg/ml after protocol day were considered "subtherapeutic" for this reagent and also excluded from further consideration. graft survival with αcd + αcd (median > d) and proportion of grafts surviving to days ( / ) was significantly increased relative to αcd alone (median d, p= . ; / > d). graft survival with csa + αcd (median > d) and proportion of grafts surviving to days ( / ) was increased relative to csa alone (median d, / > d). with therapeutic αcd (trough level > µg/ml until graft explant), / ( %) developed ab vs. / with αcd + αcd ( %) (p= . ). average cav score for the αcd group was . ± . vs. . ± . in the αcd + αcd group (p= . using unpaired t test). preliminary cav scoring suggests that added αcd inhibited cav (scores ranging . - . ) relative to csa alone (median . ; range . - . ); ab analysis is in progress for these groups. conclusions: using αcd is associated with significant attenuation of cav when used with either "therapeutic" αcd or csa. our findings demonstrate for the first time that αcd reduces the severity of cav in conjunction with both conventional immunosuppression and costimulation pathway blockade. mechanisms include inhibition of ab production, and perhaps others that remain to be defined. subsaturating concentrations of anti-hla antibody ( sensitized recipients having donor specific anti-hla abs have been successfully transplanted following a conditioning regimen employing plasmapheresis with or without pooled human immunoglobulin. in vitro studies have shown that exposure of ecs to subsaturating concentrations (ssc) of anti-hla ab (priming) followed by subsequent exposure to high concentrations (hc) of anti-hla induces the expression of protective genes, bcl and ho- and confers protection to complement mediated lysis of ecs. however, the molecular events following priming with exposure to ssc of anti-hla ab still remains undefined. to determine the priming events and to define the kinetics of protective gene expression, we exposed human aortic ecs to ssc of anti-hla ab for hrs and re-exposed them to saturating concentrations of anti-hla abs. ecs were collected at , , and hrs after exposure to ssc as well as hrs after exposure to hc. expression profile of signaling intermediates (mapk, wnt, nf-kb, hedgehog, pi kinase, stress pathway, tnf family) in the ecs were analyzed by gene array. analysis of the bcl and ho- expression showed no significant increase in expression following exposure to ssc of w / (priming) or control ab alone at any of the time points ( background/aims: microcirculation disturbance, endothelial injury and cytokine overproduction are implicated in the pathophysiology of hepatic ischemia reperfusion injury (iri). thrombomodulin (tm) is a membrane-bound endothelial thrombin receptor that accelerates thrombin-catalyzed protein c activation, inhibits thrombin-induced fibrin formation, and also regulates inflammation. in the present study, we investigated the effects of recombinant human soluble tm (rhstm) on hepatic iri in the rat. methods: wister hannover rat was used for preparing ischemia/reperfusion model. hepatic iri was induced by subjecting % area of the rat liver to minutes of ischemia followed by h of reperfusion. the rats were randomly assigned to a group receiving an intravenous injection of rhstm ( mg/kg body weight) and to a group treated with saline minutes prior to the beginning of reperfusion. sinusoidal endothelial cells (secs) and kuppfer cells (kcs) were isolated using centrifugal elutriation. the plasma levels and the concentration in cultured cell supernatant of il- and tnfα were measured by specific enzyme immunoassays. results: the plasma alt, ast and hyaluronic acid levels were significantly decreased, and the histological damage of the liver was attenuated in rhstm-treated rats as compared to control rats. using laser doppler flow-meter we found that rhstm treatment improved hepatic microcirculation. the intrasinusoidal fibrin deposition, injury of secs and liver dysfunction during hepatic iri were weaker in rhstm-treated rats than in control rats. tm activity in secs was significantly recovered, and plasma il- and tnfα were significant decreased in rhstm-treated rats as compared to control rats. further, il- and tnfα production in isolated kcs was also significant decreased in rhstm-treated rats as compared to control rat . conclusion: the present results suggest that rhstm is useful for the prevention of secs damage and kcs activation induced by iri. our present study also suggests that disturbance of hepatic microcirculation is induced in part by intrasinusoidal microthrombus formation and by locally released inflammatory cytokines from kcs. protective effects of preservation solution including activated protein c in small-for-size liver transplantation in rats. background: small-for-size liver graft is a serious obstacle of partial orthotopic liver transplantation (olt). however, various therapeutic strategies including surgical innovations, pharmacological agents and gene therapies to protect small-for-size liver graft have not yet been developed. aims: activated protein c (apc) is known to have cell protective properties via its anti-inflammatory and anti-apoptotic activities. this study aimed to examine the cytoprotective effects of preservation solution containing apc on olt using smallfor-size rat liver graft ( % partial liver). methods: liver grafts were assigned to two groups: in the control group, the grafts were flushed and stored in histidine-tryptophan-ketoglutarare (htk) solution alone for h; in the apc group, in htk solution containing apc for h. results: the apc group significantly increased -day graft survival from % to %, decreased levels of transaminase, and improved histological features of hepatic iri compared to the control group. myeloperoxidase activity demonstrated that the apc group markedly suppressed the infiltrations of neutrophil. hepatic expressions of tumor necrosis factor-α and il- of the apc group were remarkably decreased. the apc group significantly reduced serum hyaluronic acid levels, indicating attenuated sinusoidal endothelial cell injury. moreover, the apc group markedly increased hepatic levels of nitric oxide caused by upregulated endothelial nitric oxide synthesis (nos) together with downregulated inducible nos, and decreased hepatic levels of endothelin- . finally, hepatocellular apoptosis of the apc group was remarkably suppressed by downregulated hepatic caspase- and caspase- activities. conclusions: preservation solution containing apc inhibited pro-inflammatory cytokine synthesis, which leads to hepatocellular apoptosis and liver injury. one of the cytoprotective effects of the apc treatment was to upregulate hepatic enos, followed by increased expression of hepatic no, and to decrease expression of hepatic et- , resulting in the prevention of microcirculatory disturbance. preservation solution containing apc is a potential novel and safe product for small-for-size liver transplantation to improve liver graft function and animal survival. deletion of cd on natural killer cells attenuates hepatic ischemia/ reperfusion injury. living donor liver transplantation (ldlt) has emerged as a solution to ease organ shortage in orthotopic liver transplantation. however, ldlt is often complicated by small-for-size liver graft that is highly susceptible to injury and shows decreased liver regeneration. suppression of liver regeneration in small-for-size grafts correlates with impaired priming as a result of limited nf-κb activation and decreased production of the priming cytokines tnf and il- . we have shown that the hepatoprotective protein a promotes liver regeneration, partly through blockade of the cyclin dependent kinase inhibitor p . however the impact of a expression in livers on il- production and/ or signaling were still unknown. in this study we demonstrate that secretion of il- (elisa) following treatment with lps or tnf was moderately lower in hepatocytes transduced with a recombinant a adenovirus (rad) as compared to non-transduced or rad.β-galactosidase transduced cells. this indicates that il- production in hepatocytes is not solely nf-κb dependent (not totally blocked by the nf-κb inhibitor a ). despite similar or lower il- levels, il- signaling as evaluated by phosphorylation of stat (western blot; wb) was enhanced in a expressing hepatocytes. this was confirmed in experiments showing that a increases stat- phosphorylation in response to exogenous human il- . accordingly, hepatocyte proliferation was significantly higher in rad.a transduced hepatocytes as opposed to controls. the pro-proliferative function of a mapped to the zn domain. since the balance of il- signaling in hepatocytes is finely regulated through a negative feed-back loop provided by the il- /stat- dependent induction of suppressor of cytokine signal- (socs ), we investigated whether a affects il- signaling by modulating socs expression. our results indicate that a indeed decreased il- mediated upregulation of socs (wb). this later result was confirmed in vivo. improved regeneration and survival in a treated livers correlated with a substantial decrease in socs levels before and hours following extended ( %) liver resection in mice. these results suggest that a enhances priming of hepatocytes by il- likely through down-regulation of socs . this added to the effect of a on p would further enhance its pro-proliferative function in hepatocytes to benefit survival and function of small-for-size liver grafts. background: we have previously demonstrated that silencing inflammatory, apoptosis and complement genes can prevent ischemia-reperfusion (i/r) injury occurring in heart transplantation. however, the method for efficiently delivering sirna into donor organ has not been established. this study was designed to develop a new method to induce gene silencing by coronal artery infusing with sirna solution for prevention i/r injury in heart transplantation. methods: multiple sirnas that specifically target tnfa, caspase , and c a receptor (c ar) genes were generated and selected. sirna protection of donor organs was evaluated in a rat heart transplantation model. heart grafts from lawis rats were infused with sirna solution via coronal artery and preserved in hkt solution at ° c for hrs, and subsequently transplanted into syngeneic lawis rats. cardiac functions were assessed by heart beating rate. gene expression at mrna level was determined by qpcr. the i/r injury was assessed by immunohistochemistry. results: after donor heart perfusion with sirna solution, sirna was found to enter the myocardial cells, indicated by the fluorescence emitted from the dye labeled sirna. the levels of tnfa, caspase and c ar genes were significantly up-regulated in the grafts after ex vivo preservation for hrs. these up-regulated tnfa, caspase , and c ar genes were significantly knocked down by sirna infusion. using a sirna infused organ as a donor, the graft survival was significantly prolonged in heart transplantation. while sirna solution-treated heart grafts retained strong heartbeat up to the end point of observation (> days), the control grafts lost function within days. in addition, an improved cardiac function was observed in the graft preserved in sirna solution. the protection of graft by sirna solution is associated with prevention of i/r injury. sirna solution-treated organs exhibited almost normal histological structures as well as less neutrophil and lymphocyte infiltration, compared with control solution-treated organs. conclusions: this study developed a novel ex vivo sirna delivery system using coronal artery infusion, which can effectively silencing genes in donor hearts and prevent cardiac i/r injury. background: ischemia/reperfusion (i/r) insult is a prime factor leading to liver dysfunction. apoptosis plays key role in the early graft loss following orthotopic liver transplantation (olt). bcl-xl has been showed to exert an anti-apoptotic function both in vitro and in vivo. this study was designed to evaluate potential cytoprotective effects and mechanisms for bcl-xl in liver i/r injury by ad-bcl-xl gene transfer. methods: a mouse model of partial min warm hepatic ischemia followed by h of reperfusion was used. balb/c wide-type (wt) mice (n= /gr) were injected with ad-bcl-xl or adβ-gal reporter gene ( . x pfu, i.p. at day - ). sham control wt mice underwent the same procedure, but without vascular occlusion. mice were sacrificed after h of reperfusion; liver tissue and blood samples were collected for future analysis. results: ad-bcl-xl treated mice showed significantly lower sgot levels (iu/l), as compared with ad-β-gal or wt controls ( ± vs. ± , and ± , respectively; p< . ). these correlated with histologic suzukis grading of hepatic i/r injury, with wt/ad-β-gal controls showing significant edema, sinusoidal congestion/cytoplasmic vacuolization, and severe hepatocellular necrosis. in contrast, wt mice treated with ad-bcl-xl revealed minimal sinusoidal congestion without edema/vacuolization or necrosis. ad-bcl-xl gene transfer significantly reduced local neutrophil accumulation and apoptosis ( . ± . of tunel+ cells in ad-bcl-xl vs. . ± . and . ± . of tunel+ cells in wt or ad-β-gal treated mice; p< . ). unlike in controls, intragraft expression of mrna coding for tnf-α, e-selectin/icam- , and ip- /mcp- remained depressed in the ad-bcl-xl group. ad-bcl-xl gene transfer markedly depressed the activation of nf-κb, caspase- , and increased ho- , a , and bcl- /bcl-xl expression, as compared with wt/ad-β-gal controls. conclusion: this study demonstrates that inhibition of nf-κb activation contributes to the cytoprtective effects after bcl-xl gene transfer in hepatic i/r injury. the induction of anti-oxidant ho- and anti-apoptotic a , bcl- by bcl-xl gene transfer exerts synergistic cytoprotective effect against antigen-independent hepatic inflammatory injury induced by i/r. hepatocyte background: primary graft non-function (pnf) affects survival and function of renal allografts. pnf, secondary to the ischemic and inflammatory injury in the peri-transplant period, leads to acute tubular necrosis and predisposes to acute rejection. defining new preconditioning regimens to reduce pnf are desirable. a is part of a negative antiinflammatory loop aimed at inhibiting nf-κb in renal proximal tubular epithelial cells (rptec). hepatocyte growth factor (hgf) is a pleiotropic growth factor upregulated in acute kidney injury and acute rejection likely to modulate inflammation and promote repair. in the present study we evaluated the effect of hgf on rptec and hypothesized that some of its protective functions may relate to the upregulation of a . methods and results: treatment of rptec with hgf ( ng/ml) led to a . ± . (n= ; p= . ) fold increase in a mrna (real time-pcr), which translated into a significant . ± . fold increase (n= ; p= . ) in a protein by h, as shown by western blot (wb). two lines of evidence suggested that upregulation of a by hgf was nf-κbindependent. hgf did not degrade iκbα in rptec (wb) nor upregulated the nf-κb dependent molecule icam- , as shown by flow cytometry analysis (facs). further, a was still upregulated in rptec expressing the nf-κb inhibitor iκbα, both at the mrna and protein levels. upregulation of a by hgf protected rptec from a subsequent inflammatory insult, here mimicked by the addition of tnf. pretreatment of rptec with hgf for hours blunted tnf-induced ( u and u/ml) upregulation of icam- , as analyzed by facs. conclusion: to our knowledge this is the first demonstration that a could be upregulated in rptec, in a non-nf-κb dependent manner. further studies are carried out to elucidate the transcription factors involved in hgf-induced upregulation of a . from a clinical standpoint, these results highlight the unique ability of hgf to protect rptec from inflammation by inducing the anti-inflammatory protein a , remarkably without triggering other pro-inflammatory signals. we propose that hgf-based therapies could serve in preconditioning regimens to prevent ischemia/reperfusion injury and reduce pnf and acute rejection in renal transplantation. background. liver ischemia reperfusion injury (iri) is one of the main causes of graft dysfunction and rejection in liver transplantation. it has been documented that iri is associated with inflammatory and complement pathway activation. this study was designed to investigate the efficacy of small interfering rna expression vector (shrna) targeting tnf-α and complement (c ) genes in the protection of mouse liver iri. methods. shrna expression vectors were constructed for tnf-α and c genes. mice received shrna by hydrodynamic injection prior to iri, which consisted of interrupting blood supply to the left lateral and median lobes of the liver for minutes followed by reperfusion. iri was evaluated using liver histopathology, as well as levels of serum alanine transferase (alt) and aspartate transaminase (ast). neutrophil accumulation was determined by a myeloperoxidase (mpo) assay. lipid peroxidation was assessed by malondialdehyde (mda) levels. realtime pcr was used to test gene silencing efficacy in vitro and in vivo. result. we demonstrated that iri is associated with an increase in tnf-α and c mrna levels in liver tissue hours after reperfusion. shrna-treatment effectively down-regulated tnf-α and c expression in iri livers. in comparison with vehicle control, the serum levels of alt ( . ± . u/l vs . ± . u/l) and ast ( . ± . u/l vs . ± . u/l), were significantly reduced in mice treated with tnf-α and c shrna. additionally, the neutrophil accumulation and lipid peroxidase-mediated tissue injury, detected by mpo and mda respectively, were improved after shrna treatment. tissue histopathology showed an overall reduction of injury area in shrna-treated mice. conclusion. this is the first demonstration that liver iri can be prevented through gene silencing of inflammatory genes and complement genes, showing potential for shrna-based clinical therapy. kidney -acute rejection: antibody-mediated rejection the ( ) in the first three days after transplantation, a temporary decrease in dsa was observed in all amr cases, and all of them quickly rebounded thereafter; ( ) c d can be detected very early (can be seen on day , % in day protocol biopsies, frequently in the absence graft dysfunction, and % in index biopsies); ( ) the pathologic changes observed in sequential biopsies were c d deposition followed by acute tubular injury, then interstitial inflammation and peritubular capillary margination seen in index biopsies; ( ) pure amr occurred early ( % at day ), usually evolving into mixed amr with accompanying cellular rejection ( % in index biopsies); ( ) most recipients ( %) had initial graft function before developing amr; ( ) background: antibody-mediated rejection (amr) has been recognized as a major problem in abo-incompatible (abo-i) renal transplantation (rtx). however, little is known about the long-term impact of amr in the abo-i renal transplant setting, especially after the introduction of a tacrolimus (fk)-based immunosuppressive regimen. the aim of this study was to assess the long-term impact of amr on the clinical and pathological outcomes in abo-i rtx. methods: fifty-eight patients who underwent abo-i rtx at our institution between march and december under an fk-based immunosuppressive regimen were enrolled in this study. protocol biopsies were performed regardless of renal function at one month and one year after rtx. fifty-six of the patients received the biopsy at one month and of patients underwent biopsy at one year posttransplant. amr was diagnosed by morphological features based on the banff ' update and other characteristic findings for amr previously reported, such as mesangiolysis, interstitial hemorrhage, and cortical infarction. we evaluated graft survival, incidence of chronic rejection characterized by transplant glomerulopathy (tgp) at one year posttransplant, and renal function using serum creatinine at three years posttransplant according to the incidence of amr at one month posttransplant. the overall graft survival rate at , , and years after rtx was %, %, and %, respectively. the incidence of amr at one month was % ( / ). the graft survival rate of the patients with amr was significantly lower than that of the patients without amr (p< . , years: % vs %, years: % vs %). the incidence of tgp in the patients with amr was significantly higher than that of the patients without amr (p< . , % vs %). the serum creatinine concentration at three years after rtx was significantly higher in the patients with amr than in those without amr (p< . , . mg/dl vs . mg/dl). in this study, we revealed that amr in abo-i rtx is associated with not only graft loss but also the progression of chronic renal impairment, functionally as well as pathologically, even after the introduction of an fk-based immunosuppressive regimen. further studies are needed to establish a more effective immunosuppressive regimen, such as rituximab induction therapy; against amr in abo-i rtx. conclusions: de novo dsa in ar is an independent predictor of graft loss and its degree of influence is comparable to other established risk factors (aa race, dgf, increased baseline creatinine). additional studies are warranted to: ) confirm the predictive ability of dsa and ) determine whether reduction/eliminattion of dsa will allow improvements in graft survival. was performed in all the recipients before and six months after lrkt. graft biopsies were performed as well within and after six months of the transplantation (tx). all the data of recipients were collected prospectively during the period of follow-up. humoral rejection rate, donor specificity, and time of appearance of the de novo abs were retrospectively studied. results among the lrkt recipients, ( %) showed negative/negative results, ( %) showed positive/positive results, ( %) showed positive/negative results, and ( %) showed negative/positive results (de novo abs) in the pre-/post-transplant flow-pra analysis. among the cases with de novo abs, ( %) had donorspecific abs (dsa) and the remaining ( %) had non-donor specific abs (ndsa) as determined by lab single antigen analysis. four of the five recipients ( %) with dsa showed evidence of both vascular and humoral rejection in the graft biopsies performed within months of the transplantation, while one of the eight recipients ( %) with ndsa showed evidence of cellular rejection during the same period. a -year graft survival rate of the recipients with de novo abs was %, compared with %, % and % in other groups without de novo abs (p= . ). conclusions lrkt recipients with developing de novo abs has much higher incidence of humoral rejection and worse prognosis, especially those with donor-specific de novo abs. cautious monitoring for the appearance of anti-hla antibodies should be adopted after transplantation, even in patients without anti-hla ab prior to the transplantation. despite significantly higher response than the males w/o amr (p< . ), the other females did not experience amr. conclusions: ) cfc is a novel assay to measure allo/ do cd -cell responses, assess the degree of sensitization, and predict amr in hs, ) allo/do cd -cell numbers are elevated in many hs, but not nc, ) hs w/ high(+) cfc are at increased risk for amr and may need additional pre-tx desensitization, ) allo/do reactivity are higher in hs females, which may explain their higher rate of amr, ) cfc cut off levels for amr prediction may be higher in females than males, ) monitoring hs using the cfc pre-and post-desensitization may help determine the efficacy of desensitization and risk for amr. were treated with plasmapheresis, ivig and rituximab, and pts with l-amr received ivig and rituximab. the -year gs post-amr in pts with e-amr and focal c d staining was % vs. % in pts with diffuse staining; while cases of l-amr with focal c d deposition had a gs of % vs. % in cases with l-amr and diffuse staining. the number of cases with focal staining was low, and the numerically evident differences were not statistically significant (log-rank p=ns). notably, when losses due to death with a functional graft were censored, post-amr gs was significantly lower in pts with e-amr and focal staining than in their counterparts with diffuse c d deposition ( % vs. %, log-rank p= . ). in this retrospective single center study, focally positive c d amr carries a worse prognosis than previously thought, and causes a significant reduction of gs. whether any degree of c d staining in the context of kt dysfunction should be treated as amr remains a pending question. association time of biopsy was . ± . mo after kt. however, cases were biopsied in the st year posttransplant. the extent of c d staining was graded as < % ( ), - % ( ), and ≥ % ( ) of ptc, and the intensity was graded as none ( ), light ( ), and strong ( ) staining. these findings demonstrate the significant discordance between detection of dsa and c d, which is a relatively specific histological evidence of ab-mediated injury. this factor should be taken into account when clinical decisions for treatment of patients with either c d or dsa positivity are made. the observed discrepancy could be partially due to the technique used for staining of the biopsy specimens, inability to detect anti-hla dsa with the available technology, or non-hla dsa. long term follow up data are needed to evaluate the impact of these markers on graft outcomes. introduction epithelial to mesenchymal transition (emt) is a potential mechanism of tissue fibrogenesis. in a previous study, we had reported that the early expression of emt markers was associated with the progression of renal grafts towards interstitial fibrosis and tubular atrophy (if/ta). here, we report the long-term follow-up of this cohort, paying a special attention to the evolution of graft function. patients and methods patients engrafted with a kidney from a cadaveric (n= ) or a living (n= ) donor, and in whom sequential protocol biopsies had been performed at and months, were included. the phenotype of epithelial cells was studied at three months according to the expression of vimentin (an intermediate filament normally expressed by fibroblast-like cells) and to the cellular localization of β-catenin. grafts in which these two markers were abnormally expressed by more than % of tubular cells were considered as emt+ grafts. serum creatinine and creatinine clearance (estimated abstracts by gault and cockcroft index) were collected from to months post-transplant and compared according to the emt status of the graft. results multivariate analysis demonstrated that the early expression of emt markers was an independent risk factor of the progression of graft fibrosis between and months. more importantly, these early phenotypic changes were associated with a progressive and sustained deterioration of the graft function : emt+ patients had a statistically higher serum creatinine from twelve months after transplantation, and a significantly lower creatinine clearance from months after transplantation (emt+ . ± . ml/min vs emt- . ± . ml/min, p= . ). the difference was persistent at months. conclusion the expression of emt markers by tubular epithelial cells at an early time point post-transplant (three months) is highly suggestive of an ongoing fibrogenic process, and has repercussions on the long-term graft function. therefore, these epithelial phenotypic changes are relevant and promising biomarkers for an early detection of if/ta. we recently reported that % of transplant glomerulopathy (tg) has evidence of alloantibody-mediated injury in biopsies for cause. we found that / of tg is c d+ab+ and / is c d-ab+ suggesting that c d staining is not sensitive enough to detect all biopsies with antibody-mediated injury. we aimed to develop a new laboratory test to detect biopsies with antibody-mediated rejection (abmr) which are missed by c d. using affymetrix microarrays, we analyzed gene expression in renal allograft biopsies for cause. we previously reported that both abmr and t cell-mediated rejection (tcmr) biopsies show increased expression of transcript sets associated with cytotoxic-t cells (cats) and gamma-interferon effects (grits) compared to biopsies without rejection (p< . ). however, abmr biopsies were discriminated by a selective increased expression of "endothelial cell-associated transcripts" (endats). these genes included established endothelial markers such as vwf, pecam , sele, cd , and cadherin , which are involved in endothelial-cell activation. hierarchical clustering of biopsies with ab+ using endats identified a group of c d-ab+ biopsies (n= ) clustered with c d+ ab+ biopsies (n= ). thus % of biopsies with antibody ( of ) had increased endat-scores despite being negative for c d. these c d-ab+ biopsies with high endats, had higher scores for cats and grits, increased incidence/severity of tg, tubular atrophy/interstitial fibrosis, and worse future graft function (p< . ), but similar incidence of tcmr or borderline lesions, in comparison to c d-ab+ biopsies with no increase in endats. the c d-ab+ cases with endothelial activation show extensive inflammation in the allograft, as measured by the gene sets, which is similar to c d+ abmr. there are a significant number of cases with alloantibody and no c d that show increased expression of endothelial genes. thus the transcriptomics detects deteriorating c d-allografts with ongoing alloantibody mediated injury. we conclude that increased expression of endothelial genes provides a new feature of abmr, and can be used as a new diagnostic test to detect and treat c d-abmr. probabilistic ( ) . the bayesian network model was analyzed and, interestingly, cd was critically related to tg, suggesting a b-cell mediated process. tg was also predicted by upregulation of ccl , ccl , ccl , cxcl , il- , il- , and icam gene expression. ten percent of the samples were excluded randomly from the initial model, and subsequently used for cross validation. in the validation analysis, the model effectively predicted tg (auc of . , % ppv) and sf (auc of . , % ppv). this study provides a compelling and clinically relevant example of the combination of quantitative gene expression with probabilistic bayesian modeling to predict renal allograft pathology. potentially important molecular pathways associated with transplant glomerulopathy were also identified. the application of this integrated approach has broad implications in the field of transplant diagnostics and interpretation of large data sets. , , , , , and months respectively. the daily dose and blood levels of tac were significantly lower in tac/slr group compared to tac/mmf group. renal function is shown. despite lower prevelance of cai in tac/slr group long-term graft function and patient and graft survival are comparable between tac/mmf and tac/slr groups. objective: the aim of this study was to determine if ethinicity impacts graft outcomes in kidney transplant patients converted to sirolimus (srl) and either maintained on calcineurin inhibitors (ci) or mycophenolate (mmf) with steroids. methods: this was a retrospective analysis of all kidney transplants converted to srl and transplanted from / to / . patients were divided into groups: group : aas converted to srl + continued on ci; group : non-aas converted to srl + continued on ci; group : aas converted to srl + continued on mmf; group : non-aas converted to srl + continued on mmf. pediatrics and multiorgan transplants were excluded. results: a total of patients were included ( % aa). demographics, baseline immunosuppression, and reason for srl conversion were similar between groups. table displays characteristics and outcomes. patients converted to srl+ci regimens had higher rates of acute rejection before srl conversion (p< . ), but equal rates after conversion. development of proteinuria was similar across groups. figure displays the graft survival rates for each group. aa patients converted to srl+mmf tended to have poorer outcomes compared to aa patients converted to srl+ci. non-aa patients converted to srl+mmf tended to have better graft outcomes compared to non-aa patients coverted to srl+ci, although this did not reach statistical significance(p= . ). conclusion: aas converted to srl may benefit from continued ci, while non-aas converted to srl appear to have better outcomes with mmf. further prospective studies are warranted to confirm these findings. aa srl+ci (n= ) there are no large registry studies evaluating the correlation of allograft failure for recipients of kidneys from the same deceased donor. we examined outcomes in such recipient pairs using data from the united states renal data system. methods: we studied the correlation of graft failure events within , pairs of same-donor recipients transplanted during through . analyses were limited to patients with functioning grafts months post-transplant (tx) and adjusted for known donor, recipient, and tx management factors. we estimated odds ratios to measure the increased risk for , , and -year graft failure and death-censored graft failure when the contralateral kidney had such an event. we also evaluated the effect of recipient pairs transplanted at the same center vs different centers. results: there is a strong correlation in outcomes for recipients with the same donor (table) . the correlation was stronger within pairs transplanted at the same center than for those transplanted at different centers. differences in the correlation of graft failures within pairs transplanted at the same versus separate centers diminished over and were absent by years post-tx. results for death-censored allograft failure were similar. conclusion: unmeasured donor factors contribute significantly to the correlated graft failure outcomes in paired recipients of deceased donor kidneys and need further study. kidney tx outcomes in the first year may be affected by differences in management between transplant centers, more so than in subsequent years post-tx. odds ratio of death-censored graft failure and graft failure in recipients of a donor pair, given that the outcome occurred in the recipient of the contralateral kidney, with both recipients being transplanted at the same (s) center or at different (d) centers. all odds ratios significant with p< . . the deterioration of kidney allograft function (dekaf) study is a nih-funded multicenter observational study of late allograft (ktx) loss. the study examines two cohorts: a "long-term cohort" (ltc) of prevalent ktx with scr < . mg/dl with deterioration of function ( % increase in scr or proteinuria) and a "prospective cohort" (pc) of incident ktx developing a persistent > % increase in scr. we examined the pathologic features of the first renal biopsy (bx) obtained for new onset deterioration in each cohort. all bx were read and scored centrally using a modified banff schema. on average, bx were obtained at (pc) and (ltc) months post-tx. mean scr was similar, but more patients (pts) in ltc had proteinuria. moderate to severe interstitial fibrosis and tubular atrophy (ta) were more prevalent in ltc than pc ( vs % ci score ≥ ; vs % for ta). the rate of vascular sclerosis > % was similar ( . % pc vs . % ltc); however, hyaline arteriolar sclerosis > % was more common in the ltc ( vs . %). interstitial inflammation (i) and tubulitis (t) scores were similar in both cohorts. however, more pts in ltc had peritubular capillary infiltrates (> cells) and evidence of tx glomerulopathy. while rates of interstitial inflammation and tubulitis sufficient to warrant diagnosis of acute rejection are similar in the prospective and long-term cohorts, long term cohort pts had more proteinuria, interstitial fibrosis, tubular atrophy, hyaline arteriolar sclerosis, and transplant glomerulopathy. analyses of histologic findings and renal outcome are ongoing. the term chronic allograft nephropathy (can) has been abolished by the last banff meeting report (am j transplant, ) and categories have been introduced for chronic changes: chronic active t cell-mediated rejection and chronic active humoral rejection (cahr). aim of the study was to review all cases of can diagnosed in the last years and to identify immunohistochemical markers of chronic rejection. a cohort of cad pts with biopsy-proven can was analyzed. each case was reviewed and assigned into groups according to banff criteria: chronic rejection (cr), chronic calcineurin toxicity (cnit) or chronic lesions not otherwise specified (nos). cd +, cd +, cd +, cd + cells and c d deposits were assessed by immunohistochemistry. twenty-eight pts were classified as cnit, as cr, of which were cahr, and as nos. serum creatinine and h proteinuria at renal biopsy, extent of interstitial fibrosis and glomerulosclerosis were not significantly different among groups (table ).the number of cd + cells was higher at ti level in cr compared to cnit (table ;*p=. ). cd + cells were higher at ti and g level in cr compared to cnit (table ;*p=. ). ti and g cd + cells were not different among the groups (table ). the number of g cd + cells was increased in cr compared to cni and nos (table ;*p=. ). no significant difference in cd , cd , cd , cd expression was found at ti and g level between c d + and c dcases of cr. cd , cd , cd but not cd expression at ti level correlated with ti fibrosis (r =. , . , . , respectively, p<. ) at the univariate analysis. only ti cd + cells independently correlated with fibrosis at multiple regression analysis. in conclusion, our data suggest that: morbid obesity limits access to kidney transplantation and predicts adverse transplant outcomes. there are limited data on the safety and efficacy of gastric bypass (gb) as a weight reduction therapy among transplant candidates and recipients. methods: we examined usrds registry data to identify medicare-insured kidney transplant candidates and recipients with billing claims for gb procedures. gb were categorized according to occurrence before listing, on the waitlist, or after transplant. we studied the clinical characteristics of gb-treated patients, and subsequent outcomes including progression from listing to transplant and -day mortality. usrds surveys bmi data at dialysis start, waitlist entry, transplant, and transplant anniversaries.we computed changes between most recently reported body mass index (bmi) values preceding and following gb, when available. results: we identified transplant candidates treated with gb before listing, who underwent gb on the waitlist, and gb cases after transplant. patients treated with gb were most commonly female, white race, and without diabetic or hypertensive renal failure (table) . -day mortality after gb, calculable for listed and transplanted patients, was . % and . %, respectively. transplant recipient experienced graft failure within days of gb. of patients treated with gb on the waitlist, proceeded to transplant. post-gb weight loss was detected for % with gb pre-listing, % with gb on the waitlist, and . % with gb after transplant. among patients listed for transplant in the same era and bmi > at first dialysis who were not treated with gb, % had lost weight between dialysis start and listing. conclusions: gb has been performed in small numbers of kidney transplant candidates and recipients, and is followed by weight-loss in the majority of cases. peri-operative mortality is comparable to reports in patients without kidney disease. gb warrants prospective study as a strategy for reducing complications of obesity in esrd. introduction: the present study investigated the incidence of posttransplant diabetes mellitus (ptdm) and calculated the risk of developing ptdm under a tacrolimus and mycophenolate mofetil (mmf)-based immunosuppression based on clinical characteristics, tacrolimus-pharmacokinetics, and genetic polymorphisms related to tacrolimus-pharmacokinetics, cytokines and diabetes mellitus. methods: seventy-one non-diabetic adult kidney recipients (male , female ) were studied. patients with continuous high plasma glucose levels, over . mg/dl of hemoglobin a c, or requiring insulin and/or oral anti-diabetic agents for more than months after transplantation at -year after transplantation were diagnosed as having ptdm. fifteen genomic polymorphisms were assessed. results: one year after transplantation, recipients ( . %) developed ptdm. positive risk factors were age (p= . ) and body mass index (p= . ). there were no significant differences in acute rejection rate, total steroid doses, tacrolimuspharmacokinetics or its related to genetic polymorphisms between the two groups. the frequencies of ptdm were significantly higher in patients with adiponectin t g tt genotype than in those with the g allele (p= . ), and in patients with glucocorticoid receptor (nr c ) bcl i cc genotype than in those with the g allele (p= . ). conclusions: the incidence of ptdm at -yr after transplantation was . % in our cohort. elder or obese patients were risky for the development of ptdm. the presence of the adiponectin t g tt or nr c bcl i cc genotype may also be risk factors for ptdm, suggesting that insulin and glucocorticoid sensitivity-related genes are associated with the development of ptdm. analysis of these genotypes is a possible method of predicting a patient's risk for developing ptdm and would be a valuable asset in selecting appropriate immunosuppressive regimens for individuals. pharmacokinetics persistent hyperparathyroidism (hpt) with hypercalcemia is common after renal transplantation. studies have shown that treatment with cinacalcet corrects hypercalcemia and lowers pth levels in these patients. so far cinacalcet's steadystate pharmacokinetics and their correlation with pharmacodynamics (pk/pd) have only been studied in hemodialysis patients, but not in renal transplant recipients with persistent hpt. to gain further insight into cinacalcet's effects on calcium-phosphate homeostasis, we determined its steady-state pharmacokinetics and pharmacodynamic effects in these patients. in a prospective, single center, open label study we examined the effect of a -week treatment with mg and subsequent -week treatment with mg cinacalcet daily on calcium-phosphate homeostasis over hours and determined the steady-state pharmacokinetics of cinacalcet in stable renal allograft recipients. the urinary calcium excretion was determined in timed urine samples. median auc - was . ng*h/ml and c max was . ng/ml for mg cinacalcet which is higher, and oral clearance (cl/f) was . l/h which is lower in renal transplant recipients compared to previously published data of hemodialysis patients ( mg cinacalcet auc - , c max . , cl/f ). we also observed a non-proportional increase of auc - after doubling of the cinacalcet dose. the once daily administration of cinacalcet dose-dependently reduced ipth and serum calcium. cinacalcet and parathyroid hormone (pth) concentrations showed an inverse correlation and were fitted to a simple emax model (e max = % reduction vs. baseline, ec = ng/ml). the -hour fractional urinary excretion of calcium was increased after mg cinacalcet (baseline . ± . %, mg . ± . %, mg . ± . %). renal function remained stable. cinacalcet's higher and non-proportional increase of auc - in transplant recipients compared to hemodialysis patients evokes the possibility of a pharmacokinetic interaction with concomitant cyclosporine treatment. cinacalcet effectively corrected the biochemical abnormalities of persistent hpt. the transient calciuria could potentially favor nephrocalcinosis and reduce bone mineral density, suggesting that higher doses of cinacalcet need to be used with caution in renal transplant recipients with severe persistent hyperparathyroidism. screening for proteinuria in the kidney transplant clinic. bryce a. kiberd, romuald panek. dalhousie university, halifax, ns, canada. proteinuria is a predictor of progression in kidney disease. it is not clear whether measuring albuminuria will have greater clinical utility over measurement by dipstick or total proteinuria in kidney transplant recipients. there has also been a trend away from using hour collections to using spot urine albumin/creatinine (ac) and protein/creatinine (pc) ratios. we compare the prevalence of proteinuria estimated by dipstick, ac and pc in prevalent patients (> months post transplant) in the kidney transplant clinic. significant albuminuria defined as ≥ mg/g was present in % ( / ). albuminuria was seen in % ( / ) with negative and % ( / ) with trace dipstick proteinuria. significant predictors of albuminuria in a mulitvariate logistic analysis were egfr (or . per ml/min/ . m , % ci . - . p= . ), diastolic bp (or . per mmhg, % ci . - . p= . ), and mmf use (or . , % ci . - . p= . ). macroalbuminuria (ac> mg/g) was seen in . % ( / ) and significant predictors in a mulivariate logistic analysis were lower egfr and higher systolic bp. sirolimus use was associated with more macroalbuminuria and mmf use with less macroalbuminuria. in a subset of patients followed for > years prior gfr loss was considerably greater (p= . ) in patients with albuminuria (- . ml/min/ . m /year) compared to those without (- . ml/min/ . m /year). however other measures of proteinuria were also significantly (p for trend) associated with prior gfr loss (ml/min/ . m /year) as shown in the < . (n= ) . - . (n= ) > . (n= ) ∆ egfr/year - . - . - . . * a pc cut point of . g/g had a sensitivity and specificity for albuminuria > mg/g of % and % respectively (c= . , % ci . - . ), and a pc cut point of . g/g had a sensitivity and specificity for macroalbuminuria > mg/g of % and % respectively (c= . , % ci . - . ). ac may be more sensitive and therefore have more clinical utility than other measures of proteinuria for progression. however prospective follow up of renal function change and cv outcomes is required. serum creatinine is a crude marker of gfr in renal transplant recipients and changes in gfr are frequently not accompanied by commensurate changes in serum creatinine concentration. serum cystatin c and estimates of gfr (egfr) based on cystatin c have been shown to be more accurate than serum creatinine and creatinine-based egfr in renal transplant recipients. the purpose of this study was to determine whether the filler, lebricon and rule cystatin c-based egfr equations were better able to detect changes in true gfr than the mdrd and cockcroft gault creatinine-based egfr equations. we performed two measures of m tc-dtpa gfr, serum creatinine and serum cystatin c on each of stable renal transplant recipients at least months apart. we calculated and compared the percent annual change in the measured gfr and the estimated gfr using the various gfr estimation equations. we also determined the sensitivity, specificity, positive predictive value and negative predictive value of each prediction equation for the detection of decline in measured gfr. results are presented below: the cystatin c and creatinine-based egfr equations all demonstrated poor sensitivity and diagnostic performance to detect a decline in gfr. novel equations derived and validated in the transplant population are needed to accurately assess kidney function over time. background: hypercalcemia, hypophosphatemia and renal phosphate wasting are common after kidney transplantation and are related to persistent hyperparathyroidism and hyperphosphatoninism. animal data suggest that these alterations in mineral metabolism may contribute to nephrocalcinosis and progressive graft dysfunction. supporting clinical data are limited. aim: to test the hypothesis that nephrocalcinosis is highly prevalent in the early posttransplant period and is related to a disturbed mineral metabolism. methods: biomarkers of mineral metabolism (including albumin-corrected serum calcium [ca c ], serum phosphorus [p], biointact pth, calcidiol, calcitriol and alkaline phosphatase) and renal calcium and phosphorus excretion parameters were prospectively assessed in renal transplant recipients ( % male, mean age ± yrs) at the time of their -month protocol biopsy. these protocol biopsies were screened for the presence of microcalcifications. intratubular, interstitial and/or cytoplasmatic microcalcifications were observed in . % of biopsies. calcifications were more prevalent in recipients of a living related donor as compared to cadaveric donor. high serum ca c levels, high serum pth levels, a high urinary ca×p product and high fractional excretion of p and low serum p levels were significantly associated with renal microcalcifications (see figure below). microcalcifications were not related to the fractional excretion of ca, use of diuretics, immunosuppressive regimen, serum alkaline phosphatase level and history of delayed graft function. the extent of microcalcifications correlated significantly with the severity of mineral metabolism disturbances. conclusion: our data demonstrate that nephrocalcinosis is highly prevalent in the early posttransplant period and suggest that a disordered mineral metabolism is implicated in its pathogenesis. polymorphism in abcb , the gene encoding for p-glycoprotein, predicts recovery of graft function early after kidney transplantation. the pharmacokinetics of cyclosporine (csa) is characterized by wide inter-individual variability. this might be particularly relevant in the early post-transplant period, due to the detrimental effects of the drug on the kidney. p-glycoprotein (p-gp), the product of the abcb gene, plays a key role in the distribution of csa at cellular level. single nucleotide polymorphisms (snps) of abcb might potentially influence the response of patients to csa. in particular, the snp in position of the exon , despite its silent nature, has been recently associated with altered specificity for its ligand, such as csa (kimchi-sarfaty et al, science , : ) . whether p-gp pharmacogenetics would help to guide csa treatment early post-transplant remains ill defined. we sought to evaluate the effects of the snps in the exon on the rate of recovery of graft function, as estimated gfr early postoperatively, in kidney transplant patients given csa as part of their immunosuppressive regimen. the frequency of dgf (as need for post-operative dialysis) among the different abcb genotypes was also estimated. of the kidney transplant recipients, % had the abcb wild type (c/c) genotype in exon , % were heterozygous (c/t) and % were homozygous (t/t) for the polymorphic variant in position . gfr values were significantly lower in patients carrying one of the two mutant alleles than in the wild type ( figure) . the frequency of dgf was %, % and % in patients with the cc, ct and tt genotypes, respectively. these findings demonstrate that in patients carrying the ct or tt mutant alleles in exon of the abcb gene and given csa, the recovery of graft function is less prompt, and the risk to develop dgf higher than in wild-type cc genotype. pre-transplant screening for abcb polymorphism would help to identify patients who may safely receive csa early post kidney transplantation. cigarette cigarette smoking has shown to reduce graft and patient survival in renal transplant patients. however, whether it could directly produce allograft disfunction has not been investigated. the aim of this study was to assess the smoking influence on renal graft function. we studied a cohort of adult renal transplant patients, transplanted from jan/ to dec/ and followed until dec/ . smoking habits were recordered at the time of transplant (never, former, current smoker). during summer of , a telephonical survey allowed us to obtain complete information about smoking habits in patients (aged . ± , % male): status (never, former, current), years of habit, years of quit, and number of cigarette smoked per day. number of "pack-years" was calculated. renal function was measure by inverse serum creatinine at rd month and then annually. time to decline a thirty percent in inverse serum creatinine was registered. patients were divided in two groups: those who always smoked during all transplant period (smokers, n= ) and those who did not (n= renal insufficiency occurs frequently after extrarenal transplantation as a result of acute tubular necrosis at transplantation, high blood pressure, and cni toxicity. we performed renal biopsies in patients after heart ( ), lung ( ) , liver ( ), bone marrow ( ), and cornea ( ) transplantation since . the time from transplantation to biopsy was ± months in general and was longest after liver ( ± months) and shortest after bone marrow transplantation ( ± months). the histologic changes were: tubular atrophy/interstitial fibrosis of ≥ % in % of biopsies (heart biopsies %, lung %, liver %, bone marrow %); acute tubular changes in % (heart %, lung %, liver %, bone marrow %); arteriolar hyalinosis in % (heart %, lung %, liver %, bone marrow %); arterionephrosclerosis in % (heart %, lung %, liver %, bone marrow %); glomerular sclerosis of ≥ % in % (heart %, lung %, liver %, bone marrow %); glomerulonephritis in % (heart %, liver %, bone marrow %; that means iga-nephropathy after heart and liver, immune complex nephritis twice after liver, mpgn after liver, and membranous glomerulonephritis and minimal changes after bone marrow transplantation); thrombotic microangiopathy in % (lung %, liver %, bone marrow %); finally one case of polyoma nephritis after lung transplantation. among heart and lung transplantations, patients needed kidney transplantation ( . %) after ± months; and among liver transplantations, needed kidney transplantation ( . %) after ± months (sign. later, p= ). conclusion: as expected, most histologic changes were those of cni toxicity and hypertension. surprising is the high number of glomerulonephritis under immunosuppression. thrombotic microangiopathy without the typical clinical signs were interpreted as cni-related toxicity and seems to occur more often after extrarenal than after renal transplantation. patients with heart and lung transplantation reach end-stage renal failure more often and earlier than patients with liver transplantation. the context: living kidney transplantation, a superior therapy to deceased donor kidney transplantation, is underutilized. states have enacted legislation and the federal government has launched initiatives to compensate living organ donors, but the effect of policy on improving living kidney donation rates in the united states is unknown. objective: to determine whether public policies are associated with changes in living kidney donation rates in the continental u.s. design, setting, and study subjects: series of cross-sectional analyses using records of state legislatures in continental states and living kidney donation rates from the united network for organ sharing. main outcome measures: living kidney donation rate during each year from - and change in donation rates before and after legislation enactment in each state and launch of federal initiatives. results: from january through december , states enacted legislation for living donors ( mandating paid leave, tax deductions, unpaid leave, encouraging paid leave). few states (n= ) enacted legislation prior to . there was a steady increase in the mean living kidney donation rate in the continental u.s during the study period (mean (standard deviation) annual increase in donations . ( . ) donations per , , population). in analyses accounting for length of time state legislation had been enacted, the types of legislation enacted, and the incidence and prevalence of esrd in each state, there was a slightly (but not statistically significantly) greater average annual increase in donations after compared to before state legislation enactment (annual increase in donations per , , population [ % confidence interval ( introduction: accurate and precise renal function assessment is essential in the evaluation of prospective kidney donors. while direct measurement of gfr is the "gold standard", it is not widely available. moreover, creatinine (scr)-based estimation equations are suboptimal to assess kidney function in this setting. ct scans are increasingly being used to study renovascular anatomy in donors and has replaced angiographic exams in many institutions. d imaging reconstruction allows for kidney volumes (kv) measurements which have been shown to highly correlate with measured gfr in this population. thus, the purpose of this study was to develop a model to estimate measured gfr that not only incorporates scr and demographic data but also kv as measured by d ct scans. methods: individuals who underwent donor evaluation were identified. an automated segmentation algorithm was used to measure renal parenchymal volume from preoperative abdominal cts. patient demographics and scr values were obtained from the medical records. gfr (normalized for bsa) was measured by i iothalamate renal clearances (igfr). an analysis of covariance model was created to correlate measured igfr with kv, patient age, sex, race, weight, height and scr. pearson's correlation coefficient was calculated for each variable. results: kv (p< . ), age (p< . ), scr (p< . ) and weight (p< . ) significantly correlated with igfr. sex ( . ), race ( . ) and height ( . ) were not statistically significant. the new fitted regression model is: kv-egfr (ml/min/ . m ) = . -( . *age) + ( . *weight) + ( . *volume) -( . *scr). we then compared the performance of the kv-egfr model to the re-expressed mdrd equation using calibrated scr assay. the r was . vs . , respectively; signed median % difference was + . % vs - . %, respectively (% difference b/w estimated gfr and igfr); and accuracy within % (% of estimated gfr values that fall within % of igfr) of . % vs . %, respectively. finally, the kv-egfr model was closer to igfr (in absolute values) than the mdrd eq. in / ( . %) cases vs / ( . %) cases, respectively. conclusions: kidney volumes highly correlate with igfr and the proposed gfr estimation model outperforms the mdrd equation in potential living kidney donors. the kv-egfr model could be used to estimate donor gfr in lieu of i iothalamate gfr which is less clinically available. for donor selection, current reports identify unsuspected renal pathology by time -biopsy. aim: to explore whether the findings at time -renal biopsy (bx) correlates with pre-donation clinical data including renal function. methods: kt databases from institutions were reviewed. time -renal bx are routinely performed from the upper pole during back-table and evaluated by nephropathologist for interstitial fibrosis (if), tubular atrophy (ta), arteriolar hyalinosis (ah), mesangial increase (mi), and glomerulosclerosis (gs). pre-donation data gathered from the donors were demography, body weight, bmi, systolic/diastolic bp, scr, proteinuria, and egfr by levey equation clinical data is summarized in the table. and gs showed no correlation. multivariate analysis failed to sustain the significant associations found on bivariate analysis, most likely due to a low event/parameter relation. conclusions: a significant correlation was observed between time -bx findings and clinical pre-donation parameters. whether these histological findings at the time of kidney donation represent a higher burden/risk for the remaining kidney ought to be evaluated during follow-up. in an era where living donation is increasing, we should advise a closer surveillance of these donors in order to modify risk factors that participate in kidney damage progression. predictors of poor early graft function following laparoscopic donor nephrectomy (ldn). matthew cooper, abdolreza haririan, stephen jacobs, michael phelan, benjamin philosophe, stephen bartlett, joseph nogueira. dept of surgery, urology, and medicine, university of maryland, baltimore, md. ldn has become the standard of care in many transplant centers. poor early graft function remains an important complication. we conducted a retrospective study to evaluate the risk factors for slow or delayed graft function following ldn methods: donor and recipient records from the first ldn were reviewed ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) . results: slow graft function (sgf) was defined as cr> . mg/dl at pod and dgf as the need for dialysis within the first week following transplantation. donor variables examined included age, sex, race, bmi, egfr, and number of renal arteries. recipient variables included age, sex, race, bmi, prior tx, pre-tx dm, history of smoking and drug usage. additional variables evaluated included degree of relationship (lurt), hla mismatch, antilymphocyte induction, de novo cni usage, r v. l nephrectomy, wit, total or time, and performance of simultaneous deceased donor pancreas tx (splk). univariate analysis was performed with significance defined as p< . . significant variables were then included in the multivariate analysis. background: a kidney exchange program is the logistic solution for patients with positive cross match (x + ) or abo incompatible donors. a major problem in all kidney transplantation programs is the sensitized recipient. we analyzed the success rate for immunized recipients in the dutch kidney exchange program. methods: from january till december donor-recipient pairs were registered. there were couples in the x + group while pairs were abo incompatible. in the x + group the median pra was % ( - %). to create new combinations a match program was run times (every months) with a median of ( - ) participating couples. allocation criteria included bloodtype (first identical, then compatible), hla match probability within the actual exchange donor pool to ensure that highly sensitized recipients have the best chance to receive a kidney, and the waittime on dialysis. cross matches between new donor and recipient were performed centrally in our reference laboratory with cdc-tests. results: after match runs, we found matching couples for / ( %) x + pairs, for / ( %) abo incompatible pairs with o recipients and for / ( %) abo incompatible pairs with non-o recipients. median pra of the recipients in the x + group was % ( - %). after match runs chances for success became small. the overall success rate for abo incompatible and x + pairs in the dutch kidney exchange program after years is %. however, the success rate for immunized patients in the x + group is significantly higher ( %) as our match program gives priority to those recipients with the smallest chance of finding a compatible donor in each match run. thus our kidney exchange program is especially suited for immunized patients. although paired kidney donation (pkd) program is an established method to overcome incompatibilities between kidney donor-recipient pairs (drp), significant proportion of the incompatible drp participating in such program could remain unmatched. domino-kidney transplantation (kt) in which altruistic living non-directed donor kidney (lndk) is offered to a pool of incompatible drp, and is used to initiate a chain of pkd transplants, could provide more opportunities of kidney transplantation to drp in pkd program. we introduce our experience of multicenter domino kt for the last years sixteen hospitals participated in the domino-kidney transplantation between february, and july, . domino-kidney transplants were performed with domino-kt chains initiated by altruistic lndk. -pair chains were , -pair chains , -pair chains , -pair chains , and -pair chains . the development of a multi-regional kidney paired donation program. using an optimization matching algorithm the new england program for kidney exchange (nepke) efficiently matches incompatible donor/recipient pairs. in , the mid-atlantic paired exchange program (mapep) and other individual centers began sharing data with nepke. this is a report of the success of two regional programs working together to increase the probability of participants in both programs finding a compatible match. methods: incompatible pairs and non-directed donors (ndd) are referred to nepke through transplant centers. donor and recipient abo, hla and recipient hla antibody screening are entered into the computer database. utilizing the optimization program, searches for compatible matches are conducted every to days. the program identifies potential and -way matches, ndd chains, and list exchange chains. following the determination of compatibility nepke notifies transplant centers involved of the potential match and centers accept or decline the offer. transplant centers notify their pairs and preliminary crossmatches are performed. a conference call is scheduled to coordinate simultaneous donor nephrectomies and recipient transplants. surgeons speak prior to incision to ensure simultaneous donation. results: from july to december , pairs and ndds have entered nepke. during this time frame over one thousand possible matches were identified, with the majority of these matches involving the same pairs in multiple matches. after "optimizing" and eliminating multiple matches two-way exchanges; three-way exchanges; three-way list chain exchanges; and ndd chain matches were offered to transplant centers as possible matches. most common reason offers were declined include: positive crossmatch ( . %); donor factors ( %), and recipient inactive or transplanted ( %). four offers occurred in mapep alone pairs, in nepke, and offers were cross-regional. three matches are pending for additional transplants. one previous and one pending transplant are the result of cross-regional exchanges. five transplants performed and pending involve ndd chains. one pending match involves a list exchange chain. conclusion: using a computerized optimization algorithm to match and way exchanges, ndd and list exchange chains has lead to a substantial increase in the number of kpd matches and transplants performed. cross-regional coordination is feasible and expands the number of transplants performed beyond the ability of individual exchange programs. as living donors become an increasingly important source of life-saving organs, there is growing concern about the lack of comprehensive research on donor outcomes. in addition to possible long term consequences, there is a risk that donors will experience complications during or following surgery. of the , living kidney donors in - , none died during surgery, and . % (n= ) needed blood transfusions during surgery. in the six weeks following donation, . % (n= ) had at least one serious adverse event (sae): . % (n= ) needed readmission following initial discharge, . % (n= ) needed an interventional procedure, . % (n= ) needed re-operation, . % (n= ) had vascular complications, and . % (n= ) had other complications. when all saes were considered in combination, the rate was . %. because over % of ldr forms were submitted by transplant centers fewer than weeks post-donation, all complication rates should be considered minimum estimates. one donor was reported to have died from donation-related causes within weeks of donation. the number of living donor kidney transplants performed by a transplant center in - ranged from to transplants. additionally, the risk of donor complications is not equal across transplant centers. for example, there was a significant correlation between the number of living donor kidney transplants performed at a transplant center and the percentage of that center's patients who were readmitted within weeks of donation, with greater donor volume associated with a lower rate of readmission. living kidney donation is relatively safe, but prospective donors should be made aware that there is a non-trivial risk ( . %) of short-term complications post-donation. as with many other major surgical procedures, complication rates are lower, on average, at institutions that perform a larger number of these procedures. we sought to determine if intensive screening improves detection of polyomaviral reactivation in asymptomatic patients and pre-emptive stepwise modification can improve outcome of polyomaviral nephropathy (pvn). methods:this is a prospective single center study. we randomly assigned de novo kt (cluster randomization) to intensive screening (is:n= ) and routine care (rc: n= ) for the detection of decoy cells. is was initiated at week- of kt and rc at the time of increase in serum creatinine. this was complemented with urine and blood nucleic acid testing. all patients had biopsies performed for detection of polomya nephritis (pvn). both groups were treated with pre-specified stepwise modification of it based on cell cytology and viremia (step : decrease dose of cellcept by %, step : decrease dose of tacrolimus by %, or switch to sirolimus therapy, step : discontinue cellcept). primary outcome included persistence of decoy cells/viremia following each step in modification of it every three months and secondary outcomes included acute rejection, graft function and graft loss. results: polyomaviral reactivation developed in . % in is group and % had pvn without changes in serum creatinine. the estimated cumulative rate of primary outcome in the is versus rc groups, -months ( % vs. %) relative risk (rr), . ; %ci, . - . ;p= . ); -months ( %vs. %) (rr= . ; % ci, . - . ; p= . ); and -months ( %vs. %) (rr= . ; % ci, . - . ; p= . ). secondary outcomes: despite similar degrees of step-wise is modification, rate of acute rejection non-significant (p= . ), but % of patients in rc loss the graft vs no graft loss in is group. patients who continue to remain on tacrolimus vs. those who were switched to sirolimus therapy had persistent viremia (or= . ; %ci, . - . ; p= . ). conclusion: is for poloymaviral reactivation allows early detection of pvn in the presence of stable graft function. stepwise modification in it resulted in early resolution of decoy cells and viremia in both groups, albeit slowly in rc group, and it did not prevent the graft loss in rc group. background: dna sequencing of the bk viral (bkv) genome non-coding control region (nccr) from individual patient isolates demonstrate divergent sequence and alterations in the arrangement of modularly conserved sequence blocks (p-q-r-s) ranging in size from to base pairs. aim: primary aim to molecularly clone and analyze patient-derived bk virus nccr sequence variants. our secondary aim is to determine if reporter gene constructs of patient-derived nccr variants differ in their promoter activity upon transfection into a mammalian cell line (vero) and a human primary tubular epithelial cell line. methods: bkv dna was amplified and sequenced from blood and urine samples of renal transplant recipients. via sequence alignment, unique nccrs were determined. these sequences were pcr amplified and cloned into reporter plasmids containing the renilla luciferase gene. promoter activity was measured via luminometer hours after transfection into vero cells and human tubular epithelial cells. results: variation in naturally occurring bkv nccr promoter regions exist as single basepair insertions and deletions, and insertions or deletions of partial sequence blocks (p-q-r-s). single basepair substitutions were most commonly seen ( % of analyzed samples). promoter activity within vero cells ranged from % to % as compared to nccr activity of an archetypal strain (wwb). low promoter activity (< %) was seen in isolates with duplications of the p block and large deletions of the r block. conclusion: these sequence blocks are rich in regulatory elements and control the expression of both bkv structural and regulatory genes. variation in these cisacting eukaryotic transcriptional promoter binding sites corresponds to differential promoter activity in naturally occurring bkv isolates. current plans include repeating the promoter activity studies in human primary tubular epithelial cells. humoral and cellular immunity to polyomavirus bk large t and vp antigens after pediatric kidney transplantation. polyomavirus bk-associated nephropathy (bkvn) has emerged as a cause of graft failure after kidney transplantation (ktx). in a cohort of pediatric renal recipients undergoing prospective three-monthly monitoring for bk by blood and urine q-pcr, we evaluated antibody response, measured by enzyme immunoassay using bk vlp, and cellular immune response, reported as frequency of ifnγ-secreting cells in a elispot assay after -day stimulation with bkv large t (lt) and vp peptides. we could not observe any influence of recipient pre-transplant bkv-specific igg or t-cell levels, which were generally low, on bkv infection after allografting. after transplantation, both specific igg levels, and frequency of bkv-specific t cells increased according to the degree of viral exposure. in detail, bkv-seropositive patients who never reactivate the virus (group , n= ) did not show significant increase in igg levels (from a median od of . at month + to . at the end of follow-up), while patients with urinary shedding alone (group , n= ) or with viremia (group , n= ) increased from a median od of . to . (p= . ), and . to . (p> . ). in the case of cellular immunity, vp -specific t-cells increased in the three groups. conversely, lt-specific t-cells, which were high (median sfu/ cells) and remained unchanged throughout the follow-up period in group patients, had a significant increase in recipients belonging to both group and . interestingly, patients with urinary shedding who do not progress to viremia show a median -fold increase in lt-specific t-cell levels at peak viruria, compared to no increase observed in patients who develop viremia. the latter group mount a significant response to lt only after therapeutic reduction of immunosuppression. at peak viruria, viremic patients already show a -fold rise in specific igg compared to the . increase observed in group recipients. our data suggest that inability to reach protective levels of bkv lt-directed t cells, rather than specific igg, predispose ktx recipients to bkv replication. introduction:the antibody response to human bkv virus (bkv) is incompletely characterized. antibody responses to the vp- protein have been detected in kidney transplant patients, but it is not known if these have virus neutralizing activity. methods:recombinant bk, jc, and sv virus like particles were used to produce a panel of monoclonal antibodies. these antibodies were characterized for isotype and for ability to bind the respective antigens in elisa assays. to test neutralizing activity, bkv gardner strain (atcc# vr ) viral particles were incubated with the corresponding antibodies for hours at degrees c, and used to infect wi cells. bkv infection was monitored by quantitative real time pcr using primers directed against the vp- gene. neutralizing activity was defined as greater than % inhibition of viral yield. results:the monoclonal antibodies were of the igg a or igg b class with the exception of one igg and one igm antibody. all anti-bkv monoclonal antibodies bound to bkv capsids in-vitro in elisa assays. this binding affinity was species specific, as only antibody showed weak binding activity to jcv and sv capsids. neutralization of infectious bk virus was shown for / antibodies. denaturation of capsid proteins indicated that the monoclonal antibodies recognized primarily conformational epitopes, with only monoclonal antibody appearing to have a linear component. paucity of linear epitopes was further suggested by lack of reactivity of sera from bkv seropositive subjects in elisa assays based on genotype-specific short peptide sequences derived from the bkv vp- loop region. four monoclonal antibodies each generated from jcv capsids and sv capsids did not show any bkv neutralizing activity. conclusions: bkv vp- protein capsids contain species specific conformational epitopes which can elicit virus neutralizing and non-neutralizing antibody responses. measurement of these antibodies in renal transplant recipients may have diagnostic and prognostic applications. bkv specific monoclonal antibodies deserve further study as potential therapy of acute infections in the viremic phase. impact of immunosuppression reduction in bk viremic patients: year follow-up. s. kuppachi, a. guasch, c. p. larsen, k. e. kokko. transplant center, emory university, atlanta, ga. background: development of bk nephropathy is a risk factor for allograft loss. bk viremia (bkv) precedes the development of bk nephropathy. it has been reported with -year follow-up that reduction of immunosuppression leads to control of bkv. here, we report our -year follow-up experience of bkv patients that were identified by a prospective screening protocol and managed by sequential reduction of immunosuppression. methods: all kidney or kidney-pancreas transplant recipients at emory university between / - / were screened prospectively for bkv by real time pcr during follow-up visits ( - , , , , ) . patients with a bk viral load of greater than , copies/ml blood received a kidney biopsy to screen for bk virus nephropathy by immunohistochemistry. bkv without nephropathy resulted in reduced immunosuppression by a % reduction in mycophenolate dose. bkv with nephropathy resulted in discontinuation of mycophenolate. all identified bk patients were monitored every - weeks until viral load was below , copies/ml. immunosuppression was further reduced if viral loads failed to decrease. results: recipients were followed over a -month period. patients had received simultaneous kidney and pancreas, a liver and kidney and the rest kidney alone. of ( %) patients developed bkv within a year from time of transplantation. average time to diagnosis of bkv was . months. average dose of mycophenolate at years was . g/d in the bkv negative population as compared to . g/d in the bkv positive population. average hour trough blood level of tacrolimus at years was . ng/ml in the bkv negative population as compared to . ng/ml in the bkv positive population. survival rates for both patient and organ were comparable at years in bk viremic vs bk negative patients ( % vs %) and ( % vs %) respectively. while bk viremia is a historic risk factor for organ loss, prospective monitoring and reduction of immunosuppression is associated with comparable year patient and organ survival to patients that never develop bk viremia. background: there are currently no bk virus (bkv) specific therapies available for clinical use. this study evaluates viral large t antigen as a potential target for drug development, since (a) this is a key molecule that participates in several different stages of viral replication, (b) has no homologous human protein, and (c) offers multiple functional domains for chemical binding, particularly the atp binding site, the dna binding site, the hexamerization surfaces, and the hinge region. methods: virtual screening and protein modeling techniques were applied to bkv large t antigen using a model developed from the known crystal structure of sv large t antigen. two different structural states of large t antigen (monomer and dimer structure) in three different states (with the nucleotide pocket empty, with bound adp and bound atp), were evaluated for a total of large t antigen receptor conformations. results: a computational solvent mapping analysis of small molecular probes allowed identification of multiple functional sites, which represent potential drug binding pockets on the large t antigen molecule. it was possible to classify molecular conformations centered on the atp binding site, hexamerization surface and hinge region of the viral protein. we docked medium sized fragments (< da) to confirm the results obtained by computational solvent mapping, and further characterize chemical properties of the atp binding site. in another approach, known chemical structures of hsp and rho-kinase inhibitors were used to search compound databases and obtain a subset of compounds (mean size of da) capable of docking large t antigen. cross-referencing the top solutions obtained by energy ranking, we were able to identify compounds that bind large t antigen in all conformational states. a subset of compounds simultaneously binds the atp binding site, hexamerization surface and hinge region of bkv large t antigen. conclusions: virtual screening and three dimensional homology modeling technology has allowed us to identify compounds that can bind multiple sites on the large t antigen. these compounds are predicted to preferentially inhibit viral replication without the toxicity expected from simultaneous inhibition of host cell kinases. bk virus (bkv), a human polyomavirus, causes bkv nephritis, which often leads to graft loss after renal transplantation. currently, the only efficient therapy against bkv nephritis appears to be a reduction/change of immunosuppressive agents, and this may increase the inherent risk of rejection. since human renal proximal tubular epithelial cells (hrptec) represent a main natural target of bkv nephropathy, the analysis of bkv infection of hrptec is likely to provide necessary additional insight into bkv biology and contribute to the development of strategies for treatment of bkv nephritis. here we report the ability of -hydroxy- -methyl-glutaryl coenzyme a (hmg-coa) reductase inhibitor pravastatin, which is routinely used to treat hypercholesterolemia, to repress bk virus entry pathways in hrptec and, correspondently, prevent bkv infection. the percentage of hrptec infected with bkv was assessed by immunofluorescent analysis in the absence and presence of pravastatin. both, the percentage of bkv infected cells and the intensity of bkv infection, assessed by western blotting using antibodies against large t antigen, were significantly decreased in hrptec treated with pravastatin. it is likely, that pravastatin's inhibitory effect is explained by depletion of caveolin- , a critical element of caveolae. we demonstrate that bkv enters hrptec by caveolarmediated endocytosis and disruption of caveolin mrna and protein inhibits bkv infection of hrptec. we provide evidence that pravastatin dramatically decreased caveolin- expression in hrptec and interfered with internalization of labeled bkv particles. our data suggest that pravastatin, acting via depletion of caveolin- , prevented caveolar-dependent bkv internalization and repressed bkv infection of hrptec. our data represent the first report of inhibitory action of statins upon bkv infection. results: . % of patients were caucasian, . % hispanic, . % african-american and . % asian. overall -and -yr patient survivals were % and %. stratified by race, only african-american survivals differed from caucasian ( -year survivals of % vs. %; figure ). compared to caucasians, african-american patients were younger ( yrs ± . vs. yrs ± . ), were more likely to be status ( . % vs. . %), to have a serum creatinine ≥ . mg/dl ( % vs. %), to receive an multi-organ transplant ( % vs. %), to have fulminant hepatic failure ( % vs. %), to have a higher meld score ( . ± . vs. . ± . ) , to be in the icu ( % vs. %), and to be ventilated pre-transplant ( % vs. %); all p-values < . . after adjustment for each of these variables, race was still an independent predictor of mortality (p= . , hr: . , ci: . - . ). multivariate analysis of the african-american group (n= ) alone revealed that a bmi greater than (p= . , hr: . , ci: . - . ), a creatinine greater than . mg/dl (p= . , hr: . , ci: . - . ), and icu admission pretransplant (p= . , hr: . , ci: . - . ) are independent predictors of mortality in this subset of patients. conclusion: in the current meld era, race is still a predictor of worse outcomes, even after adjustment for multiple clinical variables. further work is necessary to elucidate why this disparity exists. the purpose of this study was to analyze the effects of successive pregnancies in female liver transplant recipients on newborn and maternal outcomes. data were collected from the national transplantation pregnancy registry via questionnaires, phone interviews and hospital records. analyses for linear trends (proportions and continuous variables) were done by chi square and least squares regression. there were outcomes of pregnancies, including twins. of the liver recipients who had a first pregnancy, had between one and four subsequent pregnancies. there were no significant differences in the variables analyzed as noted in the conclusions: successive pregnancies in liver transplant recipients are not associated with adverse fetal outcomes and/or increased maternal graft loss. female liver recipients with excellent allograft function without significant recurrent disease or chronic rejection who wish to have more than one pregnancy should not be discouraged to conceive. recorded and categorized in a blinded fashion. univariate, multivariate and survival analyses were performed. over a . -year period ( ) ( ) ( ) ( ) ( ) ( ) , olt recipients were randomized to receive a cca with biliary stent (n= ) or cca alone (n= ). patients with hepatic artery thrombosis (n= ; . %) were excluded. there was no significant difference in demographic or graft-related variables. the mean age at transplant was years, % were male, the mean meld was and % had hepatitis c. the mean donor age was years, % of donors were male, and the mean cold ischemia time was . hrs. the only complication related to the biliary stent was one occlusion. the rate of overall bc in the stented patients was . % vs. . % in non-stented patients, (p=ns). however, stented patients had significantly less bc in the first -days post-olt ( . % vs. . %, p< . ) and significantly less anastomotic leaks ( . % vs. . %, p< . ). over the year following olt, stented patients also required less biliary therapeutic interventions (mean . vs. . interventions/patient, p< . ) and fewer readmissions (mean . vs. . readmissions/patient, p< . ). we also observed improved late graft survival (> mo) in the stented group. intraoperative stenting of the cca at olt does not appear to reduce the long-term rate of bc but it decreases the incidence of biliary leaks and significantly improves many facets of early patient management. is background: long-term outcomes after retransplantation of the liver (re-olt) is inferior compared to primary olt. however, the survival benefit of re-olt based on model for end stage liver disease (meld) is not known. a single-center analysis of adult patients who underwent re-olt between february to february was performed. survival benefits, at a given meld score, were calculated by comparing re-olt survival at months to expected -month survival without retransplantation results: of pts, underwent re-olt, and received transplants. the figure shows re-olt survival benefit at any meld score with increased significance in patients with meld scores > . although meld scores - predicted the highest mortality after re-olt, they also demonstrated survival benefit. multivariate cox regression identified cold ischemia time > hrs (rr . , p . ), meld - (rr . , p . ), time from first olt ( days - year, rr . , p . ) and third transplant (rr . , p . ) as independent predictors for mortality following re-olt. conclusions: re-olt should be considered in all patients even with low meld scores. although patients with high meld scores ( - ) exhibit poor survival outcomes, a survival benefit is achieved. survival benefit that considers both probability of death without re-olt and expected survival with re-olt, should be used for selection of retransplantation candidates. this study analyzed posttransplant complications, meld score, and donor ages and their effect on length of stay (los). methods: this irb approved retrospective review of our prospectively maintained database included liver transplant recipients transplanted between - . los < days, - d, and > d were analyzed. primary analysis to look at los, meld, and donor age was performed using wilcoxon two-sample test. complication groups were analyzed using logistic regression and odds ratio estimates were determined. kaplan-meier for patient survival for the los groups was performed. univariate analysis: allograft dysfunction, vascular complication of liver allograft, intra-abdominal (other than liver), biliary, cardiac, pulmonary, neurologic, sepsis, renal, and endocrine were statistically significant (p< . ) using fisher exact test. multivariate analysis: using logistic regression, significant complications were analyzed to determine the complications linked with los > d and > d. analysis of liver transplants demonstrated increasing los with increasing meld in each donor age group ( - , - ) p< . . for donor age > , this relationship was not significant (p= . ). multivariate analysis using logistic regression determined odds ratio estimates for each complication resulting in los > days and > days. los > days and los > days were associated with different complications (as shown above). allograft dysfunction (including pnf) and renal complications were not significant factors in multivariate analysis. patient survival significantly decreased (p< . ) with increased los > days. introduction: some patients with primary biliary cirrhosis (pbc) may require longterm corticosteroid (cs) therapy following liver transplantation (olt) due to recurrent inflammation in the graft. our center has attempted to minimize cs use in all of our olt recipients. we reviewed our experience in this cohort of patients to determine ) patient outcome including recurrent disease and ) long-term requirement for cs use in pbc patients. methods: from to , , olts were performed in , adults at the university of colorado of which patients ( . %) with pbc received allografts. recurrence was defined by characteristic histologic changes on biopsy. bivariate and multivariate analyses were used to evaluate predictors of cs withdrawal. potential predictors of cs discontinuation were considered: age, gender, bmi, race, presence of inflammatory bowel disease (ibd), type of graft (cadaver or living donor (ld), recurrence of aih, warm ischemia time, follow up time (time since transplant), and immunosuppressant (is). results: overall survival at years was %. the , and year recurrence-free survival was , , and %, respectively. disease recurred in patients ( . %). of these patients, none received a second transplant because of recurrent disease. cs was withdrawn in % of patients at time of review. independent predictors of cs discontinuation are age (>median) (p = . ) and ld graft type (p= . ). conversely, cyclosporine (csa) (p= . ), female gender (p= . ), and bmi > (p= . ) were negatively associated with cs withdraw. interestingly, cs withdrawal did not influence pbc recurrence. conclusions: ) long-term outcomes in pbc patients are favorable and disease recurrence can be managed medically without abstracts re-transplantation. ) using an aggressive cs minimization approach, almost / of the patients were cs-free at the time of last follow-up. ) increasing age and ld grafts were associated with successful cs withdraw; while csa, female gender, and increasing bmi were associated with unsuccessful cs withdraw. incidence cholestatic disease (cd), either chronic rejection or recurrent primary sclerosing cholangitis (psc), post liver transplantation (lt) occurs in - % of psc grafts. the study objectives were to evaluate the incidence and long-term outcome of cd. from to , grafts in consecutive psc patients and grafts in concurrent alcoholic liver disease (ald) patients were compared. cd was diagnosed by biliary imaging and/or histology. median follow-up was months. the groups were similar including meld score and cold ischemic time; however, roux-en-y biliary anastomosis was more common in the psc group ( % vs. %, p< . ). the psc group had more cmv hepatitis ( % vs. %, p< . ) and acute rejection ( % vs. %, p< . ), and fewer biliary anastomotic strictures ( % vs. %, p< . ). cd occurred in psc grafts and ald grafts (p< . ). the incidence of cd was greater in the psc group (p= . , fig. ). no significant risk factors for cd were identified. in the psc group the graft survival was lower in the cd group (p= . , fig. ) ; however, patient survival at and years was not effected by cd: % and % vs % and % because the re-lt rate was greater in this group ( % vs. %, p< . ). at years, patient survival in the psc group was better than for ald ( % vs %, p< . ). long-term outcome post lt for psc is good and better than for ald; however, cd continues to develop beyond the first years with a prevalence of % at years. graft loss in patients with cd is high, but with re-tx, patient survival is similar to non-cd patients. further studies to distinguish chronic rejection vs. recurrent psc may provide insight into the prevention of cd following lt for psc. discussion: our analysis showed that patients with aih have a worse long term survival compared to pbc and psc after ddlt. this may be explained by possibly more advanced disease at the time of presentation or more septic complications due to pretransplant salvage therapy with immunosuppressants. also,pbc had the worst relative outcome after ldlt in this group-possibly explained by the older age of the recipients. overall, ldlt offered better outcomes than ddlt in terms of survival in patients with aih,pbc and psc. this study highlights an important and previously unvisited aspect of transplantation for autoimmune and cholestatic liver diseases. inflammatory bowel disease course in patients transplanted for primary sclerosing cholangitis. ariana wallack, joel s. levine, lisa forman. internal medicine, univ. of colorado hsc, aurora, co; gastroenterology and hepatology, univ. of colorado hsc, aurora, co. the natural history of ibd following liver transplant (lt) for psc is unknown. prior studies have not shown factors consistently associated with disease activity, but were limited by small sample sizes. the aim of the study is to describe our experience with ibd post-lt in patients with psc and determine factors predictive of disease activity. a survey was mailed to liver recipients transplanted for psc between and asking about medications, ibd activity and quality of life after lt. responses were linked to our lt database. results ( %) recipients responded. % were male with a median of ( - ) months since transplant. % were caucasian with a median transplant age of ( - ) years. % developed recurrent psc post-lt. % had a diagnosis of ibd ( % uc). immunosuppression included tacrolimus in % and cyclosporine in %. % experienced at least one episode of acute rejection. % rated their quality of health - on a scale of - . there was no significant difference in demographic variables between ibd and non-ibd cohorts. % of respondents had ibd pre-lt. of the patients without pre-existing ibd, developed ibd post-lt. of the de novo ibd cohort, % were male, median transplant age was years ( - ), and % were caucasian. ibd developed in % at more than years post-lt. there was no statistical difference between the de novo ibd cohort and those with pre-existing ibd except for ibd type ( % uc vs %, p= . ). significantly more patients were not on any ibd medications post-lt compared to pre-lt ( % vs %, p= . ). fewer post-lt patients were on aminosalicylates ( % vs %, p= . ) and prednisone ( % vs %, p= . ) compared to pre-lt. post-lt recipients developed fewer ibd flares requiring hospitalization ( % vs %, p= . ). % reported improvement in ibd activity post-l. % and % reported no change and worsening activity, respectively. there was no significant difference between reported disease activity and immunosuppression, cmv status, rejection rate, recurrent psc, transplant age, gender or race. background: symptomatic cmv continues to be a significant problem. the costs associated with its development remain high without an optimal method for prevention. the aim of this study was to measure the pharmacoeconomic impact of implementing an abbreviated pre-emptive monitoring strategy versus valganciclovir (vgc) prophylaxis in a large teaching hospital. methods: costs for this analysis were based on a societal perspective, including drug, personnel, hospital, outpatient infusion and monitoring costs related to resources needed to perform pre-emptive monitoring and treatment of cmv related events. time per hr costs were nurse/data coordinator $ , physician $ , and pharmd $ . cmv pcr cost was $ . vgc cost per mg was calculated for prophylaxis, treatment and days of consolidation as needed based on an awp ($ . per mg). estimated cost of cmv syndrome was based on cost of picc line placement, drug, personnel and supply costs based on days of therapy and was estimated to be $ , . . inpatient admission cost per day were $ . results: a total of patients were included in this analysis. baseline and transplant demographics were well matched. table displays the total direct and indirect costs accumulated for each group. cmv syndrome occurred in three patients for each group. there was no cmv disease in either group. % of patients in the pre-emptive group had dnaemia, but only patients required oral anti-viral therapy. provider time and lab monitoring costs were significantly higher in the preemptive group, while direct medication cost was significantly higher in the prophylactic group. conclusions: frequency of disease severity and outcomes were equal in each group. although the overall costs between strategies is equivocal, allocation of resources to provide pre-emptive monitoring places the burden of disease prevention on the health care system versus the patient necessitating further abbreviation of these strategies. we propose a non-simultaneous form of kidney paired donation that starts with a living, non-directed donor (lnd). a paired donation matching algorithm was developed to allow for lnds to start potentially never-ending altruistic donor (nead) chains in addition to closed loops of -, -and -way exchanges. results: in july , a lnd from michigan traveled miles to donate a kidney to a woman whom he had never met in arizona. the following week, the arizona recipient's husband donated one of his kidneys to a -year-old woman in ohio. the following month, the mother of this recipient traveled to a city hours away to donate her kidney to a patient whose incompatible donor simultaneosly gave a kidney to the fourth patient in the chain. the incompatible donor for this fourth recipient is now slated to give her kidney to a patient in maryland. over the past months, transplant programs have partnered to perform paired donation transplants. demonstrating the advantage of nead chains over classic paired donation, of transplants resulted from altruistic donor chains. conclusion: in order to fully realize the potential of the above approach, one must be willing to supplant two prevalent ideas: ) that kidneys from altruistic donors should be given to the top candidate on the deceased donor waiting list, and ) that paired exchanges must be done simultaneously. while there are certain pitfalls to using chains of donors as opposed to traditional "swaps" (i.e. the possibility that a donor could renege, or the accumulation of type ab donors who are not likely to be able to begin another chain), this proposed paradigm shift could result in a very significant increase in both the number and quality of paired donation kidney transplants. purpose: medical literature and national best practices correlate families' understanding of brain death with organ donation rates. analysis of hospital data demonstrated variability in family communication. although surgical residents frequently interact with family members of potential donors and play a critical role in the donation process, they receive no formal cst. we sought to determine if pre-training residents improved performances in cst involving explanation and notification of brain death to family members and aided efforts to achieve the national donation rate goal of %. methods: in collaboration with a regional organ procurement organization (opo), an educational model for end of life cst was developed. surgical residents were divided into groups. the first group (n= ) attended a -hour didactic session at the opo that included role-playing exercises explaining brain death to families. opo staff, trained to serve as family role-players and skills station coaches, debriefed residents after each simulation. the second group (n= ) received no specialized training. six weeks later both resident groups participated in formal videotaped family communication simulations. independent observers (hospital faculty and senior opo staff), blinded to training, evaluated residents' communication skills using a -parameter assessment tool. all residents reviewed their videotaped performances and evaluations, then repeated the simulations after six months. results: during the study period, the pre-trained resident group assessment scores increased by % (p= . ), while the untrained group increased by % (p<. ). while evidence of improvement existed in both groups, pre-trained residents consistently scored higher when compared to untrained ( % vs. %, p= . ). during this same time period, donation rates in the surgical intensive care unit (sicu) increased by %. conclusion: our educational model demonstrated effective training in communication skills during end of life discussions. although a direct relationship cannot be established, donation rates in the sicu increased after resident training. incorporation of this training program into resident education has the potential to improve donation rates and increase the number of organs available for transplantation. program. saverio mirarchi, graeme n. forrest, benjamin philosophe. medicine, university of maryland, baltimore, md; surgery, university of maryland, baltimore, md. objective: to improve the efficiency of care for solid organ transplant patients by having internists work in conjunction with transplant surgeons to manage transplant patients admitted to the hospital more than days post organ transplant. this includes patients who have undergone kidney, pancreas, and liver transplants which our center transplants over of these organs/year. in , the organ transplant program was divided into a surgical transplant service (sts) and medical transplant hospitalist service (mths). the mths consists of four full time physicians, one part time physician, and one nurse practictioner with daily rounds from a transplant surgeon on a weekly rotating schedule. methods: we analyzed our data from the past five years since the inception of the mths at a large university medical center. the length of stay (los) for the mths and sts were compared to data from the previous combined program as well as data from the university health consortium (uhc). in addition, we looked at the cost of care to determine if there were any savings related to reduced los and adjusted for the combined salary of the mths. results: in the review period, the total admissions for both the mths and sts averaged admissions/year. over the past five years the mths showed a major decrease in the los index, defined as the ratio between the observed los and the expected los based on uhc data. this index dropped from . to . for patients on the mths. there was also a parallel drop in the sts los index from . to . . this was associated with a significant cost savings for the hospital. on average, the program has realized an average savings of approximately $ , , per year. when accounting for the combined salary for the mths group which is , dollars/year, the total savings over the year period this amounts to $ . million. conclusion: the creation of a mths working within an organ transplant program at a large university center has been able to demonstrate a major decrease in los for transplant patients which has resulted in decreased costs and improved efficiency. this has also allowed more focused care for a complex medical population. we believe that our program can serve as a model for similar programs at other busy transplant sites. with increasing demand for kidney transplants(tx), more patients are opting to travel outside the us to obtain transplantation. we describe the characteristics and outcomes of kidney tx recipients followed at our center who traveled abroad for a kidney tx between and . methods: data were obtained via chart review. we compared demographics to all tx recipients at our center during the same period and compared post-tx outcomes to a cohort of patients transplanted at our center matched for age, race, tx year, dialysis time, prior tx, and donor type. median follow-up time was days (range - ). results: demographics are outlined in table. patients transplanted abroad were more likely to be asian and had shorter dialysis times. most patients were transplanted in china ( %) followed by iran ( %), the philippines ( %), and india ( %). living unrelated tx were most common. all patients were discharged on a calcineurin inhibitor, pred, and either mmf ( %), aza ( %), or rapamycin ( %). patients received induction. only patients received cmv prophylaxis. the median duration of hospitalization was days. the median time post-tx to initial visit at our center was days. patients required urgent admission to hospital, of whom lost their grafts. patients ( graft survival at -year was % for both "tourists" and matched recipients at our center. median time to graft loss was ( - ) days. mean scr and rejection -year post tx was not significantly different between recipients transplanted abroad and at our center. conclusion: compared to patients transplanted locally, patients transplanted abroad had similar graft survival and renal function post tx, but had a high incidence of infectious complications. [background] the primary benefits anticipated following successful induction of allograft tolerance are avoidance of the complications of long-term immunosuppression and prevention of chronic rejection. we have previously reported successful induction of renal allograft tolerance in recipients of hla mismatched combined kidney and bone marrow transplantation (ckbmt). no evidence of chronic rejection has been observed in these recipients after immunosuppression-free periods of to years. in the current study, we have evaluated the longer-term economic impact of this approach. [method] the conditioning regimen for ckbmt included cyclophosphamide, thymic irradiation, anti-cd mab, and a calcineurin inhibitor, which was discontinued after - months. stable renal transplant recipients receiving ongoing triple or double drug immunosuppressive therapy with compatible follow up times were compared with the four tolerant recipients. tolerant patients and stable patients on maintenance immunosuppression were also comparable with respect to their age, their original disease and donor-recipient histocompatibility. [results] the perioperative charges for ckbmt were approximately $ , higher than those for conventional living donor kidney transplantation. after - months, continuing medications in ckbmt recipients included only occasional over-thecounter analgesics. in contrast, the stable conventionally treated recipients were taking an average of pills daily. these included treatments required for de novo diabetes ( %), hypertension ( %), hyper lipidemia ( %) and gastro-intestinal symptoms/ prophylaxis ( %) in addition to their maintenance immunosuppression. these needs resulted in annual maintenance charges of over $ , /year/allograft recipient and do not include unmeasured costs related to issues such as quality of life or absences from employment. [conclusion] even with this admittedly expensive approach to tolerance induction, the overall medical costs for conventional kidney transplantation with ongoing immunosuppression and treatment for complications will exceed the cost of tolerance after approximately years. increasing african american donation rates in a midwest metropolitan community. susan gunderson, susan mau larson, david m. radosevich, clarence jones, bill tendle, tiffany scott. lifesource, st. paul, mn; transplant information services, university of minnesota, minneapolis, mn; southside community health services, minneapolis, mn. purpose: african americans are underrepresented in deceased organ donation in this + million community. historically minimal educational outreach had occurred and this study was designed to understand the community's disposition toward donation and to increase support for donation. methods: television, newspaper, and radio advertising aired over a month period beginning in using the nationally produced donate life-african american campaign as the primary intervention. other components included related faith-based and community outreach. the opo partnered with a minority focused community health clinic to survey african americans pre-and post-intervention. a mailed, selfadministered survey was sent to a sample drawn from organizational lists with a high likelihood of including african american households. for the evaluation, the sample was separated into ) community members and ) church members exposed to faithbased campaigns. results: african americans in this community have a sophisticated understanding of the importance of donation (average of knowledge questions scored correctly) in pre-intervention survey. in both community and church groups media exposure and donation knowledge increased after the media campaign (p< . and p= . respectively). similarly, donor designation rates for the entire population on the drivers licenses increased ( . % versus . %, p= . ). among all african americans the propensity to donate was, however, unchanged following the campaign. propensity to donate increased (p= . ) in the community sample whereas there was a reduced propensity to donation (p= . ) in the church sample. during the study time period the opo also experienced significant increases in donation authorization rates among african-americans, increasing from % to %. summary: a media based campaign combined with grassroots outreach is an effective tool to increase knowledge and awareness. partnership between the opo and community and faith based leadership was a significant positive byproduct of the project and should be included in future outreach efforts. background: it has been hypothesized that the clinical benefits of anti-thymocyte globulin (atg) induction therapy do not completely result from immunodepletion, but may also result from induction of immunoregulatory t-cells (treg). in this prospective, controlled study we investigated the effect of atg-induction therapy on the frequency and phenotype of peripheral cd + foxp + cd -/low t-cells in kidney transplant patients. methods: after transplantation, patients received atg-induction therapy (thymoglobulin ® ) and triple therapy consisting of tacrolimus, mmf and steroids. the control group (n= ) received triple therapy only. by flow cytometry, t-cells were analyzed for markers associated with immune regulation: cd , foxp and cd . within the foxp + t-cell population, the cd ro (memory) and ccr (homing receptor) markers were characterized. results: pre-transplant levels of cd + foxp + cd -/low t-cells in all patients were - % (median %) of cd + t-cells. one wk post atg induction therapy, no measurable numbers of treg were present. at wks post atg-induction therapy, a higher proportion of the first detectable t-cells expressed foxp compared to the control-group (atg vs. control-group; vs. %, median, respectively, p= . ) and then returned to pre-transplant levels at wks. this increased proportion of cd + foxp + cd -/ low t-cells resulted in a significantly higher foxp + /foxp neg ratio than in the controlgroup at wks; . vs. . , median, p= . ) . at wks, we found a decline in the proportion of naive foxp + treg (cd ro neg ccr + pre-transplant vs. post-transplant; vs. %, median, p= . ) which was associated with a rise in the proportion of memory foxp + treg (cd ro + pre-transplant vs. wks post-transplant; vs. %, p= . ). moreover, the proportion of memory t-cells exceeded that in the control-group at wks (atg vs. control-group; % vs. %, median, p= . ) which was mainly due to an increase in the proportion of effector memory foxp + treg (cd ro + ccr neg ). conclusion: after atg-induced immune cell depletion, a shift towards cd + foxp + cd -/low peripheral regulatory t-cells with the memory phenotype was measured in kidney transplant patients. this finding suggests that atg treatment triggers the generation of de novo peripheral regulatory t-cells by homeostatic proliferation. introduction in a prospective study, we investigated whether donor-specific regulatory cd + cd bright+ foxp + t cells develop in kidney transplant patients. methods we analyzed the percentage and function of peripheral regulatory cd + cd bright+ foxp + t cells of patients before, , and months after kidney transplantation. the immune regulatory capacities of cd + cd bright+ foxp + t cells were assessed by their depletion from pbmc and reconstitution to cd neg/dim responder t cells at a : ratio in the mlr. in the first year after transplantation, peripheral cd + cd bright+ foxp + t cells decreased from , % ± , pre-transplantation to , % ± , at months (p< . ). while mlr reactivity to rd party-ag ( rd p) significantly improved (p< . ), the reactivity against donor antigens remained low. functional analysis demonstrated potent donor-specific regulatory activities by cd + cd bright+ foxp + t cells after transplantation. depletion of cd + cd bright+ foxp + t cells from pbmc resulted into increased proliferation upon stimulation by donor antigens (p< . ). upon reconstitution, the capacity of cd + cd bright+ foxp + t cells to control the proliferation of anti-donor reactive cd neg/dim t cells increased over time: from % (median) pre-transplant to % post-transplant at month (p< . ). moreover, the anti-donor regulatory activities by the cd + cd bright+ foxp + t cells were significantly more vigorous than those controlling rd p-ag stimulated responder t cells ( %, p< . ). the generation of potent donor-specific regulatory cd + cd bright+ foxp + t cells in the periphery of kidney transplant patients prevents the development of adequate alloreactivity. conclusions: these results indicated that anti-donor responses could be detected even in a significant proportion of "stable" long-term hla identical kidney transplant recipients. speculatively this may be the cause of late graft failures in this group of patients. expression klotho is a gene almost exclusively expressed in renal distal tubules and loss of expression is associated with accelerated aging. in various models of acute and chronic renal injury, and with normal aging, renal klotho expression has been found to decrease. increased donor age is associated with poorer long term allograft function. we hypothesized that klotho mrna expression in renal implant biopsies would correlate with donor kidney quality, as determined by donor chronologic age and peri-transplant renal injury. our recent unsupervised microarray analysis of renal implant biopsies revealed a continuum of organ quality across all samples ranging from the best living donor (ld) kidneys at one end to the worst performing deceased donor (dd) kidneys at the other (am j transpl , nov ,epub). three predominant groups were identified of ld, dd kidneys with low risk ( . %) of delayed graft function (dgf) and dd kidneys with high risk ( %) of dgf (p < . ). analysis of klotho gene expression among these groups also revealed a spectrum of expression from highest levels in ld to lowest in dd kidneys, especially those who developed dgf. differences were highly significant among ld vs dd kidneys (p < . ), although donor age was not different between these groups. klotho transcript levels were significantly different among kidneys that developed dgf compared to those with immediate graft function (igf) (p < . ). in conclusion, reduced klotho gene expression is associated with grafts at risk of poorer function and appears to be independent of donor age. decreases in klotho expression likely reflect other factors impacting the renal tissue, which may impact potential for repair and ultimately allograft function. anti renal allograft rejection episodes produce a stereotypical response in the allograft characterized by infiltration by cytotoxic t lymphocytes (ctl), potent ifng response by donor and recipient cells, and decreased transcripts associated with the epithelium. we previously identified pathogenesis based transcript sets (pbts) that reflect the disturbance in rejecting allografts (qcats -ctl, grits -ifng response, kts -decreased function, ajt : , . we hypothesized that anti-rejection treatment would reverse the transcriptome changes of rejection more than histopathologic lesions. using microarray analysis we measured expression of these pbts in antibody mediated rejection (abmr)( untreated, treated) and t-cell mediated rejection (tcmr) ( untreated, treated) biopsies, normalized to nephrectomy samples. histopathology scoring of the primary rejection lesions were analyzed; interstitial inflammation(i), tubulitis(t), intimal arteritis(v), and glomerulitis(g) (fig a) . of these, only i and t differentiated between abmr and treated abmr (p< . ) yet none differentiated between tcmr and treated tcmr. pbts on the other hand differentiated treated from untreated for both abmr (p< . ) and tcmr (p< . ) with all pbts (fig b/c) . the changes in transcript expression in treated cases were dramatic. particularly impressive was the consistent correction of the disturbances in pbt expression despite the heterogeneous treatment following abmr episodes. unlike abmr treatment, tcmr treatment always included steroids which may contribute to the more pronounced changes compared to abmr. the time of treatment before the biopsy, which also varied within the groups, did not affect the changes in pbt expression since values in treated cases approached those of nephrectomy samples. thus, histologic rejection lesions persist following anti-rejection treatment whereas transcript disturbances of rejection are greatly reduced compared to untreated cases. this study suggests that assessment of anti-rejection treatment is more sensitively monitored by transcript expression than histopathology. blood deciphering the mechanisms of tolerance and chronic immune-mediated rejection remains a major goal in transplantation. data in rodents suggests that toll-like-receptors (tlr), regulators of innate immune responses, play a role in determining graft outcome. however, few studies have focused on tlr in human kidney transplant recipients. we addressed this issue by analyzing the peripheral blood (n = ) and graft biopsies (n = ) of renal transplant patients and healthy volunteers. we analyzed, for the first time, the expression of tlr in pbmc from kidney recipients with contrasted situations: operational tolerance and chronic immune-mediated rejection (banff ), compared to patients with normal histology and stable graft function, non transplant patients with renal failure and healthy volunteers. we found that myd and tlr were significantly contrasted in the pbmc, and in particular in monocytes, of patients with chronic immune-mediated rejection vs. operational tolerance. chronic rejection patients had significantly increased tlr and myd compared to operationally tolerant patients, who resembled healthy volunteers and non transplant patients with renal failure. interestingly, analysis of tlr transcripts in graft biopsies from patients with normal histology or chronic immune-mediated rejection reflected the blood findings, with a significant increase of tlr in chronic immune-mediated rejection. thus, we provide data to support a link between tlr expression and long-term graft outcome. the role of tlr and their endogenous ligands in mediating allograft rejection or acceptance therefore warrants further investigation and could give rise to new strategies of therapeutic intervention. our results suggest that peripheral blood tlr shows potential as a biomarker of chronic immune-mediated rejection and that measuring blood tlr levels may help to identify patients experiencing chronic rejection who require a biopsy. moreover, our data suggest that absence of tlr signaling may be a feature of operational tolerance to kidney grafts. identification of immune identification of immunological tolerance is an important prerequisite in order to establish an individually-tailored approach to the post-transplant management of allograft recipients. it will also provide new insight into the mechanism underlying the balance between tolerance and rejection. here we present data from a multi-centre study aimed at identifying tolerance to renal allografts. we have collected samples from five selected groups of renal transplant recipients: drug-free tolerant patients that were functionally stable despite remaining immunosuppression-free for more than one year; functionally stable patients on minimal immunosuppression (< mg/ day prednisone); stable patients maintained with calcineurin inhibitors (cni); stable patients maintained on cni-free immunosuppression regimen; and patients showing signs of chronic rejection. a group of age and sex matched healthy volunteers was also included as control. several biomarkers and bioassays, were combined to provide an immunological 'fingerprint' of the tolerant state. immunophenotype showed a selective expansion of peripheral blood b and nk lymphocytes in drug-free tolerant patients. this group of patients was also characterized by the absence of anti-donor specific antibodies. the differential expression of several immune relevant genes and a high ratio of foxp / α- , -mannosidase expression in these patients was observed. tcr landscape analysis highlighted differences between the vβ repertoires of drug-free tolerant recipients and chronic rejection patients. additionally, direct pathway donor-specific hyporesponsiveness by ifnγ elispot and lack of indirect pathway anti-donor responses assessed by trans-vivo dth were detected in drug-free patients. the diagnostic capabilities of the combined results of several of the above mentioned biomarkers and bioassays are as follows: specificity . , sensitivity of . and a positive predictive value of . %. these biomarkers could be used to inform drug weaning protocols of kidney transplant recipients. bile acid aspiration stimulates lung allograft immunity. bile acids detected in the broncho alveolar lavage (bal) as a marker of aspiration has been associated in a dose dependent fashion to earlier development of bronchiolitis obliterans syndrome. we sought to study the relationship between bile acids and active immune molecules as detected in the bal. methods: bal collected prospectively from lung transplant recipients at routine surveillance bronchoscopies were assayed for bile acids. samples were then assayed by luminex for cytokines , and by elisa for pulmonary collectins (sp-a, sp-d). results were analyzed according to levels of bile acids as per roc testing for accuracy for bronchiolitis obliterans syndrome diagnosis (high levels ≥ . µmol/l). results: we prospectively examined lung transplant recipients and a total of bal samples were collected. in no bile acids were detected, low levels were present in , and high levels were detected in samples. samples with high bile acids had significantly greater innate (tnf-a, il- b, il- , il- , il- ) and adaptive (ifn-g, il- ) cytokines as well as greater chemokines (mcp- , il- ) compared to the other samples. in contrast pulmonary collectins sp-a and sp-d were significantly reduced in samples with high bile acids. the figures shows the median and interquartile range for each molecule according to bile acid levels. conclusion: bile acids detected in the bal as markers of aspiration stimulate the lung allograft immunity in a dose dependent fashion. in particular high levels of bile acids are associated with an impaired lung specific innate defense system provided by the pulmonary collectins and with a broncho-alveolar district "cytokine storm". tolerance/immune deviation iii the results of using ex vivo cd + t-cells converted dn t-cells in nod mouse models support the concept and the feasibility of potentially utilizing this novel cell-based therapeutic approach clinically for the treatment of autoimmune type i diabetes. horng-ren yang, gouping jiang, john j. fung, shiguang qian, lina lu. immunology and general surgery, cleveland clinic, cleveland, oh. liver transplant tolerance was recognized by spontaneous acceptance of liver allograft in many species. the underlying mechanism remains unclear. interestingly, although liver allografts are accepted, hepatocyte transplants in the same combination are promptly rejected, indicating a crucial role of liver tissue cells in immune suppression. we have demonstrated a profound t cell inhibitory activity of hepatic stellate cells (hpsc), which are known to participating in repairing and fibrosis during liver injury. addition of activated (a) hpsc, but not quiescent hpsc, significantly inhibited allo-dc induced-t cell proliferative responses (mlr) in a dose dependent manner, which was associated with enhanced t cell apoptosis (tunel). neutralization of b -h by anti-b -h mab significantly reduced the hpsc-induced t cell apoptosis and reversed the inhibition of t cell proliferation, suggesting a key role of b -h . to evaluate this in vivo, balb/c islets ( ) were co-transplanted with x activated hpsc (b ) into stz-induced diabetic b recipients. co-transplant with hpsc effectively protects islet allografts from rejection. this was associated with reduction of graft infiltrating t cells and enhancement of apoptotic activity. co-transplant with hpsc from b -h -/livers markedly lost their islet graft protective capacity, associated with less apoptosis of infiltrating cells. to determine the subsets of apoptotic t cells, t cells were isolated from spleen, draining lymph nodes, and grafts for phenotype and function analyses. on pod , a marked reduction of graft infiltrating cd + ( . %) and cd + t cells ( . %) was seen in hpsc co-transplant group, as compared to islets alone, which was further progressed thereafter. cd t cells dropped ∼ folds on pod . cd + / cd + ratio was increased from . at the early pod to . in the long-term survival grafts. adoptive transfer of cfse-labeled des transgenic t cells was used to track the response and fate of antigen-specific cd + t cells. the results showed active division of des + cells within the allograft in both the islet only and hpsc co-transplantation groups. however, accumulation of these des + cells was significantly lesser in the hpsc co-transplantation group as compared to that in the islet only group ( . × vs. × cells per graft). these findings suggest that hpsc induce antigen-specific cd + t cell death, and may not inhibit their activation. donor-specific memory t cells are potent mediators of allograft rejection due to their ability to proliferate and give rise to cytotoxic and inflammatory cytokine-secreting effectors within hours of stimulation. furthermore, memory t cells have been shown to be relatively resistant to the effects of many tolerance-induction protocols, including blockade of the cd and cd pathways. while seminal studies have shown that donor-reactive memory cells, generated through pre-sensitization with donor tissue or infection with pathogens with cross-reactive epitopes, contribute to costimulation blockade-resistant rejection of fully mhc disparate allografts, the role of memory t cells specific for minor antigens in costimulation blockade-resistant rejection has not been well studied. we addressed the ability of memory t cells specific for a single donorderived class i epitope to mediate this process. tcr transgenic t cells (ot-i) specific for siinfekl/kb were adoptively transferred into naive b recipients, which were then infected with ovalbumin-(siinfekl) expressing listeria monocytogenes (lm-ova). at memory, mice received a skin graft expressing ovalbumin (mova), and therefore the siinfekl epitope. results showed that the transferred ot-i t cells proliferated in response to lm-ova, but not in response to a control infection with wild-type listeria (lm). at day , ot-i t cells comprised ∼ % of the total cd + t cell compartment. during memory, the siinfekl-specific cells comprised ∼ - % of the total cd + t cell compartment. engraftment of mova skin on lm-ova memory recipients resulted in rejection with accelerated kinetics relative to lm infected controls (mst= d vs d). following treatment with ctla- ig and anti-cd , / lm-ova infected recipients experienced costimulation blockade-resistant rejection, while lm-infected controls went onto long-term graft survival (p< . ). lm-ova-infected recipients also resisted the engraftment of mova-expressing donor bone marrow following a tolerance induction protocol containing busulfan, ctla- ig, and anti-cd . these results suggest that memory t cells specific for a single surrogate minor antigen are sufficient to induce costimulation blockade-resistant rejection, and support the feasibility of using this model to study the specific requirements for donor-reactive memory t cell activation and tolerance induction during transplantation. the during an immune response, cd helper t cells can be instructed by non-antigenspecific signals to differentiate into functionally distinct subsets with mutually exclusive patterns of cytokine production. we find that ikaros, a zinc finger transcription factor required for lymphocyte development, is crucial for the development of polarized t helper subsets. in the absence of ikaros dna binding activity, cd t cells induced to undergo th differentiation in vitro or in vivo produce high levels of il- , but fail to silence expression of ifn-gamma and il- , cytokines that contribute to inflammatory disease processes such as autoimmunity and organ transplant rejection. similarly, ikaros is required for repression of il- and il- expression by th cells, and inhibition of ifn-gamma and il- production by th cells. our results show that ikaros controls the expression of transcription factors such as gata- , c-maf, stat- , and runx , and in polarized th cells, ikaros inhibits ifn-gamma gene expression through direct repression of the t-bet locus. these studies place ikaros, a dna binding protein previously recognized only as a regulator of lymphocyte development, as a master regulator of peripheral t cell differentiation and function. introduction as the fastest growing subpopulation seeking organ transplantation is > yrs of age, it will be imperative to discern how aging impacts the acquisition of transplantation tolerance. prior work has demonstrated that viral infections induce the development of alloreactive t cells, which impede the induction of transplantation tolerance. in this study, we tested the hypothesis that aging alters host defense against viruses leading to the development of cross-reactive t cells, which impair transplantation tolerance induction. we first examined how aging modifies the function of plasmacytoid dcs (pdcs), as ifnα production by pdcs is essential for control of viral infections. using both in vitro and in vivo murine systems, we found that aged pdcs produced lower levels of ifnα in response to tlr activation with cpg sequences or herpes simplex (hsv)- virus (elisa). aged mice ( - mths) failed to the clear this virus as effectively as young ( - mths) mice. this was associated with increased liver inflammation, the release of systemic th -skewing cytokines, il- and il- , and augmented splenic il- levels in aged mice (elisa). prior to transplantation, unmanipulated aged mice (b or cba background) produced more donor-specific il- effector-memory t cells as compared to unmanipulated young mice (elispot). furthermore, mlr assays demonstrated that aged memory cd + t cells from unmanipulated mice produced significantly more il- in response to donor antigen compared to young t cells (elisa). to determine if aged recipients manifest an altered response to therapies that prolong allograft survival, aged and young b mice received balb/c skin allografts and perioperative anti-cd and anti-cd . we found that aged mice rejected their allografts significantly faster (median survival days) than young mice (mst, days, p < . ). similar results were noted when we employed perioperative treatment with anti-cd + dst and when we altered the donor-recipient (b to cba) strain combination. pre-treating aged mice with an anti-il- mab improved the efficacy of the graft-prolonging therapy compared to aged mice that received control mab (p = . ). conclusion our results suggest that impaired control of viral infections with aging leads to the generation of alloreactive il- producing t cells that impair therapies that may induce transplantation tolerance. prevention of type diabetes by thymus genetic modification with protective mhc class ii molecules. jesus r. paez-cortez, michela donnarumma, chaorui tian, john iacomini. transplantation research center, boston, ma. introduction. susceptibility to type diabetes is determined by multiple genetic factors, among the strongest of which is the inheritance of at-risk genes that lead to disease development. here we examined whether diabetes can be prevented by providing protective mhc class ii genes through directly infecting the thymus of diabetes prone nod mice. methods. after direct exposition of the thymus, lentiviruses encoding control (phage-cmv-dsred-ires-zsgreen-w) or protective mhc class ii iaβ d (phage-cmv-iaβ d -ires-zsgreen-w) genes were injected in a single thymic lobe of to week old female euglycemic nod mice. gene expression was determined by microscopic examination at different time points after injection and blood glucose levels were monitored weekly to examine whether this approach prevents the development of diabetes. the presence of diabetogenic cd + t cells were detected by flow cytometry via tetramer staining of splenocytes. pancreatic islet integrity and insulin production was assessed by immunohistochemistry. results. viral gene expression was observed exclusively in thymic epithelial cells beginning at days post-injection in both groups. nod mice injected with control lentivirus developed diabetes by weeks post injection, similar to non-treated animals ( - weeks). in contrast, phage-cmv-iaβ d -ires-zsgreen-w injected animals remained normoglycemic at months post-injection. diabetogenic cd + t cell population were not detected in splenocytes of iaβ d injected animals using mhc class i tetramers. islet integrity and insulin production was preserved in the treated group, in marked contrast to controls, which exhibited characteristic lymphocytic infiltration of islet cells and low insulin storage. conclusions. thymic genetic modification with protective a mhc class ii iaβ d molecule can be used to prevent diabetes in nod mice. central deletion of diabetogenic t cell populations may be involved in prevention of autoimmunity in these animals. role of invariant nkt cells in liver sinusoidal endothelial cell-induced immunosuppression of t cells with indirect allospecificity. masayuki shishida, hideki ohdan, yuka tanaka, masataka banshodani, yuka igarashi, toshimasa asahara. surgery, hiroshima university, hiroshima, japan. we have reported that liver sinusoidal endothelial cells (lsecs) endocytose portally injected allogeneic splenocytes and can negatively regulate t cells with indirect allospecificity via the fas/fasl pathway. as a result of in vitro transmigration across the lsecs from balb/c mice treated with a portal injection (pi) of b mhc class iideficient (c d) splenocytes, the naive balb/c cd + t cells lost their responsiveness to the stimulus of balb/c splenic antigen presenting cells (apcs) that endocytosed the donor-type alloantigens. however, they maintained a normal response to the stimulus of balb/c apcs that endocytosed third-party c h alloantigens. in the present study, we examined whether invariant nkt (inkt) cells influence the ability of lsecs to endocytose irradiated allogeneic cells. balb/c wild-type (wt) mice or balb/c cd d-deficient (cd d -/-) mice that lacked inkt cells were portally injected with × irradiated b c d splenocytes labeled with pkh- . only . ± . % lsecs endocytosed the labeled splenocytes in the balb/c cd d -/mice at h after pi, whereas . ± . % lsecs endocytosed the labeled splenocytes in the wt control mice (p < . , n = each). when balb/c wt mice intraperitoneally received µg α-galcer, before pi of b c d splenocytes, the expression of mhc class ii on lsecs and endocytic activity of lsecs were enhanced. thus, we found that the endocytic activity of lsecs was regulated by the inkt cells. intraportal adoptive transfer of lsecs isolated from balb/c wt mice, treated with a pi of b c d splenocytes, into balb/c mice significantly prolonged the survival of subsequently transplanted heart allografts (n = ) as compared to the adoptive transfer of lsecs isolated from balb/c cd d -/mice, treated similarly, into the balb/c mice (n = ). however, intraportal adoptive transfer of lsecs isolated from balb/c wt mice, which received µg α-galcer intraperitoneally prior to pi of b c d splenocytes, into balb/c mice did not result in a further prolonging of the effect (n = ). these findings indicate that inkt cells are required for such lsec-induced immunosuppression of t cells with indirect allospecificity; however, α-galcer-induced activation of inkt cells does not promote such suppressive effects on these t cells. in conclusion, naive inkt cells play a pivotal role in the lsec-induced immunosuppression of t cells with indirect allospecificity. notwithstanding the considerable amounts of in-vitro data supporting the nonimmunogenicity and immunomodulatory effects of mesenchymal stem cells (msc), scanty and conflicting data are available on their in-vivo immunomodulatory capacities. in this study we formally investigated whether msc had immunomodulatory properties in solid organ transplantation, using a semi-allogeneic heterotopic heart transplant mouse model, and studied the underlying mechanism(s). msc, isolated from bone marrow by adherence, were depleted from cd +cd b+ cells before injection. bone marrow-induced hematopoietic mixed chimerism is the most robust mechanism for the induction of transplantation tolerance. however, immunogenicity of bone marrow cells requires harsh immunosuppressive regimens that can lead to severe sideeffects including death. here, we examined whether embryonic stem (es) cells can be successfully coaxed to form hematopoietic progenitor cells (hpc) which potentially could be less immunogenic than bone marrow cells. here, we transduced es cells with hoxb , a hematopoietic transcription factor that confers self-renewal properties to hematopoietic cells and differentiated them into hematopoietic cells. transduced cells had a - fold greater proliferation capacity than controls. at the end of the differentiation procedure, most cultures were > % cd + . hpcs were purified using immunomagnetic bead separation. the separated cells were further characterized for leukocyte markers and showed a high percentage of cd , cd , cd and low class i, but no class ii expression. further, they poorly express co-stimulatory molecules such as cd and cd . when transplanted in rag -/γ c _/_ mice, hpcs fully reconstituted bone marrow, forming multi-lineage hematopoietic cells. to now determine whether these cells engraft in allogenic recipients, the cells were transplanted in syngeneic and allogenic mrl mice. all transplanted animals became chimeric (n> ), reaching - % after days. thereafter donor cells declined as a result of out-competition by resident bone marrow cells. this pattern was identical in both syngeneic and allogenic recipients. unexpectedly, allogenic chimeric mice became tolerant to donor-type cardiac allografts as monitored over days. grafts showed no mononuclear cell infiltration or signs of chronic rejection. interestingly, the t cells in tolerant animals showed responses to mrl alloantigen similar to that of controls, suggesting that our protocol was likely non-deletional, but could involve regulatory t cells. indeed, when stained for cd + foxp + cells, the allografts showed a high percentage of these cells, but not in controls, confirming our hypothesis. thus, these data show for the first time the potential of es-derived hpcs to regulate engraftment of allografts, providing an alternative approach for the induction of transplantation tolerance. we had previously shown that a is part of the regulatory atheroprotective response of endothelial (ec) and smooth muscle (smc) cells to injury. a is a nf-κb dependent gene with potent anti-inflammatory effects in ec and smc, through blockade of nf-κb. a also serves an anti-proliferative function in smc and opposite anti-apoptotic or pro-apoptotic functions in ec and neointimal smc. based on these functions, a would be a good candidate to prevent transplant arteriosclerosis (ta) and chronic rejection in vascularized organ grafts. this is supported by a expression in ec and smc correlating with the absence of ta in rat kidney allografts and long-term functioning human kidney allografts. fully mismatched c bl/ (h b ) and balb/c (h d ), were used as donors and recipients of an aortic to carotid allograft. in this combination, ta lesions start at weeks and become occlusive by weeks when left without immunosuppression. a expression in the graft was achieved by recombinant adenoviral (rad) mediated gene transfer prior to retrieval. control mice were infused with saline or control rad beta-galactosidase. the grafts were harvested at weeks and analyzed for ta lesions by measuring intima to media ratios (i/m) and for markers of inflammation and of the immune response by immunohistochemistry. a expressing vessels were significantly protected from intimal hyperplasia with i/m reaching . ± . as compared to saline ( . ± . ) and beta-gal ( . ± . ) treated vessels. this effect of a did not associate with a decrease in infiltrating cd , cd or cd t cells in a vessels as compared to controls. a possible modification of the phenotype of these t cells (t-regs vs. effector t cells) is being explored. rather, protection from ta correlated with increased expression of endothelial and inducible nitric oxide synthases (nos) in ec and smc of a expressing vessels, suggesting that this effect was, at least in part, related to increased in situ production of nitric oxide. in conclusion, we present the first direct evidence that expression in the vessel wall of the anti-inflammatory and atheroprotective protein a prevents transplant arteriosclerosis through a mechanism implicating increased expression of nos. carbon monoxide inhalation reverses established chronic allograft nephropathy through the no pathway. g. faleo, a. nakao, j. kohmoto, r. sugimoto, k. tomiyama, a. ikeda, m. a. nalesnik, d. b. stolz, n. murase. thomas e starzl transplantation institute, university of pittsburgh, pittsburgh, pa. chronic allograft nephropathy (can) is the most common cause of graft loss; however an established therapeutic strategy in preventing/treating can is not yet available. we have previously shown that carbon monoxide (co) effectively inhibits can development. here, we examine the mechanisms of co in overturning can through vascular endothelial cell protection. methods: orthotopic kidney transplantation (ktx) was performed in lewis to binephrectomized bn rats under brief tacrolimus ( . mg/kg, d - , im). by d after ktx, bn developed can with decreased creatinine clearance (ccr, . ± . ml/min), significant proteinuria ( . ± . mg/ h), and increased banff scores for intimal arteritis, interstitial fibrosis, and tubular atrophy. recipients were then treated with inhaled co ppm from d to d . results: inhaled co effectively reversed the severity of can and markedly improved renal function at d (table) and recipient survival (> d vs. d air control). co treatment resulted in reduced cytokine mrna levels (tnf-α, ifn-γ) and improved banff scores compared to untreated controls. in untreated allografts, cd expression on peritubular capillaries (ptc) was markedly diminished, while co-treated grafts showed normal cd expression, suggesting significant improvement in maintaining ptc integrity with co. interestingly, enos and inos protein expression was significantly upregulated in untreated grafts, while it was maintained at steady levels in co-treated grafts. immunohistochemistry revealed enos expression on vascular endothelial cells while inos on infiltrates. further, serum nitrate/nitrite levels were significantly higher in untreated than in co-treated recipients. elevated levels of mda, a marker for oxidative stress, in air control group were accordingly reduced in co-treated grafts. renal cortical blood flow data showed a better perfusion in co-treated group at d ( . chronic allograft vasculopathy (cav) is a component of chronic rejection and a major cause of graft loss. non-immunologic factors and indirect allorecognition participate in the pathogenesis of cav. new therapies with tolerogenic dendritic cells (dcs) are based on in situ-delivery of alloag to "quiescent" dcs of the recipient's lymphoid organs via apoptotic cells, vesicles or particles. we have shown that the ability of apoptotic cells to deliver alloag and an inhibitory signal to dcs down-regulates the indirect alloresponse and prolongs allograft survival in mice. aims: to test if targeting of recipient's dcs in situ with donor apoptotic cells ameliorates cav by down-regulating indirect pathway allo-immunity. methods: we performed functional aortic (abdominal) transplantation in mice [balb/c→c bl/ (b )]. b mice were injected i.v. with balb/c uvb-induced early apoptotic splenocytes (d- ). sixty days later, grafts were evaluated in sections with h&e, vangieson's (elastic fibers) and masson's (collagen) techniques. cfse-labeled h . tcrtg cd t-cells specific for ia b (b ) loaded with ieα - (balb/c) were used to evaluate the indirect pathway t-cell response. results: pkh + balb/c apoptotic cells injected (i.v.) in b mice were captured by splenic cd and cd α + dcs, but not plasmacytoid dcs. splenic dcs with apoptotic cells remained quiescent in vivo (mhc-i/ii lo , cd / lo , icosl + , pdl- / + ) and were unable to up-regulate mhc-ii and cd upon culture with gm-csf. injection of donor apoptotic cells induced defective activation and deletion of indirect pathway h . cd t-cells. therapy with donor apoptotic splenocytes reduced intimal thickness in aortic allografts ( ± vs. ± mm in controls; p< . ) and proliferation of α-smooth muscle cells and collagen deposition. the effect of apoptotic cells was allospecific, superior than that of cells alive, and depended on the physical properties of apoptotic cells, since necrotic cells did not achieve the effect. treatment with donor apoptotic cells decreased significantly the indirect pathway t-cell response (assessed by elispot for ifn-γ) and reduced the level of circulating alloab. conclusion: in situ-targeting of recipient's dcs with (early) apoptotic cells carrying donor alloag is a novel approach to prevent cav by down-regulating the indirect pathway alloresponse. the antifibrotic agent, pirfenidone, has direct inhibitory effects on t cell activation, proliferation, and cytokine and chemokine production, leading to suppression of host alloresponses. gary a. visner, fengzhi liu, hanzhong liu, liqing wang, wayne w. hancock. medicine, children's hospital boston, boston, ma; pathology, children's hospital of philadelphia, philadelphia, pa. there is an urgent need to develop new therapies effective against the fibrotic and other complications of chronic allograft rejection. while pirfenidone (pfd) is an established anti-fibrotic agent, we previously showed that pfd treatment reduced acute rejection in a rat lung transplant model suggesting that it might have direct immune modulating properties. accordingly, in this study, we tested the effects of pfd on t cell responses. we first evaluated whether pfd alters t cell proliferation and cytokine release in response to t cell receptor (tcr) activation in vitro. since pfd can inhibit tgf-β by mononuclear cell fractions, we also examined whether pfd affects the suppressive effects of regulatory t cells (cd +cd +). the effects of pfd on alloantigen-induced t cell proliferation in vivo were then assessed by adoptive transfer of cfse-labeled t cells across a parent->f mhc mismatch, as well as by using a murine heterotopic cardiac allograft model (balb/c->c bl/ ). pfd was found to significantly inhibit tcr-stimulated cd + t proliferation in vitro (p< . ), whereas cd + t cell proliferation was not significantly affected. while the beneficial effects of pfd were not associated with increased cd + t cell apoptosis, pfd use inhibited tcr-induced production of multiple cytokines and chemokines, including . interestingly, there was no change on tgf-β production by purified t cells, and pfd also had no effect on the suppressive properties of naturally occurring regulatory t cells. similar to the in vitro studies, pfd inhibited allo-antigen-induced t cell proliferation in vivo (parent->f model), and showed synergistic effects with low dose rapamycin in this model. lastly, though pfd alone did not affect the tempo of acute cardiac allograft rejection across a full mhc mismatch, use of pfd plus a subtherapeutic regimen of rapamycin significantly prolonged allograft survival (p< . ), decreased mononuclear cell infiltration and prevented development to chronic rejection, including arteriosclerosis and myocardial fibrosis. we conclude that pfd may be an important new agent in transplantation, with particular relevance to combating chronic rejection by inhibiting both fibroproliferative and alloimmune responses. we have previously reported studies in miniature swine showing that transplantation (tx) of prevascularized donor islets as part of composite islet-kidney (i-k) reversed diabetic hyperglycemia across fully allogeneic barriers, while free islets did not. in order to test the potential clinical applicability of this strategy, we have extended it to a fully allogeneic nonhuman primate model. methods: two diabetic baboons received composite iks and one diabetic baboon received free islets across fully allogeneic barriers. ( ) i-k preparation in donors: two i-ks were prepared by isolating islets from % partial pancreatectomies and injecting them under the autologous renal capsule, allowing for vascularization before allogeneic tx. these i-ks were harvested at days and for allogeneic ik tx. ( ) induction of insulin-dependent diabetes (iddm) and allogeneic i-k or free islet tx: all recipients received streptozotocin at mg/m x (body surface area). iddm was induced successfully in two animals, while one animal required total pancreatectomy to induce iddm. after confirming iddm by fasting blood sugar (fbs) > mg/dl for consecutive days, either i-ks or free islets were transplanted (single donor to each recipient) with atg (day - ) followed by mmf and low-dose tacrolimus. free islets were injected into the liver through the ileocolic vein. islet function was assessed by fbs and renal function was assessed by serum creatinine. immunologic status was examined by cml/mlr assays. results: all three recipients had strong ctl/mlr responses to donors pretx, indicative of a fully allogeneic combination. fbs decreased immediately after i-k tx and no insulin therapy was required throughout the experimental period (days and ). bs levels averaged . +/- . mg/dl in the first baboon and . +/- . mg/dl in the second. normal creatinine levels (< . mg/dl) were maintained by these life-supporting ik grafts. in contrast, the recipient of allogeneic free islets had unstable bs levels and required insulin from day (bs at day ) conclusions: life-supporting i-ks from single donors achieved glucose regulation without insulin therapy and maintained normal renal function. these results demonstrate the feasibility of composite i-k tx in a non-human primate allogeneic model, with possible clinical applicability for the cure of diabetic nephropathy. donor cell infusion without immunosuppression as a novel therapy for induction of donor-specific tolerance in islet cell transplantation. xunrong luo, kathryn pothoven, derrick mccarthy, matthew degutes, aaron martin, xiaomin zhang, guliang xia, dixon kaufman, stephen miller. medicine, northwestern university; microbiology and immunology, northwestern university; surgery, northwestern university, chicago, il. background in autoimmune models, peptide-pulsed splenic antigen presenting cells that are chemically fixed with ethylcarbodiimide (ecdi) have been used as a powerful and safe method to induce antigen specific t cell tolerance. ecdi fixed donor cells for allo-antigen specific transplant tolerance has not been well studied. material and methods c bl/ mice were rendered diabetic by stz. kidney subcapsular allogeneic islet transplant was performed days after diabetes stabilization (day ). x ecditreated donor splenocytes were injected i.v. either once (day - ) or twice (day - and day + ). animals were analyzed for graft outcome. results control mice receiving islet graft alone rejected the graft between day to day (mst = days, n= ). mice receiving one dose of ecdi-treated donor splenocytes (on day - ) showed similar graft survival as controls (mst = days, n= ). in contrast, mice receiving doses of ecdi-treated donor splenocytes (on day - and day + ) showed significant prolongation of graft survival with . % functional grafts at day (n= ), and some remained functional > days. this protection is donor-specific as mice receiving ecdi-treated sjl cells rejected balb/c islet grafts as controls (mst = days, n= ). immunohistochemistry of protected grafts showed positive insulin staining within well-defined islet architecture. peri-islet infiltrates were composed of cd +, cd +, and cd c+ cells, with occasional foxp + cells. anti-donor antibody production (igg ,g a,b, g ) was completely abolished in long-term graft survivers. this tolerance was undisturbed by anti-cd antibody treatment during maintenance stage, but could not be established if treatment was given around the time of the first donor cell infusion. in addition, lack of pd-l also impaired tolerance induction evidenced by using pd-l -/as recpients. conclusion . multiple infusions of ecdi-treated donor splenocytes significantly prolonged allo-graft survival in the islet transplant model. . the protective effect is donor-specific and is dependent on regulatory t cells as well as the pd-l signaling pathway. therapy with ecdi-treated donor cells may emerge to be a novel and potent agent for induction of donor-specific transplant tolerance. mice. rebecca stokes, k. cheng, c. scott, w. hawthorne, p. o'connell, j. e. gunton. garvan institute, darlinghurst, nsw, australia; nptu, westmead, nsw, australia. the aim was to investigate the effects of increasing hif- α protein in human islets upon islet-transplant outcomes. hif- α is a transcription factor which co-ordinates a program of cellular responses to stressors including hypoxia. in other cell-types hif- α improves survival following hypoxic-challenge. hif- α functions as a heterodimer with arnt which we have shown to be important for normal β-cell function ( ) . islet transplantation subjects islets to hypoxia. it is thought up to % of islets die within week of transplantation and this is at least partly due to hypoxia. the role of hif- α in β-cell function is unknown. we hypothesized that increasing levels of the protective factor hif- α in islets before transplantation would improve survival and engraftment and thus improve islet transplant outcomes. isolated human pancreatic islets from separate donors were cultured overnight in control media, or media supplemented with desferrioxamine (dfo), a small molecular stimulator of hif- α protein. islets were transplanted into diabetic scid mice. there were transplant groups, with mice receiving: .supra-physiological-mass transplant of control-cultured ieq (islet equivalents) .minimal-mass-transplant of control-cultured ieq, or .minimal-mass-transplant of ieq cultured with dfo. for each human donor, at least of each of the transplant groups was performed to avoid the confounder of inter-donor variability. recipients of control ieq cured in % of cases. minimal-mass-transplantation was ineffective: ieq cured % mice at -days. however, minimal-mass-transplant of dfo treated islets had % success (p< . vs group and p=ns vs -control-ieq). blood glucose levels were markedly improved in the dfo treated group compared to ieq control group (p< . ) and equivalent to control ieq transplants (p=ns). this data demonstrates increasing hif- α in human islets prior to transplantation markedly improves islet transplant outcomes. hif- α and dfo may have a therapeutic role in human islet transplantation. long-term disappearance of neovascularization of transplanted islets. eba hathout, nathaniel chan, annie tan, john chrisler, john hough, naoaki sakata, john mace, ricardo peverini, richard chinnock, lawrence sowers, andre obenaus. loma linda university, loma linda, ca. we recently reported an in vivo time-line for neovascularization of transplanted islets using dynamic contrast enhanced (dce) magnetic resonance imaging (mri) over a -day period. however, vascularization of transplanted islets must be maintained for extended periods to provide long-term function. in this dataset, we investigated whether vascularization was maintained in transplanted feridex-labeled syngeneic murine subcapsular islets ( ieq per kidney) using dce imaging on an . t mr scanner and subsequent immunohistochemistry over days. sub-capsular transplants could be visualized at post-transplant days and using t weighted imaging. however, the islets could not be seen on mri at post-transplant day . injection of the contrast agent gadolinium (gd)-dtpa for dce at , and days showed increased signal in the transplant area. at days, there was no change in signal intensity after contrast injection during dce. immunohistochemistry confirmed mri and dce findings. these results suggest that islet neovascularization occurs early after transplantation but is likely not maintained for the -day duration of our experiments. this work was supported by nih/niddk grant # r dk . a) t imaging at day clearly identifies iron-labeled islets (arrows) in the subcapsular region. no iron-labeled islets are observed at day (arrows). b) dce imaging for neovascularization of transplanted islets in the subcapsular region demonstrates a temporal decline in signal intensity. oleanolic acid, a natural triterpenoid, significantly improves islet survival and function following transplantation. n. angaswamy, d. saini, s. ramachandran, n. benshoff, w. liu, n. desai, w. chapman, t. mohanakumar. , surg, wusm; path & immunol, washington univ sch med, st. louis, mo. oleanolic acid (oa), a triterpenoid in medicinal herbs, is an integral part of normal human diet. oa has anti-oxidant, anti-inflammatory properties (inhibits inos & cox ) & lowers plasma glucose levels. we hypothesis that these properties of oa will prevent early islet cell loss following transplantation & also benefit long term function of allograft. c bl/ mice, made diabetic by streptozotocin ( mg/kg) were transplanted with balb/c islets (isolated by collagenase digestion) under kidney capsule. oa ( . mg/day) was administered i.p. in µl of pbs (with m dmso) or pbs-dmso as vehicle control daily from day - onwards. blood glucose was monitored daily. immunohistochemical analyses of grafts were performed for cd & cd markers. cellular immune responses to donor antigens & cytokines produced by cells and in sera were measured using elispot & luminex assays. effect of oa on function of transplant with suboptimal dose of islets ( - ) was also analyzed. optimal dose of islets ( ) transplanted into diabetic bl/ mice administered with oa significantly reduced time taken to reverse diabetes following transplantation (< ± vs ± days, p= . ). further, oa treatment reversed diabetes even with suboptimal dose ( ) of islets while untreated animals did not achieve normoglycemia. as expected, control diabetic mice rejected on ± days whereas, oa administration alone prolonged islet allograft survival to ± days (p< ). oa treatment resulted in > fold increase in serum kc, il- & vegf (p< . ) & fold decrease in mcp- , ip- & il- (p< . ) in luminex assay. stimulation of splenocytes from oa treated mice with donor balb/c cells resulted in significantly reduced ifng ( . fold), il- ( . ), il- ( . ) & il- ( ). in addition, proliferation in mlr was also reduced . fold. immunohistochemical analysis of grafts showed significant reduction in cellular infiltration in oa treated animals with reduction in both cd and cd t cells. daily administration of oa markedly improved islet engraftment & function with reversal of diabetes even when suboptimal dose of islet were transplanted. further, oa treatment allowed significant long term survival of allograft with no other immunosuppression. we demonstrate that prevention of inflammatory signaling cascades by oa resulted in marked reduction of cellular infiltration into graft allowing long term function of allograft. endoplasmic reticulum stress may be an important cause of cell loss after human islet isolation. soon hyang park, michel tremblay, steven paraskevas. surgery, mcgill university health center, montreal, qc, canada; mcgill cancer center, mcgill university, montreal, qc, canada. purpose: to evaluate the presence of endoplasmic reticulum (er) stress, induced by conditions to which islets are subjected during isolation (ischemia, nutrient deprivation, thermal stress and cytokine release) in human islets and to determine if this leads to the unfolded protein response (upr), which could alter cell survival. methods: human islets were purified from cadaveric pancreata by collagenase dissociation and continuous density gradient purification. islet preparations were cultured in serum-free medium and sampled at the end of isolation and daily thereafter. total mrna was purified and gene expression evaluated by rt-pcr. activity in upr signaling pathways was evaluated by immunoblot. apoptosis was measured by a caspase- activity assay. representative trends observed in > isolations are described. results: following isolation, a rapid increase in upr signaling was observed in the perk and ire- modules of the upr. these include the phosphorylation of perk target eif ? and splicing of mrna for the transcription factor xbp- . these changes occurred concurrently with a rapid spike in jnk activity and a rise in expression of the upr target gene chop. after these signals peaked, caspase- activity increased with time (apoptotic cells), as did expression of er chaperone bip (surviving cells). conclusion: we consistently observed upr activation in human islets. er stress and the upr may be one important and unrecognized cause of apoptosis in this context. current investigations focus on upr modification and determination of a causal relationship with apoptotic cell death. immunosuppression and the risk of renal transplant failure due to recurrent glomerulonephritis. atul mulay, carl van walraven, greg knoll. the ottawa hospital, ottawa, on, canada. glomerulonephritis (gn) is the most common cause of end-stage renal disease among those who undergo kidney transplantation. recurrent gn is a major cause of kidney transplant failure. immunosuppressive medication is used to treat gn in the native kidney prior to the development of end-stage renal disease but the impact of different immunosuppression on recurrent gn post-transplantation is unknown. we used the united states renal data system to determine the association of routine post-transplantation immunosuppressant use with time to renal allograft failure due to recurrent gn. immunosuppressants were treated as time-varying covariates. the study-cohort included patients with kidney failure due to gn who received first kidney transplant between and . the study cohort included , patients with a median follow-up of months. ten-year overall graft survival (including death as graft loss) and death-censored graft survival was . % and . % respectively. use of cyclosporine (hazard ratio . ; % ci . - . ), tacrolimus (hazard ratio . ; % ci . - . ), azathioprine (hazard ratio . ; % ci . - . ) or mycophenolate mofetil (hazard ratio . ; % ci . - . ) was not associated with risk of graft failure due to recurrent gn after adjusting for important covariates. there was no difference of recurrent gn causing graft failure between cyclosporine and tacrolimus (p= . ) or between azathioprine and mycophenolate mofetil (p= . ). however, change in any immunosuppressant during follow-up was independently associated with graft loss due to recurrence (hr . , % ci . - . , p= . ).when we restricted the analysis to patients who had no change in immunosuppression during follow-up we again found no association between any of the immunosuppressive medications and the risk of graft loss due to recurrent gn. despite the increased use of tacrolimus, cyclosporine and mycophenolate mofetil to treat gn in native kidney disease, the use of these medications following kidney transplantation had no impact on the risk of graft loss due to recurrent gn. glomerulosclerosis. junichiro sageshima, gaetano ciancio, alessia fornoni, linda chen, carolyn abitbol, jayanthi chandar, warren kupin, giselle guerra, david roth, sherry shariatmadar, gaston zilleruelo, george w. burke iii. university of miami miller school of medicine, miami, fl. background: disease recurrence is a major obstacle of kidney transplant for focal segmental glomerulosclerosis (fsgs). anti-cd antibody (rituximab) has been used for nephrotic syndrome of native kidney. the significant reduction of proteinuria in transplant recipients with fsgs recurrence was also reported after rituximab use for posttransplant lymphoma. we hypothesized that rituximab induction could alter the posttransplant course of fsgs recipients, particularly in those patients with rapid progression to end-stage renal disease who are higher risk of recurrence. methods: we compared the outcome of transplants for primary fsgs treated with and without rituximab. from jan. to dec. received renal allografts along with our "standard" immunosuppressive protocol, consisting of tacrolimus, mycophenolate, corticosteroids, antithymocyte globulin and/or daclizubab. from jan. to dec. received rituximab in addition to the "standard" immunosuppression. posttransplant proteinuria was treated with plasmapheresis (pp) and maintenance angiotensin blockade (ab). results: there was no adverse event related to rituximab infusion. the overall incidence of posttransplant proteinuria was significantly lower in recipients with rituximab induction (p < . ). four recipients treated with "standard" immunosuppression developed massive proteinuria (u-protein/creat. > ) immediately following transplantation; they responded poorly to pp and ab. four other recipients had moderate proteinuria. in contrast to this, of the patients induced with rituximab, only had massive proteinuria and had mild to moderate proteinuria which responded well to pp and ab. with a median follow-up of months, there was no significant difference of graft survival between groups ( -year survival: % without rituximab vs. % with rituximab). a half of the graft loss was related to non-compliance. conclusion: while the mechanism of action is unclear, our observation indicates that rituximab induction may decrease the incidence and severity of recurrence of fsgs following kidney transplantation. a larger-scale study is desirable to confirm this observation. mesangial chimerism in recurrent iga nephropathy. geoffrey talmon, dylan miller. department of pathology and laboratory medicine, mayo clinic, rochester, mn. background iga nephropathy (in) is the most common primary glomerulonephritis and nearly % of patients who undergo a renal transplant for in recur. data support that bone abstracts marrow-derived cells are capable differentiating into various mesenchymal cells within the kidney. the extent to which this phenomenon versus proliferation of resident mesenchymal cells is involved in populating mesangium is not well understood. the mesangial injury and/or hypercellularity seen in in provides a robust in vivo model for determining if this phenomenon is prevalent in human kidneys. design follow-up biopsies from male patients receiving female renal allografts for in that showed recurrent disease were selected. fluorescent in-situ hybridization and immunofluorescent staining was performed on unstained slides from the paraffinembedded tissue for smooth muscle actin, x, and y chromosome centromeres. cells within nonsclerotic glomeruli with triple positivity (y+) were assumed to be mesangial cells derived from the recipient. results four cases of recurrent in with nonsclerotic glomeruli were obtained, each displaying at least minimal mesangial proliferation by light microscopy (one "minimal", two "mild", one "moderate"). mesangial cells with y chromosome centromeric material were observed in each case ( %). between and mesangial cells were present in each glomerulus (mean . ) with no to three y+ cells seen (mean . ). these accounted for between % and % of mesangial cells in individual glomeruli. the ratio of y+ to total mesangial cells in each case ranged from : . . to : . (mean : . ). the case exhibiting minimal mesangial hypercellularity had a ratio of : . , those with mild had ratios of : . and . . , and that with moderate : . . conclusions recipient-derived mesangial cells make up a fraction of the population of glomerular cells in renal allografts affected by recurrent in. although the number of cases is small, the number of recipient-derived cells does not seem to be directly related to the degree of mesangial hypercellularity seen by light microscopy. the consistent presence of these "colonizing" cells in patients with recurrent in does, however, suggest that there may be a role for targeted therapy directed against circulating recipient cells. in the mid 's, patients developing esrd secondary to systemic lupus (sle) were deemed to be poor transplant candidates because of concern for early recurrent lupus nephritis (rln) leading to allograft loss. subsequently, rln was considered an unusual complication of kidney transplantation, occurring in < % of allografts. however, over the last decade, several reports have shown the frequency of rln to range from - %. we sought to determine the frequency of rln at our center and to identify any clinical variables associated with rln. between / between / - / allografts in patients with esrd due to sle functioned for more than days after engraftment. immunosuppression consisted of azathioprine (aza), or cyclosporine (csa) and aza, or mycophenolate (mmf) and csa, or tacrolimus and mmf depending on the date of transplant. all received steroids. proteinuria was defined as + on dipstick or urine protein/creatinine ratio > . . medical charts were reviewed. we found pathologic evidence of rln in ( %) of patients who underwent biopsy due to allograft dysfunction or proteinuria, comprising % of all patients transplanted for sle. characteristics of these patients are shown below: randomized studies have shown little or no increase in ar in kidney tx recips on p-free is; but concern remains about long-term outcome. we present -yr f/u of a protocol incorporating rapid (< days) discontinuation of p, with now over patients transplanted using this protocol. between / between / and / between / , adult tx recips were treated with thymoglobulin ( doses)(extended in dgf), p ( days), a cni, and either mmf or srl. of these, were ld ( lurd); dd. of the , % were female; % white; mean recipient age was ± years and mean donor age was . ± . years. diabetes was present in . % of the recipients and . % of the transplants were retransplants. the peak pra was > % in % of recipients; % had tx pra > %. table shows actuarial survival rates. graft survival rates were significantly better in ld vs dd transplants (p= . ) and and acute rejection rates lower (p= . ). compared to national data from srtr, overall outcomes were not significantly different. with mean follow-up of . years, a total of ( %) recipients have died -the most common cause being cerebrovascular accident ( %) followed by malignancy ( %). there were only ( . %) patient deaths due to cardiac causes. of ( . %) graft losses, ( %) were from dwf (death with function); ( %) from cr/can. renal function has been stable with mean serum cr of . mg/dl at and years posttransplant with cretinine clearance of and respectively. at years posttx, compared to ppretransplant values, recipients showed a . % increase in weight, a % decrease in serum cholesterol, and a . % decrease in serum lipid values. % of the kidney recips remain p-free; the most common reason for restarting p was acute rejection (ar). conclusion: short-term data suggests kidney tx recips do well with rapid discontinuation of p. our intermediate-term data suggests that patient and graft survival rates remain good and renal function remains stable. ongoing long-term follow-up is necessary. background. since / our program has employed a steroid-free, rapamycin and neoral maintenance immunosuppression regimen for kidney transplant recipients. prior to that time recipients were treated with prednisone, mmf and neoral. we noted a significant reduction in acute cellular rejection (acr) after implementing this regimen. this retrospective analysis was performed to examine the impact, if any, on the incidence, character, and outcomes of early ahr. results. this study includes consecutive kidney recipients transplanted between / and / . there were recipients in the prednisone, mmf, and neoral era (grp ) and in the steroid-free, rapamycin and neoral era (grp ). recipient age, gender, african-american race, ab and dr mismatch, and frequency of pra> % was not statistically significantly different between the groups. however, % of grp pts vs % of grp pts received a living donor kidney due to a more recent volume increase in this procedure (p< . ). there were a total kidney biopsies in pts performed in the first months post-transplant; in pts when excluding pre-perfusion biopsies. nineteen percent ( / ) of these showed > % peritubular capillary (ptc) c d deposition. comparison of grp to grp demonstrated the following: ) the incidence of clinical acute rejection in the first months was . % ( / ) in grp and . % ( / ) in grp (p< . ), ) the overall incidence of a c d+ biopsy was similar in the groups ( conclusions. despite a significant reduction in the incidence of acute rejection using our newer, steroid-free immunosuppression protocol, there has been no reduction in the incidence of early ( - months) ahr evidenced by c d+ kidney biopsy. however, the percentage of ahr unassociated with acr has significantly increased. the poor graft survival in pts with early ahr has not improved with our newer immunosuppression regimen. conclusions: four risk factors for ar were identified in the rdp study population: retx, aa race, age - (vs. > ) and pra > (vs. < ). four risk factors for gl were identified: pre-tx t dm, ar, dgf and dd tx (when ar and dgf omitted). these risk factors for ar and gl are the same as we observed in prednisone-containing protocols. additionally, many of these factors are not modifiable. identification of high risk groups allows for individualization of is. increasing lds and utilization of is protocols to decrease or minimize dgf and ar are goals for improving graft outcome. effect with the advent of more potent immunosuppression hla matching has been deemphasized in the allocation of deceased donor kidneys due to the limited impact on acute rejection and graft survival. an unforeseen consequence of poorer matching could be an increase in sensitization of patients in need for a repeat transplant. our study examined candidates listed in the us from - from the srtr database that were re-listed following loss of a primary kidney transplant (n= , ). the primary outcome of the analysis was change in pra from the listing prior to recipient's initial transplant to the subsequent listing. absolute change in peak and current pra levels were examined in general linear models as well as the proportion of patients with a rise in pra level in a logistic model. results hla(a,b,dr)-matching in the primary transplant was strongly associated with change in pra level (p<. , figure ). among recipients with -hla mm, over % had a rise in pra at re-listing as compared to % of -hla mm recipients. younger recipient and donor age, males, deceased donor transplants and african american recipients were also significantly associated with elevation in pra. in addition, the effect was apparent stratified by primary donor type. while there might be a limited impact of hla matching on graft survival, many patients might be negatively impacted from poor hla matching from their first transplant when needing a second transplant. as high pra is one of the strongest risk factors for not getting transplanted, this should be taken into account when evaluating the impact of hla matching in kidney transplantation. this might be particularly important in younger patients and in patients with a long life expectancy in general because of the high likelihood of needing a second transplant during their lifetime. the were about % less likely to die or lose an ecd kidney from a donor aged - and % more likely with kidneys from donors older than . a similar trend was seen with older recipients, but the risks seemed to be lower. logistic regression indicates recipients older than age were times more likely to have a donor older than age than recipients younger than , hypertension and creatinine > . were less likely in older donor kidneys. interestingly, the use of these kidneys relative to the total number of ecd kidneys has decreased since (odds ratio= . , . - . , p< . ). conclusion: an increase in the supply of kidneys might be achieved with increased utilization from deceased donors older than age . outcomes were similar to those from donors age - in recipients that were older than age . outcomes , but approximates non-dcd survival thereafter. dcd listing for retransplantation and graft failure progressed continuously over days versus days in non-dcd. when retransplanted, dcd recipients waited longer and received higher risk allografts (p= . ) more often from another region. more dcd recipients remain waiting for retransplantation with fewer removed for death, clinical deterioration, or improvement. conclusions: dcd utilization is impeded by early outcomes and a temporally different failure pattern that limits access to retransplantation. allocation policy that recognizes these limitations and increases access to retransplantaton is necessary for expansion of this donor population. orthotopic liver transplantation with allografts from dcd donors. roberto c. lopez-solis, background: in the current era of liver transplantation, organ shortage continues to be a significant problem. the use of extended criteria allografts from donation after cardiac death (dcd) donors to increase transplantation rates is widely practiced. this study is a review of one of the largest single center experiences utilizing dcd donors in the world with a follow-up of almost years. methods: from / / to / / , , liver transplants were performed at our institution, ( . %) of which were liver allografts from dcd donors. patient and donor demographics, recipient and graft survival, and the incidence of primary non function, hepatic artery thrombosis, retransplantation, and bile duct complications were analyzed for this subset of recipients. results: kaplan meier analysis showed a -and -year patient and graft survival of % and %, and % and %, respectively. the mean age of recipients was ± years with an average meld score of . ± . (range, to ), and there were male patients ( %). donor mean age was ± years and cold ischemia time was ± minutes. one hundred and three patients ( . %) are alive and ( . %) underwent retransplantation. the incidence of primary non-function was . % ( patients) and hepatic artery thrombosis was . % ( subjects the fda warns against using sirolimus (srl) in liver transplants, reporting increased hepatic artery thrombosis (hat), excess mortality and graft loss when srl is used as initial immunosuppression (is) with calcineurin inhibitors. we report the largest experience to date of patients with srl used as initial is, assessing hepatic artery complications and survival outcomes. materials and method all olt pts from - were reviewed. those using srl as initial is were identified, and the remaining olt pts from that time period were used as controls. ultrasound assessed graft vascular status and any issues were verified by angiogram. . there were no significant difference in demographics variables, lt indication or pre-lt meld score between the two gr. mean follow-up was . ± . months. all the enrolled pts were treated with abstracts an initial dose of cs of mg/kg/day, to target ng/ml for the first days. pts were randomized on day into one of the two following gr on a : basis. ev gr: initial dose of ev was mg/day, to reach blood level of ng/ml. the dose was increased on day , when cs was discontinued, in order to reach an ev blood level between and ng/ml. cs gr: after the th post-operative day the dose of cs was adjusted to a target level of ng/ml until day , then to ng/ml until the end of month . all pts received basiliximab induction on day and after lt. pts were weaned off prednisone by weeks. pt survival at and months was similar in the ev and cs gr ( . % and . % vs . % and . % respectively; p= ns). causes of death were sepsis ( ), hcv recurrence ( ), pulmonary embolism ( ) in the ev gr, and sepsis ( ), rupture of splenic artery aneurysm ( ) the overall incidence of infection episodes was comparable between two gr ( . % ev gr vs . % cs gr; p=ns). cholesterol but not triglycerides increased in the ev gr compared with the cs gr (p<. ); ev dose reduction decreased such parameters without the need for statin implementation. conclusion ev monotherapy in de novo lt showed similar patient survival and incidence of morbidity compared to a cs immunosuppressive protocol. the primary endpoint was achieved inasmuch as renal function was statistically better in the ev gr. background: sir is a potent immunosuppressive agent that inhibits t-cell activation and proliferation. in lt recipients, sir has primarily been used as a renal-sparing agent, but its toxicity and tolerability in this population has not been well defined. aims: to identify the adverse effects and predictors of discontinuation of sir in lt recipients. methods: records from adult lt recipients transplanted between / and / were reviewed. reasons for starting and discontinuing sir were captured, as were all significant adverse effects and laboratory abnormalities. factors predicting sir discontinuation in univariate analysis were further analyzed by multivariable logistic regression (mlr). results: mean age of the study group was ± years, and % were male. underlying liver disease was hcv ± alcohol in %, % had hepatocellular carcinoma, and % received living donor grafts. calcineurin inhibitors (cni) were started post-operatively in % ( % tacrolimus/ % cyclosporine), with or without mycophenolate and prednisone. patients ( %) started sir a median of days (iqr: - ) post-lt primarily for renal insufficiency ( %) or cni neurotoxicity ( %). sir was overlapped with tacrolimus and cyclosporine in % and %, respectively. prior to starting sir, total cholesterol was ± mg/dl, ldl-cholesterol ± , triglycerides ± . peak lipids after sir were ± , ± , and ± mg/dl, respectively, despite lipid-lowering therapy. serum creatinine was . ± . and . ± . mg/dl before and after sir, respectively. before sir, % of patients had no proteinuria, but only % had no proteinuria after sir. high range proteinuria (> mg/dl) was noted in % before and % after sir. finally, sir was discontinued in total of ( %) patients, for indications of cytopenias ( %), hyperlipidemia ( %), mouth ulcers ( %), sepsis ( . %), skin reactions ( . %), nephrotic syndrome ( %), gi intolerance ( %), pneumonitis/boop ( . %), myopathy ( . %), and combinations of above ( %). mlr failed to identify any pretreatment predictors of discontinuation. conclusions: immunosuppression with sir improves azotemia at the expense of considerable hematologic, metabolic, dermatologic, renal, pulmonary and muscle toxicity. considering the high incidence of proteinuria after sir treatment, the use of sir as a less nephrotoxic agent must be re-considered. and to identify the most effective protocol. peripheral blood was obtained from ebvseronegative and ebv-seropositive pediatric heart (h) tx patients. lcl vs dc-based methods were compared as follows: (i) lcl (ii) lcl + il- (iii) type- polarized dc (treated with il -b, tnf-a, il- and ifn-g) loaded with mhc class i-restricted ebv-peptide pool (dc/pep.) and (iv) dc/pep. + il- . the ebv-specific cd + t cell phenotype and function were screened using flow cytometry, ifng elispot and cytotoxicity assays. the yields and the functional activities of in vitro co-cultures differed based on the induction method employed, and on the ebv status of the patients tested. for the ebv-seropositive pediatric htx patients, all four methods resulted in the successful expansion of functional type- ebv-specific cd + t cells, suggesting that memory cd + t cell are readily reactivated in vitro. for the ebv-seronegative pediatric tx patients however, only the lcl + il- approach resulted in significant augmentation of type- ebv-specific ctls that were competent to secrete ifn-g ( ± / cells) and to kill ( ± lu/ cells) ebv + targets. we found that il- secreted by lcl (and not by dc) was critical in triggering expression of il- rb on naive cd + t cells, and rendering these cells responsive to il- p . further addition of exogenous il- p (which is generally not produced by lcl) proved to be essential for effective type- priming. however, blocking il- during ebv-priming has abolished il- rb expression and subsequent ifng production. these results demonstrate that the inducible expression of il- rb on naïve cd + t cells was dependent on il- , and support the critical early role of ebv infected b cells in the in vivo priming of naïve precursors into potent ebv type- cd + t cell in children. serial ebv load monitoring in pediatric heart transplant patients (phtx) has identified a group of asymptomatic children that exhibit persistently high ebv loads in peripheral blood (≥ , copies/ml on at least % samples over a period of at least months). these patients have a high rate of progression to late ptld. our goal is to characterize the deficiency of ebv-specific cd + t-cell immunity that allows this state to occur and be maintained. twenty-one stable ebv + phtx patients were categorized as follows: group (n= ) no detectable viral load; group (n= ) low viral load (≤ , copies/ ml); group (n= ) high viral load (≥ , copies/ml). twelve healthy subjects were recruited as controls. flow cytometric analysis with hla-a ebv-tetramer (tmr) probes in conjunction with mabs against memory/activation markers was performed on peripheral blood cd + t cells, and their ebv-specific ifn-γ production was measured by elispot. ebv-"latent" specific cd + t cells in g patients were mostly cd l + / cd ro + (central memory) and expressed heterogeneous levels of pd- and high cd (il- receptor α), the ebv-lytic-specific cd + t cells were more frequent, and biased toward cd l -/cd ro + (effector memory) and cd l -/cd ra + (stable effector memory), corresponding to terminally differentiated memory compartments. this cell population also expressed heterogeneous levels of pd- and down-regulated cd . in contrast, both ebv-lytic and -latent specific tmr + cd + t cells from g patients were homogeneously cd l + /cd ro + (effector memory), cd + and cd -, suggestive of "recently activated" phenotype. interestingly, although patients in groups g and g had high frequencies of ebv-specific tmr + cd + t cells (g . %± . , g . %± . , p= . ), only g patients exhibited a direct correlation between tmr + cd + t cells and ebv-specific ifn-γ production. these results demonstrate that different levels of chronic ebv-antigenic pressure trigger significant differences in the phenotypic and functional features of ebv-specific cd + t cells from phtx, suggesting that the immunologic characterization of high ebv load carrier state is a combined "activated" phenotype with "exhausted" function of ebv-specific cd + t cells. ebv-encoded lmp indirectly activates the jak/stat pathway through induction of ifnγ. abstracts evidence of ptld. children were enrolled at sites. mean age at transplant . years, mean time to ptld months . organs transplanted were lung , heart , kidney . all ptld were of b cell origin and expressed cd and all were ebv positive. histology was: polymorphic , monomorphic , hodgkins-like . patients received doses, patients doses and patient doses. treatment was associated with minimal side effects in , mild-moderate infusion related reactions in and moderate reaction in . no patient had treatment discontinued because of side effects. twelve patients ( %) showed complete response after - doses, had progressive disease and one had stable disease. at months, ( %) were alive with one graft loss (kidney) and none with residual disease. at latest follow-up (mean . months, - ), remain alive with further graft loss (lung) and no ptld. the deaths occurred between . and months and were associated with progressive disease ( ), chronic rejection ( ), and complications of elective surgery ( ) . conclusions: these findings support prior registry data and suggest that rituximab without chemotherapy is a successful second line treatment in approximately two-thirds of children with refractory ptld. cancer after organ transplantation in france. jean michel rebibou, fabienne pessione, francois aubin, bernard loty. agence de la biomedecine, france. cancer prevention appears as a major challenge in transplantation. estimating cancer frequency is a major step toward designing prevention policy. we report on patterns of cancer incidence among transplantations registered in the french data base. ). the risk of lung cancer is higher for recipients of a thoracic organ and the risk of kidney cancer appeared higher for kidney recipients. ten year cumulative incidence was . % for all cancer and transplantation types, . % for nhl. multivariate analysis demonstrated that cancer risk increased with recipient age (p< - ), year cumulative incidence was % for recipients older than year. it was higher in male (p< - ) and in thoracic organ recipients when compared with kidney recipients (p< - ). cancer incidence did not vary according to the transplantation period ( ( - ( vs ( - . p= . ) and nhl risk was significantly lower during the period - (rr= . , p= . ). this work does not report any increase in cancer incidence among transplant recipients while cancer incidence increased in the general population. the observed decrease of nhl risk is of particular interest. the both costimulatory and co-inhibitory signals are delivered by b ligands through the cd family of receptors on t lymphocytes determining the ultimate immune responses. although b -h , a recently discovered member of the b family, is known to negatively regulate t cell immunity in autoimmunity and cancer, its role in transplantation rejection and tolerance has not been established. to study its role in physiologic rejection processes, we first treated b wt recipients of balb/c hearts with a blocking mab against b -h or isotype igg control and found no difference in graft survival (mst days, n= vs. , n= ). however, b -h blockade resulted in accelerated allograft rejection in cd deficient b recipients (mst . vs. , n= , p= . ) , indicating that b -h signaling can mediate negative regulation in the absence of cd costimulation. we next studied b - /b - double deficient (dko) b recipients of balb/c heart allografts, as these mice are truly independent of cd /ctla- :b signals. while cardiac allografts were accepted in control dko recipients (mst> , n= ), blocking b -h precipitated rejection (mst , n= , p= . ) demonstrating non-redundant functions of these two negative pathways. based on these results, we next evaluated the role of b -h in acquired transplantation tolerance by blocking cd :b using ctla -ig. b -h blockade abrogated prolongation of allograft survival by ctla -ig ( µg on day ) in the fully mhc-mismatched cardiac allograft model (mst vs. . , n= , p= . ) . we conclude that the novel b -h molecule can regulate alloimmune responses independent of an intact cd /ctla- :b costimulatory pathway. the interplay between positive (stimulatory) and negative (regulatory) costimulatory signals is an important determinant of the outcome of the alloimmune response and could be exploited to induce tolerance. specific in a model of mhc-mismatched kidney allograft in the rat, treatment with anti-cd antibodies induced a form of tolerance independent of treg cells but associated with a two-fold accumulation of mdsc in the blood. to further characterize these cells, we analyzed their phenotype and mechanism of action by flow cytometry, western blotting and suppression assays. mdsc expressed cd and cd , nkrp- , cd a (sirpa), cd a, cd b, his and for a fraction of them cd but did not express mhc class ii molecules. mdsc dose-dependently suppressed the proliferation of t cells in mlr and after stimulation with anti-cd + anti-cd antibodies. although detected in blood, bone marrow, spleen and lymph nodes, mdsc were only suppressive in blood and bone marrow. this suppression was lost after physical separation from the responding t cells by semi-permeable membranes in transwell assays, as well as after addition of l-nmma, a selective inhibitor of inducible nitric oxide synthase (inos), suggesting a role for no in the suppression. western blot analyses revealed that inos was expressed only after contact between mdsc and activated cd + cd effector t cells and to a much lesser extent after contact with activated cd + cd high t reg cells. mdsc affected the viability of stimulated cfse-labeled effector t cells by blocking their proliferation but not their activation. in contrast, mdsc did not block the response of t reg cells stimulated by anti-cd + anti-cd antibodies. this selective suppression of effector but not of regulatory t cells was confirmed by cytokine profile analyses. in vivo, the expression of inos was higher in the blood of tolerant recipients, as well as in the graft, as compared with isografted recipients. in addition, the injection in stable tolerant animals of aminoguanidine, which inhibits inos, induced graft rejection within weeks. in conclusion, these results suggest that mdsc, accumulated in the blood of tolerant recipients of kidney allografts, release high levels of no after contact with activated effector t cells and specifically control their proliferative response. liver non-parenchymal components inhibit dendritic cell differentiation and maturation. ching-chun hsieh, horng-ren yang, guoping jiang, john j. fung, shigunag qian, lina lu. immunology and general surgery, cleveland clinic, cleveland, oh. the inherent tolerogenicity of liver allografts could be due to comparatively large numbers of potentially tolerogeneic antigen presenting cells, in particular dendritic cells (dc). it is not clear whether the unique antigen presenting function of liver dc is intrinsic, or is altered by the microenvironmental factors within the liver. in the present study, we investigated the effect of hepatic stallet cells (hpsc), a unique tissue cells in the liver which were actively expanded in the liver allografts, on generation and function of bone marrow derived dc (bm dc). we hypothesize that liver hpsc may modulate immune response via inhibition of liver dendritic cells (dc) which are known of bone marrow (bm) origin. in this study, dc were propagated from b bm cells with gm-csf for days. irradiated hpsc isolated from b mouse liver were added at the beginning into the culture at hpsc : bmdc progenitor ratio of : . the differentiation, maturation and function of propagated dc were determined by characterizing their surface molecule expression and functions of instructing t cell activation / differentiation. the results showed that addition of hpsc markedly blocked the differentiation of dc from bm precursors (most cells remained in cd b + cd cprecursors stages). the incidence of cd c + cells was % vs. . % in normal bm dc culture (without hpsc). the presence of hpsc also prevented maturation of cd c + dc, as evidenced by low expression of cd , cd and cd , but high of b -h . the inhibitory effect appeared to be mediated by soluble factor (s) produced by hpsc, since addition of the hpsc culture supernatant or transwell culture provided comparable inhibitory activity. culture of allogeneic cd + t cells with hpsc-dc elicited poor proliferative response in a d mlr assay, with low il- and ifn-γ production. three color staining of t cells stimulated by hpsc-dc showed that cd + foxp + t cells were preferentially expanded, suggesting that hpsc-dc were capable of inducting treg. in contrast, bm dc induced vigorous cd + t cells proliferation, most of activated cd + t cells were cd + foxp associated with high levels of ifnγ and il- in the culture, indicating induction of th cells. in conclusion, liver tissue cells, such as hpsc markedly inhibit dc differentiation and maturation, suggesting that the tolerogenic property of liver dc may not be intrinsic, but is altered by the microenvironmental factors in the liver. . allograft rejection was also significantly accelerated when bm hearts were transplanted into cd ko recipient (mst days). to investigate the mechanisms of these findings, lymphocytes harvested at day post-tx from spleens and regional lymph nodes were assessed by flow cytometry for phenotypic differentiation of the activation status, regulatory t cell markers and effector cell generation. the ratio of effector to regulatory cells was . : . : . in the wt, cd ko and b dko recipients, respectively. cytokine production was evaluated by elispot using donor specific antigen stimulation of recipient splenocytes. the mean ifn-γ production was spots per , splenocytes in wt, in cd ko and in b dko. this study for the first time demonstrates the paradoxical role of b -cd co-stimulation in fully allogeneic and class ii mismatched grafts and proposes a possible mechanism through the re-alignment of the effector to regulatory t cell ratio in favor of a highly alloreactive immune response. the major concern regarding kidney donation has been whether the occurrence of hyperfiltration, on the background of increasing prevalence of hypertension with aging and the decline in glomerular filtration rate (gfr) noted in some people as they get older, may put donors at a higher risk for progressive kidney disease. we have previously reported on donors > years after donation and found them to incur no excessive risk of hypertension or kidney disease. herein, we report on renal and non-renal outcomes on the world's largest experience of kidney donors to date (n= ) who donated more than years ago. methods: kidney donors were asked to fill out a survey detailing their medical history since donation and obtain a physical exam and laboratory testing by their local physician. results are expressed as mean±standard deviation (sd the graph below depicts the cross-sectional distribution of last serum creatinine available years or more after donation regardless whether the donor is presently alive or not though the majority is from currently alive donors. conclusion: in the longest follow-up of kidney donors to date, this data indicates that - decades of living with one kidney has no serious adverse renal effects and a prevalence of hypertension that is probably similar to the general population considering the age of these donors. centers ) . before and after adjustment for comorbidity, ( . %) and ( . %) centers, respectively, met all criteria for review, but only met criteria for review both before and after comorbidity adjustment. we conclude that failure to adjust for pre-existing recipient comorbidity results in grossly inaccurate estimation of expected gf per ktx center, more often resulting in expected gf that is too low. using data that are not adjusted for comorbidity to judge the quality of tx programs could encourage denial of access to high-risk patients. introduction: the purpose of our study was to examine temporal trends in the regionalization patterns and center volume-outcomes relationship for lung transplantation in the united states over the past decade. a retrospective analysis of all adult single-organ lung transplants included in the scientific registry of transplant recipients for three consecutive time periods between and was performed. for each time period, lung transplant centers were divided into three groups based on each center's annual volume of the procedure (low-volume group = - procedures per year, medium-volume group = - procedures per year, high-volume group = greater than procedures per year). oneyear observed-to-expected patient death ratios were then calculated and compared for each group in each time period. a temporal analysis of the percentage of transplants being performed relative to center volume was also performed. statistical comparisons were made using chi square testing. results: a total of , lung transplant procedures were included in the analysis. in period , there was not a significant difference in the one-year observed-to-expected patient death ratio of low-volume lung transplant centers when compared to high-volume centers (ratio . for low-volume centers vs. . for high-volume centers, p = . ). by period , however, a significant relationship between center volume and outcomes had emerged (ratio . for low-volume centers vs. . for high-volume centers, p = . ). over this same time period, the percentage of lung transplants within the united states that are performed at low-volume centers has decreased significantly (from . % of all lung transplants in period to . % in period , p < . ), while the percentage being performed at high-volume centers has increased significantly (from . % of all lung transplants in period to . % in period , p< . ). conclusions: a significant relationship between center volume and patient outcomes has emerged for lung transplantation over the past decade. at the same time, the percentage of these procedures being performed at high-volume centers has increased. these findings suggest that regionalization patterns for a given procedure may be influenced by the presence or absence of a volume-outcomes relationship for that procedure. liver transplantation (lt) has emerged as one of the few curative treatment modalities for patients with hepatocellular carcinoma (hcc). however, the increase in the incidence of hcc recurrence due to immunosuppressants administered after lt is a serious issue. we have recently proposed a novel strategy of adjuvant immunotherapy for preventing the recurrence of hcc after lt: intravenous administration of il- -stimulated natural killer (nk) cells extracted from donor liver graft to liver transplant recipients. since the immunosuppressive regimen currently used after lt reduces the adaptive immune components but well maintains the innate components of cellular immunity, the augmentation of nk cells response might be a promising immunotherapeutic approach. we confirmed that the il- -stimulated donor liver nk cells exhibited a significantly high level of trail and showed vigorous cytotoxicity against an hcc cell line without cytotoxicity against normal cells. after obtaining approval from the ethical committee of our institute, we successfully applied this therapy to cirrhotic patients with hcc from january . the average number of nk cells that had been administered to lt recipients at days after lt was . ± . × cells/body. the lt recipients were categorized as follows: ( ) based on the milan criteria, recipients met the criteria while did not, and ( ) based on the tnm stage, recipients were categorized as pathological tnm stage i; , stage ii; , stage iii; and , stage iv. in our institute, the -year recurrence-free survival rates of the lt recipients treated with and without this therapy were % and . %, respectively. kinetic studies revealed that in the early postoperative period, the peripheral blood obtained from the treated lt recipients exhibited a significant improvement in cytotoxicity against hcc cell line as compared to the untreated lt recipients (p < . ). furthermore, flow cytometric analyses revealed that the frequency of trail + nk cells increased remarkably in the peripheral blood of the treated lt recipients (p < . ). in conclusion, the administration of il- -stimulated donor liver nk cells contributes to the promotion of host anticancer activity and has the potential to regulate hcc recurrence after lt. abstract# vegf, a well-established angiogenesis factor, is expressed within allografts at high levels in association with acute and chronic rejection. in previous studies, we have reported that vegf possesses potent proinflammatory properties in part via its ability to mediate leukocyte trafficking into allografts. recently, we discovered that vegf mediates cd + and cd + t cell migration via interaction with its receptor kdr (also called vegfr ). blockade of t cell kdr significantly inhibits transendothelial migration. these observations suggest that kdr may be a novel t cell receptor for allogeneic lymphocyte recruitment. here, we first examined the expression of kdr on peripheral human cd + and cd + t cells by facs analysis. we found that ≤ % of circulating t cells express kdr, and its expression was at low levels on individual unactivated t cells. further, we found that the expression of kdr on t cells increased markedly following activation with mitogen and following interactions with activated allogeneic endothelial cells. induced expression of kdr on cd + and cd + t cells was at a similar level as that observed on endothelial cells. therefore, kdr appears to be selectively expressed on t cells, that traffick into allografts. to test the pathophysiological significance of these observations, we analyzed the expression of kdr in a total of cardiac, and renal, human allograft biopsies. we correlated the expression of kdr with cd + t cell infiltrates; and by double immunofluorescence staining, we determined co-expression of kdr on individual cd + t cell infiltrates. in cardiac allografts we found that kdr was expressed throughout the endomyocardium, and was most notable on endothelial cells in all biopsies examined. by grid counting of - areas of each biopsy, we found that the mean number of cd + t cell infiltrates ranged from to cells/hpf (x mag.). by double staining, we noted that kdr was expressed on ± % (mean±sem) of these cd + t cell infiltrates. similarly, we found that kdr was co-expressed on cd + t cells within renal allografts. while infiltrates were more focal, again ± % (mean±sem) of graft infiltrating t cells expressed kdr. collectively, these observations for the first time identify kdr as a novel receptor on allogeneic t cells. we suggest that intragraft vegf may interact with t cell kdr to facilitate homing and recruitment of allospecific lymphocytes into allografts. interaction of infiltrating cd + t cells and tissue cells in tolerant liver allografts: using tcr transgene approach. guoping jiang, qiwei zhang, horng-ren yang, kathleen brown, john j. fung, lina lu, shiguang qian. immunology and general surgery, cleveland clinic, cleveland, oh. the liver allografts are accepted without requirement of immunosuppression in mice. the underlying mechanisms are not completely understood. we hypothesized that it resulted from an abortive t cell response within the liver due to hyporesponsiveness or apoptosis. to test this, we examined the activation and fate of allo-ag specific cd + t cells following liver transplants (ltx) compared with heart transplants (htx) that were acutely rejected. following transplantation [b (h b )→c h (h k )], cfse labeled cd + t cells ( x ) from des tcr tg mice (h k b specific tcr) were adoptively transferred into recipients. animals were sacrificed two days following des t cell administration for analyses of t cells in the grafts or draining lymph nodes (d-ln). host cd + leukocytes were quickly infiltrated following ltx. among cd population ∼ % were cd + and ∼ % cd + . cd + t cells were further increased thereafter in grafts and d-ln, associated with high inf-g production. cd + t cells in the liver grafts rapidly reduced to % by pod , and to % by pod . however, the incidence of cd + t cells remained high. cfse dilution assay and elispot showed an active division of des + t cells in liver allografts either on pod or . these ag-specific cd + t cells functioned well evidenced by ifn-g production in response to allo-ag. however, compared to htx, the accumulation of des + cells in grafts was significantly lower in ltx [ . % ( x /heart), and . % ( . × /liver)] on pod , and further dropped to . % ( . × /liver) on pod . the expended cohorts of adoptively transferred cells followed by their elimination suggested elimination of ag-specific cd + cells. to examine the role of liver environment, graft cd non-parenchymal cells (npc) were isolated tested fro regulatory effect on des + t cell response. liver allografts showed significantly expansion of cd -npc, which were donor mhc class i + (h b + ), b -h + , trail + and low for cd and cd . these cells did not inhibit des + t cell proliferation in response to b spleen stimulation, but significantly enhanced their death, which was dependent on b -h /trail. this was confirmed by using cd -npc from b h -/or trail -/mice. in conclusion, activated t cells in liver grafts may stimulate tissue cells to express inhibitory and death inducing molecules, resulting in t cell death and graft acceptance. foxp + graft-infiltrating lymphocytes (gil) have been detected in the rejecting allografts of transplant patients. foxp is a marker for regulatory t cells (t reg ). published reports suggest that human foxp can be upregulated following tcr stimulation without induction of regulatory function. to investigate whether foxp + gil during acute rejection are t reg , we analyzed the phenotype of gil harvested from acutely-rejected non-human primate kidney allografts. methods: renal allografts with histologically-confirmed acute rejection were harvested at the time of necropsy from macaques that had undergone experimental transplantation. following digestion of kidney fragments using collagenase, gil were isolated using lymphocyte-separation media and cryopreserved. axillary lymph nodes (ax ln) were also isolated either at the time of necropsy or by biopsy. for seb-stimulated lymphocytes, ax ln cells were stimulated for days in the presence of ng/ml seb for - days. to perform intracellular interferon-gamma (ifng) analysis, cells were stimulated with pma and ionomycin in the presence of brefeldin a for - hours at °c. samples were prepared for flow cytometry by first staining for extracellular antigens. cells were then fixed and permablized (kit from ebiosciences) prior to intracellular staining for foxp , ki and ifng. results: the percentage of foxp + in cd + gil was similar to that found in ax ln ( table ). the majority of these cells were cd + . while % of the cd + /cd + /foxp + population of both ax ln and gil were cd + , a significantly higher frequency of cd + and ki + cells were found in gil (p < . ; student's t-test). simlar levels of cd and ki expression were found in seb stimulated lymphocytes. unlike the seb-stimulated lymphocytes, few ifng -producing cells were demonstrated following pma/ionomycin stimulation of gil. conclusion: our current data indicates that the majority of foxp + gil from acutely rejecting renal allografts are recently activated cd t cells that lack effector function. this suggests that foxp t cells within rejecting allografts may indeed be t reg . findings in mouse models of transplantation often fail to translate well in humans. three variables may account for the discrepancy: ( ) evolutionary divergence between mice and humans, ( ) influence of infection history on alloimmunity, and ( ) use of highly inbred strains of laboratory mice. here, we investigated whether the use of inbred mouse strains skews the rejection phenotypes and their response to treatment due to decreased genetic diversity and/or fixation of undesirable genetic loci, known as inbreeding depression. we examined heterotopic cardiac allograft survival in outbred and inbred mouse populations in the presence or absence of immunosuppression. in the absence of immunosuppression, heart transplantation within or between outbred stocks of mice (n = ) resulted in three distinct rejection phenotypes that resemble accelerated ( - days), acute ( - days), and chronic rejection (> days), respectively. in contrast, all fully allogeneic grafts transplanted between inbred mice (n = ) were rejected acutely ( - days) as were historical controls (n > ). the accelerated phenotype, present in % of outbred to outbred transplantations, was characterized by extensive hemorrhagic necrosis of the heart with thrombosis, neutrophil margination and neutrophilic arteritis, and did not correlate with donor:recipient mhc ii disparity. immunosuppression with t cell costimulation blockade did not prevent accelerated rejection (incidence = % in the treated group, n = ) but did convert the acute rejection phenotype into longterm allograft survival in all groups studied. the same accelerated phenotype was observed if transplantation was performed from outbred to inbred mice (incidence = %; n = ) but could not be duplicated if inbred to outbred transplantation was performed (n = ). finally, c depletion with cobra venom factor abrogated the accelerated rejection phenotype in outbred to outbred transplantations (n = ), suggesting a role for complement in the pathogenesis of this phenotype. in summary, our data ( ) indicate that the use of outbred mouse stocks may uncover clinically-relevant rejection phenotypes not observed in inbred mouse strains, and ( ) underscore the importance of the donor background in determining the phenotype of rejection. outbred mouse stocks may provide a platform to uncover mhc-unlinked genetic loci that play an important role in the outcome of solid organ transplantation. th are limited in their ability to reject allografts. elderly recipients represent the most rapidly growing segment of patients on the waiting list. however, little is known about age-dependent alterations of the immune response in organ transplantation. we examined age dependent t-cell functions in a transgenic mouse transplant model. effector t-cell phenotype, -function, cytokine production and regulatory t-cell function were analyzed in and mths old b mice. in an in vivo transplant model, bl/ nude mice were reconstituted with x young or old transgenic alloantigen-specific cd + tcells and engrafted with bm skin grafts. t-cell phenotype and cytokine secretion were sequentially analyzed in all lymphatic compartments. splenocytes of naïve old b mice contained significantly higher frequencies of t-cells with an effector/memory phenotype (cd + cd high cd l low and cd + cd h igh cd l low; p< . ). in vitro proliferation and ifnγ-production were significantly reduced in aged mice indicating an impaired t-cell response with increasing age as assessed by mlr (p< . ) and elispot (p< . ). in parallel, regulatory functions remained age-independent as alloantigen-specific cd + cd + foxp + t-cells isolated from sensitized old mice demonstrated a dose-dependent well preserved suppressor function. next, we tested the age-dependent alloantigen -specific cd + t-cell function in a transgenic skin transplant model: age did not significantly impact rejection kinetics (young vs. old: . vs. . days, n.s.) however, t-cell migration and activation were significantly different: fewer numbers of activated cd + cd + and effector/memory phenotype t-cells (cd + cd high cd l low ) were found in recipient spleens (p< . ) and draining lymph nodes (dln) (p< . ) after transfer of old t-cells. chemokine receptor staining revealed less cxcr + and ccr + t-cells in dln following the transfer of old t-cells (total cell numbers x : cxcr + : . ± . vs. . ± . , ccr + : . ± . vs. . ± . , p< . ). this was paralleled by reduced intragraft t-cell infiltration as observed by immunohistochemistry. in summary, native elderly mice showed an increased frequency of effector memory t-cells but an overall impaired t-cell response. regulatory -t-cell function remained preserved. in vivo allospecific cd + t-cell activation and migration was impaired in elderly transplant recipients. the background: the sensitized transplant recipients may undergo an "accelerated" form of rejection, which is mediated by t cell-dependent mechanisms. these patients often experience increased rate of early rejection episodes, which are difficult to control with currently used immunosuppressive agents. methods: in our model of cardiac graft rejection in sensitized recipients, b mice are first challenged with b/c skin, followed - days later by b/c heart transplant (htx). unlike in naive hosts, htx rejection and alloreactive cd activation in this model are cd blockade-resistant. we first performed systemic analysis at the intragraft transcriptional level by microarray to identify disparities in local immune responses in naive vs. sensitized hosts. aiming to improve the efficacy of costimulation blockade in the sensitization settings, we then determined the role of cd t cells in costimulation blockade-resistant alloimmune response by using cd depleting (gk . ) vs. cd blocking (yts ) ab, in conjunction with cd blockade (mr ). results: htx harvested from groups of naïve (day - ), sensitized (day - ), control ig or mr ab treated mice (n= /gr) were subjected to microarray analysis. mr treatment suppressed htx expression of proinflammatory genes (il- β, il- , tnf-α), and t cell-targeted chemokines (rantes, mig, cxcl ) early after htx in naïve, but not sensitized recipients. five groups of sensitized mice treated at the time of htx with: ( ) ). ctl activation was determined by facs phenotyping at day and . the simultaneous blockade of cd costimulation and cd help, but not a single blockade with mr ab or anti-cd ab, was required to inhibit peripheral alloreactive cd activation in sensitized mice. additionally, cd activation in the absence of cd help showed defective cytotoxic molecule profile, with suppressed perforin but upregulated granzyme b expression at the graft site. conclusion: cd blockade-resistant cd activation is critically dependent on cd t cells. this study provides novel immunological basis to study the potential synergy between adjunctive cd and cd targeted therapies to control accelerated graft rejection in sensitized hosts. mediators. g. einecke, l. g. hidalgo, p. f. halloran. department of medicine, university of alberta, edmonton, canada. the hallmark of t cell mediated rejection (tcmr) are interstitial inflammation and tubulitis. the mechanisms of tubulitis and epithelial deterioration during tcmr are unknown. we previously showed that tubulitis in mouse allografts is independent of cytotoxic molecules (gzma/b, prf) and is preceded by molecular changes with loss of epithelial genes, reflecting epithelial dedifferentiation. human tcmr is associated with loss of the same epithelial genes and re-expression of embryonic pathways (wnt, notch). we hypothesized that tcmr is mediated through soluble factors released by effector t cells or macrophages in the interstitium, and that supernatants of effector t cells would simulate these changes in epithelial cultures. we established an in vitro model in which cultured primary human renal epithelial cells are incubated with supernatants from effector t cell/monocyte co-cultures. the transcript changes in this model, analyzed by microarrays, closely simulated those in human and mouse tcmr (fig ), with loss of epithelial transcripts, activation of wnt/ notch pathways, and increased expression of ifng-inducible and injury-related transcripts previously defined in our mouse model. some of these changes were reproduced by incubation of epithelial cells with ifng or tgfb. the in vitro model identified additional epithelial transcript changes not previously identified in vivo (not affected by ifng or ischemic injury, not expressed in t cells, macrophages, b cells, or nk cells). expression of these transcripts (n = ) was highly altered in human tcmr compared to nonrejecting biopsies of human renal allograft biopsies and distinguished tcmr from antibody-mediated rejection in a hierarchical cluster analysis. thus we have established an in vitro model that closely simulates the epithelial events during human tcmr and confirms that these changes are independent of direct contact with inflammatory cells, supporting the hypothesis that interstitial effector t cells mediate allograft deterioration by soluble mediators. together with previous mouse and human data these results provide the first in vitro model of the epithelial consequences of tcmr. objective. c split product deposition to hla antigen-coated microparticles ([c d] flowpra) was previously shown to be a specific marker of c d-positive antibodymediated rejection (amr). the objective of this study was to assess the predictive value of [c d]flowpra reactivity in a cohort of non-biopsied patients with stable graft function during the first year. methods. a total of kidney transplant recipients were enrolled (inclusion criteria: functioning graft at months; prospective collection of sera taken before and at - , , and months after transplantation). included patients were serially screened for humoral panel reactivity applying [igg] and [c d]flowpra screening. results. fifty-four of the included recipients had stable graft function within the first year and were not subjected to diagnostic renal biopsy. in this particular patient group, detection of complement-fixing hla reactivity tended to be less frequent than in the patients with biopsied graft dysfunction (≥ % [c d]flowpra before transplantation: % vs. % of recipients, p= . ; ≥ % [c d]flowpra after transplantation: % vs %, p= . ). in line with our previous results, within the group of biopsied patients, pre-and/or post-tx [c d]flowpra reactivity was tightly associated with the immunohistochemical detection of peritubular capillary c d deposition (p= . ) reflecting ongoing amr. remarkably, in initially stable patients, detectable [c d] flowpra reactivity was not associated with inferior long-term outcomes. within this patient group, recipients with and without (pre-and/or post-transplant) c d-fixing anti-hla reactivity did not differ with respect to yr allograft survival (p= . ), yr serum creatinine levels ( . vs. . mg/dl; p= . ), and proteinuria at yrs ( . vs. . g/ h; p= . ). similar results were obtained for a comparison of [igg]flowpra positive vs. negative subjects. conclusion. our data suggest that a considerable number of patients with initially stable graft function may have excellent long-term graft function despite serologically detectable levels of (complement-fixing) alloreactivity. for these antibody-positive recipients, a potential role of graft accommodation can be speculated. the immunoproteasome subunit beta as a novel peripheral blood and intragraft biomarker of chronic antibody mediated allograft rejection in clinical transplantation. joanna ashton-chess, in an attempt to identify non-invasive biomarkers of specific histological scarring, we compared publicly available gene sets derived from microarray studies of human renal transplant biopsies published in the literature with our own microarray data derived from studies of rat heart allografts. in this way we identified an immunoproteasome subunit (proteasome subunit beta -psmb ) as a potentially interesting candidate. psmb is one of three members of the immunoproteasome that are induced by abstracts interferon gamma. messenger rna profiling in renal transplant biopsies (n = ) with normal histology, interstitial fibrosis and tubular atrophy, calcineurin inhibitor toxicity, transplant glomerulopathy or chronic antibody-mediated rejection (banff ) revealed psmb to be strongly and significantly increased in chronic antibody mediated rejection vs. the other three histological diagnoses. receiver operator characteristic (roc) curve analysis showed that psmb mrna could diagnose chronic antibody-mediated rejection with an auc of . , a sensitivity of . and a specificity of . . moreover, psmb mrna was significantly increased in the pbmc (n = ) of patients with chronic antibody-mediated rejection compared to those with normal histology. roc analyses revealed an impressive auc of . with all patients being correctly classified. similar results were also observed in a rat allograft model where psmb was significantly increased at day post transplantation in both the heart allograft and the pbmc of animals presenting chronic transplant vasculopathy vs. syngeneic grafts. moreover, inhibition of the proteasome by administration of velcade® at . mg/kg every other day for the first days post transplantation significantly and dose-dependently prolonged allograft survival (mst . days in velcade-treated vs. . days in untreated animals).together our data point towards psmb as a blood and intragraft biomarker of chronic antibody-mediated rejection as well as a potential therapeutic target. furthermore, our results suggest that using a threshold of psmb in the blood could help in guiding the decision to biopsy in the clinic. baff monitoring after b-cell depletion therapy for acute renal transplant rejection. valeriya zarkhin, snehal mohile, li li, jonathan martin, minnie sarwal. pediatrics, stanford university, stanford, ca. introduction: the objective of this study was to investigate the interaction between b-cell activation factor of the tnf family (baff) level and circulating b-cell repopulation in pediatric patients with acute kidney transplant rejection treated with the b-cell-depleting agent rituximab. methods: pediatric patients ( - yrs) with biopsy proven b-cell positive ar were treated with steroids and rituximab ( x mg/m /dose/week). all patients were followed up for months. peripheral blood cd cells and donor specific antibodies (dsa) were monitored monthly. serum level of baff was measured by elisa at ar, , , , and months post-ar treatment and correlated with clinical outcomes. results: complete depletion of circulating and intragraft b-cells was observed with rituximab, with improvement in ar grade in all patients. the median time of peripheral b-cells repopulation was months (range - months, fig. a) . no correlation was found between pre-treatment peripheral b-cell number and the b-cell repopulation time (r= . , p= . ). baff levels rose significantly with b-cell depletion with maximum values at months post-treatment ( . fold increase, p= . ) and returned to pre-treatment levels, with b-cell recovery, at months (fig. b) . serum baff levels correlated positively with b-cell depletion > months (r= . , p= . , fig b). a lack of depletion of dsa i, but not dsa ii correlated with higher baff levels (r= . , p= . ). the timing of b-cell repopulation and depletion of dsa i may be dependant on serum baff level. anti-baff treatment may be considered in addition to rituximab or standard immunosuppressive treatment protocols in patients with persistent and/or antibody mediated rejection. the background: the development of donor specific hla antibodies (dsa) post-transplant has been associated with graft failure. we have shown in a longitudinal study that increases in dsa may precede rejection by months. this retrospective analysis evaluates changes in maintenance immunosuppression (mi) and appearance of dsa in stable transplant recipients. methods: sera from stable renal transplant recipients were collected at - month intervals and tested for the presence of dsa. the types and doses of immunosuppression were correlated with the appearance of dsa. two hundred eighty stable renal transplant recipients who received either a deceased or a living donor kidney were monitored post-transplantation for the development of dsa. patients have been followed for to years and had a minimum of serum samples analyzed. all recipients received anti-lymphocyte induction therapy. maintenance immunosuppression (mi) consisted of a calcineurin inhibitor, prednisolone, and mycophenolic acid. hla single antigen beads analyzed in the luminex instrument were used to establish donor specificity of the antibodies. a chart review was undertaken to determine the doses of the mi posttransplantation. all mi was managed by the transplant team and changed according to clinical indications without regard to dsa. results: of the patients monitored developed dsa post-transplantation with a functioning graft. dsa was against hla-class ii antigens in of ( %); class i antigens in of ( %); and against both class i and ii antigens in of ( %). dsa against class ii was against dq in all except one case. in the majority of the recipients the appearance of dsa was preceded with dose reduction of the mi, either calcineurin inhibitor or mycophenolic acid or both. conclusions: our data show that dsa developed predominantly against hla-class ii antigens and that the appearance of dsa was often preceded with reduction of one or more of the mi. this data shows the importance of monitoring dsa with mi decreases in a stable allograft recipient. antibody production and antigen presentation are directly inhibited by mycophenolate mofetil. anat r. tambur, joe leventhal, nancy d. herrera, joshua miller. division of organ transplantation, northwestern university, chicago, il. immunosuppressive medications are primarily designed to target t cell proliferation. mycophenolate mofetil (mmf) exerts its effect by inhibiting de-novo synthesis of guanine, a dna building block. we, and others, have previously shown that mpa (active metabolite of mmf) affects the differentiation of monocytes into dendritic cells (dc). we further demonstrated that cell-surface receptors associated with antigen up-take and antigen-processing and presentation (cd and cd ) are down regulated when cells are matured in the presence of mpa. this phenotype translated into a decreased uptake of alloantigens and reduced stimulation of t cells. we concluded that mmf inhibits also cell functions requiring mrna synthesis. we now present data regarding the role of mpa in maturation and function of b-lineage cells. pbmcs from subjects were cultured in the presence of cpg, il- , il- and cd l for days to induce memory b and plasma cell maturation in-vitro. cultures were performed in the presence or absence of mpa ( ugr/ul) for the length of the incubation period. in-vitro stimulation of b cells increased the memory population (cd + cd +) from . +/- . % to +/- %. similarly, plasma cells were increased from . +/- . % to . +/- . %. the addition of mpa to the culture inhibited stimulation for both memory and plasma cells ( . +/- % p= . ; and . +/- . % p=ns, respectively). we have further analyzed the effects of mpa on antibody secretion using an elisa (measuring soluble antibodies) as well as a b-cell elispot assay (assessing the number of b cells that produce antibodies). while an expected increase in od values was observed for stimulated samples compared with non-stimulated samples, a significant decrease was observed when stimulation occurred in the presence of mpa. nonstimulated cells: . +/- . ; stimulated cells: . +/- . ; mpa treated stimulated cells: . +/- . ; p< . ). the number of antibody producing cells was also significantly lowered when cultures were done in the presence of mpa (a mean of cells were counted for the stimulated cells compared with - cells for non-stimulated and mpa-treated-stimulated cells). to our knowledge this is the first time where in-vitro experimental data document the inhibitory effect of mpa on b lineage cells and antibody secretion, although clinically known for some time. these results confirm our previous observations regarding the effects of mmf on non-proliferating immune cells. a non-allogeneic stimulus triggers the production of de novo hla and mica antibodies. luis e. morales-buenrostro, , lluvia a. marino-vazquez, anh nguyen, paul i. terasaki, josefina alberu. nephrology and transplantation., instituto nacional de ciencias medicas y nutricion salvador zubiran, mexico city, df, mexico; terasaki foundation laboratory, los angeles, ca; one lambda inc., canoga park, ca. background: in a previous study, we found that healthy people developed hla abs after immunization against hepatitis b virus. the aim of this prospective study was to establish if stimulation with influenza vaccine is capable of triggering the production of hla and mica abs. methods: we determined the presence of hla and mica abs (de novo and preformed abs) in groups of patients vaccinated against influenza: a) healthy adults, b) esrd patients, and c) tr. additionally, we followed healthy unvaccinated people without exposure to sensitizing factor: d) control group. sera samples were collected at baseline (pre influenza shot), at week, and monthly up months after immunization. hla abs were assessed with labscreen single antigen beads for luminex. all samples of each patient were tested simultaneously. a luminescence value higher than was considered positive only if it was times the baseline value. we analyzed the data using chi square, one way anova test, and logistic regression. results: the table shows the types of abs in each group. interestingly, we found preformed abs across all four groups, including the control group (which is free of any known sensitizing factors). the proportion of de novo abs was higher in the group b and c. multivariate analysis shows that the only independent factor associated with development of de novo abs was the presence of preformed abs. we observed a nonspecific immunologic response triggered by external stimulus and was not necessarily associated to the vaccine in people previously sensitized. a introduction. decisions about the minimization and ultimate withdrawal of immunosuppression (is) would be facilitated by the identification of biomarkers associated with operational tolerance (ot). methods. as part of an itn/nih supported study tolerant kidney transplant recipients (off all is for > yr with stable function, n= ) were compared to recipients with stable function on is (sis, n= ), recipients with chronic allograft nephropathy (can, n= ), and healthy volunteers (hv, n= ). pbmc, whole blood total rna, and urine samples from each group were examined using flow cytometry, microarrays, and rt-pcr respectively. results. analysis of microarrays revealed significantly higher expression of b cell differentiation genes in tolerant recipients compared to the sis and can groups. consistent with this finding, tolerant recipients also displayed higher numbers of naïve b cells in peripheral blood and increased expression of cd in urine relative to the sis and can groups. no differences in treg or genes associated with regulatory cells were observed in tolerant recipients relative to other groups. these analyses failed to demonstrate significant differences between tolerant recipients and hvs although support vector machine learning methods suggested potential differences in a number of genes including nfat and calcineurin. finally, relative to tolerant patients, those with can showed decreased numbers of t and nk cells and expressed lower levels of genes associated with immune cell activation in peripheral blood. conclusions. differences in b cell numbers may be useful in identifying tolerant renal transplant recipients or those predisposed to developing tolerance and could potentially provide insights into the mechanisms of tolerance. erythrocyte development of antibodies (abs) to mismatched donor hla antigens has been associated with acute and chronic rejection. complement activation, and c d deposition, has been correlated with humoral rejection of allografts. however, utility of c d staining in ltx has been controversial. a recent study (arthritis rheum. nov; ( ) : - ) shows a strong correlation between erythrocyte bound c d (e-c d) in diagnosis and monitoring of sle. goal of our study is to determine the utility of measuring e-c d in the diagnosis of humoral rejection following human ltx. ltx recipients were analyzed post-ltx for e-c d using facs of rbcs incubated with anti-c d (quidel) followed by fitc-goat anti-mouse. normals were also analyzed. the serum was analyzed for development of anti-hla abs by solid phase assays and for the presence of autoabs to kα tubulin and collagenv (elisa). biopsies from patients were stained immunohistochemically for c d deposition. summary of results are presented in table . infection= / ar= / % e-c d in normals- . %; mfi in normals- . ; mfi=mean fluorescence intensity; ar=acute rejection; dsa=donor specific abs out of patients show significant increase in the % bound e-c d (p< . ) as compared to controls. / had anti-hla and / had autoabs which were significantly different from those with low e-c d (p= . ). staining of the biopsies showed c d deposition in recipients with increased e-c d. all patients with acute humoral rejection had elevated e-c d. we conclude that there is a significant correlation between increase in % e-c d in ltx recipients and development of abs to either hla antigens or auto-antigens during the post-ltx period. biopsies from patients with increased e-c d showed deposition of c d in the allografts. preliminary data suggest that measurement of e-c d using a non-invasive method of flow cytometry may be of value in monitoring ltx patients for humoral rejection. innate immunity: chemokines, cytokines innate immunity is emerging as an important initiator and modulator of the adaptive immune response. in the setting of transplantation, ischemia-reperfusion injury and tissue trauma appear to potentiate the alloimmune response. one of the mechanisms through which the innate immune system modulates an adaptive immune response is dendritic cells (dc). in this study, we examined dc activation in a mouse model of skin transplantation by monitoring the expression of mhc ii and p chain of the proinflammatory cytokine il- . the p expression was detected in live cells using the yet reporter mouse, in which a transgene for yellow fluorescent protein (yfp) was placed downstream of the endogenous il- p gene, thus faithfully "reporting" p expression. skins from c bl/ or balb/c donors were grafted on the dorsal thorax of c bl/ .yet mice. draining and non-draining lymph nodes (ln) were harvested at hours and examined for yfp expression by fluorescent microscopy. a marked increase in the numbers of yfp-expressing dc was observed in draining ln in both syngeneic and allogeneic graft recipients. surprisingly, yfp-expressing dc also increased in nondraining ln when compared to non-transplanted controls. similarly, dc in draining and non-draining ln showed higher mhc ii expression than those in non-transplanted controls. upregulation of mhc ii was highest at hours and decreased significantly by - hours. to assess whether dc activation in non-draining ln was functionally significant, we monitored the activation of adoptively transferred ovalbumin-specific ot-ii tcr transgenic t cells in response to footpad antigen challenge in mice with or without a syngeneic skin transplant on the contralateral upper thorax. otii t cells in transplanted animals proliferated approximately -to -fold better and a higher percentage of the otii cells produced il- than those from non-transplanted animals. thus, local surgical trauma results in a widespread, time-limited, functional activation of dc that appears to act as a partial adjuvant for t cell responses. together, these data suggest that surgical trauma may incite a systemic barrier to transplantation via the activation of dc and therapeutic interventions that reduce the surgical trauma and dc activation may help to improve survival of transplanted grafts. tolerance of cardiac allografts: studies with cx cr -deficient mice. takaya murayama, katsunori tanaka, takuya ueno, mollie jurewics, guleria indira, fiorina paolo, paez jesus, smith n. rex, sayegh mohamed, reza abdi. transplantation research center, renal division, brigham and women's hospital, harvard medical school, boston, ma; department of pathology, massachusetts general hospital, harvard medical school, boston, ma. although donor/tissue dendritic cells (ddc) have long been known to play a key role in mounting alloimmune responses, however, their generation, trafficking and role in tolerance have not rigorously examined. we have used b .fvb-tg (itgax-dtr/ egfp) mice which has a gfp linked to the cd c promoter. using these mice as the donors of heart allograft transplantation provided us a unique model to study ddc posttransplantation. our trafficking data indicate there is a rapid migration of ddc into spleen ( hours post transplantation) but not lymph nodes and that ddc were unexpectedly detected in the spleen of the recipients long after rejection of heart allografts suggesting ddc could escape from the immunosurveillance of host immune system. our data also show that ddc proliferate in the lymphoid tissue of the recipients and co-express class ii molecule of the recipients. we then show that cx cr pathway regulates generation abstracts of heart tissue dc constitutively. as compared to wt hearts, cx cr -/hearts contain lower number of dc and transplanting cx cr -/donor hearts into wt balb/c mice led to significant prolongation of allograft survival without immunosuppression (mst of vs. days, respectively). increasing cx cr -/heart dc by implanting donors with flt -producing hybridoma cells has restored the time of rejection. unexpectedly, induction of long-term survival with anti-cd blockade (mr ) and ctla- ig (but not low dose rapamycine) was abrogated when cx cr -/hearts were used as donors, with concomitant lesser tregs in the cx cr -/heart allografts as compared to wt. furthermore, co-transplanting hearts from wt and cx cr -/into the same recipient treated with mr resulted in significant prolongation of cx cr -/heart allograft survival. depleting the ddc of heart donors prior to transplantation with diphtheria toxin also worsened markedly chronic rejection in the recipients at day post-transplantation. our data indicate that, in contrast to the widely accepted dogma, the presence of donor dc in graft tissue is not only central to allograft rejection but also is necessary for the induction and maintenance of peripheral tolerance. local c a interaction with c ar on dcs modulates dc function, subsequently up-regulating allospecific t cell responses. qi peng, ke li, steven h. sacks, wuding zhou. mrc centre for transplantation, department of nephrology and transplantation, king's college london, guy's campus, london, united kingdom. the innate system of immunity plays an important role in ischemia-reperfusion injury and allograft rejection. the early stages of inflammatory processes are accompanied by complement activation. one biological consequence of this activation is the release of potent inflammatory anaphylatoxins, c a and c a, which have been reported to regulate a range of inflammatory responses. we previously reported that dcs express c ar and c ar, and c a-c ar interaction has a positive impact on murine bm dcs, in terms of activation phenotype and capacity for ag uptake and allostimulation. however, the role of c a in modulating dc function remains unclear. the aim of this study is to investigate the role of local c ar signalling in modulating murine bm dc function and subsequent regulation of the allospecific t cell response. we first evaluated if c a-c ar interaction could result from local expression of factors. our results showed that c ar mrna was detected in wt dcs at different stage of dc culture by rt-pcr, and c a was detected by elisa in the culture supernatants from different stages of dc culture. we next determined if c a-c ar interaction modulates dc function in allospecific t cell stimulation in vitro and in vivo. we found that bm dcs cultured from c ar-/-mice or treated with c ar antagonist (c ara, w ) exhibited a less activated phenotype (producing significantly less il- and more il- , in response to lps stimulation); both c ar-/-and antagonist-treated dcs (lps stimulated) showed reduced capacity to stimulate naïve alloreactive t cells, as measured by ifn-γ production and thymidine uptake. as regards interaction in vivo, following i.p. administration of the c ara-treated dcs into allogeneic mice for days, ex vivo mixed lymphocyte reaction showed that cd + t cells from those recipients have reduced thymidine uptake, but increased il- production compared to that with untreated dcs. conversely, dcs treated with c ar agonist (c a) exhibited a more activated phenotype (producing more il- and less il- ) and were more potent in allospecific t cell stimulation. our findings demonstrate that murine bm dcs can express c ar and c a can be generated locally; c a-c ar interaction up-regulates murine bmdc activation and their allostimulatory capacity. thus, targeting c a-mediated signal may be able to prevent allograft injury. role of tnfα in early chemokine production and leukocyte infiltration into heart allografts. daisuke ishii, austin d. schenck, robert l. fairchild. immunology, glickman urological and kidney institute., cleveland clinic, cleveland, oh. objectives: the acute phase cytokines il- and tnfα are produced early during inflammatory processes, including wound healing and ischemia/reperfusion. the goal of this study was to investigate the role of these cytokines in the induction of early chemokine production and leukocyte infiltration into heart allografts. methods: c bl/ (h- b), balb/c (h- d), and balb/c.il- -/-mice received vascularized syngeneic or complete mhc mismatched, a/j (h- a), cardiac grafts. grafts were retrieved at different time points and total rna and tissue protein were prepared and analyzed by quantitative rt/pcr and elisa to test expression levels of tnf-α, il- , cxcl /kc, cxcl /mip- , and ccl /mcp- in the grafts. anti-tnfα mab ( ug) was given at the time of transplantation with or without anti-cd mab ( ug on days and ). infiltration of cd +, cd + t cells, neutrophils and macrophages was assessed by flow cytometry and immunohistochemistry. donor-reactive t cell priming to ifn-g producing cells in the recipient spleen was measured by elispot. results: expression of tnfα and il- mrna reached an initial peak at hrs post-transplant and a second peak at - hrs with equivalent levels in both iso-and allo-grafts. the neutrophil and macrophage chemoattractants cxcl /kc, cxcl / mip- and ccl /mcp- reached peak levels at hrs post-transplant in both sets of grafts and then declined to background levels. il- deficiency in the recipient or the cardiac allograft did not prolong allograft survival. in untreated mice, heart allografts were rejected at . ± . days after transplantation. anti-tnfα mab decreased neutrophil and macrophage chemoattractant levels % at hrs post-transplant and subsequent neutrophil, macrophage and cd + cell infiltration into the allografts as well as extended graft survival to . ± . days. anti-tnfα mab also decreased the number of donor-reactive ifn-g producing cd t cells almost % on day post-transplant. whereas anti-cd mab prolonged survival to day , administration of anti-tnfα and anti-cd mab delayed rejection to day and resulted in the long-term (> days) survival of % of the heart allografts. conclusions; these data indicate that anti-tnfα antibodies can delay donorreactive cd t cell priming and leukocyte infiltration into heart allografts rejection. as a conjunctive therapy, tnfa antibodies can promote long-term survival of the allografts. introduction recent evidence indicates that inflammation impairs immune regulation, yet the mechanisms behind this effect are not clear, in particular in regards to transplantation tolerance. in this study, we investigated the role of inflammatory cytokines, il- and tnfα, in transplantation tolerance induction. we first examined the impact of il- + tnfα on in vitro t cell alloimmune responses. t cells that were stimulated by allogeneic apcs in conditioned media harvested from lps-activated dcs proliferated more than apc-stimulated t cells that were cultured in conditioned media derived from non-lps-activated dcs. the ability of cd and cd t cells to respond to allogeneic apcs in the lps-activated conditioned media was significantly impaired by the addition of either anti-il- or anti-tnfα mabs. the addition of both mabs further diminshed t cell proliferation, indicating that il- and tnfα synergize to augment in vitro t cell allostimulation. in support of these findings, we noted reduced t cell proliferation during the mlr when t cells were cultured in conditioned media derived from either il- -/-or tnfα-/-lpsactivated dcs. furthermore, these diminished responses was restored by the addition of recombinant il- or tnfα, respectively. to examine the in vivo implications of these findings, we employed a murine skin allograft model, with recipients that were either b wild type, il- -/-or tnfα-/-. these groups were transplanted with a balb/c skin graft and treated with (or without) perioperative costimulatory blockade (ctla ig and anti-cd ). in the absence of immune modulation, all groups rejected balb/c skin allografts at a similar tempo (< days). in the presence of costimulatory blockade, il- -/-recipients (median survival time, mst = days, p = . ) rejected their allografts at a slower tempo compared to wt (mst = days) or tnfα-/-recipients (mst = days). however, this response in il- -/-recipients was further delayed by administering a tnfα inhibiting mab (mst = days), indicating that synergy between il- and tnfα occurs in vivo and prevents the ability of costimulatory blockade to delay the onset of allograft rejection. conclusions we conclude that synergy between il- and tnfα augments t cell alloimmune responses and impairs the effects of costimulatory blockade to delay allograft rejection. abstract# background: calcineurin inhibitors (cni) are involved in the development of post transplant diabetes mellitus (ptdm). changes in insulin secretion and sensitivity are central mechanisms involved in the development of ptdm. in addition alterations in endothelial function seem to be involved. the present study investigated the effect of cni's on these factors. methods: in a predefined sub-study of a previously published randomized trial it was aimed to compare the effect of cni treatment (n= ) with complete cni-avoidance (n= ) on insulin secretion and sensitivity as well as endothelial function. an oral glucose tolerance test and endothelial function investigation with laser doppler flowmetry was performed in patients, weeks and months following transplantation. results: insulin sensitivity differed already weeks posttransplant and was significantly better after months in patients never treated with cni drugs (p= . ). endothelial function was significantly correlated with insulin sensitivity (n= , r = . , p= . ) at weeks posttransplant, but not after months (p= . ). insulin secretion tended to be higher in cni treated patients both at week and month (p= . ). conclusions: findings in the present study indicate that long-term cni treatment reduce insulin sensitivity which was associated with impaired endothelial function. in response to this peripheral insulin resistance a tendency towards a compensatory increase in insulin secretion was seen. these effects combined may indicate a future risk for premature cardiovascular disease in cni treated renal transplant recipients, but this hypothesis needs further study. group(n) ktx ptx(kptx) ecd re-tx pra(> %) race(aa) low immunologic risk alem ( ) overall pt, ktx, and ptx survival are , , and % at months median follow-up. actuarial survival rates, initial length of stay, delayed graft function, steroid free rates, major infection, and incidence of ptld ( ratg pt) were similar for alem and ratg groups, but treated acute rejection (ar) occurred in ( %) alem pts compared to ( %) ratg pts (p= . ) and biopsy proven rejection (bpar) in ( %) alem pts compared to ( %) ratg pts (p= . ). only alem bpar has occurred after months. total daily mmf doses were similar for alem and ratg groups at months ( ± mg vs ± mg). neupogen use was greater in the alem group, ( %), than in the ratg group ( ( %), p= . ). excluding pak, chronic allograft nephropathy (can) was observed in ( %) alem pts and ( %) ratg pts. (p= . ). conclusions: alem and ratg induction both provide excellent and yr pt, ktx, and ptx survival. alem is associated with lower acute rejection rates and perhaps less can, but requires increased neupogen administration to help maintain mmf dosing. thymoglobulin dosing intensity and density: effects on induction efficacy ruth-ann m. lee, adele h. rike, background: alemtuzumab (campath h) has been used as induction therapy for kidney transplant recipients with acute rejection rates reported by us and others of to % at one year. the histologic type of rejection and the time frame for occurrence after treatment with alemtuzumab have not been well established. this study is a retrospective single center review of acute rejection episodes of kidney transplant recipients treated with alemtuzumab induction with respect to the kinetics and histologic patterns of acute allograft rejection. methods: from / / to / / , kidney transplants were done meeting the inclusion criteria for this review. all patients had negative t and b cell flow cytometric crossmatches and received induction therapy with alemtuzumab mg iv intra-operatively, methylprednisolone - mg during the first hours, and were then maintained on tacrolimus (target level - ) and mycophenolate mofetil without steroids. all episodes of biopsy-proven acute rejection (ar) were reviewed. patients in pre-transplant desensitization protocols or with documented non-adherence with medications were excluded from the analysis. results: a total of of patients ( . %) experienced ar during the study period, with a mean follow up of months (range - months). of the ar episodes, ( %) occurred within the first months post-transplant, ( %) occurred between - months, ( %) occurred between - months, ( %) occurred between - months, and ( %) occurred more than one year post-transplant. of the rejection episodes within the first months post-transplant, / occurred within the first days. histologic analysis showed that / rejection episodes ( %) within the first months included an antibody mediated component ( / within the first days.) in contrast, / rejection episodes ( %) which occurred greater than months post-transplant were antibody mediated. of the patients with antibody mediated rejection, only patients had panel reactive antibody (pra) levels > % at the time of transplant. conclusion: this large experience with alemtuzumab induction therapy with a steroidfree maintenance protocol, demonstrates that the majority of rejection episodes occur within the first months post-transplant, with the largest fraction in the first months. a significant number of early rejection episodes are antibody mediated and occur in unsensitized recipients. in a randomized, international, multicenter study, comparing the use of thymoglobulin (tmg) and basiliximab (bas) in recipients at high risk for delayed graft function (dgf) or rejection, tmg was associated with less acute rejection ( . % vs . %, p= . ) and a lower triple endpoint (rejection, death or graft loss, . % tmg vs . % bas, p= . ) but not a significantly lower quadruple endpoint including dgf. the purpose of this study was to compare the efficacy of tmg and bas for induction stratified by donor source: standard criteria donor (scd), extended criteria donor (ecd) or donor with hypertension (htn). methods: retrospective review of data collected in the original randomized trial. data-capture limitations necessitated defining ecd as donor age > or donor age between and with both a donor history of htn and donor renal insufficiency (history of atn or creatinine above . mg/dl during the hours prior to organ recovery/start of cold ischemia time). results: recipients received ecd kidneys [tmg n= ( . %), bas n= ( . %), p=ns]. outcomes are presented below. there were no differences in the rates of dgf between the groups examined. conclusion: standard and non-htn donor recipients had a tremendous benefit of tmg compared to bas with less acute rejection and death. contrary to the perceived niche of tmg in ecd recipients, tmg has its most beneficial effect in scd recipients and recipients of donors without htn at risk for acute rejection or dgf. evaluation we have changed our immnosuppressive protocol in abo-incompatible kidney transplantations and attempted to determine whether the changes in agents have resulted in better outcomes. we used tacrolimus(fk), mycophenolate mofetil(mmf) and methylprednisolone(mp) in immunologically high risk patients between and . moreover, we performed splenectomy at the time of the transplant surgery in patients (group ) with aboincompatibilities between ansd , and administered rituximab as an alternative to splenectomy in patients(group ) with abo-incompatibilities between and . in this study, we compared the graft survival rates as well as the incidence of acute rejection in these two treatment eras. the graft survival rate at one year was % in group and % in group (p=ns). the graft was lost in one case of the cases of group due to insufficient doses of the immunosuppressive drugs. the incidence rate of acute rejection was % ( / ) in group , and % ( / ) in group (p< . ). there were no significant differences in serum creatinine level one year after transplanatation between two groups( . ± . in group vs. . ± . mg/dl in group ). no serious adverse events associated with rituximab or splenectomty were encountered in either groups. in abo-incomaptible kidney transplantation, rituximab under fk/mmf combination as an alternative to splenectomy seems to yield an excellent result in terms of the incidence rate of acute rejection. introduction: the choice of immunosuppression in the elderly kidney transplant recipient remains unclear. the objective of this study was to compare outcomes with different t cell-depleting induction agents in the elderly. method: all solitary kidney transplant recipients over the age of years that received induction therapy with either alemtuzumab or thymoglobulin from to were included in this unos analysis. overall graft survival, the risk of graft loss, and the risk of rejection were compared using kaplan meier, cox proportional hazards, and logistic regression, respectively. results: patients receiving alemtuzumab had a significantly lower -year graft survival ( . %) than patients receiving thymoglobulin ( . %), p= . ) (figure) after adjusting for other risk factors, alemtuzumab had a higher risk of graft loss compared to patients given purpose. the aim of this prospective randomized study was the comparison of efficacy and incidence of adverse events in two induction therapy regimens (atg versus basiliximab) in patients receiving a dual immunosuppression. methods. recipients of first or second deceased donor kidney transplants were prospectively randomized to receive either atg (fresenius) or basiliximab (novartis) as induction therapy. dual immnosuppression consisted of tacrolimus (astellas) and methylprednisolone. cmv prophylaxis was not applied on a regular basis. statistical analysis was performed with fisher's exact or chi-squared test, anova or mann-whitney u test, kaplan meier curves and log-rank test. results. patient characteristics of populations treated with atg versus basiliximab were similar concerning average age ( years), gender and dialysis time prior to transplantation ( vs. months). average donor age and cold ischemia were also comparable ( vs. years and vs. minutes). the actuarial -year patient survival for the atg subpopulation is , % in comparison to % in the basiliximab group (n.s.). analyzing graft survival after years, rates of , % in atg patients compared to % in the basiliximab group can be observed (n.s.). the incidence of acute rejection episodes was similar in both groups (atg: n= vs. basiliximab: n= ). ( %) patients in the atg group and ( , %) in the basiliximab group showed a delayed graft function. serum creatinine was not significantly different at and years (atg: , ± , mg/dl and , ± , mg/dl vs. basiliximab: , ± , mg/dl and , ± , mg/dl). patients in the atg group had a higher rate of cmv infections (n= vs. n= ; p= , ), whereas patients treated with basiliximab had significantly more hematological complications like anaemia, leukopenia and thrombocytopenia. conclusion. comparing induction therapy with atg and basiliximab, our data shows similar patient and graft survival rates with slightly better results in the atg group. patients treated with atg had a higher rate of cmv infections but less hematological complications. predicting cardiovascular events (cvd) and the varied effects of immunosuppressive medication on cvd risk factors requires an understanding of how traditional and nontraditional risk factors impact cvd after kidney transplantation (ktx). single-center studies have generally lacked statistical power and generalizability. registry studies have lacked sufficient data on cvd risk factors. the port project is creating a multicenter international database of ktx recipients with the primary objective of developing risk prediction models for post-transplant cvd. as a preliminary data assessment, an analysis was done on , ktx from - from transplant centers representing european centers, north american centers, and centers from asia/oceania. all data were extracted from preexisting databases at each individual transplant center and processed into the consolidated port database. , major adverse cardiac events (mace) were identified, defined as non-fatal or fatal myocardial infarction, cardiac abstracts arrest, and sudden death. the mace-free survival curves by participating center are shown in the figure. in this preliminary analysis, the overall one-year cumulative incidence of mace was . %, ranging from . % to . % across centers; and the five-year cumulative incidence was . %, ranging from . % to . %. the prevalence of diabetes pre-transplant varied from % to % across centers. in cox proportional hazards models adjusted for age, gender, race, donor type, transplant center, and reported history of diabetes, hypertension (htn), and ami, patients with a reported history of ami had a % increased risk ( %- %, p< . ) for mace. for the final analysis, the definition of mace will be expanded to include major revascularization events. the final port database will be used to develop and validate an equation to predict mace and other health outcomes of interest, after accounting for differential cvd risk factors internationally. abstracts to center. the most commonly used bp medications were beta-blockers, followed by ccbs, and both were used with the same frequency at and months. in contrast, the percentage of patients on an acei or arb more than doubled between and months, suggesting reluctance to use these agents early after tx (a practice not necessarily evidence-based); however, more than % of subjects did not receive acei/arb therapy even at months. fewer than half of all patients received aspirin, including only % with dm and/or cvd. similarly, only half with dm and/or cvd received a statin at and months. these data indicate current management of ktx recipients fails to utilize optimal cvd risk reduction measures in a timely fashion, perhaps missing an opportunity to reduce long-term morbidity and mortality from cvd in this at-risk population. background: cardiovascular (cv) risk reduction has been a primary reason for pursuing early corticosteroid withdrawal (ecswd). to date, actual cardiovascular event data (cve) (rather than cv risk) has not been reported for ecswd. therefore, we analyzed and compared actual cv events (cve) and cv-related survival in ecswd (≤ days) and chronic corticosteroid (ccs) pts. methods: cve and heart failure (hf) data were prospectively collected. cve were defined as sudden death, myocardial infarction, angina, and cerebrovascular accident/ transient ischemic attack. hf events were defined as pulmonary edema or hf diagnosis. conclusions: rtx recipients receiving ecswd experienced: ) fewer cve and ) a trend toward overall better pt survival. these differences in cve and pt survival do not present until at least yrs ptx. and therefore require long term followup to become evident. abstracts immunohistochemistry was performed for cd + and cd + cells. results were compared with those of the patients on maintenance is (gr-is n= ) and the liver tissue from normal subjects (gr-normal n= ). results: the follow-up time in gr-tol was longer than that in gr-is.(gr-tol and gr-is: m and m, p< . ) in gr-tol, typical features of neither acute nor chronic rejection were observed following banff criteria. the extent of graft fibrosis in gr-tol, however, was greater, than those in gr-is and gr-normal (gr-tol, gr-is and gr-normal ; . , . and (ishak's modified staging )(gr-tol vs. gr-is, gr-is vs.gr-normal p< . ). each number of cd + and cd + cells in graft infiltrates was increased in gr-tol, compared with that in gr-normal, but equivalent with that in gr-is (cd + gr-tol, gr-is and gr-normal ; . , . and . cells/field, gr-tol vs.gr-normal p< . , gr-tol vs.gr-is ns / cd + gr-tol, gr-is and gr-normal; . , . and . cells/field gr-tol vs.gr-normal p< . , gr-tol vs.gr-is ns). conclusions+discussion : in tolerant graft after pediatric living-donor ltx, neither acute nor chronic rejection was observed, but fibrosis developed. because of the similar extents of cd + and cd + cells infiltrates and different follow up time between tolerant and immunosuppressed patients, it remains questionable whether fibrosis in tolerant graft is antigen-dependent. serial protocol biopsy before and after starting weaning is will detect fibrosis early, and observing whether reintroduction of maintenance is reverses fibrosis in that case will answer this question. operational tolerance may not always guarantee intact graft morphology. development of "operational tolerance" after pediatric liver background : in the setting of our pediatric living-donor liver transplantation (ltx), % of all the patients (significantly higher proportion, compared with those of other transplant centers) achieved complete withdrawal of immunosuppression (is), which is reffered to as "operational tolerance". nonetheless, some patients encountered rejection while they were undergoing weaning from is. it is,therefore,essential to identify and characterize the differences that will enable patients in these two distinct populations to be distinguished reliably. methods: the study groups consisted of group tolerance(gr-tol) in which patients are successfully weaned off from is, and group rejection (gr-rej) in which patients experienced clinically evident rejection during or after weaning process. the correlation between the clinical outcome (success or failure of weaning is) and following parameters was assessed ; donor/recipient age, donor/recipient gender, abo compatibility, hla mismatch, graft size, early (< month) rejection episode and initial immunosuppression.results: there was no difference between gr-tol and gr-rej with respect to donor/recipient age (gr-tol and gr-rej; y and y ns/ m and m ns), or donor/recipient gender (gr-tol and gr-rej (female); % and % ns/ % and % ns). abo compatibility did not differ between the two groups(gr-tol and gr-rej (i dentical:compatible:incompatible); %: %: %and %: %: % ns).the presence of hla-b mismatch was more frequent in gr-tol than that in gr-rej (gr-tol and gr-rej; % and % p< . ), while the presence of hla-a or dr mismatch did not affect success or failure of weaning is(hla-a gr-tol and gr-rej; % and % ns, hla-dr ; % and % ns). graft size did not differ between the two groups(gbwr gr-tol and gr-rej; . % and . % ns). the patients in gr-rej experienced early rejection more frequently than those in gr-tol(gr-tol and gr-rej; % and % p< . ). mean trough level of tacrolimus within days after ltx was compatible between the two groups (gr-tol and gr-rej; ng/ml and ng/ml ns). conclusions: development of operational tolerance after pediatric ltx was associated with the absence of early rejection and the presence of hla-b mismatch between donors and recipients. auxiliary partial orthotopic liver transplantation (apolt) in children with fulminant hepatic failure. patients with fulminant liver failure (fhf) who undergo auxiliary partial orthotopic liver transplantation (apolt) have a chance to come off immunosuprresion (isp) when the native liver regenerates. it may be most beneficial for children with fhf; however, the literature regarding its use in children has been limited. from the beginning of the pediatric liver transplant program at our institution, patients underwent liver transplantation for fhf. of those, received apolt and the remaining standard liver transplantation (olt). seven children (age months to years) who received apot (apolt group) were compared to matched control group of patients (olt group). since apolt was offered routinely at out instituting since , of apolt cases were done since . in apolt group, either left lateral segment or left lobe graft was used. recipients left lobe was removed in all cases. in olt group, received whole liver graft and received partial liver graft. all native livers showed submassive to massive necrosis at the time of transplant in pathology. all children ( %) in apolt group are currently alive with a median follow up of days (range - days) where ( %) patients are alive in olt group (median follow up days). six of children in apolt group ( %) showed native liver regeneration. first four apolt recipients ( %) are currently off isp with fully regenerated native liver. two of those patients developed complete atrophy of the graft liver, one underwent graft removal due to sepsis caused by severe rejection. one remaining patient who is off isp is displaying progressive atrophy of the transplant liver. incidence of acute rejection was % ( / ) in apolt group vs % ( / ) in olt group. other postoperative complications included hepatic artery thrombosis (hat) (n= ), bile leak (n= ), bilary structure (n= ) and bowel obstruction (n= ) in apolt group, hat (n= ), bile leak (n= ), bowel perforation (n= ), chylothorax (n= ) and aplastic anemia (n= ). median posttransplant length of stay was days in apolt and days in olt group. conclusions: apolt was safely performed in children with fhf. significant proportion of recipients displayed native liver regeneration and came off immunosuppression. natural killer cell dysfunction in pediatric acute liver failure. nada yazigi, greg tiao, alexandra filipovich, john bucuvalas. in pediatric patients, indeterminate acute liver failure (alf) accounts for ∼ % of all cases, and carries a particularly poor prognosis without transplantation. evidence exists to suggest that acute liver failure may reflect a disproportionate immune response to a common stimulus. nk cells comprise a central component of the innate immune system. we hypothesized that nk cell dysfunction (innate or secondary to an antigenic insult) plays a pathologic role in indeterminate alf. we reviewed peripheral nk cell function in a series of consecutive children cared for at cincinnati children's hospital, who met criteria for indeterminate alf as defined by the pediatric alf study group. peripheral blood testing was carried for nk cell number, cytolytic function and perforin and granzyme activity as part of our clinical alf protocol. seven of fifteen patients had nk cell dysfunction. only the severity of cholestasis was statistically higher in the nk cell dysfunction group. there was no statistical difference between the groups with respect to age, inr, or peripheral blood cell counts. of seven patients with nk cell dysfunction died in contrast to none of eight in the normal nk cell group. of the patients with nk cell dysfunction who eventually received a liver transplant, had severe early recurrence of chronic hepatitis in the graft at a year follow up. those outcomes are in sharp contrast to the group with no nk cell dysfunction where patients needed transplantation, but all had no complications both on short or long term follow up. we documented nk cell dysfunction at the time of alf diagnosis in / pediatric patients with indeterminate alf. this subgroup of patients was found to have higher: mortality, risk of infections, as well as recurrent disease in the graft. our findings suggest that nk cell dysfunction is involved in the pathogenesis of indeterminate alf. as such, it could therefore be a prime target for therapeutic intervention, with goals to rescue the patient from liver failure and /or to improve post-transplantation outcomes. background hrs is a reversible renal failure which occurs in pts with advanced liver disease and portal hypertension and is characterized by a marked decrease in gfr and rpf in absence of other identifiable causes. vasodilation theory is currently the most accepted hypothesis to explain the pathogenesis. in decompensated cirrhotics, probability of developing hrs is - %/yr and increases to % at yrs. ideal treatment is ltx. however, there is an urgent need for effective alternative tx to increase surv chances for pts until ltx can be performed. interventions that have shown some promise are vasoconstrictors in splanchnic circulation and tips. main objective was to compare efficacy of two different regimens (albumin/terlipressin resp hes/terlipressin both w/ wo midotrine) against tips whereas grf was considered as primary efficacy endpoint. pts/tx dx of hrs was based on criteria, as proposed by international ascites club. only pts with esld on the waiting list for ltx were eligible to be enrolled. pts were assigned to tx arms and randomized w/wo midotrine. volume/vasoconstrictor tx lasted for d, mitodrine was continued; follow up for d. results iia (albumin/terlipressin); iib (hes/terlipressin); iic (tips) discussion combination of volume expansion/vasoconstriction improved effectively gfr in pts with hrs. use of albumin shows no advantage compared to (cost effective) hes. although a marked improvement was observed during iv-treatment, renal fct deteriorated upon treatment withdrawal whereas pts with continued mitodrine showed superior long term outcome. we analyze our single institution experience to quantify the long-term incidence of renal failure based on month gfr compared to subsequent determinations. methods: this is an irb approved retrospective review of the prospectively maintained database of lt recipients. exclusion: patients on renal replacement therapy (rrt) at time of transplant, combined liver kidney, fulminant hepatic failure, and < -year follow-up. gfrs (i iothalamate glofil method) were measured at initial evaluation (ie), month (m ), year (y ), (y ), (y ), (y ), and (y ). patients were grouped by gfr > (g ), - (g ), and < (g ). ie and m gfr were used as starting points for longitudinal analyses. paired data analysis for ie, m , y and y was also performed. renal failure was defined as gfr < , received kidney transplant, on dialysis, or on kidney transplant list. results: liver transplant patients were reviewed between and . paired glofil data was available for the y and y analysis in patients. m gfr correlated more with long-term renal function (p< . ). g demonstrated largest reductions in gfr over time. g and g , when corrected for patients that got kidney transplantation and rrt, demonstrated progressive reduction in gfr. g , g , and g were statistically significant (p< . wilcoxon two-sample test). this study clearly demonstrates progressive decline in gfr continuing out to years after liver transplantation. m gfr correlates better with long-term renal function compared to ie gfr (not truly reflective of renal function at time of lt). if m gfr < , data showed a high rate of renal failure in our paired data analysis by y (p< . ). by correcting for patients with renal failure, the previously reported stability in gfr between y and y is not seen. analysis of grouping demonstrates that g patients at m have lower incidence of renal failure > years after lt. g and g patients will be at higher risk for renal failure each year after transplantation. introduction: calcineurin inhibitors have demonstrated efficacy in liver transplantation. however, they have a potential to impair renal function. delayed tacrolimus (tac) administration may reduce the risk of renal dysfunction. methods: a prospective study included liver transplant pts randomised to delayed introduction of tac (day ) + daclizumab (dac) (group a) or to immediate tac administration (group b). in both groups tac t was - ng/ml until week and - ng/ml thereafter. mmf was given at g/d for months, and corticosteroids (cs) at standard doses. pts with a serum creatinine (scr) > µmol/l at hours (h ) were excluded. the primary endpoint was the rate of pts with a mean scr > mmol/l at month . month results are presented. results: pts were randomised. baseline characteristics were similar. at month , mean tac t was . (group a) and . ng/ml (group b median follow-up post-tx was years ( - ). most frequent tx indications were alcoholic ( %) and hcv ( %) cirrhosis. amdrd glomerular filtration rate (gfr) was < ml/min/ , m in % (bt), % ( m), % ( y) and % ( y) of the patients. changes in gfr were then compared according to the immunosuppressive protocol: -group "cni+mmf" = a calcineurin inhibitor (cni) + mycophenolate mofetil (mmf). -group "cni" = a cni without mmf. in this group, some patients received only cni and some cni + azathioprine. there was no difference between those sub-groups, neither on rf nor on cni doses. all those patients were thus pooled. in both groups, gfr decreased from bt: - % in "cni+mmf" vs - % in "cni" at m (p= . ), - % vs - % at y (p= . ), and - % vs - % at y (p= . ). although their mean gfr bt was lower ( vs ml/min/ . m , p= . ), the decrease in rf in "cni+mmf" patients was less severe. nearly % of the patients had renal insufficiency in the years following liver tx. the reduction in the gfr is less pronounced in patients treated with mmf even if they were significantly more at risk bt. except at m, there was no difference in cni doses between the groups, suggesting that the sustained lower decrease in rf observed in "cni+mmf" may not be only explained by a cni dose reduction. acute rejection is a complex biologic process involving multiple cell types, cytokines and chemokines/chemokine receptors. we hypothesized that an mrna panel that included genes implicated in the anti-allograft response would distinguish allografts undergoing acute rejection from normal allografts with a high degree of accuracy. we tested this hypothesis by measuring levels of urinary cell mrna and peripheral blood cell mrna for cell surface proteins cd , cd , cd , cd , and ctla ; chemokines/ chemokine receptors ip , mig, cxcr ; cytotoxic attack molecules granzyme b(gb) and perforin, and immunoregulators foxp , tgf-beta and il- . gene specific primer pairs and probes were used in pre-amplification enhanced real time quantitative pcr assays to measure mrna and transcripts for s rrna. for each cell source, we used logistic regression to identify a linear function of up to log-transformed measures that would distinguish biopsies of ar patients from those of stable transplant patients. our study demonstrates that molecular signatures developed using urinary cell levels of just genes (signature , urinary cell levels of mrna for ctla , foxp , gb, cd , and mig), or a combination of urinary and blood cell levels (signature , urinary cell level of ctla mrna and peripheral blood cell levels of cd and ctla ) differentiate ar from stable biopsies with % sensitivity and % specificity. blood cell levels alone are also informative, but less so. we conclude that molecular signatures, developed from noninvasively ascertained mrna profiles of urinary cells/ peripheral blood cells, predict acute rejection with extraordinary accuracy. clinical trials to validate the predictive value of these signatures are worthy of pursuit. we have described the association of cd + b cell infiltrates in renal transplant (tx) biopsies (bxs) with acute cellular rejection (acr) and tx dysfunction (dysfx). we have also found metabolically active plasma cells (pcs) staining for s ribosomal protein (s rp) within these txs. herein, we report the significance of cd + pcs in rejection (rj) and evaluate the impact of cd , cd , and s rp on long term tx fx by calculated creatinine clearance (crcl). we studied tx bxs from pediatric (ped) patients (pts) who were bxed for suspicion of rj from nov to nov . pts were given daclizumab and maintained on prednisone, mycophenolate mofetil, tacrolimus or cyclosporine. immunohistochemical staining and quantification for cd , cd , s rp, and c d were performed under x light microscopy. bxs were classified by modified banff criteria. crcl was followed yr post-bx. cd + pcs were associated with c d-negative acr (p= . ) but not antibody mediated rj (amr, p= . ). roc analysis confirmed > cd + cells/hpf strongly associated with acr, yielding % sensitivity, % specificity, correctly classifying % and comprising total roc area . ( % ci . , ). higher cd counts at bx correlated with worse tx fx (fig ) . a univariate regression model showed that cd , cd , s rp and time were associated with a decline in tx fx at bx. multivariate model showed that cd , cd , and time had the main effects on crcl decline, with s rp dropping out. all patients regardless of rj status had a ml/min/ . m crcl decline exerted by time (p= . ). pts with cd had an additional sustained ml/min/ . m crcl decline seen yrs post-bx (p= . ). pts with cd also had ml/min/ . m crcl decline at bx (p= . ), but there was an interaction between time and cd that negated a sustained effect (p= . ). this study identifies a numerical threshold of > cd cells/hpf that is associated with acr and tx dysfx. infiltrating cd cells had the greatest, sustained effect on tx dysfx. we conclude that cells of the b lineage, particularly cd , play a key, but undefined, role in acr. intragraft there is now evidence that foxp + cells are not indicators of tolerance, since foxp is also increased during acute rejection. however, it is unknown whether foxp + cells are present during chronic antibody mediated rejection. moreover, the relative balance of regulatory, effector and cytotoxic pathways in chronic vs. acute injury has yet to be explored. here we addressed this issue. intragraft regulatory, effector and cytotoxic transcriptional profiles were analysed within renal transplant biopsies (n = ) classified (banff ) as displaying normal histology, chronic calcineurin inhibitor toxicity (cnitox), chronic antibody mediated rejection (camr) and acute cellular rejection (acr). granzyme b, tbet and foxp mrna were measured by quantitative pcr and foxp -positive cells were additionally quantified in graft biopsies by immunohistochemistry. distinguishing mrna profiles were analyzed in the peripheral blood (n = ). our data show that foxp mrna is increased not only in acr (p< . ) but also in camr (p< . ). expression of foxp mrna correlated tightly with the density of foxp protein-positive cells by immunohistochemistry (spearman r = . ; p < . ); foxp + cells were found in aggregates and within tubules. moreover, graft cytotoxic, effector and regulatory pathways were all found to be active in chronic as well as acute graft injury. significant increases in granzymze b, tbet and foxp mrna were observed in camr, cni-tox and acr compared to normal histology (p< . , p< . or p< . ). however, differences in the relative contribution of each pathway were evident, with significant accumulation of foxp mrna predominating in acr and granzyme b predominating in camr. thus, camr can be distinguished from both acr and cni-tox by an unfavorable intragraft granzyme b/foxp mrna ratio (p< . ). interestingly, this ratio was reversed in the blood, suggesting different migratory patterns for regulatory and cytotoxic cells between the blood and the graft.our data thus confirm that intragraft and peripheral blood foxp accumulation is also a feature of camr of kidney grafts. moreover, camr can be distinguished from other graft injury types based on its intragraft or blood cytoxicity/regulatory profile. survival of solid organ grafts depends on life long immunosuppression which results in increased rates of infection and malignancy. induction of tolerance to allograft would represent the optimal solution for controlling both chronic rejection and side effects of immunosuppression. we previously showed that operational tolerance after kidney transplantation could occur in some patient. here, the potential of high throughput microarray technology allowed us to study the peripheral blood gene expression profile associated to operational tolerance and chronic rejection in a cohort of human kidney graft recipients (n= ). microarrays were used to compare the gene expression profile of pbmc from patients with chronic rejection and drug-free operationally tolerant recipients. results have been treated using a classical statistical and a non-statistical analysis based on the identification of key leader genes associated respectively to chronic rejection and operational tolerance, either as those mostly changing their expression or having the strongest interconnections. differentially expressed genes were identified between operational tolerant patients and patients with chronic rejection. abstracts defined as missing > % of prescribed doses on mam and mems, and > sd among consecutive blood serum levels. results: participants were transplant patients (m = . + . years old, % male, . % caucasian). on the mam, . % of the patients acknowledged some non-adherence but minimized how many doses they missed. using mems technology, % had some non-adherence and specifically, . % of the participants missed doses and only % of their doses were taken within the allowable time frame. using > sd criteria for blood serum levels, % of the participants were considered non-adherent. non-adherence worsened with years since transplant. more missed (r = . , p = . ) and late doses (r = . , p = . ) on the mam and > sd among blood serum levels (r = . , p = . ) was associated with higher incidence of acute rejections. adherence data was examined for patients with documented acute rejections (n = ). sensitivity and specificity of each detection method was also examined. the mam and sd detection methods each identified non-adherence in % of the patients with acute rejections; mems did not identify any additional non-adherent patients. only % of the patients with acute rejections were identified consistently by all three adherence detection methods; all patients with acute rejections were identified by at least one method. discussion: non-adherence worsening with time since transplant and was associated with acute rejections. since no single method of detecting adherence identified all the patients with acute rejections, multi-method adherence assessments should be used to accurately capture patients who are non-adherent. non na is a leading cause of allograft loss and results from multiple factors. locus of control (loc) and beliefs regarding health have been associated with adherence in other populations. randomly chosen ktr's were interviewed using a confidential questionnaire administered by an outside investigator that included questions regarding loc, health beliefs and self-efficacy. the population was % female, % black, % hispanic, % deceased donor kidney, % diabetic, % greater than high school education, % employed, % married or cohabiting, % income < k per year, % insured by medicaid. mean age . ± . yrs, time on dialysis . ± mos, months since transplant . ± . , total meds . ± . . by pearson correlation, non-adherence (na), defined as "having missed doses of immunosuppression over the preceding months", was not correlated with race, gender, income, type of insurance, age, marital status, type of txp, mos on dialysis, time since transplant, or number of medications. na was correlated with higher education level (r= . , p= . ), current employment (r= . , p= . ), and knowledge of most recent creatinine value (r= . , p= . ). na was associated with concerns regarding prednisone (long term effects, dependency) r= . , p= . , feelings of greater personal control over illness (r= . , p= . ), and inversely correlated with powerful others loc (feeling that one's health is dependent on other people), r=- . , p= . and belief in the necessity of medication for maintenance of transplant health, r=- . , p= . . we conclude, in our population of inner-city patients: . na is not associated with standard demographic factors including income, race and gender. . contrary to findings in other populations, na is associated with higher education and current employment. . na is associated with knowledge about creatinine value, concern regarding long-term effects of prednisone and disbelief in the importance of transplant medications. . na is associated with feelings of personal control and feeling that powerful others (e.g. health care providers) are not of high importance in the outcome of illness. . education programs designed to address na in this population should be targeted towards altering negative beliefs regarding medications and stress the importance of partnering with the transplant team for optimal long-term outcome. purpose: the present study aimed to prospectively examine the relationships among nonadherence, health-related quality of life (hrqol), and family factors in adolescent kidney, liver, and heart transplant recipients. method: adolescent transplant recipients aged to years (m = . , sd = . ; % female; % kidney, % liver, % heart) and their parents participated. at baseline and -month follow-up assessments, adolescents and their parents independently completed phone interviews assessing self-/proxy-reported medication adherence, hrqol, and family cohesion and conflict. medical record reviews were conducted to obtain current medications, immunosuppressant drug assays, and clinical outcomes in the past year (i.e., rejection episodes, hospitalizations, graft loss). results: at baseline, adolescents classified as nonadherent based on self-report and tacrolimus standard deviation (sd) reported significantly lower general health perceptions (f( , ) = . , p < . ), self-esteem (f = . , p < . ), mental health (f = . , p < . ), and behavior hrqol (f = . , p < . ) compared to adolescents classified as adherent. similarly, parents of adolescents classified as nonadherent reported significantly lower physical functioning (f = . , p < . ), self-esteem (f = . , p < . ), and behavior hrqol (f = . , p < . ) for their adolescents. family conflict was correlated with adolescent report of behavior (r = -. , p < . ), physical functioning (r = -. , p < . ), self-esteem (r = -. , p < . ), and mental health hrqol (r = -. , p < . ). family conflict was correlated with parent report of behavior (r = -. , p < . ) and physical functioning (r = . , p < . ). improvement and deterioration in hrqol from baseline to -month follow-up is currently being examined. it is expected that increased family conflict and decreased medication adherence will be associated with deteriorations in hrqol. the interrelationships between medication adherence, family conflict, and hrqol domains such as self-esteem and mental health suggest that interventions targeting these domains may result in improvements in medication adherence behavior. the use of cam in general is associated with non-disclosure by patients to physicians. randomly chosen ktrs were interviewed using a confidential questionnaire administered by an outside investigator, including questions on cam usage, whether it was doctor-recommended, and whether the patient disclosed use. cam was defined as ingestion of herbal or other preparations, use of mind-body techniques or manipulation of the body for healing by someone not an allopathic medical provider. use of vitamins and spirituality were excluded. the population was % female, % black, % hispanic, % deceased donor kidney, % diabetic, % > high school education, % employed, % married or cohabiting, % income < k per year, % insured by medicaid. mean age . ± . yrs, time on dialysis . ± mos, months since transplant . ± . , total meds . ± . . % of patients (n= ) used cam. by pearson r, cam use was correlated with na to immunosuppressants, p= . , r= . , blood sugar-lowering medications, p= . , r= . , and cholesterol lowering medications, p= . , r= . , worries about long-term effects of medicines, p= . , r= . , belief that doctors place too much trust in medication, p= . , r= . , and that natural remedies are safer than medicines, p= . , r= . . cam use was inversely related to belief that health depends on allopathic medicines, p= . , r= - . , medicines protect from worsening disease, p= . , r= - . , and that following doctors orders is the best way to stay healthy, p= . , r= - . , and that having a kidney transplant makes them feel happy, p= . , r= - . . we conclude, in our population of inner-city patients: . use of cam is correlated with medication non-adherence. . patients who use cam are more worried about long term effects of medication, believe that natural remedies are safer than medications and that doctors place too much trust in medication. . patients who use cam do not believe that their health depends on allopathic medication, that medicines protect from worsening disease, or that following a doctor's orders is the best way to maintain optimal health. . patients who use cam are less happy with their kidney transplant. . disussing cam use and motivation for use is important in the transplant clinic and may alert the provider to possible risk for non-adherence. by multivariate cox analysis the risk of tg related to the presence of hla-iiab death censored graft loss occurred in . % of patients without tg and in . % of patients with tg (p< . ) hla-iiab are associated with higher risk of tg and reduced graft survival. furthermore, the risk of tg and its prognosis relate to the level of hla-iiab quantitated in a solid phase assay term survival of cardiac allografts in wild-type mice by alloantigen (alloag)-specific foxp + cd + cd + natural regulatory t (nt reg ) cells. guliang xia, jie he methods: fresh naive cd + cd + nt reg were isolated from congeneic b .pl mice via automacs and enriched for alloag specificity by in vitro culture with either anti-cd / cd -coated dynabeads (d - ), then donor bone marrow-derived dendritic cells % (d+ ) for dc/beads-expanded nt reg , while total fold of expansion of nt reg remained similar ( . ∼ . for beads/dc-or . ∼ . for dc/beads-expansion) regardless of the presence or absence of tgf-β. introducing ra ( nm) into bead/dc-based, tgf-β/ il- -conditioned culture resulted in marginal improvement with . % (d+ ) nt reg being foxp + . in mlr assays, nt reg expanded with tgf-β/il- exerted more potent suppression than cells conditioned with il- alone. in vivo, beads/dc-expanded, tgf-β/ il- -conditioned nt reg synergized with transient host t cell-depletion (anti-thy . mab i.p. µg at d- & µg on d+ ) in c bl/ mice to suppress balb/c heart allograft rejection with . % (n= ) and % (n= ) allografts surviving over days when x or x cells/mouse were injected immediately post-transplant, respectively. anti-thy . treatment alone led to only . % long-term survival. infused nt reg survived long-term ( . % circulating t cells ( x cell dose) or . % ( x cell dose) at d+ post-transplant) and expressed high level foxp ( ∼ %) in vivo. long-term surviving allografts showed characteristics of 'acquired immune privilege' with cellular infiltrates that were foxp + , tgf-β + , il- + and indoleamine , -dioxygenase (ido) + , although signs of mild to moderate chronic rejection were still evident conclusion: t-bet deficiency results in up-regulation of il- expression in addition to th associated cytokines resulting in acceleration of chronic rejection despite profound deficiency of ifn-γ. t-bet deficiency may contribute to the alloimmune responses independent of ifn-γ by in situ hybridization, tir mrna level was higher (p< . ) in cortical tubuli, glomeruli, perivascular and peritubular areas of kidney grafts at , and - d post-tx, than in naive kidneys. to assess how local expression of tir affects the outcome of kidney grafts, we transplanted tir -/-b x kidneys into dba/ mice. most ( %) recipients of tir -/-kidneys rejected their grafts with a median survival of . d (n= , p< . vs wt) and had more severe graft dysfunction (bun levels) at day , and - days post-tx, than recipients of a wt allograft (p< . ) opticept trial: efficacy and safety of monitored mmf in combination with cni in renal transplantation at months. r trough-based dose adjustments were made in the mmf cc arms. antibody induction and/or corticosteroids were administered according to center practice. primary endpoints were the proportion of patients with treatment failure (biopsy-proven acute rejection [bpar], graft loss, death), and mean percent change in calculated glomerular filtration rate (gfr; nankivell equation) at months. safety endpoints were incidences of adverse events (aes) and serious aes baseline characteristics did not differ among treatment groups with living donors accounting for approximately % of grafts. % received tacrolimus (tac) and % cyclosporine (cya): cni doses and levels were significantly lower in group a. mmf doses were greater in cya-treated subjects in all groups cya treated patients and in group a (p= . ); stability of renal function over time was greatest in group a. despite higher mmf doses in group a (p< . ) at most time points, significantly fewer mmf withdrawals occurred in group a vs. groups b and c. conclusions: a concentration-controlled mmf and reduced level cni regimen is not inferior to that of fixed-dose mmf and standard-dose cni as regards bpar and other end points. this regimen facilitated higher mmf dosing without an overall increase in adverse effects, and with a trend toward preservation of kidney function versus standard-dose cni regimens comparison at one year of interstitial fibrosis (if) by automatic quantification in renal transplant recipients with cyclosporine (csa) discontinuation and sirolimus (srl) introduction introduction: we previsouly reported the clinical results of a multicentric study showing that csa conversion to srl at week (w) is associated with a significant improvement in renal function. using routine renal biopsy (rb) performed at w during this study routine rb was performed at w . for each rb, a section was imaged using a colour video camera and analyzed by a program of colour segmentation which automatically extracts green colour areas characteristic of if. results were expressed as percentage of if and grade according to banff classification. results: male donor gender was associated with higher if ( ± % vs. ± %, p = . ). if was numericaly higher in patients who had experienced acute rejection ( ± %, n = vs. ± %, n= , p= . ) there was a positive correlation between renal function and the percentage of if on rb (p= . ). despite significant improvement of renal function at w in the srl group intent to treat (n= ) mean if (%) grade i (%) grade ii (%) grade iii (%) sirolimus (n= ) conclusion: despite significant improvement in renal function after csa to srl conversion at months, we found no difference of if on rb at w . the observed improvement of renal function may be due to a hemodynamic effect. a longer delay may be necessary to observe histological improvement. the higher if score than the one previously reported by others may be explained by the use of expanded criteria donors abstract# effects of cni or mmf withdrawal on carotid intima media thickness in renal transplant recipients methods: we included stable renal transplant patients on cni-based immunosuppression, including steroids ( mg/d) and mmf ( g/d), who were randomized to mmf-withdrawal (group a: csa-auc ng*h/ml) or cni-withdrawal (group b: auc-mpa µg*h/ml). patients were treated for traditional risk factors according to stringent predefined targets. ambulatory bloodpressure (abpm), lipids, estimated creatinine clearance (mdrd) and imt were measured at baseline and after months. results: groups were comparable with respect to demographic characteristics, immunological profile, renal function, systolic and diastolic bloodpressure and lipids. mean duration of follow-up was . ± . months. only patient ( . %) in group b and patients ( . %) in group c experienced acute rejection despite adequate exposure (p= . ). imt did not change final renal function outcomes from the spare-the-nephron (stn) trial: mycophenolate mofetil (mmf)/sirolimus (srl) maintenance therapy and cni withdrawal in renal transplant recipients purpose: to compare the effect on renal function of maintenance immunosuppression with mmf and srl to that of mmf and a cni in renal allograft recipients. methods: in a -year open-label, prospective, randomized, controlled, multicenter study, subjects maintained on mmf and a cni were randomized - days posttransplantation to either mmf ( - . g bid) plus srl ( - mg followed by ≥ mg/ day results: outcomes of the first subjects receiving mmf/srl and receiving mmf/cni (tac, n= ; csa, n= ) completing year of follow-up will be reported here. final outcomes of all subjects will be presented at the congress. mean time from transplant to randomization in both groups was days. groups were similar at baseline for all reported renal function endpoints after months of therapy, maintenance immunosuppression with mmf/ srl after cni withdrawal appears to preserve renal function when compared with a mmf/cni-containing regimen improved outcomes after de novo renal transplantation: -year results from the symphony study. h. ekberg, h. tedesco-silva frei, y. vanrenterghem, p. daloze, p. halloran at years, the rate of uncensored graft loss was lowest in patients receiving tacrolimus ( % vs - % in other groups; kaplan-meier estimates). gfr at the end of the core study was slightly better in the follow-up itt patients ( - ml/ min) than in the core study itt patients ( - ml/min), suggesting inclusion of betterperforming patients in the follow-up. renal function was generally stable over year . a slight improvement in gfr in the sirolimus group (+ . ml/min) was observed, whereas the tacrolimus group still had superior gfr ( vs - ml/min in other groups). conclusions: in follow-up patients, renal function was stable during the second year and gfr differences were less marked than at year a prospective randomized study of alemtuzumab vs rabbit anti-thymocyte globulin induction in kidney and pancreas transplantation gautreaux, s. iskandar, p. adams, r. stratta. surgery; medicine; pharmacy alemtuzumab (alem) and rabbit anti-thymocyte globulin (ratg) are the most commonly used t-cell depleting induction agents in kidney (k) and pancreas (p) transplantation (tx) expanded criteria donors (ecd) were included. results: between / / and / / pts enrolled and pts were transplanted. of pts, ( %) had ktx alone, ( %) kptx, and ( %) paktx. of ktx alone, ( %) were deceased donor, and ( %) were ecds. recipient age, race, re-tx abstract# purpose: to determine the impact of alginduction on long-term outcomes post-renal tx. methods: between / and / , consecutive adult pts received a deceased donor renal tx at a single institution results: the incidence of acute rejection was lower in gr. ( % vs. %, p< . ). the incidence of cmv infection was % in gr. and % in gr. (p=ns). the overall incidence of cancer was abstract# single-dose induction with rabbit anti-thymocyte globulin (ratg) safely improves renal allograft function and reduces chronic allograft nephropathy clifford miles, gerald groggel, lucile wrenshall. divisions of transplantation and nephrology we conducted a prospective, randomized trial in renal transplant recipients comparing two dosing protocols [single dose ( mg/kg) vs. divided doses ( . mg/kg for doses)] of rabbit anti-thymocyte globulin (ratg; thymoglobulin®). we present herein the results of the first patients throughout the first months post-transplantation, recipients of kidneys from non-marginal deceased donors derived the greatest benefit in renal function (egfr) from the single-dose regimen (p = . ). the incidence of chronic allograft nephropathy (can) was also lower in the single-dose group, in both clinically-indicated and protocol biopsies combined (p = . ) and in -month protocol biopsies alone high risk (race, pra) ( %) in multivariable regression, allograft failure strongly predicted increased risk of subsequent cve. among listed candidates, receipt of a transplant was associated with significant time adjusted for baseline factors, cve after transplant predicted increased risk of subsequent mortality: hr . (ci . - . ) after is microalbuminuria post-renal transplantation is related to inflammation and cardiovascular risk our objective was to define the relationship between microalbuminuria and these risk factors in stable rtr. methods: over one year, we identified stable rtr who were at least months post-transplant and provided successive urine albumin-to-creatinine ratio (acr) measurements, excluding those with recent illness and overt proteinuria. microalbuminuria was defined as averaged acr ≥ . in men and . in women (cda ). framingham-based traditional as well as novel cardiovascular risk factors associated with microalbuminuria were determined by univariate (p < . ), followed by stepwise backwards elimination (p > . ) multivariate logistic regression analysis microalbuminuria did not correlate with prior acute rejection, delayed graft function, or any specific antihypertensive or immunosuppressive agents. conclusions: post-transplant microalbuminuria is highly prevalent and is associated with elevated crp, elevated bp, and smoking. its relationship to these other factors suggests that it reflects an inflammatory state in otherwise stable patients and thus may indicate graft and patient health the first year after kidney transplantation (tx) is associated with increased mortality relative to dialysis. early post-tx deaths are often cardiovascular (cv) and frequently occur after the first week post-tx. ctnt is a sensitive and specific maker of myocardial injury. in this study we investigated whether ctnt relates to early post-tx survival. methods: patients received kidney tx from / to / , % from living donors. ctnt was measured during the pre-tx workup and periodically while on the tx waiting list. patients ( %) had a dobutamine stress echo (dse) and ( %) had a coronary angiogram. the combined end point of the study was death or major cardiac events. survival was censored for graft loss. results: mean age was + , % males. pre-tx ctnt level was elevated (> . ng/ ml) in % of patients other dse derived parameters did not relate significantly to survival. ctnt further stratified the risk associated with other variables. thus, among patients with ef< %, year survival was %, % and % (p= . ) in patients with ctnt < . , . - . and > . , respectively. similarly, these ctnt ranges stratified risk in patients with low albumin conclusion: an elevated pre-tx ctnt is a strong and independent predictor of reduced early post-tx survival. ctnt allows stratification of risk in patients who have other risk factors such as low ef, low serum albumin and dialysis> years. in all patients, independent of any other variables, a normal ctnt was an excellent predictor ( %) of survival abstract# validation of framingham risk assessment by actual cardiovascular event data in renal transplant recipients alloway, michael cardi, gautham mogilishetty, shazad safdar excellent outcome after liver transplantation in children with cystic fibrosis some studies have reported benefits of liver transplantation (lt) in cf patients, but large outcome studies are not available. we report the outcomes of a large cohort of cf patients undergoing lt. methods: pre and post-lt patient characteristics, post-lt morbidity and mortality, and patient and graft survival were patients age < yr) received a st isolated lt. cf patients were listed for st lt, neither waitlist deaths nor the probability of death from time of listing was different from non-cf. ( . %) cf patients underwent lt with an average followup of yrs ( - yrs) average peld: . ( . % had a peld < ), median age: . yrs ( . - . ) graft survival in cf patients was . %, . %, and . % at , , and yrs compared to . %, . %, and . %. rejection rates were not different ( . % cf vs . % non-cf @ yrs with % of these patients requiring dialysis. standardized height and weight scores showed no improvement over years followup in the cf patients (height z - . at tx to - . at yrs., weight z - . to - . ), but tended to improve in the non-cf group in addition, death rates from time of listing are not increased compared to non-cf patients. these data support lt as a treatment for cf liver disease, but studies investigating the lack of growth improvement and increased renal complications in these patients may further improve outcomes. abstracts full cni group. conclusion: compared to full cni, low cni/mmf a) allows renal function to recover in patients with impaired renal function at the time of ltx and b) preserves long term renal function. cni sparing in combination with mmf may become cellular islet autoimmunity influences clinical outcome of islet cell transplantation methods: twenty-one t d patients received cultured islet cell grafts prepared from multiple donors and transplanted under anti-thymocyte globulin (atg) induction and tacrolimus plus mycophenolate mofetil (mmf) maintenance immunosuppression. immunity against auto-and alloantigens was measured before and during one year after transplantation. cellular auto-and alloreactivity was assessed by lymphocyte stimulation tests against autoantigens and cytotoxic t lymphocyte precursor assays, respectively. humoral reactivity was measured by auto-and alloantibodies. clinical outcome parameters remained blinded until their correlation with immunological parameters. results: all patients showed significant improvement of metabolic control and out of became insulin-independent. multivariate analyses showed that presence of cellular autoimmunity before and after transplantation was associated with delayed insulinindependence (p= . and p= . , respectively) and lower circulating c-peptide levels during the first year after transplantation (p= . and p= . , respectively). / patients without pre-existent t-cell autoreactivity became insulin-independent, versus / patients reactive to both islet autoantigens gad and ia- before transplantation. autoantibody levels and cellular alloreactivity were not associated with outcome. conclusions: cellular islet-specific autoimmunity affects clinical outcome of islet cell transplantation under atg-tacrolimus-mmf immunosuppression bmp- is downregulated & tgfβ to bmp- ratio favors emt during acute rejection of human renal allografts allospecific cd + t-cells predict rejection risk and measure immunosuppressive effect after abdominal organ transplantation in recipients methods: allospecific cd +t-cells were measured in < hours with polychromatic flow cytometry to identify rejectors (who had experienced acute cellular rejection within days post-transplantation) in single mixed leukocyte responses (mlr) from cross-sectional recipients- children with liver or intestine allografts, and adults with renal allografts. where possible, results were correlated with proliferative alloresponses measured by cfse-dye dilution (n= ), allograft biopsies (n= ), and expression of ctla , a negative t-cell costimulator, which antagonizes cd -mediated effects (n= ). results: in the first children, logistic regression identified donor-specific, memory cd + t-cytotoxic cells (tc) as enhanced among rejectors, compared with non-rejectors ( ± vs ± per , cells, p= . ), relatively drug-resistant (r with drug levels =- . , p=ns), with greatest sensitivity/specificity (> %) for rejectors noninvasively developed molecular signatures accurately predict acute rejection of human renal allografts greater emotional well-being (sf- ) and felt that their transplant interfered significantly less with various aspects of their life (iirs). conclusions: findings highlight the potential utility of assessing attachment style in transplant populations cni sparing in de novo renal transplantation: -year results from the symphony study one background: single center non-randomized results with steroid avoidance have shown patient and graft benefits. methods: unsensitized, primary kidney recipients, - yrs of age, were enrolled from us transplant programs ( )( )( ), in a prospective : randomized multicenter study of steroid-free (sf) vs. steroid-based (sb) immunosuppression with matched demographics. . % of sf and . % of sb were african americans and . % of sf vs. . % of sb had esrd from fsgs. sf patients received extended ( mo) vs. standard ( mo) daclizumab induction in the sb group. patients in both arms received tacrolimus and mmf maintenance. protocol biopsies were performed at , , and mo, and for renal dysfunction. primary end-points were differences for standardized height scores and biopsy proven acute rejection (bpar) at year. results at year: sf and sb patients were enrolled; sf and sb were - yrs of age. patient survival was % in both arms. graft survival was similar ( . % in sf vs. . % in sb). intent to treat median delta height sds scores from baseline for different age groups were: . for sf and . for sb in the - yr old (p= . ); . for sf and . for sb in the - yr old (p= . protection of liver ischemia reperfusion injury by silencing of tnf-α and complement genes. roberto hernandez-alejandro, xusheng zhang, dong chen, xiufen zheng, hongtao sun, weihua liu, marianne beduhn, aminah shunnar, motohiko suzuki, norihiko kubo, bertha garcia, anthony jevnikar, , living kidney donation is rapidly increasing worldwide to offer a partial (?) solution for the numerous esrd wait-listed pts. in spite of properly followed guideline criteria conclusion: dcd donors are a viable source of liver allografts for transplantation. patients who receive dcd livers have outcomes comparable to subjects who receive grafts from brain dead donors. use of dcd livers from donors over years of age is accompanied by a higher incidence of retransplantation and biliary complications. background: hypothermic machine perfusion (hmp) is in its infancy in liver transplantation (ltx). potential benefits include diminished reperfusion injury and improved early function. methods: the study was designed as a phase trial of liver hmp. exclusion criteria included: multiple organ recipients, meld> , icu patients, and patients > years of age. donor livers > years, biopsy with > % macrosteatosis and dcd were also ineligible for hmp. seventeen patients were enrolled transplanted with livers that underwent hmp for - hours using dual centrifugal perfusion with vasosol solution at - °c. patient, operative and early outcome variables were recorded. we compared outcomes to matched cold stored (cs) controls from the same era. results: all hmp grafts functioned immediately by usual clinical criteria with intraoperative bile production. results are summarized in table . synergy between il- and tnfα promotes t cell alloreactivty and impairs the graft-prolonging effects of costimulatory blockade. hua shen, bethany m. tesar, wendy e. walker, daniel r. goldstein. internal medicine, yale university, new haven, ct. a novel role of th cells in allograft rejection and vasculopathy. francesca d'addio, jesus paez-cortez, m. javeed ansari, laurie glimcher, john iacomini, mohamed sayegh, xueli yuan. transplantation research center, renal division, brigham and women's hospital, boston, ma; harvard school of public health, boston, ma. introduction: transcription factor t-bet plays a crucial role in th /th development. here, we investigated the role of t-bet in th differentiation and function of th cytokines in allograft rejection using an mhc class ii mismatched model of cardiac allograft vasculopathy. methods/results: cardiac allografts from bm mice were transplanted into wild-type as well as t-bet and ifn-γ deficient c bl/ recipients. t-bet-/-mice showed significantly accelerated allograft rejection (mst= . ± . days). however, as previously reported, all ifn-γ-/-and majority of the c bl/ mice accepted grafts for greater than days. upon in vitro stimulation of recipient splenocytes by irradiated donor cells, t-bet-/-and inf-γ-/-lymphocytes produced significantly less inf-γ and more th cytokines. interestingly, production of the proinflammatory cytokines il- and il- was significantly higher in t-bet-/-( ± . and ± . pg/ml) than c bl/ ( . ± . , . ± . pg/ml, p= . and . compared to t-bet-/-) and inf-γ-/-( . ± . , . pg/ml, p= . and . compared to t-bet-/-) mice. in vivo administration of il- neutralizing antibody (mab ) significantly prolonged survival of bm hearts (mst> days, p< . compared to the . ± . days of the control igg group) in t-bet-/-mice. immunofluorescence staining of bm hearts harvested from t-bet-/-recipients indicated that both cd and cd infiltrating lymphocytes produced il- . however, t-bet-cd double knockout mice did not reject bm heart grafts, nor did the grafts exhibit chronic vasculopathy. in contrast, t-bet-cd double knockout mice rejected (mst: . ± . days). splenocytes from t-bet-cd knockouts produced significant lower il- ( . ± . ) and il- ( . ± . pg/ml) than observed in t-bet knockouts ( ± . and . ± . pg/ml) and t-bet-cd knockouts ( . ± . and . ± . pg/ml respectively) recipients when re-stimulated with donor cells, while there was no significant difference in inf-γ production. induction in the elderly transplant recipient: an analysis of the optn/ unos database. suphamai bunnapradist, steven takemoto, jagbir gill, tariq shah. medicine-nephrology, ucla, la, ca; medicine-nephrology, national institute of transplantation, la, ca.we examined the incidence and mortality implications of cerebrovascular events (cve) after kidney transplant. we also compared variations in risk on the transplant waitlist and after allograft failure. methods: we used registry data from the us renal data system to retrospectively investigate ischemic stroke (is), hemorrhagic stroke (hs) and transient ischemic attacks (tia) among , adults who received kidney transplants in - with medicare as primary payer. patients with prior indications of cve in the registry were excluded. we ascertained events from billing claims, and estimated incidence of first events by the product-limit method. at-risk time was censored at: loss of medicare, yr transplant anniversary, non-cve death or end of study ( / / ). cox regression was used to identify independent correlates of cve, and to examine cve events as time-dependent mortality predictors. we estimated cve incidence after graft failure among patients without cve diagnoses prior to graft loss (n= , ), and amongthe association between hyperuricemia at six months after kidney transplantation and the development of new cardiovascular disease, many studies have previously reported safe withdrawal of prednisone (pw) late after kidney transplantation (ktx). to determine the best immunosuppression regimen during the pw, we performed a prospective trial with stable ktx patients randomized to either csa or 's' based regimen. methods: all patients received antibody induction therapy at the time of rtx and maintained on csa, p and cellcept®. patients excluded if they had > acute rejection, > gm/d proteinuria or serum creatinine > . mg/dl. patients were enrolled and data presented for patients with > weeks follow-up (f/u) with mean f/u of . ± . weeks. no differences observed in baseline characteristics in both groups. all patients then randomized to either csa (n= ) or 's' (n= ) and cellcept® converted to equivalent dose of ms. csa dosed by c level ( -hour) with goal level of ng/ml. sirolimus target level was ng/ml. results: patients withdrew from study, patients on s returned to csa regimen because of side effects. patients in the csa group and patient in 's' group had ar ( of them due to drug non-compliance). death censored graft survival was %. mean csa drug level acheived was ± ng/ml and 's' drug level was . ± ng/ml. no significant differences noted in hematological values or bp measurements. csa ( purpose: the clinical significance of c d positiviity in patients with acute rejection is well defined but its significance in stable graft function is undetermined. this study was performed to evaluate the clinical outcome of protocol biopsy-proven c d positive renal transplants with stable graft function in the early posttransplantation period. methods: renal allograft biopsies were included. protocol biopsies (n= ) were performed from stable allografts on day posttransplantation, and indication biopsies (n= ) were performed from dysfunctioning allografts. incidence of c d positivity was compared between protocol and indication biopsies. clinical characteristics, biopsy findings, graft function, acute rejection episodes, and graft survival rates were compared between the c d-positive and c d-negative grafts in each group. results: c d deposition in protocol biopsies was detected in of biopsies ( . %), whereas . % ( of biopsies) in indication biopsies. the histological findings of c d-positive protocol biopsies were minimal inflammation of tubulointerstitium. on the other hand, those of c d-positive indication biopsies were various including acute humoral rejection, acute cellular rejection, acute tubular necrosis and calcineurin inhibitor toxicity. in the protocol biopsy group, graft function during year after biopsy, acute rejection rate, and cumulative graft survival did not differ between the c d-positive and c d-negative grafts. all c d-positive allografts maintained stable graft function without any antirejection therapy. in the indication biopsy group, graft function during year after biopsy and acute rejection rate did not differ between the c d-positive and c d-negative grafts. however the cumulative graft survival rate was worse in the c d-positive grafts than the c d-negative ones (p= . ). conclusion: c d positivity associated with allograft dysfunction indicates a poor graft outcome. however, c d-positive allografts with stable graft function in the early posttransplantation period take an indolent course. are methods: this was a retrospective single centre study reviewing all the adult patients who had a kidney transplant biopsy between april and october at guy's hospital. results: patients had diffuse (> %) c d staining out of who had kidney transplant biopsies in this time and had been followed up within the centre. of these patients, also had dsa prior or at the time of biopsy. the fall in egfr in this group a year post biopsy was greater than those with diffuse staining for c d but no dsa. the mean change in egfr from the day of biopsy at a year was - . ml/min/ . m (+/- . ) in those with dsa compared with + . ml/min/ . m (+/- . ) for those with c d but without dsa. the changes in egfr from the pre-biopsy baseline at one year showed a fall in egfr in both groups but this was greater in those with dsa (- . compared with - . ml/min/ . m ).of patients who never had diffuse or focal c d staining on biopsy, only had had dsa tested. of these only ( %) had a positive dsa result. from these eight, four had features of rejection and four did not. one person in each of these groups is dialysis dependant and one person in the rejection group has egfr < ml/min/ . m . although small numbers, this outcome appears to be worse than that of c d negative patients with no dsa but features of rejection who in fact showed an improvement in egfr from the day of biopsy by . m l/min/ . m or an improvement from their pre-biopsy baseline of . m l/min/ . m at one year. conclusion: dsa is of additional value in evaluating risk of graft failure. this appears to be of value in those with and without diffuse c d staining on biopsy. utility of post-transplantation flow cytometry crossmatching in predicting graft outcomes. michelle willicombe, graham shirling, ray fernando, henry stephens, paul sweny, peter j. dupont. department of renal medicine, royal free hospital, london, united kingdom; histocompatibility laboratories, anthony nolan trust, london, united kingdom. de novo development of donor-specific anti-hla antibodies after renal transplantation may be associated with increased rejection and decreased graft survival. flow-cytometry crossmatches (fcxm) have been suggested as method of screening for development of donor-specific hla antibodies post-transplantation, but interpretation of crossmatch results can be confounded by antibodies directed against antigens other than hla. we assessed the impact of developing a positive fcxm post-transplantation on clinical outcomes in a cohort of live donor renal allograft recipients. methods: patients were studied. / ( %) received tacrolimus-based and / ( %) ciclosporin-based immunosuppression. median follow-up was months. all patients had negative complement-dependent cytotoxic (cdc) t cell crossmatches pretransplantation. / ( %) in the group with a positive fcxm had an acute rejection episode in the first months compared with / ( %) in the group with a negative fcxm (p=ns). graft function at months was not different between the groups (positive fcxm -median creatinine mmol/l; negative fcxm -median creatinine mmol/l; p=ns). / grafts ( %) were lost within the first year in the positive fcxm group compared with / ( %) in the group with negative post-transplant fcxm (p=ns). the development of a positive fcxm post-transplantation alone is not predictive of adverse clinical outcomes. this may be explained by the poor correlation between a positive fcxm and the presence of antibody directed against mismatched donor hla antigens. surveillance for development of donor-specific anti-hla antibodies after transplantation may be best performed using high-resolution bead technologies rather than fcxm. use of the fcxm alone, without establishing antibody profiles, is of limited predictive value. based upon the amount of antibody (ab) measured by the titer of donor specific hla antibodies, dsa, or the fluorescence intensity (fi) of the donor specific single antigen bead. it is unclear whether abs indentified by sensitive single antigen bead and solid phase assays (flow pra and luminex) correlate with and are predictive of a clinically relevant end-point (a + fcxm). we evaluated the pra, dsa, bead specific ag fi and fcxm reactivity of pre-transplant (pre-tx) sera from recipients of a deceased donor renal allograft to determine whether amount of ab (measured by fi) predicts a (+) fcxm. patients with a (+) dsa, a (+) fcxm and pre-tx class i pra ≥ % (n = , mean pra of ± %) when compared to patients with pre-tx pra < % (n = , mean pra ± %) had comparable mean fis ( , ± , vs , ± , ) , fi ranges ( , - , vs , - , ) and median fis ( , vs , ). the class ii comparisons were of the same pattern. surprisingly, patients with a (+) dsa, a (-) fcxm and pre-tx class i pra ≥ % (n = , mean ± %) compared to patients with pre-tx pra < % (n = , mean pra ± %) had comparable mean fis ( , ± , vs , ± , ) , fi ranges ( , - , vs , - , ) we have recently showed that pre-treatment of the donor with epo causes a substantial reduction of the dysfunction and injury associated with the transplantation of kidneys recovered after cardiac death. -aminoisoquinolinone ( -aiq) a potent water soluble parp inhibitor has proven to reduce renal ischemia-reperfusion (i/r) injury. the aim of our study was to determine the effects in the graft and in the receptor of the pre-treatment of the donor with epo and treatment of the recipient with -aiq, in a porcine model of dcd kidney transplantation. material/methods: landrace pigs were killed by lethal injection; their kidneys were subjected to min of warm ischemic time (wit) and then transplanted after h of cold storage in celsior. in the pre-treated group, donors received a single dose of epo ( iu/kg) min before cardiac arrest. in the treated group, recipients received a continuous dose of -aiq ( mg/kg/h) minutes before reperfusion and maintained during minutes. blood, urine and renal tissue samples were collected at the end of the experiment for biochemical, histological and immunohistochemistry (pars, inos and cox- ) evaluation. data analysis performed with graph pad prism statistical package; p< . considered statistically significant. results:transplantation of kidneys from dcd resulted in: a significant rise of the levels of creatinine, n-acetil-b-d-glucosaminidase, glutathione-s-transferase, ast, ldh, alt, fractional excretion of na+, interleucin and , malondialdehyde levels and myeloperoxidase activity (p< . ); a significant reduction in urine flow and creatinine clearance, disturbances in the histological and imunohistochemistry pattern. administration of epo before ischemia and -aiq before reperfusion reduced significantly the biochemical (p< . ), histological and imunohistochemical evidence of glomerular dysfunction and tubular injury. they also reduced systemic injury, inflammatory response and oxidative stress. conclusions:pre-treatment of the donor with epo and treatment of the recipient with -aiq causes a substantial reduction of the dysfunction and injury associated with the transplantation of kidneys recovered after cardiac death. in the hmp group perfusate ast levels strongly correlated with recipient peak ast by linear regression (p< . ). conclusions: hmp of liver grafts provides safe and reliable preservation in our pilot series. perfusate ast may allow pretransplant prediction of reperfusion injury. a larger randomized trial will be necessary to demonstrate the magnitude of benefits of hmp over cs in ltx.purpose: liver discard rates have increased in a large, urban organ procurement organization from % to %. the reason is likely the result of increased transplant surgeon willingness to consider organs close to the margin of clinical acceptability. however, the costs associated with recovering these organs are high if the liver is discarded. we endeavored to determine whether a model based on pre-recovery data could predict liver discard introduction: we report our years experience with the use of campath- h (c h) in adult liver transplantation. from december until july we administered c h induction with low dose maintenance tacrolimus immunosuppression to adult recipients of a liver allograft. most common primary diseases were laennec (n= ), cryptogenic cirrhosis (n= ) and autoimmune: psc (n= ), pbc (n= ) and aih (n= ). the first dose of c h was administered immediately before (n= ) or after (n= ) the transplant procedure. follow up was until september, . results: five year patient and graft survival was % and % respectively. there were deaths due to stroke (n= ), chronic rejection (n= ), failure to thrive/pneumonia (n= ), sepsis (n= ), hepatic artery thrombosis, hcc, prostate cancer, graft lymphoma and non-compliance (one each). seven patients were retransplanted, for primary non function (n= ), portal vein thrombosis (n= ), hepatic artery thrombosis (n= ), hepatitis b (n= ) and chronic rejection (n= ). thirty six patients had biopsy proven rejection episodes: mild (n= ), moderate (n= ) or severe (n= ). the average tacrolimus hour trough levels were . , . ng/ml and . ng/ml for the rst, nd and th year post-transplantation, respectively. there was no significant difference in the outcome of the transplant so far, between patients that received c h before or after the transplant procedure. immunosuppression-related complications included a). opportunistic infections: most common were herpes zoster (n= ), cmv (n= ), and herpes simplex (n= ), b). neoplasms: skin cancer (n= ), kaposi sarcoma (n= ), lymphoma (n= ) and c). nephrotoxicity: five patients received a kidney graft for diabetic nephropathy (n= ), nephrotic syndrome (n= ) and calcineurin nephrotoxicity (n= ). conclusion: the use of c h induction with half the usual dose of tacrolimus is an effective regimen in adult liver transplantation. the timing of c h administration does not seem to affect the clinical outcome so far. a. david mayer, james m. neuberger. the liver unit, queen elizabeth hospital, birmingham, united kingdom. introduction: in the prospective respect study, primary liver transplant patients were randomised to of groups: a) standard-dose tacrolimus (target trough level > ng/ml) for the st month; b) g mycophenolate mofetil (mmf) iv until at least day , g po thereafter + reduced-dose tacrolimus (target trough level ≤ ng/ml); and c) mmf as in group b + reduced-dose tacrolimus introduced on day (target trough level ≤ ng/ml) + daclizumab on days and . steroids were given in all groups according to local centre protocol. results at year showed that g mmf + delayed and reduced tacrolimus + daclizumab is associated with significantly less impairment of renal function compared with standard treatment. here we present the results of the per protocol (pp) population. methods: the pp population, which was defined prior to the sub-group analysis, consisted of patients from the full analysis set who had no inclusion/exclusion criteria violation, had at least one creatinine clearance (crcl) value beyond months, were treated according to the protocol and did not receive any prohibited medication during the first days and for less than week at any time during the study. a composite endpoint comprising freedom from renal dysfunction (≥ % decrease from baseline in calculated crcl), acute rejection, graft loss or death was also investigated. results: the full analysis set included , and patients, whereas the pp population only included , and patients in groups a, b and c, respectively. the mean difference in calculated crcl from baseline to year was significantly smaller in group c compared with group a (- . ml/min vs - . ml/min, p = . ), but was not significantly different between groups a and b (- . ml/min). the incidences of death (n = , , ) and graft loss (n = , , ) were similar in all groups. the incidence of the composite endpoint at year was in both the full analysis set and the pp population significantly lower in group c compared with group a (pp population: % vs %, p < . ), but was not significantly different between groups a and b ( %). the pp analysis confirms the results from the full analysis set that g mmf + delayed and reduced tacrolimus + daclizumab is associated with less impairment of renal function compared with standard treatment with no negative effect on death and graft loss. aims: post transplant lymphoproliferative disorder (ptld) is a serious complication of solid organ transplantation that is closely associated with epstein barr virus (ebv) infection. ebv + ptld lymphomas express several latent viral genes including latent membrane protein (lmp ), a proven oncogene that is essential for human b cell transformation. lmp is able to activate erk, jnk, p , nfκb and pi k. the aim of this study is to determine whether lmp isolated from ptld tumors differs in signaling ability from lmp derived from the b. strain of ebv, originally isolated from a patient with infectious mononucleosis. methods: lmp variants isolated from a panel of ebv + ptld-associated b cell lines were cloned and sequenced. inducible chimeric constructs containing the lmp c-terminus and ngfr transmembrane domain were created for each tumor variant and expressed in the burkitts b lymphoma cell line bl . lmp signaling in bl clones was induced by crosslinking of ngfr. activation of p , erk, akt and jnk was assayed by western blotting (wb) with phospho-specific antibodies. nfκb activation was assayed by wb for iκb and cfos induction was analyzed by wb and the transam cfos binding assay. results: all three tumor variants of lmp , as well as the b. lmp isoform, were able to induce p activation within min of ngfr crosslinking while akt and jnk were activated within min. all variants showed similar ability to activate nfκb. however, tumor lmp variants induced prolonged erk activation (up to hrs) while the b. lmp variant induced a transient response. cfos is induced only during the sustained phase of erk activation. indeed, the tumor variants of lmp , but not b. lmp , were able to induce cfos protein. similarly, cfos binding to the ap consensus site was only observed in tumor lmp -induced nuclear lysates. two mutations in the c-terminus-aa (s vs g) and aa (t vs s) -are conserved in the tumor variants lmp compared to b. lmp . point mutation of either of these amino acids from the b. to tumor variant version allowed for sustained activation of erk and subsequent cfos induction and binding to the ap site. conclusion: tumor-derived lmp has enhanced ability to induce the cfos oncogene and this property can be localized to two amino acids in the c terminus. these findings suggest that these specific amino acid residues of lmp are important in determining whether ebv infection is benign or results in ptld. the absence of interferon regulatory factor- (irf- ) confers protection against the liver ischemia and reperfusion injury through an il- independent pathway. elizabeth r. benjamin, xiu-da shen, feng gao, yuan zhai, genhong cheng, ronald w. busuttil, jerzy w. kupiec-weglinski. surgery, dumont-ucla transplant center, los angeles, ca. toll-like receptor (tlr ) mediated liver reperfusion damage after warm ischemia requires signaling through the myd -independent, irf -dependent pathway with cxcl- (ip- ) playing a central role in the injury development. studies using cxcl- ko mice have shown that these mice are protected through an il- dependent mechanism. we chose to investigate irf , upstream of cxcl- , to further characterize its role in the injury progression, and to better understand the involvement of il- in this pathway. methods: we used irf ko mice and their wt counterparts in a model of partial hepatic warm ischemia with , , and h of tissue reperfusion (n= ko, wt at and h; n= ko, wt at h). wt bone marrow derived macrophages were generated and stimulated with lps to determine the kinetics of il- production. tissue was analyzed for histology and mrna levels were measured by qpcr. results: kinetic studies showed peak il- production at and h post-reperfusion (pr). on pathology, irf ko mouse livers were protected from ir injury both early pr, at and h, and at hrs pr when compared to wt. consistent with these data, il- mrna induction was decreased in irf ko, as compared with wt at , , and h. although il- induction was maintained in the cxcl- ko mice, the irf ko mice showed decreased levels of il- at h pr. by h, il- levels were normalized to wt. conclusion: irf ko mice are protected from liver ir injury with evidence of this protection as early as h pr. although cxcl- ko mice are protected from ir injury with maintained il- expression, the absence of the upstream molecule, irf , confers protection in an il- independent manner. these data suggest a novel mechanism of ir injury mediated by irf in the liver. background: bone marrow (bm) transplantation may induce donor-specific tolerance to prevent rejection of allogeneic solid organs while maintaining immunity against infections and tumors. currently allogeneic bm transplantation is limited by donor t cell mediated graft-versus-host disease (gvhd), as well as a variable requirement for recipient marrow ablation and high numbers of donor bm cells. furthermore, sustained macro-chimerism has not yet been easily or predictably achieved in partially ablated patients or large animals. while rejection of allografts is mediated primarily by recipient t cells, recent studies have demonstrated the capacity of nk cells to reject allogeneic bm and to prevent long-term mixed chimerism. thus, nk cells represent a barrier to long term bm engraftment even with t cell tolerance. we have previously identified a novel type of regulatory t (treg) cell with a "double negative" (dn) phenotype (tcrab + cd + cd -cd -). dn-treg cells can effectively suppress anti-donor t and b cell responses and prolong graft survival in allo-and xenotransplantation models. we therefore tested the capacity of dn-treg to alter nk cell function. methods: c bl/ bm cells were i.v. injected into sub-lethally-irradiated ( . gy) cb f (h- b/d) in a "parent to f " model, or into allo-disparate balb/c mice. bm cells were co-transplanted with various numbers of c bl/ dn-treg cells or cd + or cd + t cells as controls. recipient spleen cells were collected days after to detect donor progenitors in a colony-forming-unit (cfu) assay. mice then received cardiac (n= ) or skin transplants (n= ) to confirm tolerance. we found that donor-derived dn-treg cells suppress nk cell-mediated allogeneic bm graft rejection in both "parent-to-f " and fully mhcmismatched bm transplantation models. adoptive transfer of dn-treg cells with donor bm cells promoted the establishment of stable mixed chimerism and donor specific tolerance to bm donor cardiac and skin grafts (mst> days), without inducing gvhd in sub-lethally irradiated mice. perforin deficient dn-treg cells were unable to efficiently inhibit nk cell function, and donor bm did not engraft. these results demonstrate a potential approach to control innate immune responses and promote allogeneic bm engraftment and donor specific tolerance through the use of dn-treg cells.framingham risk score (frs) predicts cardiovascular (cv) risk in the general population, but may underestimate cv risk in kidney transplant (txp) patients (pts). frs has not previously been validated by prospective cardiovascular event (cve) data collection in kidney txp pts. the purpose of this study was to validate frs with actual observed cve data in kidney txp pts. methods: cve data was collected at routine intervals in our kidney txp pts and entered in a cardiovascular risk database. frs was calculated from baseline to yrs posttransplant (ptx) individual frs factors of age, sex, smoking, diabetes mellitus (dm), high-density lipoprotein (hdl), total cholesterol (tc), and blood pressure (bp) were evaluated for their ability to predict acutal cve occurring after kidney txp. pts with coronary artery disease (cad) were excluded from the frs analysis. cve were defined as sudden death, myocardial infarction, angina, and cerebrovascular accident/transient ischemic attack. frs factors were evaluated by cox proportional hazards in univariate (uva) and multivariate (mva) models. rho kinase (rok) modulates calcium sensitivity of vascular smooth muscle cells and contributes to the regulation of peripheral vascular tone in man. in essential hypertension increased rok-activity contributes to the generation of vascular resistance. arterial hypertension is a common complication in renal transplant recipients. in this study we were interested in the role of rok for systemic hemodynamics in hypertensive renal transplant recipients (tx). we tested the specific inhibitor of rok fasudil. tx and matched control subjects (c) received either fasudil ( g/min) or placebo over a period of minutes intravenously. peripheral blood pressure and heart rate were recorded every min over a total of minutes. measurements for pulse wave analysis (sphygmocor vt) were performed every minutes during this period. statistics by anova for repeated measurements.compared to placebo fasudil significantly reduced peripheral mean arterial pressure p< . ; figure ) and increased heart rate (+ . . bpm, p< . ) in tx but not in c. likewise, central systolic pressure(p= . ; (figure ), augmented pressure and augmentation index were decreased in tx only.we conclude that acute inhibition of rok by fasudil consistently and effectively lowers blood pressure in tx with a calcineurin inhibitor-based immunosuppression. interestingly, rok-inhibition also reduces central blood pressure and arterial stiffness in these patients. improvement of both these parameters has been linked to a reduction in cardiovascular morbidity and mortality in large trials. hence rok inhibition might prove beneficial for the treatment of hypertension in renal transplant recipients. use death with function causes half of late ktx failure, and cardiovascular disease (cvd) is the most common cause of death. chronic kidney disease (ckd) is a cvd risk equivalent, justifying aggressive risk reduction with blood pressure (bp) control, statins, aspirin, and use of angiotensin converting inhibitors (acei) and angiotensin receptor blockers (arb). dekaf is an nih-sponsored prospective observational study examining causes of ktx failure at transplant centers in the us and canada, with current enrollment of over subjects. we examined the use of cardioprotective medications among patients transplanted after / / with at least mos follow-up, focusing on subgroups with preexisting diabetes (dm) and/or cvd. we conducted a retrospective cohort study to asses the prevalence and the predictors for the development of hyperuricemia at months after kidney transplantation and the association between hyperuricemia and clinical outcomes including patient and graft survival, new cardiovascular events and chronic allograft nephropathy (can). adult patients who underwent kidney transplantation at mount sinai medical center between . . - . . were included. patients who died or lost the allograft within months after transplantation were excluded from analysis. of the patients with a functioning allograft at months after transplantation, patients ( %) had normal uric levels and patients ( %) had hyperuricemia. after age, race, sex adjustment, receiving a cadaveric kidney, having an egfr< ml/min, and taking diuretics and cyclosporine were associated with a higher odds ratio of hyperuricemia. over a mean of . years of follow-up, patients had one, or more, of the pooled outcomes; had new cardiovascular events, developed biopsy-proven can, patients died, and had graft failure. kaplan-meier survival curves demonstrated that the pooled outcomes of events occurred more frequently in hyperuricemic patients (figure, p < . ). due to association between low egfr and hyperuricemia, we analyzed the clinical outcomes in patients with low and normal egfr. while . % of hyperuricemic patients with an egfr< ml/min had one of the pooled outcomes, it was . % in patients with normal uric acid levels (p= . ). among patients with an egfr ≥ ml/min, . % of normouricemic and % of hyperuricemic patients had one of the events. these results suggests an important association between hyperuricemia at months after transplantation and the new cardiovascular events, biopsy-proven can, and graft loss in kidney transplant recipients with decreased allograft function. background: long-term survival after liver transplantation (lt) is now the rule rather than the exception. hence, assessment of outcomes for children after lt must consider not only the quantity, but also the quality, of life years survived and restored. aim: to examine key hrqol themes after pediatric lt raised by both recipients and their parent proxies, with evaluation by time ( - yrs, - yrs, - yrs, and > yrs) since lt. methods: semi-structured : item generation interviews were conducted in person with children (c) and parents (p) at time of ambulatory lt follow-up at pediatric lt programs in canada and uk. all interviews were audio-taped, transcribed verbatim, and subjected to content analysis utilizing qsr nvivo . software for hrqol related theme generation. the participants interviewed were part of a larger research program aimed at developing a disease-specific instrument to assess hrqol for children after lt. results: data representing ( % male) pediatric lt recipients was obtained from a total of ( c, p) item generation interviews. median recipient age at lt was . (range, . to ) yrs, for primary indications including biliary atresia ( %), fulminant liver failure ( . %), metabolic liver disease ( , %), malignancy ( . %) and others ( . %). median patient age at time of interview was . (range, . to . ) yrs. themes emerging at all time points post-lt included infection risks, limitations on physical activities, side effects from immunosuppression meds, educational supports, and ongoing bloodwork. themes identified within the medium ( - yrs) follow-up included worries about rejection episodes, need for future re-transplantation, school absenteeism, and altered sibling and family dynamics. the impact of living with a surgical scar was a more frequent theme with recipients > yrs from lt. as time from lt increased to > yrs, themes suggest a focus on normalization and health promoting behaviours, along with expressed desires to be like healthy peers. conclusions: unique hrqol themes emerged from item generation interviews not captured by currently available generic hrqol tools. hrqol themes identified after pediatric lt suggest the importance of considering time trajectories from lt, and a focus on elements of 'everyday life' apart from lt. the shortage of cadaveric donors has led many transplant centers to expand their criteria for accepting life-saving organs. utilization of donation after cardiac death (dcd) donors has been estimated to increase the number of cadaveric donors. we report our experience with a recently established dcd program at a pediatric hospital and the outcome with the transplanted grafts. methods: in a protocol for dcd was established at a free standing pediatric hospital. from to all patients undergoing withdrawal of care were evaluated for dcd. patients meeting criteria for dcd underwent withdrawal by the critical care team and organ retrieval was initiated if asystole was reached in less than minutes. in addition, one dcd liver was imported and was included in the liver results. results: during the year study period patients ( % of total donors) underwent dcd resulting in organs ( kidneys and livers) transplanted. the cases had a mean donor age of yrs (range - ), wit of min ( - ), time sbp < of min ( - ), and time from asystole to aortic flush of min ( ) ( ) ( ) ( ) ( ) ( ) ( ) . four kidneys were transplanted locally with cit - hrs, no dgf, and one month creat . - . . the remaining kidneys were exported for transplant. four livers were transplanted locally with donor age mth- yrs, wit - min, recipient age mth- yrs, cit - hrs, ast peak - , ast day - , inr peak . - . , inr day . - . , total bili one month . - . , and graft survival . - . yrs. there were no vascular or biliary complications. conclusions: a protocol for dcd at a pediatric hospital increased the number of pediatric donors by %. liver and kidney grafts from pediatric dcd donors demonstrated excellent graft function and survival. liver retransplantation in children. a year single centre experience. christophe bourdeaux, andrea brunati, magda janssen, jean-bernard otte, etienne sokal, raymond reding. pediatric liver transplant program, université catholique de louvain, saint-luc university clinics, brussels, belgium. when graft failure occurs in liver recipients, secondary transplantation represents the only chance of long-term survival. in such instance however, several surgical and immunological aspects should be carefully considered, with respect to their impact on final outcome.in the present study, the epidemiology and outcome of graft loss following primary pediatric liver transplantation (lt) were analysed, with the hypothesis that early retransplantation (relt) might be associated with lower immunologial risks when compared to late relt. between march and december , liver grafts were transplanted to children at saint-luc university hospital, brussels. among them, a total of children ( %) underwent relt, and were categorized into two groups (early relt, n= ; late relt, n= ), according to the interval between both transplant procedures (< or > days).ten-year patient survival rate was % in recipients with a single lt, versus % in recipients requiring relt (p= . ). ten-year patient survival rates were % and % for early and late relt, respectively (p= . ), the corresponding graft survival rates being % and % (p= . ). along the successive eras, the rate of relt decreased from % to %, whereas progressive improvement of outcome post-relt was observed. no recurrence of chronic rejection (cr) was observed after relt for cr ( / ). two children developed a positive cross-match at relt ( / , %), both retransplanted lately for cr secondary to immunosuppression withdrawal following a post-transplant lymphoproliferative disease.in summary, the current need for relt has been decreasing over years, with a parallel improvement of its outcome. the results presented could not evidence better results for early relt when compared to late relt. the latter did not seem to be associated with higher immunological risk, except for children with immunosuppression withdrawal following the first graft. the background: a serum conjugated bilirubin greater than umol/l (cb ), in neonates who receive parenteral nutrition (pn), has been demonstrated to be a predictor of end-stage liver disease requiring transplantation. given the recent interest in the role of omega- lipids in the development of parenteral nutrition associated liver disease (pnald), we sought to examine in a multiple variable model the role of days of maximal lipid (> . g/kg/day), in the development of this outcome. method: between and , data were collected prospectively on all neonates undergoing an abdominal surgical procedure. univariate logistic regression models for the prediction of cb were developed with the following predictors: gestational age, percentile weight, percent predicted small bowel and colonic length, resection of the ileocecal valve, presence of a stoma, post-operative enteral tolerance, number of septic episodes, days of pn amino acid > . g/kg/day, days of pn lipid > . g/kg/day, and total days of pn. univariate predictors significant at the . level were entered into a backward stepwise multiple variable logistic regression. results: infants received pn post-operatively, and developed cb . predictors that met criteria for consideration in the multiple variable model were: age (p= . ), weight (p= . ), small bowel length (p= . ), presence of a stoma (p= . ), proportion of enteral feeds post-operatively (p= . ), days of pn amino acid > . g/ kg/day (p= . ), days of lipid > . g/kg/day (p= . ), and total days of pn (p= . ).the final multiple variable model which had a negative predictive value of . % and positive predictive value of . % is presented in the table below. our model suggests a key role of pn lipids and intercurrent septic events in the development of cb from pnald. these data may provide targets, such as careful line care, reduction in maximal lipid dose, or the use of alternate lipids such as omega- fatty acids, to prevent cb an identified marker for the need of subsequent liver transplantation in infants with pnald. terminal renal failure occurs in more than % of liver transplant recipients after years. we have previously shown that, beside renal toxicity of calcineurin inhibitors, renal lesions may be related to diabetes, arterial hypertension, accumulation of hydroxyethylstarch (elhoes), and the etiology of the liver disease. we made the hypothesis that these lesions may be already present at the time of liver transplantation (lt), a finding that could lead to adapt the perioperative management. this work investigated prospectively whether renal histopathological lesions were present before lt by performing systematically a renal biopsy by endovenous route in candidates to lt with end-stage liver disease. these patients were ± years old, males ; / had a diabetes, and an arterial hypertension ; the liver disease was related to alcohol in cases, hcv in cases, hbv in cases, and to a cholestatic disease in cases. at the time of the pre-lt workup, the biochemical parameters were : child score ± , meld score ± , prothrombin rate ± , creatinin serum level ± umol/l, proteinuria . ± . g/ h. severe side effects related to the procedure were limited to cases of macroscopic hematuria, lasting less than hours. in cases, the material obtained during the procedure did not allow the histological analysis. among the samples available, were considered as normal ; in cases, lesions related to mesangial iga glomerulonephritis ( cases), diabetic glomerulosclerosis ( cases), elhoes accumulation ( cases), thrombotic microangiopathy ( case) were found, often associated ; in cases, the lesions were severe and lead to combined kidney/liver transplantation in cases. in conclusion, significant renal lesions are detectable in more than % of the candidates to lt. interestingly, histological findings often combined lesions related to the liver disease and to an associated cause (diabetes, previous treatment by elhoes or interferon). results of histological analysis could help to decide either to perform a combined renal/liver transplantation, to adapt the immunosuppressive regimen, or to abandon the lt project. . because serum creatinine is one of the components of meld, liver candidates with renal insufficiency have been transplanted in increasing numbers, with some candidates receiving a kidney along with the liver transplant. we aimed to compare the liver graft outcomes for liver alone (lta) transplants with those from combined liver-kidney transplants (clkt). a propensity score analysis was used to reduce the impact of selection bias in the comparison of outcomes in the two groups. methods. demographics, clinical factors and outcomes on lta and clkt recipients from / / to / / (n= , ) obtained from the optn database were used for the analysis. univariate post-transplant survival rates were estimated using kaplan-meier survival, and multivariable post-transplant outcomes were analyzed using a cox regression model with and without stratification by categories of the propensity score. the propensity score (probability of receiving a clkt) for each recipient was estimated using a logistic regression model. several donor and recipient factors were included in both the cox and logistic regression models. in this cohort, liver graft outcomes for clkt were significantly better than those for lta based on the multivariable analysis. the results were similar, although slightly less significant, when the model was adjusted for propensity. the superior outcomes of clkt may be due to unobserved differences between these groups of recipients, reflecting data not currently captured by the optn. effect of liver the tgfβ to bmp- ratio was higher in the ar group (median ratio: ) compared to recipients with stable graft function & normal biopsy (median ratio: , p= . ).our observations that bmp- is specifically down-regulated during an episode of ar and that the balance between tgfβ & bmp- is in favor of emt advance a mechanism for the deleterious impact of ar on the long-term outcome of human renal allografts. the non-statistical bioinformatic approach identified leader genes which define the highest interaction genes derived from the sam-gene list. an interaction map between the genes identified has been calculated. this network is formed around majors clusters: a network of interleukins and a network of signal transduction which allow us the identification of key genes such as bank , a negative modulator of cd mediated akt activation, thereby preventing hyperactive b cell response in blood from patients with operational tolerance and il r, a specific marker absent on potentially regulatory cd + cd +high t cells in blood from patients with chronic rejection. we have identified by a non-statistical analysis of the peripheral blood gene expression in human kidney recipients a cluster of genes which are strongly interconnected and which could be a starting point for further analysis of the molecular mechanisms of kidney graft operational tolerance and chronic rejection. fecal algorithm based on multiparameter mixed lymphocyte reaction assay for tailoring maintenance immunosuppressants after living donor liver transplantation. yuka tanaka, hideki ohdan, toshimasa asahara. department of surgery, hiroshima university, hiroshima, japan.background: no reliable immunological parameters exist for identifying liver allograft recipients in whom immunosuppressants can be safely withdrawn. for minimizing maintenance immunosuppressants, we established an algorithm determining anti-donor alloreactivity based on multiparameter mixed lymphocyte reaction (mlr) assay, wherein the number and phenotype of alloreactive precursorscan be quantified. we enrolled adults undergoing living donor liver transplantation (lt). the initial immunosuppressive regimen comprised tacrolimus/cyclosporine and methylprednisolone, which were gradually tapered off by months after lt. thereafter, therapeutic adjustments were determined by a policy of slow tapering off in the case of normal liver function. mlr assay was performed at month intervals to monitor immune status. in this assay, cfse-labeled pbmcs from recipients were used as responders. irradiated donor and third-party pbmcs were used as stimulators. after coculture, the responder cells were stained with cd or cd mabs along with cd mab, followed by fcm analyses. the proliferation and cd expression of cd + and cd + t cell subsets in response to anti-donor and anti-third-party stimuli were analyzed; the immune status of lt patients was categorized as hypo-response, norm-response, or hyper-response for cd + t cells and as hyper-response for cd + t cells. of the patients, had normal liver function at > months after lt. we examined the fluctuation of immunosuppressants at months after mlr in these patients. in patients whose immune status was categorized as hyper-response for cd + or cd + t cells (n= ), immunosuppressants had to be increased. in patients with norm-response immune status (n= ), immunosuppressant tapering was abandoned. immunosuppressant therapy was successfully tapered off in patients with hypo-response immune status (n= ). of patients with hypo-response immune status at > years after lt, immunosuppressants were completely discontinued in . in these "operational tolerance" patients, the precursor frequency of anti-donor cd + t cells (mean= . ± . %) was not reduced compared to that in non-tolerance patients, suggesting that donor-specific immune tolerance is maintained via inhibitory/ suppressive mechanisms rather than via clonal deletion. conclusion: multiparameter mlr assay can provide a clinically validated rule predicting the success of tailoring/weaning immunosuppression. psychological factors associated with non-adherence among adolescents before and after kidney transplant. nataliya zelikovsky. dept. of pediatrics, div. of nephrology, the children's hospital of philadelphia, philadelphia, pa.purpose: little is known about psychosocial risk factors for poor adherence among pediatric transplant patients. identification of variables that impact illness management can guide targeted interventions to improve adherence. methods: a longitudinal study was conducted to determine whether quality of life (pedsql), family functioning (fad), and parent adjustment (pip) would predict adherence in adolescent transplant patients. psychological questionnaires were administered prior to and months after the transplant. medical adherence measure (mam), a semi-structured interview was used to assess adherence. adherence was calculated as % missed and % late doses of those prescribed. results: patients (m = . years + . , % male, % caucasian) and their parents were evaluated at the time of listing for kidney transplant. the rate of non-adherence prior to transplant was high, with % of patients reporting some degree of nonadherence. of these patients, % missed and % took late > % of prescribed doses. on the quality of life measure, behavior issues were associated with missed (r=-. , p=. ) and late doses (r=-. , p<. ), and mental health issues were associated with late doses (r=-. , p<. ). adolescent reports of problems in affective responsiveness among family members was associated with missed (r=. , p=. ) and late (r=. , p=. ) doses. missed doses were also associated with mother reports of difficulties with overall family functioning (r=. , p<. ), communication (r=. , p<. ) and role definitions (r=. , p<. ) among family members. of the families were re-evaluated one year after the kidney transplant. % had been on dialysis prior to transplant and % received living-related transplants. % of the patients reported some degree non-adherence post-transplant, and using more stringent criteria, % of patients reported missing and % reported taking late > % of prescribed doses. worse quality of life such as limitations due to emotional problems (r=-. , p<. ), behavioral problems (r=-. , p<. ), and difficulties with family cohesion (r=-. , p<. ) was related to worse adherence. discussion: adolescent quality of life in behavioral and emotional domains, and family functioning play a significant role in adherence both before and after transplant. programs to improve adherence among transplant patients should incorporate psychosocial supports and behavioral interventions to improve adjustment of patients and families. kidney transplantation leads to marked improvements in health, yet transplant (tx) recipients often have difficulty with sexual functioning, which can affect quality of life. specific sexual concerns of tx recipients remain under investigated. the purposes of this study were to ) further establish the psychometric properties of the sexual concerns questionnaire (scq) including reliability and preliminary construct validity and ) identify the sexual concerns of kidney tx recipients. the scq was answered by kidney tx recipients who rated each item on a (not at all) to (extremely) scale. a cronbach's alpha correlation coefficient was calculated to determine the reliability of the scq. an alpha value of . was calculated for the questionnaire indicating it was reliable. exploratory factor analysis (efa) was performed to establish preliminary construct validity of the scq. as a result from the efa, items were dropped and a factor structure was accepted. examples of items and responses include question : "how difficult is it for your vagina to get or stay wet or moist?" (for women) and "how difficult is it for you to get or keep an erection?" (for men) and question "how comfortable are you talking about sexual concerns with your doctors and nurses?"participants were also asked to indicate how important their sexuality was to them on a (not at all) to (extremely) rating scale. twenty-six percent of participants rated their sexuality as quite a bit important, % rated their sexuality as very important, and % rated their sexuality as extremely important. the findings provide evidence of a reliable questionnaire with evidence for preliminary construct validity. they also indicate that sexuality is an important issue for a majority of kidney tx recipients. a cross-sectional study of fatigue before and after liver transplantation. james r. rodrigue, timothy antonellis, p= . # (none) to (extremely high); ♣ higher score = more fatigue, poorer sleep quality, or more mood disturbance; ¶ higher score = better qol one-third of pre-lt ( %) and post-lt ( %) patients reported severe fatigue. poor sleep quality was reported by % and % of pre-and post-lt patients, respectively. pre-lt fatigue was predicted by higher bmi (ß = . ) and meld (ß = . ), depression (ß = . ), and poor sleep quality (ß = . ), adj r = . , f = . , p < . . post-lt fatigue was predicted by older age (ß = . ), tension-anxiety (ß = . ), anger-hostility (ß = . ), and poor sleep quality (ß = . ), adj r = . , f = . , p < . . more fatigue was associated with lower sf- physical (r = - . ) and mental (r = - . ) qol. conclusions. fatigue and poor sleep quality are clinically significant problems for lt candidates and recipients. bmi, psychological functioning and sleep quality are modifiable variables that predict fatigue severity and should be targets of intervention when addressing fatigue symptoms. health literacy in kidney transplant recipients. elisa j. gordon, michael s. wolf. medicine, albany medical center, albany, ny; medicine, northwestern university. background: in order to successfully manage the transplant long-term, kidney recipients must have a basic understanding of key transplant-related concepts and terms indicative of their condition and treatment to properly communicate with health care providers and manage their health. kidney recipients must also possess numeracy skills to enable proper medication-taking and to monitor serum creatinine levels, bodily temperature, etc. the objective of this study was to examine health literacy levels among kidney transplant recipients. methods: we surveyed consecutive adult renal transplant recipients using the test of functional health literacy in adults (s-tofhla), and a modified version of the rapid estimate of adult literacy in medicine, called the "realm-transplant," which measured patients' knowledge of kidney transplant-related terms that patients are expected to have familiarity with. open-ended and multiple choice questions assessed numeracy related to kidney survival. results: most kidney recipients ( %) had adequate health literacy (s-tofhla), but % were unfamiliar with at least kidney transplant-related term (realm-t). patients who were less educated (p< . ), had lower income (p< . ), and were single or without a partner (p= . ) had significantly lower health literacy levels (s-tofhla). patients less familiar with transplant-related terms (realm-t) had less education (p< . ), lower income (p< . ), and were nonwhites (p= . ). the five least familiar terms were: sensitization ( %), urethra ( %), trough level ( %), blood urea nitrogen ( %), and toxicity ( %). sixteen percent wanted more information about their transplant. numeracy levels varied: % knew the likelihood of -year survival; % knew that half of kidney recipients have problems with the transplant in the first months; % knew the normal range of creatinine for kidney recipients; and % were aware of the risk of death within the first year of transplantation. conclusion: at this clinic, kidney transplant recipients generally had high levels of health literacy. however, most had difficulty recognizing frequently used transplantrelated terms, which could impede their understanding of health information and self-care management. greater efforts are needed to educate kidney recipients about transplant concepts, which may foster better self-care management, and ultimately transplant outcomes. abstracts by a single pathologist using the banff, cadi and cnit classifications. all indication biopsies with clinical acute rejection (ar; . % for sf and . % for sb) were excluded from this analysis. the histological and clinical parameters were assessed using multivariate generalized-estimating-equations statistical analysis. results: subclinical ar was present in . % sf vs. . % sb bx at mo and % sf vs % sb bx at mo (p=ns); borderline ar was seen in . % sf vs. . % sb bx at mo and % sf vs. % sb bx at mo (p=ns). despite the pristine condition of the kidneys at implantation, regardless of steroid exposure, there was a significant trend increase (p< . ) in chronic tubulo-interstitial damage; % of mo bx and % of mo bx demonstrated ifta; with moderate/severe changes (ifta grade - ) in . % and % of and mo bx respectively. the prevalence of biopsies with ischemic glomerular changes (p< . ), tubular microcalcifications (p= . ), vascular intimal thickening (p= . ) and the number of sclerosed glomeruli (p< . ) increased over the first year after transplantation, without any difference between the sb and sf group. a critical risk factor for ifta injury by multivariate analysis, independent of time after transplantation, was smaller recipient size. in this first ever serial histological analysis, embedded in a randomized multicenter pediatric study of steroid avoidance, we found significant progression of chronic graft injury in the first year post-transplantation in both study arms. small recipient size is the primary risk factor for tubulo-interstitial damage, likely related to vascular size discrepancies between recipient and the graft, resulting in chronic graft ischemia. in the -year symphony core study, a regimen with g mycophenolate mofetil (mmf) + low-dose tacrolimus ( - ng/ml) + daclizumab + steroids resulted in less acute rejections and better glomerular filtration rate (gfr) compared with g mmf + steroids and either standard-dose cyclosporine (csa), low-dose csa ( - ng/ml) + daclizumab or low-dose sirolimus ( - ng/ml) + daclizumab. methods: patients participated in an optional follow-up of years. gfr data from % of included patients ( % of the core itt population) were available at years.here we present results in the itt population. results: at inclusion into follow-up %, % and % of patients received csa, tacrolimus or sirolimus, and at years %, % and %, respectively. many follow-up patients had been switched to tacrolimus in the st year, including % of patients randomized to sirolimus. at years % of patients were on mmf and % on steroids.over the nd and rd year all arms had a low rate of biopsy-proven acute rejection (bpar; - %) and of graft loss ( - %). low-dose tacrolimus remained clearly superior in terms of bpar ( % vs. %- % in the other arms). uncensored -year graft survival was % with low-dose tacrolimus and low-dose csa, % with standarddose csa and % with low-dose sirolimus (p= . ). patient survival was between % and % (p= . ). in the four arms, the mean gfr change over the nd and rd year was between + and - ml/min and low-dose tacrolimus still had the highest gfr ( vs - ml/min, p= . ). observational follow-up results based on approximately half of the core symphony population indicate that during the nd and rd year renal function was stable, bpar and graft loss rates were low and many patients changed treatment regimen substantially. still, the itt arm with g mmf + low-dose tacrolimus + daclizumab + steroids was superior at years with respect to renal function and graft loss but differences were less marked and statistically not significant. discovering histological evaluation of time zero donor kidney biopsies has not conclusively predicted graft outcome. we hypothesize that gene expression analysis could provide additional information to determine graft outcome in the first year of transplantation. to this end, we evaluated all implantation biopsies obtained post reperfusion in deceased donors (dd) and living donors (ld) at our center. biopsies were evaluated and scored using banff criteria. low density real time pcr arrays were utilized to measure intragraft expression of genes associated with programmed cell death, fibrosis, innate and adaptive immunity, and oxidative stress signaling. results of expression were defined as folds compared to a pool of normal kidney biopsies. in dd, histological features of atn were more common ( %) than in ld grafts ( %; p< . ), whereas arteriosclerosis was infrequent in both groups ( % and %, respectively), as well as the extent of glomerular sclerosis ( % and %). there was no association between these histological features and renal function at year post transplant. not surprisingly, dd grafts displayed a pattern of gene expression remarkably different from ld, including an increased expression of complement protein c ( background: isa is a novel calcineurin inhibitor (cni), developed using a pharmacodynamic approach for use in autoimmune disease and solid organ transplantation. in moderate to severe plaque psoriasis, a canadian phase iii trial has demonstrated that isa is efficacious with minimal changes to renal function and a european trial is presently underway comparing isa to cyclosporine a (csa).in renal transplantation, a phase iia study comparing isa to csa in stable renal transplant recipients demonstrated isa to be efficacious and well tolerated. a phase iib study in de novo renal transplant patients comparing isa to tacrolimus is ongoing and final data will be available may . we hypothesize that isa is non-inferior to tacrolimus in terms of efficacy.methods: this is a month, randomized, multicenter, open-label, concentrationcontrolled study comparing three oral isa dosing groups ( . , . , or . mg/kg bid) to tacrolimus in north american transplant centres. all cni's were titrated to target trough concentrations. inclusion criteria included males and (non-pregnant) females between the ages of - who were receiving a first deceased or living donor renal transplant. cold ischemia times were to be ≤ hours, and peak panel reactive antibodies ≤ %. the primary efficacy parameter of the trial is non-inferiority (in at least one dose group) in biopsy proven acute rejection (bpar) at months as compared to tacrolimus. secondary objectives include: renal function; pk/pd relationships; patient and graft survival; and proportion of patients with hypertension, hyperlipidemia or new onset diabetes mellitus (nodm).results: interim data, as previously presented at the atc, demonstrated that isa had rejection rates similar to tacrolimus (isa . mg/kg bid %, isa . mg/kg bid %, isa . mg/kg bid %, tacrolimus %) and confirmed previous results indicating an improved safety profile. patients have now been enrolled between january and june , with an optional extension to months added to the trial. a recent approval by both fda and health canada has allowed continued use of isa in these patients until commercialization. the six month final results will be available for presentation at atc .